# Patent

## Title
Variants of glucoamylase

## Abstract

The present invention relates to combinatorial variants of a parent glucoamylase that have altered properties for reducing the synthesis of condensation products during hydrolysis of starch. Accordingly, the variants of a parent glucoamylase are suitable such as for use within brewing and glucose syrup production. Also disclosed are DNA constructs encoding the variants and methods of producing the glucoamylase variants in host cells.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is the U.S. National Stage of International Application PCT/EP2011/061082, filed Jun. 30, 2011, which designates the U.S and was published by the International Bureau in English on Jan. 5, 2012, and which claims the benefit of U.S. Provisional Application No. 61/360,891, filed Jul. 1, 2010 and 61/444,481, filed Feb. 18, 2011, Danish Patent Application No. PA 201070337, filed Jul. 15, 2010, and International Application No. PCT/EP2010/062035, filed Aug. 18, 2010, all of which are hereby incorporated by reference in their entirety.


FIELD OF THE INVENTION
Disclosed are combinatorial variants of a parent glucoamylase that have altered properties and are suitable such as for use within brewing and glucose syrup production. Also disclosed are DNA constructs encoding the variants and methods of producing the glucoamylase variants in host cells.
BACKGROUND OF THE INVENTION
Glucoamylase enzymes (glucan 1,4-α-glucohydrolases, EC 3.2.1.3) are starch hydrolyzing exo-acting carbohydrases, which catalyze the removal of successive glucose units from the non-reducing ends of starch or related oligo and polysaccharide molecules. Glucoamylases can hydrolyze both the linear and branched glucosidic linkages of starch (e.g., amylose and amylopectin).
Glucoamylases are produced by numerous strains of bacteria, fungi, yeast and plants. Particularly interesting, and commercially important, glucoamylases are fungal enzymes that are extracellularly produced, for example from strains of Aspergillus (Svensson et al., Carlsberg Res. Commun. 48: 529-544 (1983); Boel et al., EMBO J. 3: 1097-1102 (1984); Hayashida et al., Agric. Biol. Chem. 53: 923-929 (1989); U.S. Pat. No. 5,024,941; U.S. Pat. No. 4,794,175 and WO 88/09795); Talaromyces (U.S. Pat. No. 4,247,637; U.S. Pat. No. 6,255,084; and U.S. Pat. No. 6,620,924); Rhizopus (Ashikari et al., Agric. Biol. Chem. 50: 957-964 (1986); Ashikari et al., App. Microbio. Biotech. 32: 129-133 (1989) and U.S. Pat. No. 4,863,864); Humicola (WO 05/052148 and U.S. Pat. No. 4,618,579); and Mucor (Houghton-Larsen et al., Appl. Microbiol. Biotechnol. 62: 210-217 (2003)). Many of the genes that code for these enzymes have been cloned and expressed in yeast, fungal and/or bacterial cells.
Commercially, glucoamylases are very important enzymes and have been used in a wide variety of applications that require the hydrolysis of starch (e.g., for producing glucose and other monosaccharides from starch). Glucoamylases are used to produce high fructose corn sweeteners, which comprise over 50% of the sweetener market in the United States.
In general, glucoamylases may be, and commonly are, used with alpha-amylases in starch hydrolyzing processes to hydrolyze starch to dextrins and then glucose. The glucose may then be converted to fructose by other enzymes (e.g., glucose isomerases); crystallized; or used in fermentations to produce numerous end products (e.g., ethanol, citric acid, lactic acid, succinate, ascorbic acid intermediates, glutamic acid, glycerol and 1,3-propanediol). Ethanol produced by using glucoamylases in the fermentation of starch and/or cellulose containing material may be used as a source of fuel or for alcoholic consumption.
At the high solids concentrations used commercially for high glucose corn syrup (HGCS) and high fructose corn syrup (HFCS) production, glucoamylase synthesizes di-, tri-, and tetra-saccharides from glucose by condensation reactions. This occurs because of the slow hydrolysis of alpha-(1-6)-D-glucosidic bonds in starch and the formation of various accumulating condensation products, mainly isomaltose, from D-glucose. Accordingly, the glucose yield in many conventional processes does not exceed 95% of theoretical yield. The amount of syrups produced worldwide by this process is very large and even very small increases in the glucose yield pr ton of starch are commercially important.
Glucoamylase is used in brewing mainly for production of low carb beer. In combination with other amylases (such as from the malt), glucoamylase gives a very extensive hydrolysis of starch, all the way down to glucose units. Glucose is readily converted to alcohol by yeast making it possible for the breweries to obtain a very high alcohol yield from fermentation and at the same time obtain a beer, which is very low in residual carbohydrate. The ferment is diluted down to the desired alcohol % with water, and the final beer is sold as “low carb”.
Although glucoamylases have been used successfully in commercial applications for many years, a need still exists for new glucoamylases with altered properties, such as an improved specific activity, a reduced formation of condensation products such as isomaltose and increased thermostability.
Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.
SUMMARY OF THE INVENTION
The glucoamylase variants and the use of glucoamylase variants for reducing the synthesis of condensation products during hydrolysis of starch are contemplated herein. These glucoamylase variants contain amino acid substitutions within the catalytic domains and/or the starch binding domain. The variants display altered properties, such as an altered specific activity, a reduced formation of condensation products such as isomaltose and/or altered thermostability.
In one aspect, a glucoamylase variant is described herein comprising the following amino acid substitutions: a) 44R and 539R; or b) 44R, 61I and 539R, the positions corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase, wherein the glucoamylase variant has at least 80% sequence identity with SEQ ID NO: 1 or 2, or the parent glucoamylase. In a further aspect, the use is described of a glucoamylase variant for the preparation of an enzymatic composition. In a further aspect, the enzymatic composition comprises at least one additional enzyme selected among amylase, protease, pullulanase, isoamylase, cellulase, glucanase, xylanase, arabinofuranosidase, ferulic acid esterase, xylan acetyl esterase, phytase and a further glucoamylase such as for example an pullulanase and a alpha-amylase.
In a further aspect, the use is described herein of a glucoamylase variant with a starch binding domain and a catalytic domain, said variant comprising two or more amino acid substitutions relative to the amino acid sequence of SEQ ID NO:2 or equivalent parent glucoamylase in interconnecting loop 2′, and/or in loop 1, and/or in helix 2, and/or in loop 11, and/or in helix 12 for reducing the synthesis of condensation products during hydrolysis of starch.
In a further aspect, the use is described of a glucoamylase variant comprising two or more amino acid substitutions relative to interconnecting loop 2′ with the amino acid sequence from position 518 to position 543 of SEQ ID NO:2 or equivalent sequence of residues in a parent glucoamylase, and/or loop 1 with the amino acid sequence from position 21 to position 51 of SEQ ID NO:2 or equivalent sequence of residues in a parent glucoamylase, and/or helix 2 with the amino acid sequence from position 52 to position 68 of SEQ ID NO:2 or equivalent sequence of residues in a parent glucoamylase, and/or loop 11 with the amino acid sequence from position 396 to position 420 of SEQ ID NO:2 or equivalent sequence of residues in a parent glucoamylase, and/or helix 12 with the amino acid sequence from position 421 to position 434 of SEQ ID NO:2 or equivalent sequence of residues in a parent glucoamylase for reducing the synthesis of condensation products during hydrolysis of starch.
In a further aspect, the use is described of a glucoamylase variant comprising two or more amino acid substitutions relative to the amino acid sequence from position 518 to position 543 of SEQ ID NO:2 or equivalent sequence of residues in a parent glucoamylase, and/or the amino acid sequence from position 21 to position 51 of SEQ ID NO:2 or equivalent sequence of residues in a parent glucoamylase, and/or the amino acid sequence from position 52 to position 68 of SEQ ID NO:2 or equivalent sequence of residues in a parent glucoamylase, and/or the amino acid sequence from position 396 to position 420 of SEQ ID NO:2 or equivalent sequence of residues in a parent glucoamylase, and/or the amino acid sequence from position 421 to position 434 of SEQ ID NO:2 or equivalent sequence of residues in a parent glucoamylase for reducing the synthesis of condensation products during hydrolysis of starch.
In a further aspect, the use is described of a glucoamylase variant wherein said two or more amino acid substitutions are relative to the interconnecting loop 2′ with the amino acid sequence from position 518 to position 543 of SEQ ID NO:2, and/or loop 1 with the amino acid sequence from position 21 to position 51 of SEQ ID NO:2, and/or helix 2 with the amino acid sequence from position 52 to position 68 of SEQ ID NO:2, and/or loop 11 with the amino acid sequence from position 396 to position 420 of SEQ ID NO:2, and/or helix 12 with the amino acid sequence from position 421 to position 434 of SEQ ID NO:2.
In a further aspect, the use of a glucoamylase variant which when in its crystal form has a crystal structure for which the atomic coordinates of the main chain atoms have a root-mean-square deviation from the atomic coordinates of the equivalent main chain atoms of TrGA (as defined in Table 20 in WO2009/067218) of less than 0.13 nm following alignment of equivalent main chain atoms, and which have a linker region, a starch binding domain and a catalytic domain, said variant comprising two or more amino acid substitutions relative to the amino acid sequence of the parent glucoamylase in interconnecting loop 2′ of the starch binding domain, and/or in loop 1, and/or in helix 2, and/or in loop 11, and/or in helix 12 of the catalytic domain for reducing the synthesis of condensation products during hydrolysis of starch.
In one aspect, the glucoamylase variant comprises two or more amino acid substitutions, wherein an amino acid substitution is in position 539 and an amino acid substitution is in position 44, the positions corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase, and which sequence has at least 80% sequence identity to the parent glucoamylase, and wherein the amino acid substitution in position 44 is not 44C.
The present disclosure further relates to a polynucleotide encoding a glucoamylase variant as described herein. One aspect, is a plasmid comprising a nucleic acid. Another aspect, is a vector comprising a polynucleotide as described, or capable of expressing a glucoamylase variant as described. Another aspect, is a host cell comprising, e.g. transformed with, a plasmid or a vector as described. Another aspect, is a host cell, which has stably integrated into the chromosome a nucleic acid sequence encoding the variant glucoamylase. Another aspect is a cell capable of expressing a glucoamylase variant as described. Another aspect is a method of expressing a glucoamylase variant, the method comprising obtaining a host cell or a cell and expressing the glucoamylase variant from the cell or host cell, and optionally purifying the glucoamylase variant.
A further aspect of the disclosure is an enzymatic composition comprising at least one glucoamylase variant as described herein, and the use thereof.
A further aspect of the disclosure is a method for converting starch or partially hydrolyzed starch into a syrup containing glucose, which process includes saccharifying a liquid starch solution in the presence of at least one glucoamylase variant or an enzymatic composition as described herein.
A further aspect of the disclosure is the use of a glucoamylase variant as described herein in a starch conversion process, such as in a continuous starch conversion process, in a process for producing oligosaccharides, maltodextrins or glucose syrups and in a process for producing high fructose corn syrup.
In a further aspect, the use of a glucoamylase variant as described herein in a alcohol fermentation process is provided.
A further aspect of the disclosure is a method for producing a wort for brewing comprising forming a mash from a grist, and contacting the mash with a glucoamylase variant as described or an enzymatic composition as described.
Yet a further aspect of the disclosure is a method for production of a beer which comprises: a) preparing a mash, b) filtering the mash to obtain a wort, and fermenting the wort to obtain a beer, wherein a glucoamylase variant as described is added to: step (a) and/or step (b) and/or step (c).
Yet a further aspect of the disclosure is the use of a glucoamylase variant as described to enhance the production of fermentable sugars in either the mashing step or the fermentation step of a brewing process.
Yet a further aspect of the disclosure is a beer, wherein the beer is produced by the steps of: a) preparing a mash, b) filtering the mash to obtain a wort, c) fermenting the wort to obtain a beer, and d) pasteurizing the beer, wherein a glucoamylase variant as described is added to: step (a) and/or step (b) and/or step (c).
Accordingly, it is an object of the invention to not encompass within the invention any previously known product, process of making the product, or method of using the product such that Applicants reserve the right and hereby disclose a disclaimer of any previously known product, process, or method. It is further noted that the invention does not intend to encompass within the scope of the invention any product, process, or making of the product or method of using the product, which does not meet the written description and enablement requirements of the USPTO (35 U.S.C. §112, first paragraph) or the EPO (Article 83 of the EPC), such that Applicants reserve the right and hereby disclose a disclaimer of any previously described product, process of making the product, or method of using the product.
It is noted that in this disclosure and particularly in the claims and/or embodiments, terms such as “comprises”, “comprised”, “comprising” and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean “includes”, “included”, “including”, and the like; and that terms such as “consisting essentially of” and “consists essentially of” have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.
These and other embodiments are disclosed or are obvious from and encompassed by, the following Detailed Description.



BRIEF DESCRIPTION OF THE DRAWINGS
The following detailed description, given by way of example, but not intended to limit the invention solely to the specific embodiments described, may best be understood in conjunction with the accompanying drawings, in which:
FIG. 1A depicts a Trichoderma reesei glucoamylase (TrGA) having 632 amino acids (SEQ ID NO: 1). The signal peptide is underlined, the catalytic region (SEQ ID NO: 3) starting with amino acid residues SVDDFI (SEQ ID NO: 12) and having 453 amino acid residues is in bold; the linker region is in italics and the starch binding domain (SBD) is both italics and underlined. The mature protein of TrGA (SEQ ID NO: 2) includes the catalytic domain (SEQ ID NO: 3), linker region (SEQ ID NO: 10), and starch binding domain (SEQ ID NO: 11). With respect to the SBD numbering of the TrGA glucoamylase molecule, reference is made in the present disclosure to either a) positions 491 to 599 in SEQ ID NO:2 of the mature TrGA, and/or b) positions 1 to 109 in SEQ ID NO:11, which represents the isolated SBD sequence of the mature TrGA. With respect to the catalytic domain numbering of the TrGA molecule, reference is made to SEQ ID NO: 2 and/or SEQ ID NO: 3. FIG. 1B depicts the cDNA (SEQ ID NO:4) that codes for the TrGA. FIG. 1C depicts the precursor and mature protein TrGA domains.
FIG. 2 depicts the destination plasmid pDONR-TrGA which includes the cDNA (SEQ ID NO: 4) of the TrGA.
FIG. 3 depicts the plasmid pTTT-Dest.
FIG. 4 depicts the final expression vector pTTT-TrGA.
FIGS. 5A and 5B depict an alignment comparison of the catalytic domains of parent glucoamylases from Aspergillus awamori (AaGA) (SEQ ID NO: 5); Aspergillus niger (AnGA) (SEQ ID NO: 6); Aspergillus oryzae (AoGA) (SEQ ID NO: 7); Trichoderma reesei (TrGA) (SEQ ID NO: 3); Humicola grisea (HgGA) (SEQ ID NO: 8); and Hypocrea vinosa (HvGA) (SEQ ID NO: 9). Identical amino acids are indicated by an asterisk (*). FIG. 5C depicts a Talaromyces glucoamylase (TeGA) mature protein sequence (SEQ ID NO: 384). FIGS. 5D and 5E depict an alignment comparing the Starch Binding Domain (SBD) of parent glucoamylases from Trichoderma reesei (SEQ ID NO: 11); Humicola grisea (HgGA) (SEQ ID NO: 385); Thermomyces lanuginosus (ThGA) (SEQ ID NO: 386); Talaromyces emersonii (TeGA) (SEQ ID NO: 387); Aspergillus niger (AnGA) (SEQ ID NO: 388); Aspergillus awamori (AaGA) (SEQ ID NO: 389); and Thielavia terrestris (TtGA) (SEQ ID NO: 390).
FIG. 6 depicts a comparison of the three dimensional structure of Trichoderma reesei glucoamylase (black) (SEQ ID NO: 2) and Aspergillus awamori glucoamylase (grey) (SEQ ID NO: 5) viewed from the side. The side is measured in reference to the active site and the active site entrance is at the “top” of the molecule.
FIG. 7 depicts a comparison of the three dimensional structures of Trichoderma reesei glucoamylase (black) (SEQ ID NO: 2) and Aspergillus awamori glucoamylase (grey) (SEQ ID NO: 5) viewed from the top.
FIG. 8 depicts an alignment of the three dimensional structures of TrGA (SEQ ID NO: 2) and AnGA (SEQ ID NO: 6) viewed from the side showing binding sites 1 and 2.
FIG. 9 depicts a model of the binding of acarbose to the TrGA crystal structure.
FIG. 10 depicts a TLC plate with standards containing different concentrations of glucose, maltose and isomaltose and samples containing reaction products from glucose incubated with TrGA and AnGA.



DETAILED DISCLOSURE OF THE INVENTION
Glucoamylases are commercially important enzymes in a wide variety of applications that require the hydrolysis of starch. The applicants have found that by introducing certain alterations in positions within specific regions of the amino acid sequence of a parent glucoamylase the rate of forming alpha-(1-6) bonds is reduced, and/or the formation of condensation products such as isomaltose is reduced. A reduction of the rate that glucoamylase forms alpha-(1-6) bonds relative to the rate it cleaves alpha-(1-4) bonds has practical implications.
The present inventors have provided a number of variants of a parent glucoamylase, which variants in some embodiments show a reduced condensation and/or a reduced ratio between isomaltose synthesis and starch hydrolysis activity (IS/SH ratio) as compared to the parent glucoamylase. In some embodiments using a glucoamylase variant as described herein in a saccharification process produces a syrup with high glucose percentage. In some embodiments using a glucoamylase variant as described herein results in an enhanced production of fermentable sugars in a mashing and/or fermentation step of a brewing step. In some embodiments using a glucoamylase variant as described herein results in an enhanced real degree of fermentation. These altered properties are obtained by mutating e.g. substituting selected positions in a parent glucoamylase. This will be described in more detail below.
Accordingly, in a further aspect, the use is described of a glucoamylase variant comprising two or more amino acid substitutions relative to interconnecting loop 2′ with the amino acid sequence from position 518 to position 543 of SEQ ID NO:2 or equivalent sequence of residues in a parent glucoamylase, and/or loop 1 with the amino acid sequence from position 21 to position 51 of SEQ ID NO:2 or equivalent sequence of residues in a parent glucoamylase, and/or helix 2 with the amino acid sequence from position 52 to position 68 of SEQ ID NO:2 or equivalent sequence of residues in a parent glucoamylase, and/or loop 11 with the amino acid sequence from position 396 to position 420 of SEQ ID NO:2 or equivalent sequence of residues in a parent glucoamylase, and/or helix 12 with the amino acid sequence from position 421 to position 434 of SEQ ID NO:2 or equivalent sequence of residues in a parent glucoamylase for reducing the synthesis of condensation products during hydrolysis of starch.
In a further aspect, the use is described of a glucoamylase variant comprising two or more amino acid substitutions relative to the amino acid sequence from position 518 to position 543 of SEQ ID NO:2 or equivalent sequence of residues in a parent glucoamylase, and/or the amino acid sequence from position 21 to position 51 of SEQ ID NO:2 or equivalent sequence of residues in a parent glucoamylase, and/or the amino acid sequence from position 52 to position 68 of SEQ ID NO:2 or equivalent sequence of residues in a parent glucoamylase, and/or the amino acid sequence from position 396 to position 420 of SEQ ID NO:2 or equivalent sequence of residues in a parent glucoamylase, and/or the amino acid sequence from position 421 to position 434 of SEQ ID NO:2 or equivalent sequence of residues in a parent glucoamylase for reducing the synthesis of condensation products during hydrolysis of starch.
Accordingly, in a further aspect, the use of a glucoamylase variant is described, which glucoamylase variant when in its crystal form has a crystal structure for which the atomic coordinates of the main chain atoms have a root-mean-square deviation from the atomic coordinates of the equivalent main chain atoms of TrGA (as defined in Table 20 in WO2009/067218) of less than 0.13 nm following alignment of equivalent main chain atoms, and which have a linker region, a starch binding domain and a catalytic domain, said variant comprising two or more amino acid substitutions relative to the amino acid sequence of the parent glucoamylase in interconnecting loop 2′ of the starch binding domain, and/or in loop 1, and/or in helix 2, and/or in loop 11, and/or in helix 12 of the catalytic domain for reducing the synthesis of condensation products during hydrolysis of starch. In a further aspect, the root-mean-square deviation from the atomic coordinates of the equivalent main chain atoms of TrGA (as defined in Table 20 in WO2009/067218) is less than 0.12 nm, such as less than 0.11 or such as less than 0.10.
In one aspect, the use is described herein of a glucoamylase variant with a starch binding domain and a catalytic domain, said variant comprising two or more amino acid substitutions relative to the amino acid sequence of SEQ ID NO:2 or equivalent parent glucoamylase in interconnecting loop 2′, and/or in loop 1, and/or in helix 2, and/or in loop 11, and/or in helix 12 for reducing the synthesis of condensation products during hydrolysis of starch.
In a further aspect, the use is described of a glucoamylase variant wherein said two or more amino acid substitutions are relative to the interconnecting loop 2′ with the amino acid sequence from position 518 to position 543 of SEQ ID NO:2 or equivalent sequence of residues in parent glucoamylase, and/or loop 1 with the amino acid sequence from position 21 to position 51 of SEQ ID NO:2 or equivalent sequence of residues in parent glucoamylase, and/or helix 2 with the amino acid sequence from position 52 to position 68 of SEQ ID NO:2 or equivalent sequence of residues in parent glucoamylase, and/or loop 11 with the amino acid sequence from position 396 to position 420 of SEQ ID NO:2 or equivalent sequence of residues in parent glucoamylase, and/or helix 12 with the amino acid sequence from position 421 to position 434 of SEQ ID NO:2 or equivalent sequence of residues in parent glucoamylase.
In a further aspect, the use is described of a glucoamylase variant wherein said two or more amino acid substitutions are relative to the interconnecting loop 2′ with the amino acid sequence from position 518 to position 543 of SEQ ID NO:2, and/or loop 1 with the amino acid sequence from position 21 to position 51 of SEQ ID NO:2, and/or helix 2 with the amino acid sequence from position 52 to position 68 of SEQ ID NO:2, and/or loop 11 with the amino acid sequence from position 396 to position 420 of SEQ ID NO:2, and/or helix 12 with the amino acid sequence from position 421 to position 434 of SEQ ID NO:2.
In a further aspect, the two or more amino acid substitutions are at least one such as one, two or three amino acid substitution in the interconnecting loop 2′ and at least one such as one, two, three, four, five or six amino acid substitution in loop 1 and/or helix 2 and/or loop 11 and/or helix 12.
In a further aspect, the two or more amino acid substitutions are one, two, three or four amino acid substitutions in the interconnecting loop 2′ and one, two, three or four amino acid substitutions in loop 1 and/or helix 2 and/or loop 11 and/or helix 12. In a further aspect, there are one, two, three or four amino acid substitutions in the interconnecting loop 2′. In a further aspect, there are one, two, three or four amino acid substitutions in loop 1. In a further aspect, there are one, two, three or four amino acid substitutions in helix 2. In a further aspect, there are one, two, three or four amino acid substitutions in loop 11. In a further aspect, there are one, two, three or four amino acid substitutions in helix 12.
In a further aspect, the two or more amino acid substitutions are at least one amino acid substitution in interconnecting loop 2′ and at least one amino acid substitution in loop 1.
In a further aspect, the two or more amino acid substitutions are at least one amino acid substitution in interconnecting loop 2′ and at least one amino acid substitution in helix 2.
In a further aspect, the two or more amino acid substitutions are at least one amino acid substitution in interconnecting loop 2′ and at least one amino acid substitution in loop 11.
In a further aspect, the two or more amino acid substitutions are at least one amino acid substitution in interconnecting loop 2′ and at least one amino acid substitution in helix 12.
In a further aspect, the two or more amino acid substitutions are at least one amino acid substitution in interconnecting loop 2′ and at least one amino acid substitution in loop 1 and at least one amino acid substitution in helix 2.
In a further aspect, the glucoamylase variant has at least one amino acid substitution within position 520-543, 530-543, or 534-543 of interconnecting loop 2′, the positions corresponding to the respective position in SEQ ID NO:2 or equivalent positions in a parent glucoamylase.
In a further aspect, the glucoamylase variant has at least one amino acid substitution within the amino acid sequence of position 30-50, 35-48, or 40-46 of loop 1, the positions corresponding to the respective position in SEQ ID NO:2 or equivalent positions in a parent glucoamylase.
In a further aspect, the glucoamylase variant has at least one amino acid substitution within the amino acid sequence of position 50-66, 55-64, or 58-63 of helix 2, the positions corresponding to the respective position in SEQ ID NO:2 or equivalent positions in a parent glucoamylase.
In a further aspect, the glucoamylase variant has at least one amino acid substitution within the amino acid sequence of position 405-420, 410-420, or 415-420 of loop 11, the positions corresponding to the respective position in SEQ ID NO:2 or equivalent positions in a parent glucoamylase.
In a further aspect, the glucoamylase variant has at least one amino acid substitution within the amino acid sequence of position 421-434, 425-434, or 428-434 of helix 12, the positions corresponding to the respective position in SEQ ID NO:2 or equivalent positions in a parent glucoamylase.
In a further aspect, the glucoamylase variant has at least 80%, 85%, 90%, 95%, 98%, or 99.5% sequence identity to the parent glucoamylase, such as at least 80%, 85%, 90%, 95%, 98%, or 99.5% sequence identity to SEQ ID NO: 1, 2, 3, 5, 6, 7, 8, or 9. In one aspect, the glucoamylase variant has at least 80%, 85%, 90%, 95%, 98%, or 99.5% sequence identity to SEQ ID NO:2.
In a further aspect, the parent glucoamylase or the glucoamylase variant has a starch binding domain that has at least 96%, 97%, 98%, 99%, or 99.5% sequence identity with the starch binding domain of SEQ ID NO: 1, 2, 11, 385, 386, 387, 388, 389, or 390. In a further aspect, the parent glucoamylase or the glucoamylase variant has a catalytic domain that has at least 80%, 85%, 90%, 95%, or 99.5% sequence identity with the catalytic domain of SEQ ID NO: 1, 2, 3, 5, 6, 7, 8, or 9.
In one aspect, the glucoamylase variant has an amino acid substitution in position 539 and one or more amino acid substitutions in a position selected from position 44, 61, 417 and 431, the positions corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase. In one aspect, the glucoamylase variant has an amino acid substitution in position 539 and a) an amino acid substitution in position 44 and/or b) amino acid substitutions in both positions 417 and 431, the positions corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase. In one aspect, the glucoamylase variant has an amino acid substitution in position 539 and an amino acid substitution in position 44, the positions corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase. In one aspect, the glucoamylase variant has an amino acid substitution in position 539 and amino acid substitutions in positions 417 and 431, the positions corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase. In one aspect, the glucoamylase variant has an amino acid substitution in position 539 and amino acid substitutions in positions 44 and 61, the positions corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase. In one aspect, the glucoamylase variant has an amino acid substitution in position 43, the position corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase. In one aspect, the glucoamylase variant has an amino acid substitution in position 61, the position corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase. In one aspect, the amino acid substitution in position 539 is 539R, the position corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase. In one aspect, the amino acid substitution in position 44 is 44R, the position corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase. In one aspect, the amino acid substitution in position 417 is 417R/V, the position corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase. In one aspect, the amino acid substitution in position 417 is 417R, the position corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase. In one aspect, the amino acid substitution in position 417 is 417V, the position corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase. In one aspect, the amino acid substitution in position 431 is 431L, the position corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase. In one aspect, the amino acid substitution in position 43 is 43R, the position corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase. In one aspect, the amino acid substitution in position 61 is 61I, the position corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase.
In one aspect, the condensation product is isomaltose. In one aspect, the hydrolysis of starch is in a brewing process. In for example brewing, the formation of isomaltose is undesired because it can not be converted into alcohol during fermentation.
Beer is traditionally referred to as an alcoholic beverage derived from malt, such as malt derived from barley, and optionally adjuncts, such as cereal grains, and flavoured with hops.
Beer can be made from a variety of grains by essentially the same process. All grain starches are glucose homopolymers in which the glucose residues are linked by either alpha-1,4- or alpha-1,6-bonds, with the former predominating.
The process of making fermented malt beverages is commonly referred to as brewing. The principal raw materials used in making these beverages are water, hops and malt. In addition, adjuncts such as common corn grits, refined corn grits, brewer's milled yeast, rice, sorghum, refined corn starch, barley, barley starch, dehusked barley, wheat, wheat starch, torrified cereal, cereal flakes, rye, oats, potato, tapioca, and syrups, such as corn syrup, sugar cane syrup, inverted sugar syrup, barley and/or wheat syrups, and the like may be used as a source of starch. The starch will eventually be converted into dextrins and fermentable sugars.
For a number of reasons, the malt, which is produced principally from selected varieties of barley, is believed to have the greatest effect on the overall character and quality of the beer. First, the malt is the primary flavouring agent in beer. Second, the malt provides the major portion of the fermentable sugar. Third, the malt provides the proteins, which will contribute to the body and foam character of the beer. Fourth, the malt provides the necessary enzymatic activity during mashing.
Hops also contribute significantly to beer quality, including flavouring. In particular, hops (or hops constituents) add desirable bittering substances to the beer. In addition, the hops act as protein precipitants, establish preservative agents and aid in foam formation and stabilization.
The process for making beer is well known in the art, but briefly, it involves five steps: (a) mashing and/or adjunct cooking (b) wort separation and extraction (c) boiling and hopping of wort (d) cooling, fermentation and storage, and (e) maturation, processing and packaging.
Typically, in the first step, milled or crushed malt is mixed with water and held for a period of time under controlled temperatures to permit the enzymes present in the malt to convert the starch present in the malt into fermentable sugars.
In the second step, the mash is transferred to a “Iauter tun” or mash filter where the liquid is separated from the grain residue. This sweet liquid is called “wort” and the left over grain residue is called “spent grain”. The mash is typically subjected to an extraction, which involves adding water to the mash in order to recover the residual soluble extract from the spent grain.
In the third step, the wort is boiled vigorously. This sterilizes the wort and helps to develop the colour, flavour and odour and inactivates enzyme activities. Hops are added at some point during the boiling.
In the fourth step, the wort is cooled and transferred to a fermentor, which either contains the yeast or to which yeast is added. The yeast converts the sugars by fermentation into alcohol and carbon dioxide gas; at the end of fermentation the fermentor is chilled or the fermentor may be chilled to stop fermentation. The yeast flocculates and is removed.
In the last step, the beer is cooled and stored for a period of time, during which the beer clarifies and its flavour develops, and any material that might impair the appearance, flavour and shelf life of the beer settles out. Prior to packaging, the beer is carbonated and, optionally, filtered and pasteurized.
After fermentation, a beverage is obtained which usually contains from about 2% to about 10% alcohol by weight. The non-fermentable carbohydrates are not converted during fermentation and form the majority of the dissolved solids in the final beer.
This residue remains because of the inability of malt amylases to hydrolyze the alpha-1,6-linkages of the starch. The non-fermentable carbohydrates contribute about 50 calories per 12 ounces of beer.
Further information on conventional brewing processes, as well as definitions for terms used in the field of brewing technology to be applied for the present invention, may be found in “Technology Brewing and Malting” by Wolfgang Kunze of the Research and Teaching Institute of Brewing, Berlin (VLB), 2nd revised Edition 1999, ISBN 3-921690-39-0 or 3rd edition (2004): ISBN 3-921690-49-8.
Recently, there has been a widespread popularization of brewed beverages called light beers, reduced calorie beers or low calorie beers, particularly in the U.S. market. As defined in the U.S., these beers have approximately 30% fewer calories than a manufacturer's “normal” beer.
As used herein, the term “light beers, reduced calorie beers or low calorie beers”, refers to the recent, widespread popularization of brewed beverages, particularly in the U.S. market. As defined in the U.S., these highly attenuated beers have approximately 30% fewer calories than a manufacturer's “normal beer”. Further information on conventional brewing processes may be found in “Technology Brewing and Malting” by Wolfgang Kunze of the Research and Teaching Institute of Brewing, Berlin (VLB), 3rd completely updated edition, 2004, ISBN 3-921690-49-8.”
Disclosed herein is the use of a glucoamylase variant as described herein, wherein the production of fermentable sugar(s) is enhanced as compared to the parent glucoamylase, such as TrGA. Further disclosed herein is the use of a glucoamylase variant as described herein, wherein the production of fermentable sugars is enhanced in a mashing step of the brewing process as compared to the parent glucoamylase, such as TrGA. Disclosed herein is the use of a glucoamylase variant as described herein, wherein the production of fermentable sugars is enhanced in a fermentation step of a brewing process as compared to the parent glucoamylase, such as TrGA. Disclosed herein is the use of a glucoamylase variant as described herein, wherein the fermentable sugar is glucose.
A glucoamylase that can produce glucose with a significantly reduced amount of by-products would be of great commercial interest, e.g. in production of glucose syrup or in brewing. Further disclosed herein is the use of a glucoamylase variant as described herein, wherein the hydrolysis of starch is in a process for producing glucose syrup. In one aspect, the glucoamylase exhibit a reduced ratio between isomaltose synthesis (IS) and starch hydrolysis activity (SH) as compared to the parent glucoamylase, such as TrGA. In one aspect, the glucoamylase exhibit a reduced starch hydrolysis activity, which is not more than 5%, not more than 10% or not more than 15% reduced as compared to the parent glucoamylase, such as TrGA. In one aspect, the glucoamylase exhibit an enhanced real degree of fermentation as compared to the parent glucoamylase such as TrGA. In one aspect, the glucoamylase forms a lower amount of condensation products than the amount of condensation products formed by the glucoamylase Aspergillus niger (AnGA) (SEQ ID NO: 6) under comparable conditions. In one aspect, the glucoamylase forms an amount of condensation products which amount is essentially the same as, not more than 5% higher, not more than 8% higher or not more than 10% higher than the amount of condensation products formed by Aspergillus niger (AnGA) (SEQ ID NO: 6) under comparable conditions. In one aspect, dosing of the glucoamylases are the same based on protein concentration. In one aspect, dosing of the glucoamylases are the same based on measurement of activity in activity assays.
Glucoamylase variants described herein contain amino acid substitutions within the catalytic domain and/or the starch binding domain. The variants may display altered properties such as improved thermostability, altered formation of condensation products such as isomaltose and/or an enhanced real degree of fermentation and/or a reduced ratio between isomaltose synthesis (IS) and starch hydrolysis activity (SH) and/or specific activity. The variants with reduced formation of condensation products such as isomaltose may significantly improve the ability to make desired products in the brewing industry, for example.
1. Definitions and Abbreviations
1.1. Definitions
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Singleton et al., Dictionary Of Microbiology And Molecular Biology, 2nd ed., John Wiley and Sons, New York (1994), and Hale & Markham, The Harper Collins Dictionary Of Biology, Harper Perennial, N.Y. (1991) provide one of skill with the general meaning of many of the terms used herein. Certain terms are defined below for the sake of clarity and ease of reference.
As used herein, the term “glucoamylase (EC 3.2.1.3)” refers to an enzyme that catalyzes the release of D-glucose from the non-reducing ends of starch and related oligo- and polysaccharides.
The term “parent” or “parent sequence” refers to a sequence that is native or naturally occurring in a host cell. Parent glucoamylases include, but are not limited to, the glucoamylase sequences set forth in SEQ ID NOs: 1, 2, 3, 5, 6, 7, 8, and 9, and glucoamylases with at least 80% amino acid sequence identity to SEQ ID NO: 2.
As used herein, an “equivalent position” means a position that is common to two parent sequences that is based on an alignment of the amino acid sequence of the parent glucoamylase in question as well as alignment of the three-dimensional structure of the parent glucoamylase in question with the TrGA reference glucoamylase amino acid sequence (SEQ ID NO: 2) and three-dimensional structure. Thus either sequence alignment or structural alignment may be used to determine equivalence.
The term “TrGA” refers to a parent Trichoderma reesei glucoamylase sequence having the mature protein sequence illustrated in SEQ ID NO: 2 that includes the catalytic domain having the sequence illustrated in SEQ ID NO: 3. The isolation, cloning and expression of the TrGA are described in WO 2006/060062 and U.S. Pat. No. 7,413,887, both of which are incorporated herein by reference. In some embodiments, the parent sequence refers to a glucoamylase sequence that is the starting point for protein engineering. The numbering of the glucoamylase amino acids herein is based on the sequence alignment of a glucoamylase with TrGA (SEQ ID NO: 2 and/or 3).
The phrase “mature form of a protein or polypeptide” refers to the final functional form of the protein or polypeptide. A mature form of a glucoamylase may lack a signal peptide, for example. To exemplify, a mature form of the TrGA includes the catalytic domain, linker region and starch binding domain having the amino acid sequence of SEQ ID NO: 2.
As used herein, the terms “glucoamylase variant” and “variant” are used in reference to glucoamylases that have some degree of amino acid sequence identity to a parent glucoamylase sequence. A variant is similar to a parent sequence, but has at least one substitution, deletion or insertion in their amino acid sequence that makes them different in sequence from a parent glucoamylase. In some cases, variants have been manipulated and/or engineered to include at least one substitution, deletion, or insertion in their amino acid sequence that makes them different in sequence from a parent. Additionally, a glucoamylase variant may retain the functional characteristics of the parent glucoamylase, e.g., maintaining a glucoamylase activity that is at least about 50%, about 60%, about 70%, about 80%, or about 90% of that of the parent glucoamylase. Can also have higher activity than 100% if that is what one has selected for.
“Variants” may have at least about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 88%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% sequence identity to a parent polypeptide sequence when optimally aligned for comparison. In some embodiments, the glucoamylase variant may have at least about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 88%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% sequence identity to the catalytic domain of a parent glucoamylase. In some embodiments, the glucoamylase variant may have at least at least about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 88%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% sequence identity to the starch binding domain of a parent glucoamylase. The sequence identity can be measured over the entire length of the parent or the variant sequence.
Sequence identity is determined using standard techniques known in the art (see e.g., Smith and Waterman, Adv. Appl. Math. 2: 482 (1981); Needleman and Wunsch, J. Mol. Biol. 48: 443 (1970); Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85: 2444 (1988); programs such as GAP, BESTHT, FASTA, and TFASTA in the Wisconsin Genetics Software Package (Genetics Computer Group, Madison, Wis.); and Devereux et al., Nucleic Acid Res., 12: 387-395 (1984)).
The “percent (%) nucleic acid sequence identity” or “percent (%) amino acid sequence identity” is defined as the percentage of nucleotide residues or amino acid residues in a candidate sequence that are identical with the nucleotide residues or amino acid residues of the starting sequence (e.g., SEQ ID NO 2). The sequence identity can be measured over the entire length of the starting sequence.
“Sequence identity” is determined herein by the method of sequence alignment. For the purpose of the present disclosure, the alignment method is BLAST described by Altschul et al., (Altschul et al., J. Mol. Biol. 215: 403-410 (1990); and Karlin et al, Proc. Natl. Acad. Sci. USA 90: 5873-5787 (1993)). A particularly useful BLAST program is the WU-BLAST-2 program (see Altschul et al, Meth. Enzymol. 266: 460-480 (1996)). WU-BLAST-2 uses several search parameters, most of which are set to the default values. The adjustable parameters are set with the following values: overlap span=1, overlap fraction=0.125, word threshold (T)=11. The HSP S and HSP S2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched. However, the values may be adjusted to increase sensitivity. A % amino acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues of the “longer” sequence in the aligned region. The “longer” sequence is the one having the most actual residues in the aligned region (gaps introduced by WU-Blast-2 to maximize the alignment score are ignored).
The term “optimal alignment” refers to the alignment giving the highest percent identity score.
As used herein the term “catalytic domain” refers to a structural region of a polypeptide, which contains the active site for substrate hydrolysis.
The term “linker” refers to a short amino acid sequence generally having between 3 and 40 amino acids residues that covalently bind an amino acid sequence comprising a starch binding domain with an amino acid sequence comprising a catalytic domain.
The term “starch binding domain” refers to an amino acid sequence that binds preferentially to a starch substrate.
As used herein, the terms “mutant sequence” and “mutant gene” are used interchangeably and refer to a polynucleotide sequence that has an alteration in at least one codon occurring in a host cell's parent sequence. The expression product of the mutant sequence is a variant protein with an altered amino acid sequence relative to the parent. The expression product may have an altered functional capacity (e.g., enhanced enzymatic activity).
The term “property” or grammatical equivalents thereof in the context of a polypeptide, as used herein, refers to any characteristic or attribute of a polypeptide that can be selected or detected. These properties include, but are not limited to oxidative stability, substrate specificity, catalytic activity, thermal stability, pH activity profile, resistance to proteolytic degradation, KM, KCAT, KCAT/KM ratio, protein folding, ability to bind a substrate and ability to be secreted.
The term “property” or grammatical equivalent thereof in the context of a nucleic acid, as used herein, refers to any characteristic or attribute of a nucleic acid that can be selected or detected. These properties include, but are not limited to, a property affecting gene transcription (e.g., promoter strength or promoter recognition), a property affecting RNA processing (e.g., RNA splicing and RNA stability), a property affecting translation (e.g., regulation, binding of mRNA to ribosomal proteins).
The terms “thermally stable” and “thermostable” refer to glucoamylase variants of the present disclosure that retain a specified amount of enzymatic activity after exposure to a temperature over a given period of time under conditions prevailing during the hydrolysis of starch substrates, for example, while exposed to altered temperatures.
The term “enhanced stability” in the context of a property such as thermostability refers to a higher retained starch hydrolytic activity over time as compared to another reference (i.e., parent) glucoamylase.
The term “diminished stability” in the context of a property such as thermostability refers to a lower retained starch hydrolytic activity over time as compared to another reference glucoamylase.
The term “specific activity” is defined as the activity per mg of glucoamylase protein. In some embodiments, the activity for glucoamylase is determined by the ethanol assay described herein and expressed as the amount of glucose that is produced from the starch substrate. In some embodiments, the protein concentration can be determined using the Caliper assay described herein.
The terms “active” and “biologically active” refer to a biological activity associated with a particular protein. It follows that the biological activity of a given protein refers to any biological activity typically attributed to that protein by those skilled in the art. For example, an enzymatic activity associated with a glucoamylase is hydrolytic and, thus an active glucoamylase has hydrolytic activity.
The terms “polynucleotide” and “nucleic acid”, used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. These terms include, but are not limited to, a single-, double- or triple-stranded DNA, genomic DNA, cDNA, RNA, DNA-RNA hybrid, or a polymer comprising purine and pyrimidine bases, or other natural, chemically, biochemically modified, non-natural or derivatized nucleotide bases.
As used herein, the terms “DNA construct,” “transforming DNA” and “expression vector” are used interchangeably to refer to DNA used to introduce sequences into a host cell or organism. The DNA may be generated in vitro by PCR or any other suitable technique(s) known to those in the art. The DNA construct, transforming DNA or recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. Typically, the recombinant expression cassette portion of an expression vector, DNA construct or transforming DNA includes, among other sequences, a nucleic acid sequence to be transcribed and a promoter. In some embodiments, expression vectors have the ability to incorporate and express heterologous DNA fragments in a host cell.
As used herein, the term “vector” refers to a polynucleotide construct designed to introduce nucleic acids into one or more cell types. Vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, cassettes, and the like.
As used herein in the context of introducing a nucleic acid sequence into a cell, the term “introduced” refers to any method suitable for transferring the nucleic acid sequence into the cell. Such methods for introduction include but are not limited to protoplast fusion, transfection, transformation, conjugation, and transduction.
As used herein, the terms “transformed” and “stably transformed” refers to a cell that has a non-native (heterologous) polynucleotide sequence integrated into its genome or as an episomal plasmid that is maintained for at least two generations.
As used herein, the terms “selectable marker” and “selective marker” refer to a nucleic acid (e.g., a gene) capable of expression in host cells that allows for ease of selection of those hosts containing the vector. Typically, selectable markers are genes that confer antimicrobial resistance or a metabolic advantage on the host cell to allow cells containing the exogenous DNA to be distinguished from cells that have not received any exogenous sequence during the transformation.
As used herein, the term “promoter” refers to a nucleic acid sequence that functions to direct transcription of a downstream gene. The promoter, together with other transcriptional and translational regulatory nucleic acid sequences (also termed “control sequences”) is necessary to express a given gene. In general, the transcriptional and translational regulatory sequences include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences.
A nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA encoding a secretory leader (i.e., a signal peptide), is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide. Generally, “operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase.
As used herein the term “gene” refers to a polynucleotide (e.g., a DNA segment), that encodes a polypeptide and includes regions preceding and following the coding regions, as well as intervening sequences (introns) between individual coding segments (exons).
As used herein, “ortholog” and “orthologous genes” refer to genes in different species that have evolved from a common ancestral gene (i.e., a homologous gene) by speciation. Typically, orthologs retain the same function during the course of evolution. Identification of orthologs finds use in the reliable prediction of gene function in newly sequenced genomes.
As used herein, “paralog” and “paralogous genes” refer to genes that are related by duplication within a genome. While orthologs retain the same function through the course of evolution, paralogs evolve new functions, even though some functions are often related to the original one. Examples of paralogous genes include, but are not limited to genes encoding trypsin, chymotrypsin, elastase, and thrombin, which are all serine proteinases and occur together within the same species.
As used herein, the term “hybridization” refers to the process by which a strand of nucleic acid joins with a complementary strand through base pairing, as known in the art.
A nucleic acid sequence is considered to be “selectively hybridizable” to a reference nucleic acid sequence if the two sequences specifically hybridize to one another under moderate to high stringency hybridization and wash conditions. Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex or probe. For example, “maximum stringency” typically occurs at about Tm−5° C. (5° C. below the Tm of the probe); “high stringency” at about 5-10° C. below the Tm; “intermediate stringency” at about 10-20° C. below the Tm of the probe; and “low stringency” at about 20-25° C. below the Tm. Functionally, maximum stringency conditions may be used to identify sequences having strict identity or near-strict identity with the hybridization probe; while an intermediate or low stringency hybridization can be used to identify or detect polynucleotide sequence homologs.
Moderate and high stringency hybridization conditions are well known in the art. An example of high stringency conditions includes hybridization at about 42° C. in 50% formamide, 5×SSC, 5×Denhardt's solution, 0.5% SDS and 100 μg/ml denatured carrier DNA followed by washing two times in 2×SSC and 0.5% SDS at room temperature and two additional times in 0.1×SSC and 0.5% SDS at 42° C. An example of moderate stringent conditions include an overnight incubation at 37° C. in a solution comprising 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate and 20 mg/ml denaturated sheared salmon sperm DNA, followed by washing the filters in 1×SSC at about 37-50° C. Those of skill in the art know how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.
As used herein, “recombinant” includes reference to a cell or vector, that has been modified by the introduction of a heterologous or homologous nucleic acid sequence or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found in identical form within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all as a result of deliberate human intervention.
In an embodiment of the disclosure, mutated DNA sequences are generated with site saturation mutagenesis in at least one codon. In another embodiment, site saturation mutagenesis is performed for two or more codons. In a further embodiment, mutant DNA sequences have more than about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 98% identity with the parent sequence. In alternative embodiments, mutant DNA is generated in vivo using any known mutagenic procedure such as, for example, radiation, nitrosoguanidine, and the like. The desired DNA sequence is then isolated and used in the methods provided herein.
As used herein, “heterologous protein” refers to a protein or polypeptide that does not naturally occur in the host cell.
An enzyme is “over-expressed” in a host cell if the enzyme is expressed in the cell at a higher level than the level at which it is expressed in a corresponding wild-type cell.
The terms “protein” and “polypeptide” are used interchangeability herein. In the present disclosure and claims, the conventional one-letter and three-letter codes for amino acid residues are used. The 3-letter code for amino acids as defined in conformity with the IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN). It is also understood that a polypeptide may be coded for by more than one nucleotide sequence due to the degeneracy of the genetic code.
Variants of the disclosure are described by the following nomenclature: [original amino acid residue/position/substituted amino acid residue]. For example, the substitution of leucine for arginine at position 76 is represented as R76L. When more than one amino acid is substituted at a given position, the substitution is represented as 1) Q172C, Q172D or Q172R; 2) Q172C, D, or R, or 3) Q172C/D/R. When a position suitable for substitution is identified herein without a specific amino acid suggested, it is to be understood that any amino acid residue may be substituted for the amino acid residue present in the position. Where a variant glucoamylase contains a deletion in comparison with other glucoamylases the deletion is indicated with “*”. For example, a deletion at position R76 is represented as R76*. A deletion of two or more consecutive amino acids is indicated for example as (76-78)*.
A “prosequence” is an amino acid sequence between the signal sequence and mature protein that is necessary for the secretion of the protein. Cleavage of the pro sequence will result in a mature active protein.
The term “signal sequence” or “signal peptide” refers to any sequence of nucleotides and/or amino acids that may participate in the secretion of the mature or precursor forms of the protein. This definition of signal sequence is a functional one, meant to include all those amino acid sequences encoded by the N-terminal portion of the protein gene, which participate in the effectuation of the secretion of protein. They are often, but not universally, bound to the N-terminal portion of a protein or to the N-terminal portion of a precursor protein. The signal sequence may be endogenous or exogenous. The signal sequence may be that normally associated with the protein (e.g., glucoamylase), or may be from a gene encoding another secreted protein.
The term “precursor” form of a protein or peptide refers to a mature form of the protein having a prosequence operably linked to the amino or carbonyl terminus of the protein. The precursor may also have a “signal” sequence operably linked, to the amino terminus of the prosequence. The precursor may also have additional polynucleotides that are involved in post-translational activity (e.g., polynucleotides cleaved therefrom to leave the mature form of a protein or peptide).
“Host strain” or “host cell” refers to a suitable host for an expression vector comprising DNA according to the present disclosure.
The terms “derived from” and “obtained from” refer to not only a glucoamylase produced or producible by a strain of the organism in question, but also a glucoamylase encoded by a DNA sequence isolated from such strain and produced in a host organism containing such DNA sequence. Additionally, the term refers to a glucoamylase that is encoded by a DNA sequence of synthetic and/or cDNA origin and that has the identifying characteristics of the glucoamylase in question.
A “derivative” within the scope of this definition generally retains the characteristic hydrolyzing activity observed in the wild-type, native or parent form to the extent that the derivative is useful for similar purposes as the wild-type, native or parent form. Functional derivatives of glucoamylases encompass naturally occurring, synthetically or recombinantly produced peptides or peptide fragments that have the general characteristics of the glucoamylases of the present disclosure.
The term “isolated” refers to a material that is removed from the natural environment if it is naturally occurring. A “purified” protein refers to a protein that is at least partially purified to homogeneity. In some embodiments, a purified protein is more than about 10% pure, about 20% pure, or about 30% pure, as determined by SDS-PAGE. Further aspects of the disclosure encompass the protein in a highly purified form (i.e., more than about 40% pure, about 60% pure, about 80% pure, about 90% pure, about 95% pure, about 97% pure, or about 99% pure), as determined by SDS-PAGE.
As used herein, the term, “combinatorial mutagenesis” refers to methods in which libraries of variants of a starting sequence are generated. In these libraries, the variants contain one or several mutations chosen from a predefined set of mutations. In addition, the methods provide means to introduce random mutations that were not members of the predefined set of mutations. In some embodiments, the methods include those set forth in U.S. Pat. No. 6,582,914, hereby incorporated by reference. In alternative embodiments, combinatorial mutagenesis methods encompass commercially available kits (e.g., QuikChange® Multisite, Stratagene, San Diego, Calif.).
As used herein, the term “library of mutants” refers to a population of cells that are identical in most of their genome but include different homologues of one or more genes. Such libraries can be used, for example, to identify genes or operons with improved traits.
As used herein the term “dry solids content (DS or ds)” refers to the total solids of a slurry in % on a dry weight basis.
As used herein, the term “initial hit” refers to a variant that was identified by screening a combinatorial consensus mutagenesis library. In some embodiments, initial hits have improved performance characteristics, as compared to the starting gene.
As used herein, the term “improved hit” refers to a variant that was identified by screening an enhanced combinatorial consensus mutagenesis library.
As used herein, the term “target property” refers to the property of the starting gene that is to be altered. It is not intended that the present disclosure be limited to any particular target property. However, in some embodiments, the target property is the stability of a gene product (e.g., resistance to denaturation, proteolysis or other degradative factors), while in other embodiments, the level of production in a production host is altered. Indeed, it is contemplated that any property of a starting gene will find use in the present disclosure. Other definitions of terms may appear throughout the specification.
As used herein, the “process for making beer” may further be applied in the mashing of any grist.
As used herein, the term “grist” refers to any starch and/or sugar containing plant material derivable from any plant and plant part, including tubers (e.g. potatoes), roots (e.g. cassava [Manihot esculenta] roots), stems, leaves and seeds. The grist may comprise grain, such as grain from barley, wheat, rye, oat, corn/maize, rice, milo, millet and sorghum, and e.g. at least 10%, or at least 15%, or at least 25%, or at least 35%, such as at least 50%, at least 75%, at least 90% or even 100% (w/w) of the grist of the wort is derived from grain. In some embodiments the grist may comprise the starch and/or sugar containing plant material obtained from cassava [Manihot esculenta] roots. The grist may comprise malted grain, such as barley malt. Often, at least 10%, or at least 15%, or at least 25%, or at least 35%, such as at least 50%, at least 75%, at least 90% or even 100% (w/w) of the grist of the wort is derived from malted grain. The grist may comprise adjunct, such as up to 10%, or at least 10%, or at least 15%, or at least 25%, or at least 35%, or at least 50%, at least 75%, at least 90%, or even 100% (w/w) of the grist of the wort is adjunct.
The term “adjunct” is understood as the part of the grist which is not barley malt. The adjunct may be any carbohydrate rich material. In term “adjunct” includes starch and/or sugar containing plant material as e.g. defined above under “grist”.
The term “fermentation” means, in the context of brewing, the transformation of sugars in the wort, by enzymes in the brewing yeast, into ethanol and carbon dioxide with the formation of other fermentation by-products.
As used herein the term “malt” is understood as any malted cereal grain, such as barley.
As used herein, the term “malt beverage” includes such foam forming fermented malt beverages as full malted beer, ale, dry beer, near beer, light beer, low alcohol beer, low calorie beer, porter, bock beer, stout, malt liquor, non-alcoholic malt liquor and the like. The term “malt beverages” also includes non-foaming beer and alternative malt beverages such as fruit flavoured malt beverages, e.g., citrus flavoured, such as lemon-, orange-, lime-, or berry-flavoured malt beverages, liquor flavoured malt beverages, e.g., vodka-, rum-, or tequila-flavoured malt liquor, or coffee flavoured malt beverages, such as caffeine-flavoured malt liquor, and the like.
The term “mash” is understood as aqueous starch slurry, e.g. comprising crushed barley malt, crushed barley, and/or other adjunct or a combination hereof, mixed with water later to be separated into wort+spent grains.
As used herein, the term “wort” refers to the unfermented liquor run-off following extracting the grist during mashing.
As used herein, the term “spent grains” refers to the drained solids remaining when the grist has been extracted and the wort separated from the mash.
Included within the term “beer” is any fermented wort, produced by the brewing and fermentation of a starch-containing material, mainly derived from cereal grains, such as malted barley. Wheat, maize, and rice may also be used.
As used herein, the term “extract recovery” in the wort is defined as the sum of soluble substances extracted from the grist (malt and adjuncts) expressed in percentage based on dry matter.
As used herein, the term “pasteurization” means heating (e.g. beer) at certain temperatures for certain time intervals. The purpose is normally killing of micro-organisms but pasteurization can also cause inactivation of enzyme activity. Implementation of pasteurisation in the brewing process is typically through the use of a flash pasteuriser or tunnel pasteuriser. As used herein, the term “pasteurisation units or PU” refers to a quantitative measure of pasteurisation. One pasteurisation unit (1 PU) for beer is defined as a heat retention of one minute at 60 degrees Celsius. One calculates that:

PU=t×1.393^(T−60), where:

    
    
        t=time, in minutes, at the pasteurisation temperature in the pasteuriser
        T=temperature, in degrees Celsius, in the pasteuriser
        [^(T−60) represents the exponent of (T-60)]
    
    


Different minimum PU may be used depending on beer type, raw materials and microbial contamination, brewer and perceived effect on beer flavour. Typically, for beer pasteurisation, 14-15 PU are required. Depending on the pasteurising equipment, pasteurisation temperatures are typically in the range of 64-72 degrees Celsius with a pasteurisation time calculated accordingly. Further information may be found in “Technology Brewing and Malting” by Wolfgang Kunze of the Research and Teaching Institute of Brewing, Berlin (VLB), 3rd completely updated edition, 2004, ISBN 3-921690-49-8.
As used herein, the term “non-alcoholic beer” or “low-alcohol beer” refers to a beer containing a maximum of 0.1% to 3.5% or 0.1% to 2.5% such as 0.1% to 0.5% alcohol by volume. Non-alcoholic beer is brewed by traditional methods, but during the finishing stages of the brewing process the alcohol is removed by vacuum evaporation, by taking advantage of the different boiling points of water and alcohol.
As used herein, the term “low-calorie beer” or “beer with a low carbohydrate content” is defined as a beer with a carbohydrate content of 1.5 g/100 g or less and with a real degree of fermentation of at least 80%.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.
Before the exemplary embodiments are described in more detail, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, exemplary methods and materials are now described.
As used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a gene” includes a plurality of such candidate agents and reference to “the cell” includes reference to one or more cells and equivalents thereof known to those skilled in the art, and so forth.
The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior invention.
1.2. Abbreviations
GA glucoamylase
GAU glucoamylase unit
wt % weight percent
° C. degrees Centigrade
rpm revolutions per minute
H2O water
dH2O deionized water
dIH2O deionized water, Milli-Q filtration
aa or AA amino acid
bp base pair
kb kilobase pair
kD kilodaltons
g or gm grams
μg micrograms
mg milligrams
μl and μL microliters
ml and mL milliliters
mm millimeters
μmicrometer
M molar
mM millimolar
μM micromolar
U units
V volts
MW molecular weight
MWCO molecular weight cutoff
sec(s) or s(s) second/seconds
min(s) or m(s) minute/minutes
hr(s) or h(s) hour/hours
DO dissolved oxygen
ABS Absorbance
EtOH ethanol
PSS physiological salt solution
m/v mass/volume
MTP microtiter plate
N Normal
DP1 monosaccharides
DP2 disaccharides
DP>3 oligosaccharides, sugars having a degree of polymerization greater than 3
ppm parts per million
SBD starch binding domain
CD catalytic domain
PCR polymerase chain reaction
WT wild-type
2. Parent Glucoamylases
In some embodiments, the present disclosure provides a glucoamylase variant. The glucoamylase variant is a variant of a parent glucoamylase, which may comprise both a catalytic domain and a starch binding domain. In some embodiments, the parent glucoamylase comprises a catalytic domain having an amino acid sequence as illustrated in SEQ ID NO: 1, 2, 3, 5, 6, 7, 8 or 9 or having an amino acid sequence displaying at least about 80%, about 85%, about 90%, about 95%, about 97%, about 99%, or about 99.5% sequence identity with one or more of the amino acid sequences illustrated in SEQ ID NO: 1, 2, 3, 5, 6, 7, 8, or 9. In yet other embodiments, the parent glucoamylase comprises a catalytic domain encoded by a DNA sequence that hybridizes under medium, high, or stringent conditions with a DNA encoding the catalytic domain of a glucoamylase having one of the amino acid sequences of SEQ ID NO: 1, 2 or 3.
In some embodiments, the parent glucoamylase comprises a starch binding domain having an amino acid sequence as illustrated in SEQ ID NO 1, 2, 11, 385, 386, 387, 388, 389, or 390, or having an amino acid sequence displaying at least about 80%, about 85%, about 90%, about 95%, about 97%, about 99%, or about 99.5% sequence identity with one or more of the amino acid sequence illustrated in SEQ ID NO 1, 2, 11, 385, 386, 387, 388, 389, or 390. In yet other embodiments, the parent glucoamylase comprises a starch binding domain encoded by a DNA sequence that hybridizes under medium, high, or stringent conditions with a DNA encoding the starch binding domain of a glucoamylase having one of the amino acid sequences of SEQ ID NO: 1, 2, or 11.
Predicted structure and known sequences of glucoamylases are conserved among fungal species (Coutinho et al., 1994, Protein Eng., 7:393-400 and Coutinho et al., 1994, Protein Eng., 7: 749-760). In some embodiments, the parent glucoamylase is a filamentous fungal glucoamylase. In some embodiments, the parent glucoamylase is obtained from a Trichoderma strain (e.g., T. reesei, T. longibrachiatum, T. strictipilis, T. asperellum, T. konilangbra and T. hazianum), an Aspergillus strain (e.g. A. niger, A. nidulans, A. kawachi, A. awamori and A. orzyae), a Talaromyces strain (e.g. T. emersonii, T. thermophilus, and T. duponti), a Hypocrea strain (e.g. H. gelatinosa, H. orientalis, H. vinosa, and H. citrina), a Fusarium strain (e.g., F. oxysporum, F. roseum, and F. venenatum), a Neurospora strain (e.g., N. crassa) and a Humicola strain (e.g., H. grisea, H. insolens and H. lanuginose), a Penicillium strain (e.g., P. notatum or P. chrysogenum), or a Saccharomycopsis strain (e.g., S. fibuligera).
In some embodiments, the parent glucoamylase may be a bacterial glucoamylase. For example, the polypeptide may be obtained from a gram-positive bacterial strain such as Bacillus (e.g., B. alkalophilus, B. amyloliquefaciens, B. lentus, B. licheniformis, B. stearothermophilus, B. subtilis and B. thuringiensis) or a Streptomyces strain (e.g., S. lividans).
In some embodiments, the parent glucoamylase will comprise a catalytic domain having at least about 80%, about 85%, about 90%, about 93%, about 95%, about 97%, about 98%, or about 99% sequence identity with the catalytic domain of the TrGA amino acid sequence of SEQ ID NO: 3.
In other embodiments, the parent glucoamylase will comprise a catalytic domain having at least about 90%, about 93%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the catalytic domain of the Aspergillus parent glucoamylase of SEQ ID NO: 5 or SEQ ID NO: 6.
In yet other embodiments, the parent glucoamylase will comprise a catalytic domain having at least about 90%, about 95%, about 97%, or about 99% sequence identity with the catalytic domain of the Humicola grisea (HgGA) parent glucoamylase of SEQ ID NO: 8.
In some embodiments, the parent glucoamylase will comprise a starch binding domain having at least about 80%, about 85%, about 90%, about 95%, about 97%, or about 98% sequence identity with the starch binding domain of the TrGA amino acid sequence of SEQ ID NO: 1, 2, or 11.
In other embodiments, the parent glucoamylase will comprise a starch binding domain having at least about 90%, about 95%, about 97%, or about 99% sequence identity with the catalytic domain of the Humicola grisea (HgGA) glucoamylase of SEQ ID NO: 385.
In other embodiments, the parent glucoamylase will comprise a starch binding domain having at least about 90%, about 95%, about 97%, or about 99% sequence identity with the catalytic domain of the Thielavia terrestris (TtGA) glucoamylase of SEQ ID NO: 390.
In other embodiments, the parent glucoamylase will comprise a starch binding domain having at least about 90%, about 95%, about 97%, or about 99% sequence identity with the catalytic domain of the Thermomyces lanuginosus (ThGA) glucoamylase of SEQ ID NO: 386.
In other embodiments, the parent glucoamylase will comprise a starch binding domain having at least about 90%, about 95%, about 97%, or about 99% sequence identity with the catalytic domain of the Talaromyces emersoniit (TeGA) glucoamylase of SEQ ID NO: 387.
In yet other embodiments, the parent glucoamylase will comprise a starch binding domain having at least about 90%, about 93%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the starch binding domain of the Aspergillus parent glucoamylase of SEQ ID NO: 388 or 389.
In some embodiments, the parent glucoamylase will have at least about 80%, about 85%, about 88%, about 90%, about 93%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the TrGA amino acid sequence of SEQ ID NO: 1 or 2.
In further embodiments, a Trichoderma glucoamylase homologue will be obtained from a Trichoderma or Hypocrea strain. Some typical Trichoderma glucoamylase homologues are described in U.S. Pat. No. 7,413,887 and reference is made specifically to amino acid sequences set forth in SEQ ID NOs: 17-22 and 43-47 of the reference.
In some embodiments, the parent glucoamylase is TrGA comprising the amino acid sequence of SEQ ID NO: 2, or a Trichoderma glucoamylase homologue having at least about 80%, about 85%, about 88%, about 90%, about 93%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the TrGA sequence (SEQ ID NO: 2).
A parent glucoamylase can be isolated and/or identified using standard recombinant DNA techniques. Any standard techniques can be used that are known to the skilled artisan. For example, probes and/or primers specific for conserved regions of the glucoamylase can be used to identify homologs in bacterial or fungal cells (the catalytic domain, the active site, etc.). Alternatively, degenerate PCR can be used to identify homologues in bacterial or fungal cells. In some cases, known sequences, such as in a database, can be analyzed for sequence and/or structural identity to one of the known glucoamylases, including SEQ ID NO: 2, or a known starch binding domains, including SEQ ID NO: 11. Functional assays can also be used to identify glucoamylase activity in a bacterial or fungal cell. Proteins having glucoamylase activity can be isolated and reverse sequenced to isolate the corresponding DNA sequence. Such methods are known to the skilled artisan.
3. Glucoamylase Structural Homology
The central dogma of molecular biology is that the sequence of DNA encoding a gene for a particular enzyme, determines the amino acid sequence of the protein, this sequence in turn determines the three-dimensional folding of the enzyme. This folding brings together disparate residues that create a catalytic center and substrate binding surface and this results in the high specificity and activity of the enzymes in question.
Glucoamylases consist of as many as three distinct structural domains, a catalytic domain of approximately 450 residues that is structurally conserved in all glucoamylases, generally followed by a linker region consisting of between 30 and 80 residues that are connected to a starch binding domain of approximately 100 residues. The structure of the Trichoderma reesei glucoamylase with all three regions intact was determined to 1.8 Angstrom resolution herein (see Table 20 in WO2009/067218 (Danisco US Inc., Genencor Division) page 94-216 incorporated herein by reference and Example 11 in WO2009/067218 (Danisco US Inc., Genencor Division) page 89-93 incorporated herein by reference). Using the coordinates (see Table 20 in WO2009/067218 (Danisco US Inc., Genencor Division) page 94-216 incorporated herein by reference), the structure was aligned with the coordinates of the catalytic domain of the glucoamylase from Aspergillus awamori strain X100 that was determined previously (Aleshin, A. E., Hoffman, C., Firsov, L. M., and Honzatko, R. B. Refined crystal structures of glucoamylase from Aspergillus awamori var.×100. J. Mol. Biol. 238: 575-591 (1994)). The Aspergillus awamori crystal structure only included the catalytic domain. As seen in FIGS. 6-7, the structure of the catalytic domains overlap very closely, and it is possible to identify equivalent residues based on this structural superposition. It is believed that all glucoamylases share the basic structure depicted in FIGS. 6-7.
The catalytic domain of TrGA thus has approximately 450 residues such as residues 1-453 of TrGA SEQ ID NO:2 and is a twelve helix double barrel domain. The helices and loops of the catalytic domain can be defined in terms of the residues of TrGA with SEQ ID NO:2 forming them:















helix 1
residues 2-20,



loop 1
residues 21-51,



helix 2
residues 52-68,



loop 2
residues 69-71,



helix 3
residues 72-90,



loop 3
residues 91-125,



helix 4
residues 126-145,



loop 4
residues 146,



helix 5
residues 147-169,



helix 6
residues 186-206,



loop 6
residues 207-210,



helix 7
residues 211-227,



loop 7
residues 211-227,



helix 8
residues 250-275,



loop 8
residues 260-275,



helix 9
residues 276-292,



loop 9
residues 293-321,



helix 10
residues 322-342,



loop 10
residues 343-371,



helix 11
residues 372-395,



loop 11
residues 396-420,



helix 12
residues 421-434,



loop 12
residues 435-443,



helix 13
residues 444-447,



loop 13
residues 448-453









The linker domain has between 30 and 80 residues such as residues 454-490 of TrGA with SEQ ID NO: 2.
The starch binding domain of TrGA has approximately 100 residues such as residues 496-596 of TrGA with SEQ ID NO:2 consisting of the beta sandwich composed of two twisted three stranded sheets. The sheets, helices and loops of the starch binding domain can be defined in terms of the residues of TrGA with SEQ ID NO:2 forming them:















sheet 1′
residues 496-504,



loop 1′
residues 505-511,



sheet 2′
residues 512-517,



interconnecting loop 2′
residues 518-543,



sheet 3′
residues 544-552,



loop 3′
residues 553,



sheet 4′
residues 554-565,



loop 4′
residues 566-567,



sheet 5′
residues 568-572,



inter-sheet segment
residues 573-577,



sheet 5a′
residues 578-582,



loop 5′
residues 583-589,



sheet 6′
residues 590-596,









It is possible to identify equivalent residues based on structural superposition in other glucoamylases as described in further detail below.
FIG. 6 is a comparison of the three dimensional structures of the Trichoderma reesei glucoamylase (black) of SEQ ID NO: 2 and of Aspergillus awamorii glucoamylase (grey) viewed from the side. In this view, the relationship between the catalytic domain and the linker region and the starch binding domain can be seen.
FIG. 7 is a comparison of the three dimensional structures of the Trichoderma reesei glucoamylase (black) of SEQ ID NO: 2 and of Aspergillus awamorii glucoamylase (grey) viewed from the top. The glucoamylases shown here and indeed all known glucoamylases to date share this structural homology. The conservation of structure correlates with the conservation of activity and a conserved mechanism of action for all glucoamylases. Given this high homology, changes resulting from site specific variants of the Trichoderma glucoamylase resulting in altered functions would also have similar structural and therefore functional consequences in other glucoamylases. Therefore, the teachings of which variants result in desirable benefits can be applied to other glucoamylases.
A further crystal structure was produced using the coordinates in Table 20 in WO2009/067218 (Danisco US Inc., Genencor Division) page 94-216 incorporated herein by reference for the Starch Binding Domain (SBD). The SBD for TrGA was aligned with the SBD for A. niger. As shown in FIG. 8, the structure of the A. niger and TrGA SBDs overlaps very closely. It is believed that while all starch binding domains share at least some of the basic structure depicted in FIG. 8, some SBDs are more structurally similar than others. For example, the TrGA SBD can be classified as within the carbohydrate binding module 20 family within the CAZY database (cazy.org). The CAZY database describes the families of structurally-related catalytic and carbohydrate-binding modules (or functional domains) of enzymes that degrade, modify, or create glycosidic bonds. Given a high structural homology, site specific variants of the TrGA SBD resulting in altered function would also have similar structural and therefore functional consequences in other glucoamylases having SBDs with similar structure to that of the TrGA SBD, particularly those classified within the carbohydrate binding module 20 family. Thus, the teachings of which variants result in desirable benefits can be applied to other SBDs having structural similarity.
Thus, the amino acid position numbers discussed herein refer to those assigned to the mature Trichoderma reesei glucoamylase sequence presented in FIG. 1 (SEQ ID NO: 2). The present disclosure, however, is not limited to the variants of Trichoderma glucoamylase, but extends to glucoamylases containing amino acid residues at positions that are “equivalent” to the particular identified residues in Trichoderma reesei glucoamylase (SEQ ID NO: 2). In some embodiments of the present disclosure, the parent glucoamylase is a Talaromyces GA and the substitutions are made at the equivalent amino acid residue positions in Talaromyces glucoamylase (see e.g., SEQ ID NO: 12) as those described herein. In other embodiments, the parent glucoamylase comprises SEQ ID NOs: 5-9 (see FIGS. 5A and 5B). In further embodiments, the parent glucoamylase is a Penicillium glucoamylase, such as Penicillium chrysogenum (see e.g., SEQ ID NO: 13).
“Structural identity” determines whether the amino acid residues are equivalent. Structural identity is a one-to-one topological equivalent when the two structures (three dimensional and amino acid structures) are aligned. A residue (amino acid) position of a glucoamylase is “equivalent” to a residue of T. reesei glucoamylase if it is either homologous (i.e., corresponding in position in either primary or tertiary structure) or analogous to a specific residue or portion of that residue in T. reesei glucoamylase (having the same or similar functional capacity to combine, react, or interact chemically).
In order to establish identity to the primary structure, the amino acid sequence of a glucoamylase can be directly compared to Trichoderma reesei glucoamylase primary sequence and particularly to a set of residues known to be invariant in glucoamylases for which sequence is known. For example, FIGS. 5A and 5B herein show the conserved residues between glucoamylases. FIGS. 5D and 5E show an alignment of starch binding domains from various glucoamylases. After aligning the conserved residues, allowing for necessary insertions and deletions in order to maintain alignment (i.e. avoiding the elimination of conserved residues through arbitrary deletion and insertion), the residues equivalent to particular amino acids in the primary sequence of Trichoderma reesei glucoamylase are defined. Alignment of conserved residues typically should conserve 100% of such residues. However, alignment of greater than about 75% or as little as about 50% of conserved residues is also adequate to define equivalent residues. Further, the structural identity can be used in combination with the sequence identity to identify equivalent residues.
For example, in FIGS. 5A and 5B, the catalytic domains of glucoamylases from six organisms are aligned to provide the maximum amount of homology between amino acid sequences. A comparison of these sequences shows that there are a number of conserved residues contained in each sequence as designated by an asterisk. These conserved residues, thus, may be used to define the corresponding equivalent amino acid residues of Trichoderma reesei glucoamylase in other glucoamylases such as glucoamylase from Aspergillus niger. Similarly, FIGS. 5D and 5E show the starch binding domains of glucoamylases from seven organisms aligned to identify equivalent residues.
Structural identity involves the identification of equivalent residues between the two structures. “Equivalent residues” can be defined by determining homology at the level of tertiary structure (structural identity) for an enzyme whose tertiary structure has been determined by X-ray crystallography. Equivalent residues are defined as those for which the atomic coordinates of two or more of the main chain atoms of a particular amino acid residue of the Trichoderma reesei glucoamylase (N on N, CA on CA, C on C and O on O) are within 0.13 nm and optionally 0.1 nm after alignment. In one aspect, at least 2 or 3 of the four possible main chain atoms are within 0.1 nm after alignment. Alignment is achieved after the best model has been oriented and positioned to give the maximum overlap of atomic coordinates of non-hydrogen protein atoms of the glucoamylase in question to the Trichoderma reesei glucoamylase. The best model is the crystallographic model giving the lowest R factor for experimental diffraction data at the highest resolution available.




  
    R
    ⁢
    
      
    
    ⁢
    factor
  
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          ∑
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        ⁢
        
          
        
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          
          
            Fo
            ⁡
            
              (
              h
              )
            
          
          
        
      
      -
      
        
        
          Fc
          ⁡
          
            (
            h
            )
          
        
        
      
    
    
      
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        h
      
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Equivalent residues that are functionally analogous to a specific residue of Trichoderma reesei glucoamylase are defined as those amino acids of the enzyme that may adopt a conformation such that they either alter, modify or contribute to protein structure, substrate binding or catalysis in a manner defined and attributed to a specific residue of the Trichoderma reesei glucoamylase. Further, they are those residues of the enzyme (for which a tertiary structure has been obtained by X-ray crystallography) that occupy an analogous position to the extent that, although the main chain atoms of the given residue may not satisfy the criteria of equivalence on the basis of occupying a homologous position, the atomic coordinates of at least two of the side chain atoms of the residue lie with 0.13 nm of the corresponding side chain atoms of Trichoderma reesei glucoamylase. The coordinates of the three dimensional structure of Trichoderma reesei glucoamylase are set forth in Table 20 in WO2009/067218 (Danisco US Inc., Genencor Division) page 94-216 incorporated herein by reference and can be used as outlined above to determine equivalent residues on the level of tertiary structure.
Some of the residues identified for substitution are conserved residues whereas others are not. In the case of residues that are not conserved, the substitution of one or more amino acids is limited to substitutions that produce a variant that has an amino acid sequence that does not correspond to one found in nature. In the case of conserved residues, such substitutions should not result in a naturally-occurring sequence.
4. Glucoamylase Variants
The variants according to the disclosure include at least one substitution, deletion or insertion in the amino acid sequence of a parent glucoamylase that makes the variant different in sequence from a parent glucoamylase. In some embodiments, the variants of the disclosure will have at least about 20%, about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 97%, or about 100% of the glucoamylase activity as that of the TrGA (SEQ ID NO: 2), a parent glucoamylase that has at least 80% sequence identity to TrGA (SEQ ID NO: 2). In some embodiments, the variants according to the disclosure will comprise a substitution, deletion or insertion in at least one amino acid position of the parent TrGA (SEQ ID NO: 2), or in an equivalent position in the sequence of another parent glucoamylase having at least about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, or about 99% sequence identity to the TrGA sequence (SEQ ID NO: 2).
In other embodiments, the variant according to the disclosure will comprise a substitution, deletion or insertion in at least one amino acid position of a fragment of the parent TrGA, wherein the fragment comprises the catalytic domain of the TrGA sequence (SEQ ID NO: 3) or in an equivalent position in a fragment comprising the catalytic domain of a parent glucoamylase having at least about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, or about 99% sequence identity to the catalytic-domain-containing fragment of the SEQ ID NO: 3, 5, 6, 7, 8, or 9. In some embodiments, the fragment will comprise at least about 400, about 425, about 450, or about 500 amino acid residues of TrGA catalytic domain (SEQ ID NO: 3).
In other embodiments, the variant according to the disclosure will comprise a substitution, deletion or insertion in at least one amino acid position of a fragment of the parent TrGA, wherein the fragment comprises the starch binding domain of the TrGA sequence (SEQ ID NO: 11) or in an equivalent position in a fragment comprising the starch binding domain of a parent glucoamylase having at least about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, or about 99% sequence identity to the starch-binding-domain-containing fragment of SEQ ID NO: 11, 385, 386, 387, 388, 389, and 390. In some embodiments, the fragment will comprise at least about 40, about 50, about 60, about 70, about 80, about 90, about 100, or about 109 amino acid residues of TrGA starch binding domain (SEQ ID NO: 11).
In some embodiments, when the parent glucoamylase includes a catalytic domain, a linker region, and a starch binding domain, the variant will comprise a substitution, deletion or insertion in at least one amino acid position of a fragment comprising part of the linker region. In some embodiments, the variant will comprise a substitution deletion, or insertion in the amino acid sequence of a fragment of the TrGA sequence (SEQ ID NO: 2).
Structural identity with reference to an amino acid substitution means that the substitution occurs at the equivalent amino acid position in the homologous glucoamylase or parent glucoamylase. The term equivalent position means a position that is common to two parent sequences that is based on an alignment of the amino acid sequence of the parent glucoamylase in question as well as alignment of the three-dimensional structure of the parent glucoamylase in question with the TrGA reference glucoamylase amino acid sequence and three-dimensional sequence. For example, with reference to FIG. 5A, position 24 in TrGA (SEQ ID NO: 2 or 3) is D24 and the equivalent position for Aspergillus niger (SEQ ID NO: 6) is position D25, and the equivalent position for Aspergillus oryzea (SEQ ID NO: 7) is position D26. See FIGS. 6 and 7 for an exemplary alignment of the three-dimensional sequence.
Accordingly, in one aspect, a glucoamylase variant is described, which glucoamylase variant when in its crystal form has a crystal structure for which the atomic coordinates of the main chain atoms have a root-mean-square deviation from the atomic coordinates of the equivalent main chain atoms of TrGA (as defined in Table 20 in WO2009/067218) of less than 0.13 nm following alignment of equivalent main chain atoms, and which have a linker region, a starch binding domain and a catalytic domain, said variant comprising two or more amino acid substitutions relative to the amino acid sequence of the parent glucoamylase in interconnecting loop 2′ of the starch binding domain, and/or in loop 1, and/or in helix 2, and/or in loop 11, and/or in helix 12 of the catalytic domain. In a further aspect, the root-mean-square deviation from the atomic coordinates of the equivalent main chain atoms of TrGA (as defined in Table 20 in WO2009/067218) is less than 0.12 nm, such as less than 0.11 or such as less than 0.10.
In one aspect, a glucoamylase variant is described, which glucoamylase variant comprises a starch binding domain and a catalytic domain, said variant comprising two or more amino acid substitutions relative to the amino acid sequence of SEQ ID NO:2 or equivalent parent glucoamylase in interconnecting loop 2′, and/or in loop 1, and/or in helix 2, and/or in loop 11, and/or in helix 12 for reducing the synthesis of condensation products during hydrolysis of starch.
In a further aspect, a glucoamylase variant is described, which glucoamylase variant comprises two or more amino acid substitutions relative to interconnecting loop 2′ with the amino acid sequence from position 518 to position 543 of SEQ ID NO:2 or equivalent sequence of residues in a parent glucoamylase, and/or loop 1 with the amino acid sequence from position 21 to position 51 of SEQ ID NO:2 or equivalent sequence of residues in a parent glucoamylase, and/or helix 2 with the amino acid sequence from position 52 to position 68 of SEQ ID NO:2 or equivalent sequence of residues in a parent glucoamylase, and/or loop 11 with the amino acid sequence from position 396 to position 420 of SEQ ID NO:2 or equivalent sequence of residues in a parent glucoamylase, and/or helix 12 with the amino acid sequence from position 421 to position 434 of SEQ ID NO:2 or equivalent sequence of residues in a parent glucoamylase.
In a further aspect, a glucoamylase variant is described, which glucoamylase variant comprises two or more amino acid substitutions relative to the amino acid sequence from position 518 to position 543 of SEQ ID NO:2 or equivalent sequence of residues in a parent glucoamylase, and/or the amino acid sequence from position 21 to position 51 of SEQ ID NO:2 or equivalent sequence of residues in a parent glucoamylase, and/or the amino acid sequence from position 52 to position 68 of SEQ ID NO:2 or equivalent sequence of residues in a parent glucoamylase, and/or the amino acid sequence from position 396 to position 420 of SEQ ID NO:2 or equivalent sequence of residues in a parent glucoamylase, and/or the amino acid sequence from position 421 to position 434 of SEQ ID NO:2 or equivalent sequence of residues in a parent glucoamylase.
In one aspect, the two or more amino acid substitutions are relative to the interconnecting loop 2′ with the amino acid sequence from position 518 to position 543 e.g. in one or more of positions 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542 and/or 543 of SEQ ID NO:2, and/or loop 1 with the amino acid sequence from position 21 to position 51 e.g. in one or more of positions 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 and/or 51 of SEQ ID NO:2, and/or helix 2 with the amino acid sequence from position 52 to position 68 e.g. in one or more of positions 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67 and/or 68 of SEQ ID NO:2, and/or loop 11 with the amino acid sequence from position 396 to position 420 e.g. in one or more of positions 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419 and/or 420 of SEQ ID NO:2, and/or helix 12 with the amino acid sequence from position 421 to position 434 e.g. in one or more of positions 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433 and/or 534 of SEQ ID NO:2.
In a further aspect, the two or more amino acid substitutions are at least one amino acid substitution in the interconnecting loop 2′ and at least one amino acid substitution in loop 1 and/or helix 2 and/or loop 11 and/or helix 12. In a further aspect, the two or more amino acid substitutions are 1, 2, 3 or 4 amino acid substitutions in the interconnecting loop 2′ and 1, 2, 3 or 4 amino acid substitutions in loop 1 and/or helix 2 and/or loop 11 and/or helix 12.
In a further aspect, the two or more amino acid substitutions are at least one amino acid substitution in interconnecting loop 2′ and at least one amino acid substitution in loop 1. In a further aspect, the two or more amino acid substitutions are at least one amino acid substitution in interconnecting loop 2′ and at least one amino acid substitution in helix 2. In a further aspect, the two or more amino acid substitutions are at least one amino acid substitution in interconnecting loop 2′ and at least one amino acid substitution in loop 11. In a further aspect, the two or more amino acid substitutions are at least one amino acid substitution in interconnecting loop 2′ and at least one amino acid substitution in helix 12. In a further aspect, the two or more amino acid substitutions are at least one amino acid substitution in interconnecting loop 2′ and at least one amino acid substitution in loop 1 and at least one amino acid substitution in helix 2. In a further aspect, the glucoamylase variant has at least one amino acid substitution within position 520-543, 530-543, or 534-543 of interconnecting loop 2′, the positions corresponding to the respective position in SEQ ID NO:2 or equivalent positions in a parent glucoamylase. In a further aspect, the glucoamylase variant has at least one amino acid substitution within the amino acid sequence of position 30-50, 35-48, or 40-46 of loop 1, the positions corresponding to the respective position in SEQ ID NO:2 or equivalent positions in a parent glucoamylase. In a further aspect, the glucoamylase variant has at least one amino acid substitution within the amino acid sequence of position 50-66, 55-64, or 58-63 of helix 2, the positions corresponding to the respective position in SEQ ID NO:2 or equivalent positions in a parent glucoamylase. In a further aspect, the glucoamylase variant has at least one amino acid substitution within the amino acid sequence of position 405-420, 410-420, or 415-420 of loop 11, the positions corresponding to the respective position in SEQ ID NO:2 or equivalent positions in a parent glucoamylase. In a further aspect, the glucoamylase variant has at least one amino acid substitution within the amino acid sequence of position 421-434, 425-434, or 428-434 of helix 12, the positions corresponding to the respective position in SEQ ID NO:2 or equivalent positions in a parent glucoamylase.
In one aspect, the glucoamylase variant comprises two or more amino acid substitutions, wherein an amino acid substitution is in position 539 and an amino acid substitution is in position 44, the positions corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase, and which sequence has at least 80% sequence identity to the parent glucoamylase, and wherein the amino acid substitution in position 44 is not 44C.
In a further aspect, the glucoamylase variant comprises two or more amino acid substitutions, wherein an amino acid substitution is in position 539 and an amino acid substitution is 44R, the positions corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase. In a further aspect, the glucoamylase variant comprises an amino acid substitution in position 61, the position corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase. In a further aspect, the amino acid substitution in position 539 is 539R, the position corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase. In a further aspect, the amino acid substitution in position 44 is 44R, the position corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase. In a further aspect, the amino acid substitution in position 61 is 61I, the position corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase.
In a further aspect, the glucoamylase variant comprises the following amino acid substitutions:
a) D44R and A539R; or
b) D44R, N61I and A539R,
the positions corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase.
In a further aspect, the glucoamylase variant consist of SEQ ID NO:2 and has the following amino acid substitutions:
a) D44R and A539R; or
b) D44R, N61I and A539R,
the positions corresponding to the respective position in SEQ ID NO:2.
In a further aspect, the glucoamylase variant has a starch binding domain that has at least 96%, 97%, 98%, 99%, or 99.5% sequence identity with the starch binding domain of SEQ ID NO: 1, 2, 11, 385, 386, 387, 388, 389, or 390. In a further aspect, the glucoamylase variant has a catalytic domain that has at least 80%, 85%, 90%, 95%, or 99.5% sequence identity with the catalytic domain of SEQ ID NO: 1, 2, 3, 5, 6, 7, 8, or 9.
In a further aspect, the parent glucoamylase is a fungal glucoamylase.
In a further aspect, the parent glucoamylase is selected from a glucoamylase obtained from a Trichoderma spp., an Aspergillus spp., a Humicola spp., a Penicillium spp., a Talaromycese spp., or a Schizosaccharmyces spp.
In a further aspect, the parent glucoamylase is obtained from a Trichoderma spp. or an Aspergillus spp.
In a further aspect, the glucoamylase has been purified. The glucoamylases of the present disclosure may be recovered or purified from culture media by a variety of procedures known in the art including centrifugation, filtration, extraction, precipitation and the like.
In some embodiments, the glucoamylase variant will include at least two substitutions in the amino acid sequence of a parent. In further embodiments, the variant may have more than two substitutions. For example, the variant may have 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 amino acid substitutions, deletions, or insertions as compared to a corresponding parent glucoamylase.
In some embodiments, a glucoamylase variant comprises a substitution, deletion or insertion, and typically a substitution in at least one amino acid position in a position corresponding to the regions of non-conserved amino acids as illustrated in FIGS. 5A, 5B, 5D, and 5E (e.g., amino acid positions corresponding to those positions that are not designated by “*” in FIGS. 5A, 5B, 5D, and 5E).
While the variants may have substitutions in any position of the mature protein sequence (SEQ ID NO: 2), in some embodiments, a glucoamylase variant comprises two or more substitutions in the following positions in the amino acid sequence set forth in SEQ ID NO: 2: 23, 42, 43, 44, 59, 60, 61, 65, 67, 68, 410, 417, 418, 430, 431, 433, 518, 519, 520, 527, 531, 535, 536, 537 or 539, or in an equivalent position in a parent glucoamylase. In a further aspect, the glucoamylase variant comprises one or more further substitutions in the following positions in the amino acid sequence set forth in SEQ ID NO: 2: 10, 14, 15, 72, 73, 97, 98, 99, 102, 110, 113, 114, 133, 140, 144, 145, 147, 152, 153, 164, 182, 204, 205, 214, 216, 219, 228, 229, 230, 231, 236, 239, 241, 242, 263, 264, 265, 268, 269, 276, 284, 291, 294, 300, 301, 303, 311, 338, 342, 344, 346, 349, 359, 361, 364, 375, 379, 382, 390, 391, 393, 394, 436, 442, 444, 448, 451, 493, 494, 495, 502, 503, 508, 511, 563, or 577, or in an equivalent position in a parent glucoamylase. In some embodiments, the parent glucoamylase will have at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with SEQ ID NO: 2. In other embodiments, the parent glucoamylase will be a Trichoderma glucoamylase homologue. In some embodiments, the variant will have altered properties. In some embodiments, the parent glucoamylase will have structural identity with the glucoamylase of SEQ ID NO: 2.
In some embodiments, the glucoamylase variant comprises two or more substitutions in the following positions in the amino acid sequence set forth in SEQ ID NO: 2: P23, T42, I43, D44, P45, D46, F59, K60, N61, T67, E68, R408, S410, S415, L417, H418, T430, A431, R433, N518, A519, A520, T527, V531, A535, V536, N537, and A539 or an equivalent position in parent glucoamylase (e.g., a Trichoderma glucoamylase homologue). In a further aspect, the glucoamylase variant comprises one or more substitutions in the following positions in the amino acid sequence set forth in SEQ ID NO: 2: T10, L14, N15, A72, G73, S97, L98, A99, S102, K108, E110, L113, K114, R122, Q124, R125, I133, K140, N144, N145, Y147, S152, N153, N164, F175, N182, A204, T205, S214, V216, Q219, W228, V229, S230, S231, D236, I239, N240, T241, N242, G244, N263, L264, G265, A268, G269, D276, V284, 5291, G294, P300, A301, A303, Y310, A311, D313, Y316, V338, T342, 5344, T346, A349, V359, G361, A364, T375, N379, S382, S390, E391, A393, K394, I436, A442, N443, S444, T448, S451, T493, P494, T495, H502, E503, Q508, Q511, N563, and N577 or in an equivalent position in a parent glucoamylase. In some embodiments, the variant will have altered properties as compared to the parent glucoamylase.
In some embodiments, the glucoamylase variant may differ from the parent glucoamylase only at the specified positions.
In further embodiments, the variant of a glucoamylase parent comprises at least two of the following substitutions in the following positions in an amino acid sequence set forth in SEQ ID NO: 2: T42V, I43Q/R, D44R/C, N61I, T67M, E68C/M, L417K/R/V, T430A/K, A431I/L/Q, R433C/E/G/L/N/S/V/Y, A519I/K/R/Y, A520C/L/P, V531L, A535K/N/P/R, V536M, or A539E/R/S, or a substitution in an equivalent position in a parent glucoamylase. In a further aspect, the glucoamylase variant comprises one or more substitutions in the following positions in the amino acid sequence set forth in SEQ ID NO: 2: T10S, A72Y, G73F/W, S97N, S102A/M/R, K114M/Q, I133T/V, N145I, N153A/D/E/M/S/V, T205Q, Q219S, W228A/F/H/M/V, V229I/L, S230C/F/G/L/N/Q/R, S231L/V, D236R, I239V/Y, N263P, L264D/K, A268C/D/G/K, S291A/F/H/M/T, g294c, A301P/R, V338I/N/Q, T342V, S344M/P/Q/R/V, G361D/E/F/I/L/M/P/S/W/Y, A364D/E/F/G/K/L/M/R/S/T/V/W, T375N, K394S, I436H, T451K, T495K/M/S, E503A/C/V, Q508R, Q511H, N563C/E/I/K/K/Q/T/V, or N577K/P/R, or in an equivalent position in a parent glucoamylase.
In further embodiments, the glucoamylase variant comprises one of the following sets of substitutions, at the relevant positions of SEQ ID NO: 2, or at equivalent positions in a parent glucoamylase:
N61I/L417V/A431L/A539R;
I43Q/N61I/L417V/A431L/A539R;
N61I/L417V/A431L/A535R/A539R
I43Q/L417V/A431L/A535R/A539R;
I43Q/N61I/L417V/A431L/A535R/A539R;
I43Q/N61I/L417V/T430A/A431L/A535R/A539R;
I43Q/L417V/T430A/A431L/Q511H/A535R/A539R/N563I;
N61I/L417V/T430A/A431L/Q511H/A535R/A539R/N563I;
I43Q/N61I/L417V/T430A/A431L/Q511H/A535R/A539R/N563I;
I43R/N61I/L417V/A431L/A539R;
I43R/N61I/L417V/T430A/A431L/A535R/A539R;
G73F/L417R/E503V/A539R/N563K;
I43R/G73F/L417R/E503V/A539R/N563K; and
I43R/G73F/E503V/Q511H/N563K.
In further embodiments, the glucoamylase variant comprises one of the following sets of substitutions, at positions of SEQ ID NO: 2 or equivalent positions in a parent glucoamylase:
L417V/A431L/A539R;
I43Q/L417V/A431L/A539R;
L417V/A431L/A535R/A539R
I43R/L417V/A431L/A539R;
L417R/A431L/A539R; or
L417G/A431L/A539R;
wherein the glucoamylase variant does not have any further substitutions relative to the parent glucoamylase, and wherein the parent glucoamylase has a catalytic domain that has at least 80% sequence identity with SEQ ID NO: 1, 2, 3, 5, 6, 7, 8, or 9. Thus the parent glucoamylase may be any of those described elsewhere.
The parent glucoamylase may comprise a starch binding domain that has at least 95% sequence identity with SEQ ID NO: 1, 2, 11, 385, 386, 387, 388, 389, or 390. The parent glucoamylase may have at least 80% sequence identity with SEQ ID NO: 1 or 2; for example it may comprise SEQ ID NO: 1 or 2. Optionally the parent glucoamylase may consist of SEQ ID NO: 1 or 2.
Glucoamylase variants of the disclosure may also include chimeric or hybrid glucoamylases with, for example a starch binding domain (SBD) from one glucoamylase and a catalytic domain and linker from another. For example, a hybrid glucoamylase can be made by swapping the SBD from AnGA (SEQ ID NO: 6) with the SBD from TrGA (SEQ ID NO: 2), making a hybrid with the AnGA SBD and the TrGA catalytic domain and linker. Alternatively, the SBD and linker from AnGA can be swapped for the SBD and linker of TrGA.
In some aspects, the variant glucoamylase exhibits altered thermostability as compared to the parent glucoamylase. In some aspects, the altered thermostability may be increased thermostability as compared to the parent glucoamylase. In some embodiments, the altered property is altered specific activity compared to the parent glucoamylase. In some embodiments, the altered specific activity may be increased specific activity compared to the parent glucoamylase. In some embodiments, the altered property is increased thermostability at lower temperatures as compared to the parent glucoamylase. In some embodiments, the altered property is both increased specific activity and increased thermostability as compared to the parent glucoamylase.
In one embodiment, some variants may include the substitutions at positions:
D44R/N61I/A539R;
D44R/A539R;
I43Q/D44C/L417V/E503A/Q511H/A539R;
I43Q/L417V/E503A/Q511H/A539R;
I43Q/D44C/N61I/L417V/E503A/Q511H/A539R;
I43Q/N61I/L417V/E503A/Q511H/A539R;
I43R/L417V/E503A/Q511H/A539R;
I43R/N61I/L417V/E503A/Q511H/A539R;
I43R/L417R/E503A/A539R;
I43R/N61I/L417R/E503A/Q511H/A539R;
G73F/T430A/Q511H;
I43R/G73F/T430A;
G73F/T430A/E503V/Q511H;
D44C/G73F/N563K;
D44C/G73F/E503V/Q511H;
D44C/G73F/N563K;
D44C/G73F/L417R/N563K;
D44C/G73F/N563K;
I43R/T43 GA;
I43Q/T430A;
I43Q/T430A/Q511H;
D44C/L417R/N563K;
L417V/T430A/A431L/Q511H/A535R/A539R/N563I;
L417V/T430A/A431Q/Q511H/A535R/A539R/N563I;
L417V/T430A/Q511H/A535R/N563I;
L417V/T430A/Q511H/A539R/N563I;
G294C/L417R/A431L;
G294C/L417V/A431Q;
G294C/L417V/A431L/Q511H;
G294C/L417R/A431Q/Q511H;
L417R/A431L/Q511H;
L417V/A431Q/Q511H;
I43Q/T430A/Q511H/N61I;
I43Q/T430A/Q511H/L417V;
I43Q/T430A/Q511H/A431L;
I43Q/T430A/Q511H/E503A;
I43Q/T430A/Q511H/A539R;
I43Q/T430A/Q511H/N61I/A539R;
I43Q/T430A/Q511H/L417V/A539R;
I43Q/T430A/Q511H/A431L/A539R;
I43Q/T430A/Q511H/A431L/E503A;
I43Q/T430A/Q511H/N61I/A539R/A431L;
I43Q/T430A/Q511H/L417V/A539R/A431L;
I43Q/Q511H/N61I;
I43Q/Q511H/L417V;
I43Q/Q511H/A431L;
I43Q/Q511H/A539R;
I43Q/Q511H/A539R/N61I;
I43Q/Q511H/A539R/E503A;
I43Q/Q511H/A539R/T430M;
I43Q/Q511H/A539R/T430M/N61I;
I43Q/Q511H/A539R/T430M/N61I/L417V;
I43R/T430A/E503V/A535R/N563K;
D44R/E503A/Q511H/N563I;
E503A/N563I;
I43R/T430A/E503A/Q511H/N563K;
D44R/T430A/Q511H/A535R;
L417V/A431L/A539R;
L417V/A431L/A539R/I43Q;
L417V/A431L/A539R/N61I;
L417V/A431L/A539R/A535R;
L417V/A431L/A539R/I43Q/N61I;
L417V/A431L/A539R/N61I/A535R;
L417V/A431L/A539R/A535R/I43Q;
L417V/A431L/A539R/I43Q/N61I/A535R;
L417V/A431L/A539R/I43Q/N61I/A535R/T430A;
L417V/T430A/A431L/Q511H/A535R/A539R/N563I/I43Q;
L417V/T430A/A431L/Q511H/A535R/A539R/N563I/N61I;
L417V/T430A/A431L/Q511H/A535R/A539R/N563I/I43Q/N61I;
L417V/A431L/A539R/I43R;
L417V/A431L/A539R/143R/N61I;
L417V/A431L/A539R/I43R/N61I/A535R/T430A;
L417R/A431L/A539R;
L417G/A431L/A539R;
G73F/E503V/N563K/L417R/A539R;
G73F/E503V/N563K/I43R/L417R/A539R; and
G73F/E503V/N563K/I43R/Q511H
of SEQ ID NO: 2, or equivalent positions in parent glucoamylases and particularly Trichoderma glucoamylase homologues.
In a further embodiment, some variants may include the substitutions at positions:
D44R/N61I/A539R;
D44R/A539R;
I43Q/D44C/L417V/E503A/Q511H/A539R;
I43Q/L417V/E503A/Q511H/A539R;
I43Q/D44C/N61I/L417V/E503A/Q511H/A539R;
I43Q/N61I/L417V/E503A/Q511H/A539R;
I43R/L417V/E503A/Q511H/A539R;
I43R/N61I/L417V/E503A/Q511H/A539R;
I43R/L417R/E503A/A539R;
I43R/N61I/L417R/E503A/Q511H/A539R;
L417V/T430A/A431L/Q511H/A535R/A539R/N563I;
L417V/T430A/A431Q/Q511H/A535R/A539R/N563I;
L417V/T430A/Q511H/A539R/N563I;
I43Q/T430A/Q511H/A539R;
I43Q/T430A/Q511H/N61I/A539R;
I43Q/T430A/Q511H/L417V/A539R;
I43Q/T430A/Q511H/A431L/A539R;
I43Q/T430A/Q511H/N61I/A539R/A431L;
I43Q/T430A/Q511H/L417V/A539R/A431L;
I43Q/Q511H/A539R;
I43Q/Q511H/A539R/N61I;
I43Q/Q511H/A539R/E503A;
I43Q/Q511H/A539R/T430M;
I43Q/Q511H/A539R/T430M/N61I;
I43Q/Q511H/A539R/T430M/N61I/L417V;
L417V/A431L/A539R;
L417V/A431L/A539R/I43Q;
L417V/A431L/A539R/N61I;
L417V/A431L/A539R/A535R;
L417V/A431L/A539R/I43Q/N61I;
L417V/A431L/A539R/N61I/A535R;
L417V/A431L/A539R/A535R/I43Q;
L417V/A431L/A539R/I43Q/N61I/A535R;
L417V/A431L/A539R/I43Q/N61I/A535R/T430A;
L417V/T430A/A431L/Q511H/A535R/A539R/N563I/I43Q;
L417V/T430A/A431L/Q511H/A535R/A539R/N563I/N61I;
L417V/T430A/A431L/Q511H/A535R/A539R/N563I/I43Q/N61I;
L417V/A431L/A539R/I43R;
L417V/A431L/A539R/I43R/N61I;
L417V/A431L/A539R/I43R/N61I/A535R/T430A;
L417R/A431L/A539R;
L417G/A431L/A539R;
G73F/E503V/N563K/L417R/A539R; and
G73F/E503V/N563K/I43R/L417R/A539R
of SEQ ID NO: 2, or equivalent positions in parent glucoamylases and particularly Trichoderma glucoamylase homologues.
In a further embodiment, some variants may include the substitutions at positions:
D44R/N61I/A539R;
D44R/A539R;
I43Q/D44C/L417V/E503A/Q511H/A539R;
I43Q/L417V/E503A/Q511H/A539R;
I43Q/D44C/N61I/L417V/E503A/Q511H/A539R;
I43Q/N61I/L417V/E503A/Q511H/A539R;
I43R/L417V/E503A/Q511H/A539R;
I43R/N61I/L417V/E503A/Q511H/A539R;
I43R/L417R/E503A/A539R;
I43R/N61I/L417R/E503A/Q511H/A539R;
L417V/T430A/A431L/Q511H/A535R/A539R/N563I;
L417V/T430A/A431Q/Q511H/A535R/A539R/N563I;
L417V/T430A/Q511H/A539R/N563I;
I43Q/T430A/Q511H/A539R;
I43Q/T430A/Q511H/N61I/A539R;
I43Q/T430A/Q511H/L417V/A539R;
I43Q/T430A/Q511H/A431L/A539R;
I43Q/T430A/Q511H/N61I/A539R/A431L;
I43Q/T430A/Q511H/L417V/A539R/A431L;
I43Q/Q511H/A539R;
I43Q/Q511H/A539R/N61I;
I43Q/Q511H/A539R/E503A;
I43Q/Q511H/A539R/T430M;
I43Q/Q511H/A539R/T430M/N61I;
I43Q/Q511H/A539R/T430M/N61I/L417V;
L417V/A431L/A539R;
L417V/A431L/A539R/I43Q;
L417V/A431L/A539R/N61I;
L417V/A431L/A539R/A535R;
L417V/A431L/A539R/I43Q/N61I;
L417V/A431L/A539R/N61I/A535R;
L417V/A431L/A539R/A535R/I43Q;
L417V/A431L/A539R/I43Q/N61I/A535R;
L417V/A431L/A539R/I43Q/N61I/A535R/T430A;
L417V/T430A/A431L/Q511H/A535R/A539R/N563I/I43Q;
L417V/T430A/A431L/Q511H/A535R/A539R/N563I/N61I;
L417V/T430A/A431L/Q511H/A535R/A539R/N563I/I43Q/N61I;
L417V/A431L/A539R/I43R;
L417V/A431L/A539R/I43R/N61I;
L417V/A431L/A539R/143R/N61I/A535R/T430A;
L417R/A431L/A539R;
L417G/A431L/A539R;
G73F/E503V/N563K/L417R/A539R; and
G73F/E503V/N563K/I43R/L417R/A539R
of SEQ ID NO: 2, or equivalent positions in parent glucoamylases and particularly Trichoderma glucoamylase homologues.
In a further embodiment, some variants may include the substitutions at positions:
D44R/N61I/A539R;
D44R/A539R;
L417V/A431L/A539R;
L417V/A431L/A539R/I43Q;
L417V/A431L/A539R/N61I;
of SEQ ID NO: 2, or equivalent positions in parent glucoamylases and particularly Trichoderma glucoamylase homologues.
In a further embodiment, some variants may include the substitutions at positions:
D44R/N61I/A539R;
D44R/A539R;
of SEQ ID NO: 2, or equivalent positions in parent glucoamylases and particularly Trichoderma glucoamylase homologues.
In a further embodiment, some variants has the following substitutions: D44R/N61I/A539R or D44R/A539R of SEQ ID NO: 2.
In a further embodiment, the variant comprises SEQ ID NO:1098. In yet a further embodiment, the variant consists of SEQ ID NO:1098. In a further embodiment, the variant comprises SEQ ID NO:1099. In yet a further embodiment, the variant consists of SEQ ID NO:1099.
A number of parent glucoamylases have been aligned with the amino acid sequence of TrGA. FIG. 5 includes the catalytic domain of the following parent glucoamylases Aspergillus awamori (AaGA) (SEQ ID NO: 5); Aspergillus niger (AnGA) (SEQ ID NO: 6); Aspergillus orzyae (AoGA) (SEQ ID NO: 7); Humicola grisea (HgGA) (SEQ ID NO: 8); and Hypocrea vinosa (HvGA) (SEQ ID NO: 9). The % identity of the catalytic domains is represented in Table 1 below.







TABLE 1







Sequence homology between various fungal glucoamylases














AaGA
AnGA
AoGA
HgGA
HvGA
TrGA
















AaGA
100
95
58
53
57
56


AnGA

100
59
53
57
56


AoGA


100
55
56
56


HgGA



100
61
63


HvGA




100
91


TrGA





100









In some embodiments, for example, the variant glucoamylase will be derived from a parent glucoamylase that is an Aspergillus glucoamylase, a Humicola glucoamylase, or a Hypocrea glucoamylase.
5. Characterization of Variant Glucoamylases
The present disclosure also provides glucoamylase variants having at least one altered property (e.g., improved property) as compared to a parent glucoamylase and particularly to the TrGA. In some embodiments, at least one altered property (e.g., improved property) is selected from the group consisting of IS/SH-ratio, starch hydrolysis activity, real degree of fermentation, reduced formation of condensation products, acid stability, thermal stability and specific activity. Typically, the altered property is reduced IS/SH-ratio, enhanced real degree of fermentation, reduced formation of condensation products, increased thermal stability and/or increased specific activity. The increased thermal stability typically is at higher temperatures. In one embodiment, the increased pH stability is at high pH. In a further embodiment, the increased pH stability is at low pH.
The glucoamylase variants of the disclosure may also provide higher rates of starch hydrolysis at low substrate concentrations as compared to the parent glucoamylase. The variant may have a higher Vmax or lower Km than a parent glucoamylase when tested under the same conditions. For example the variant glucoamylase may have a higher Vmax at a temperature range of about 25° C. to about 70° C. (e.g., about 25° C. to about 35° C.; about 30° C. to about 35° C.; about 40° C. to about 50° C.; at about 50° C. to about 55° C., or about 55° C. to about 62° C.). The Michaelis-Menten constant, Km and Vmax values can be easily determined using standard known procedures. In another aspect, the glucoamylase may also exhibit a reduced starch hydrolysis activity which is not more than 5%, not more than 10% or not more than 15% reduced as compared to the parent glucoamylase such as TrGA.
5.1. Variant Glucoamylases with Altered Thermostability
In some aspects, the disclosure relates to a variant glucoamylase having altered thermal stability as compared to a parent (wild-type). Altered thermostability can be at increased temperatures or at decreased temperatures. Thermostability is measured as the % residual activity after incubation for 1 hour at 64° C. in NaAc buffer pH 4.5. Under these conditions, TrGA has a residual activity of between about 15% and 44% due to day-to-day variation as compared to the initial activity before incubation. Thus, in some embodiments, variants with increased thermostability have a residual activity that is between at least about 1% and at least about 50% more than that of the parent (after incubation for 1 hour at 64° C. in NaAc buffer pH 4.5), including about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, and about 50% as compared to the initial activity before incubation. For example, when the parent residual activity is 15%, a variant with increased thermal stability may have a residual activity of between about 16% and about 75%. In some embodiments, the glucoamylase variant will have improved thermostability such as retaining at least about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 92%, about 95%, about 96%, about 97%, about 98%, or about 99% enzymatic activity after exposure to altered temperatures over a given time period, for example, at least about 60 minutes, about 120 minutes, about 180 minutes, about 240 minutes, or about 300 minutes. In some embodiments, the variant has increased thermal stability compared to the parent glucoamylase at selected temperatures in the range of about 40° C. to about 80° C., also in the range of about 50° C. to about 75° C., and in the range of about 60° C. to about 70° C., and at a pH range of about 4.0 to about 6.0. In some embodiments, the thermostability is determined as described in the Assays and Methods. That method may be adapted as appropriate to measure thermostability at other temperatures. Alternatively the thermostability may be determined at 64° C. as described there. In some embodiments, the variant has increased thermal stability at lower temperature compared to the parent glucoamylase at selected temperature in the range of about 20° C. to about 50° C., including about 35° C. to about 45° C. and about 30° C. to about 40° C.
In some embodiments, variants having an improvement in thermostability include one or more deletions, substitutions or insertions and particularly substitutions in the following positions in the amino acid sequence set forth in SEQ ID NO: 2: 10, 42, 43, 44, 59, 61, 68, 72, 73, 97, 98, 99, 102, 114, 133, 140, 144, 152, 153, 182, 204, 205, 214, 216, 228, 229, 230, 231, 236, 241, 242, 263, 264, 265, 268, 269, 276, 284, 291, 294, 300, 301, 303, 311, 338, 342, 344, 346, 349, 359, 361, 364, 375, 379, 382, 390, 391, 393, 394, 410, 417, 430, 431, 433, 436, 442, 444, 448, 451, 493, 495, 503, 508, 511, 518, 519, 520, 527, 531, 535, 536, 537, 539, 563, or 577, or an equivalent position in a parent glucoamylase. In some embodiments, the parent glucoamylase will be a Trichoderma glucoamylase homologue and in further embodiments, the parent glucoamylase will have at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 98% sequence identity to SEQ ID NO: 2. In some embodiments, the parent glucoamylase will also have structural identity to SEQ ID NO: 2. In some embodiments, the variant having increased thermostability has a substitution in at least one of the positions: T10S, T42V, I43Q, I43R, D44C, D44R, E68C, E68M, G73F, G73W, K114M, K114Q, I133V, N153A, N153E, N153M, N153S, N153V, W228V, V229I, V229L, S230Q, S231V, D236R, L264D, L264K, A268D, S291A, S291F, S291H, S291M, S291T, G294C, A301P, A301R, V338I, V338N, V338Q, S344M, S344P, S344Q, S344R, S344V, G361D, G361E, G361F, G361I, G361L, G361M, G361P, G361S, G361W, G361Y, A364D, A364E, A364F, A364G, A364K, A364L, A364M, A364R, A364S, A364T, A364V, A364W, T375N, L417K, L417R, R433C, R433E, R433G, R433L, R433N, R433S, R433V, I436H, T495K, T495S, E503A, E503C, E503V, Q508R, Q511H, A519K, A519R, A519Y, V531L, A535K, A535N, A535P, A535R, A539E, A539R, A539S, N563C, N563E, N563I, N563K, N563L, N563Q, N563T, N563V, N577K, N577P, or N577R of SEQ ID NO: 2.
5.2. Variant Glucoamylases with Altered Specific Activity
As used herein, specific activity is the activity of the glucoamylase per mg of protein. Activity was determined using the ethanol assay. The screening identified variants having a Performance Index (PI)>1.0 compared to the parent TrGA PI. The PI is calculated from the specific activities (activity/mg enzyme) of the wild-type (WT) and the variant enzymes. It is the quotient “Variant-specific activity/WT-specific activity” and can be a measure of the increase in specific activity of the variant. A PI of about 2 should be about 2 fold better than WT. In some aspects, the disclosure relates to a variant glucoamylase having altered specific activity as compared to a parent or wild-type glucoamylase. In some embodiments, the altered specific activity is increased specific activity. Increased specific activity can be defined as an increased performance index of greater than or equal to about 1, including greater than or equal to about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, and about 2. In some embodiments, the increased specific activity is from about 1.0 to about 5.0, including about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2., about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, and about 4.9. In some embodiments, the variant has an at least about 1.0 fold higher specific activity than the parent glucoamylase, including at least about 1.1 fold, about 1.2 fold, about 1.3 fold, about 1.4 fold, about 1.5 fold, about 1.6 fold, about 1.7 fold, about 1.8 fold, about 1.9 fold, about 2.0 fold, about 2.2 fold, about 2.5 fold, about 2.7 fold, about 2.9 fold, about 3.0 fold, about 4.0 fold, and about 5.0 fold.
In some embodiments, variants having an improvement in specific activity include one or more deletions, substitutions or insertions in the following positions in the amino acid sequence set forth in SEQ ID NO: 2: 10, 14, 15, 23, 59, 60, 61, 65, 67, 68, 72, 73, 97, 98, 99, 102, 110, 113, 133, 140, 144, 145, 147, 152, 153, 164, 182, 204, 205, 214, 216, 219, 228, 229, 230, 231, 236, 239, 241, 242, 263, 264, 265, 268, 269, 276, 284, 291, 300, 301, 303, 311, 338, 342, 344, 346, 349, 359, 361, 364, 375, 379, 382, 390, 391, 393, 394, 410, 417, 418, 430, 431, 433, 442, 444, 448, 451, 493, 494, 495, 502, 503, 508, 511, 518, 519, 520, 531, 535, 536, 539, or 563, or an equivalent position in a parent glucoamylase. In some embodiments, the parent glucoamylase will comprise a sequence having at least about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% sequence identity to the sequence of SEQ ID NO: 2. In some embodiments, the parent glucoamylase will also have structural identity to SEQ ID NO: 2. In some embodiments, variants of the disclosure having improved specific activity include a substitution in the following positions in the amino acid sequence set forth in SEQ ID NO: 2: I43Q, I43R, D44C, D44R, N061I, T067M, A072Y, S097N, S102A, S102M, S102R, I133T, N145I, N153D, T205Q, Q219S, W228A, W228F, W228H, W228M, S230C, S230F, 5230G, 5230L, 5230N, S230Q, S230R, S231L, I239V, I239Y, N263P, A268C, A268G, A268K, S291A, G294C, T342V, K394S, L417R, L417V, T430K, A431I, A431L, A431Q, R433Y, T451K, T495M, A519I, A520C, A520L, A520P, A535R, V536M, A539R, N563K, or N563I, or an equivalent position in a parent glucoamylase. In some embodiments, the specific activity of the parent as compared to the variant is determined as described in the Assays and Methods.
5.3. Variant Glucoamylases with Both Altered Thermostability and Altered Specific Activity
In some aspects, the disclosure relates to a variant glucoamylase having both altered thermostability and altered specific activity as compared to a parent (e.g., wild-type). In some embodiments, the altered specific activity is an increased specific activity. In some embodiments, the altered thermostability is an increased thermostability at high temperatures (e.g., at temperatures above 80° C.) as compared to the parent glucoamylase.
In some embodiments, variants with an increased thermostability and increased specific activity include one or more deletions, substitutions or insertions and substitutions in the following positions in the amino acid sequence set forth in SEQ ID NO: 2: 10, 15, 43, 44, 59, 61, 68, 72, 73, 97, 99, 102, 140, 153, 182, 204, 205, 214, 228, 229, 230, 231, 236, 241, 242, 264, 265, 268, 276, 284, 291, 294, 300, 301, 303, 311, 338, 344, 346, 349, 359, 361, 364, 375, 379, 382, 391, 393, 394, 410, 430, 433, 444, 448, 451, 495, 503, 511, 520, 531, 535, 536, 539, or 563, or an equivalent position in a parent glucoamylase. In some embodiments, the parent glucoamylase will be a Trichoderma glucoamylase homologue and in further embodiments, the parent glucoamylase will have at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 98% sequence identity to SEQ ID NO:2. In some embodiments, the parent glucoamylase will also have structural identity to SEQ ID NO: 2. In some embodiments, the variant having increased thermostability and specific activity has a substitution in at least one of the positions: I43Q/R, D44C/R, W228F/H/M, S230C/F/G/N/Q/R, S231L, A268C/D/G/K, S291A, G294C, R433Y, S451K, E503C, Q511H, A520C/L/P, or A535N/P/R of SEQ ID NO: 2.
5.4. Variant Glucoamylases with Production of Fermentable Sugar(s)
In a further aspect, the glucoamylase exhibit an enhanced production of fermentable sugar(s) as compared to the parent glucoamylase such as TrGA. In a further aspect, the glucoamylase exhibit an enhanced production of fermentable sugars in the mashing step of the brewing process as compared to the parent glucoamylase such as TrGA. In a further aspect, the glucoamylase exhibit an enhanced production of fermentable sugars in the fermentation step of the brewing process as compared to the parent glucoamylase such as TrGA. In a further aspect, the fermentable sugar is glucose. A skilled person within the field can determine the production of fermentable sugar(s) by e.g. HPLC techniques.
5.5 Variant Glucoamylases with a Altered Ratio Between Isomaltose Synthesis and Starch Hydrolysis Activity (IS/SH Ratio)
In a further aspect, the glucoamylase exhibit a reduced ratio between isomaltose synthesis and starch hydrolysis activity (IS/SH ratio) as compared to the parent glucoamylase such as TrGA. In a further aspect, the glucoamylase exhibit a starch hydrolysis activity which is not more than 5%, not more than 10% or not more than 15% reduced as compared to the parent glucoamylase such as TrGA.
In one aspect, a screening method for identification of a glucoamylase variant having a reduced synthesis of condensation products during hydrolysis of starch and the glucoamylse variants obtained by the method is provided, the method comprising the steps of measuring the isomaltose synthesis and starch hydrolysis activity of glucoamylase variants and selecting the variants having a reduced starch hydrolysis activity which is not more than 5%, not more than 10% or not more than 15% reduced as compared to the parent glucoamylase and having a reduced ratio between isomaltose synthesis and starch hydrolysis activity (IS/SH ratio) as compared to the parent glucoamylase.
In some embodiments the glucoamylase variants are selecting for having a reduced ratio between isomaltose synthesis and starch hydrolysis activity (IS/SH ratio) as compared to the parent glucoamylase.
In some embodiments the glucoamylase variants are selecting for having the same or increased starch hydrolysis activity and reduced isomaltose synthesis, which is not more than 5%, not more than 10% or not more than 15% reduced as compared to the parent glucoamylase and thereby having a reduced ratio between isomaltose synthesis and starch hydrolysis activity (IS/SH ratio) as compared to the parent glucoamylase.
In a further aspect, the glucoamylase exhibit an enhanced real degree of fermentation as compared to the parent glucoamylase such as TrGA.
5.6 Variant Glucoamylases with an Altered Formation of Condensation Products
In one aspect, the glucoamylase forms a lower amount of condensation products than the amount of condensation products formed by Aspergillus niger (AnGA) (SEQ ID NO: 6) under the same conditions. In a further aspect, the glucoamylase forms an amount of condensation products which amount is essentially the same as, not more than 5%, not more than 8% or not more than 10% higher than the amount of condensation products formed by Aspergillus niger (AnGA) (SEQ ID NO: 6) under the same conditions. In a further aspect, the dosing of the glucoamylases are the same based on protein concentration. In a further aspect, the dosing of the glucoamylases are the same based on measurement of activity in activity assays such as a GAU activity assay as described herein or a starch hydrolysation-activity assay also as described herein.
6. Polynucleotides Encoding Glucoamylases
The present disclosure also relates to isolated polynucleotides encoding the variant glucoamylase. The polynucleotides may be prepared by established techniques known in the art. The polynucleotides may be prepared synthetically, such as by an automatic DNA synthesizer. The DNA sequence may be of mixed genomic (or cDNA) and synthetic origin prepared by ligating fragments together. The polynucleotides may also be prepared by polymerase chain reaction (PCR) using specific primers. In general, reference is made to Minshull J. et al., Methods 32(4):416-427 (2004). DNA may also be synthesized by a number of commercial companies such as Geneart AG, Regensburg, Germany.
The present disclosure also provides isolated polynucleotides comprising a nucleotide sequence (i) having at least about 50% identity to SEQ ID NO: 4, including at least about 60%, about 70%, about 80%, about 90%, about 95%, and about 99%, or (ii) being capable of hybridizing to a probe derived from the nucleotide sequence set forth in SEQ ID NO: 4, under conditions of intermediate to high stringency, or (iii) being complementary to a nucleotide sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 4. Probes useful according to the disclosure may include at least about 50, about 100, about 150, about 200, about 250, about 300 or more contiguous nucleotides of SEQ ID NO: 4. In some embodiments, the encoded polypeptide also has structural identity to SEQ ID NO: 2.
The present disclosure further provides isolated polynucleotides that encode variant glucoamylases that comprise an amino acid sequence comprising at least about 50%, about 60%, about 70%, about 80%, about 90%, about 93%, about 95%, about 97%, about 98%, or about 99% amino acid sequence identity to SEQ ID NO: 2. Additionally, the present disclosure provides expression vectors comprising any of the polynucleotides provided above. The present disclosure also provides fragments (i.e., portions) of the DNA encoding the variant glucoamylases provided herein. These fragments find use in obtaining partial length DNA fragments capable of being used to isolate or identify polynucleotides encoding mature glucoamylase enzymes described herein from filamentous fungal cells (e.g., Trichoderma, Aspergillus, Fusarium, Penicillium, and Humicola), or a segment thereof having glucoamylase activity. In some embodiments, fragments of the DNA may comprise at least about 50, about 100, about 150, about 200, about 250, about 300 or more contiguous nucleotides. In some embodiments, portions of the DNA provided in SEQ ID NO: 4 may be used to obtain parent glucoamylases and particularly Trichoderma glucoamylase homologues from other species, such as filamentous fungi that encode a glucoamylase.
7. Production of Glucoamylases
7.1. DNA Constructs and Vectors
According to one embodiment of the disclosure, a DNA construct comprising a polynucleotide as described above encoding a variant glucoamylase encompassed by the disclosure and operably linked to a promoter sequence is assembled to transfer into a host cell. In one aspect, a polynucleotide encoding a glucoamylase variant as disclosed herein is provided.
The DNA construct may be introduced into a host cell using a vector. In one aspect, a vector comprising the polynucleotide, or capable of expressing a glucoamylase variant as disclosed herein is provided. The vector may be any vector that when introduced into a host cell is stably introduced. In some embodiments, the vector is integrated into the host cell genome and is replicated. Vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, phage particles, cassettes and the like. In some embodiments, the vector is an expression vector that comprises regulatory sequences operably linked to the glucoamylase coding sequence.
Examples of suitable expression and/or integration vectors are provided in Sambrook et al. (1989) supra, and Ausubel (1987) supra, and van den Hondel et al. (1991) in Bennett and Lasure (Eds.) More Gene Manipulations In Fungi, Academic Press pp. 396-428 and U.S. Pat. No. 5,874,276. Reference is also made to the Fungal Genetics Stock Center Catalogue of Strains (FGSC, www.fgsc.net) for a list of vectors. Particularly useful vectors include vectors obtained from for example Invitrogen and Promega.
Suitable plasmids for use in bacterial cells include pBR322 and pUC19 permitting replication in E. coli and pE194 for example permitting replication in Bacillus. Other specific vectors suitable for use in E. coli host cells include vectors such as pFB6, pBR322, pUC18, pUC100, pDONRTM201, 10 pDONRTM221, pENTRTM, pGEM® 32 and pGEM®4Z.
Specific vectors suitable for use in fungal cells include pRAX, a general purpose expression vector useful in Aspergillus, pRAX with a glaA promoter, and in Hypocrea/Trichoderma includes pTrex3g with a cbh1 promoter.
In some embodiments, the promoter that shows transcriptional activity in a bacterial or a fungal host cell may be derived from genes encoding proteins either homologous or heterologous to the host cell. The promoter may be a mutant, a truncated and/or a hybrid promoter. The above-mentioned promoters are known in the art. Examples of suitable promoters useful in fungal cells and particularly filamentous fungal cells such as Trichoderma or Aspergillus cells include such exemplary promoters as the T. reesei promoters cbh1, cbh2, egl1, egl2, eg5, xln1 and x1n2. Other examples of useful promoters include promoters from A. awamori and A. niger glucoamylase genes (glaA) (see Nunberg et al., Mol. Cell. Biol. 4: 2306-2315 (1984) and Boel et al., EMBO J. 3:1581-1585 (1984)), A. oryzae TAKA amylase promoter, the TPI (triose phosphate isomerase) promoter from S. cerevisiae, the promoter from Aspergillus nidulans acetamidase genes and Rhizomucor miehei lipase genes. Examples of suitable promoters useful in bacterial cells include those obtained from the E. coli lac operon; Bacillus licheniformis alpha-amylase gene (amyL), B. stearothermophilus amylase gene (amyS); Bacillus subtilis xylA and xylB genes, the beta-lactamase gene, and the tac promoter. In some embodiments, the promoter is one that is native to the host cell. For example, when T. reesei is the host, the promoter is a native T. reesei promoter. In other embodiments, the promoter is one that is heterologous to the fungal host cell. In some embodiments, the promoter will be the promoter of a parent glucoamylase (e.g., the TrGA promoter).
In some embodiments, the DNA construct includes nucleic acids coding for a signal sequence, that is, an amino acid sequence linked to the amino terminus of the polypeptide that directs the encoded polypeptide into the cell's secretory pathway. The 5′ end of the coding sequence of the nucleic acid sequence may naturally include a signal peptide coding region that is naturally linked in translation reading frame with the segment of the glucoamylase coding sequence that encodes the secreted glucoamylase or the 5′ end of the coding sequence of the nucleic acid sequence may include a signal peptide that is foreign to the coding sequence. In some embodiments, the DNA construct includes a signal sequence that is naturally associated with a parent glucoamylase gene from which a variant glucoamylase has been obtained. In some embodiments, the signal sequence will be the sequence depicted in SEQ ID NO: 1 or a sequence having at least about 90%, about 94, or about 98% sequence identity thereto. Effective signal sequences may include the signal sequences obtained from other filamentous fungal enzymes, such as from Trichoderma (T. reesei glucoamylase, cellobiohydrolase I, cellobiohydrolase II, endoglucanase I, endoglucanase II, endoglucanase II, or a secreted proteinase, such as an aspartic proteinase), Humicola (H. insolens cellobiohydrolase or endoglucanase, or H. grisea glucoamylase), or Aspergillus (A. niger glucoamylase and A. oryzae TAKA amylase).
In additional embodiments, a DNA construct or vector comprising a signal sequence and a promoter sequence to be introduced into a host cell are derived from the same source. In some embodiments, the native glucoamylase signal sequence of a Trichoderma glucoamylase homologue, such as a signal sequence from a Hypocrea strain may be used.
In some embodiments, the expression vector also includes a termination sequence. Any termination sequence functional in the host cell may be used in the present disclosure. In some embodiments, the termination sequence and the promoter sequence are derived from the same source. In another embodiment, the termination sequence is homologous to the host cell. Useful termination sequences include termination sequences obtained from the genes of Trichoderma reesei cbl1; A. niger or A. awamori glucoamylase (Nunberg et al. (1984) supra, and Boel et al., (1984) supra), Aspergillus nidulans anthranilate synthase, Aspergillus oryzae TAKA amylase, or A. nidulans trpC (Punt et al., Gene 56:117-124 (1987)).
In some embodiments, an expression vector includes a selectable marker. Examples of selectable markers include ones that confer antimicrobial resistance (e.g., hygromycin and phleomycin). Nutritional selective markers also find use in the present disclosure including those markers known in the art as amdS (acetamidase), argB (ornithine carbamoyltransferase) and pyrG (orotidine-5′ phosphate decarboxylase). Markers useful in vector systems for transformation of Trichoderma are known in the art (see, e.g., Finkelstein, Chapter 6 in Biotechnology Of Filamentous Fungi, Finkelstein et al. (1992) Eds. Butterworth-Heinemann, Boston, Mass.; Kinghorn et al. (1992) Applied Molecular Genetics Of Filamentous Fungi, Blackie Academic and Professional, Chapman and Hall, London; Berges and Barreau, Curr. Genet. 19:359-365 (1991); and van Hartingsveldt et al., Mol. Gen. Genet. 206:71-75 (1987)). In some embodiments, the selective marker is the amdS gene, which encodes the enzyme acetamidase, allowing transformed cells to grow on acetamide as a nitrogen source. The use of A. nidulans amdS gene as a selective marker is described in Kelley et al., EMBO J. 4:475-479 (1985) and Penttila et al., Gene 61:155-164 (1987).
Methods used to ligate the DNA construct comprising a nucleic acid sequence encoding a variant glucoamylase, a promoter, a termination and other sequences and to insert them into a suitable vector are well known in the art. Linking is generally accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide linkers are used in accordance with conventional practice (see Sambrook et al. (1989) supra, and Bennett and Lasure, More Gene Manipulations In Fungi, Academic Press, San Diego (1991) pp 70-76). Additionally, vectors can be constructed using known recombination techniques (e.g., Invitrogen Life Technologies, Gateway Technology).
7.2. Host Cells and Transformation of Host Cells
1. The present disclosure also relates to host cells comprising a polynucleotide encoding a variant glucoamylase of the disclosure. In some embodiments, the host cells are chosen from bacterial, fungal, plant and yeast cells. The term host cell includes both the cells, progeny of the cells and protoplasts created from the cells that are used to produce a variant glucoamylase according to the disclosure. In one aspect, a host cell comprising, preferably transformed with a vector is disclosed. In a further aspect, a cell capable of expressing a glucoamylase variant is provided. In a further aspect, the host cell is a protease deficient and/or xylanase deficient and/or glucanase deficient host cell. A protease deficient and/or xylanase deficient and/or native glucanase deficient host cell may be obtained by deleting or silencing the genes coding for the mentioned enzymes. As a consequence the host cell containing the GA-variant is not expressing the mentioned enzymes
In some embodiments, the host cells are fungal cells and optionally filamentous fungal host cells. The term “filamentous fungi” refers to all filamentous forms of the subdivision Eumycotina (see, Alexopoulos, C. J. (1962), Introductory Mycology, Wiley, New York). These fungi are characterized by a vegetative mycelium with a cell wall composed of chitin, cellulose, and other complex polysaccharides. The filamentous fungi of the present disclosure are morphologically, physiologically, and genetically distinct from yeasts. Vegetative growth by filamentous fungi is by hyphal elongation and carbon catabolism is obligatory aerobic. In the present disclosure, the filamentous fungal parent cell may be a cell of a species of, but not limited to, Trichoderma (e.g., Trichoderma reesei, the asexual morph of Hypocrea jecorina, previously classified as T. longibrachiatum, Trichoderma viride, Trichoderma koningii, Trichoderma harzianum) (Sheir-Neirs et al., Appl. Microbiol. Biotechnol. 20:46-53 (1984); ATCC No. 56765 and ATCC No. 26921), Penicillium sp., Humicola sp. (e.g., H. insolens, H. lanuginosa and H. grisea), Chrysosporium sp. (e.g., C. lucknowense), Gliocladium sp., Aspergillus sp. (e.g., A. oryzae, A. niger, A sojae, A. japonicus, A. nidulans, and A. awamori) (Ward et al., Appl. Microbiol. Biotechnol. 39:738-743 (1993) and Goedegebuur et al., Curr. Genet. 41:89-98 (2002)), Fusarium sp., (e.g., F. roseum, F. graminum, F. cerealis, F. oxysporum, and F. venenatum), Neurospora sp., (N. crassa), Hypocrea sp., Mucor sp. (M. miehei), Rhizopus sp., and Emericella sp. (see also, Innis et al., Science 228:21-26 (1985)). The term “Trichoderma” or “Trichoderma sp.” or “Trichoderma spp.” refer to any fungal genus previously or currently classified as Trichoderma. 
In some embodiments, the host cells will be gram-positive bacterial cells. Non-limiting examples include strains of Streptomyces (e.g., S. lividans, S. coelicolor, and S. griseus) and Bacillus. As used herein, “the genus Bacillus” includes all species within the genus “Bacillus,” as known to those of skill in the art, including but not limited to B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B. coagulans, B. circulans, B. lautus, and B. thuringiensis. It is recognized that the genus Bacillus continues to undergo taxonomical reorganization. Thus, it is intended that the genus include species that have been reclassified, including but not limited to such organisms as B. stearothermophilus, which is now named “Geobacillus tearothermophilus.”
In some embodiments, the host cell is a gram-negative bacterial strain, such as E. coli or Pseudomonas sp. In other embodiments, the host cells may be yeast cells such as Saccharomyces sp., Schizosaccharomyces sp., Pichia sp., or Candida sp. In other embodiments, the host cell will be a genetically engineered host cell wherein native genes have been inactivated, for example by deletion in bacterial or fungal cells. Where it is desired to obtain a fungal host cell having one or more inactivated genes known methods may be used (e.g., methods disclosed in U.S. Pat. No. 5,246,853, U.S. Pat. No. 5,475,101, and WO 92/06209). Gene inactivation may be accomplished by complete or partial deletion, by insertional inactivation or by any other means that renders a gene nonfunctional for its intended purpose (such that the gene is prevented from expression of a functional protein). In some embodiments, when the host cell is a Trichoderma cell and particularly a T. reesei host cell, the cbh1, cbh2, egl1 and eg12 genes will be inactivated and/or deleted. Exemplary Trichoderma reesei host cells having quad-deleted proteins are set forth and described in U.S. Pat. No. 5,847,276 and WO 05/001036. In other embodiments, the host cell is a protease deficient or protease minus strain.
Introduction of a DNA construct or vector into a host cell includes techniques such as transformation; electroporation; nuclear microinjection; transduction; transfection, (e.g., lipofection-mediated and DEAE-Dextrin mediated transfection); incubation with calcium phosphate DNA precipitate; high velocity bombardment with DNA-coated microprojectiles; and protoplast fusion. General transformation techniques are known in the art (see, e.g., Ausubel et al. (1987) supra, chapter 9; and Sambrook et al. (1989) supra, and Campbell et al., Curr. Genet. 16:53-56 (1989)).
Transformation methods for Bacillus are disclosed in numerous references including Anagnostopoulos C. and J. Spizizen, J. Bacteriol. 81:741-746 (1961) and WO 02/14490.
Transformation methods for Aspergillus are described in Yelton et al., Proc. Natl. Acad. Sci. USA 81:1470-1474 (1984); Berka et al., (1991) in Applications of Enzyme Biotechnology, Eds. Kelly and Baldwin, Plenum Press (NY); Cao et al., Protein Sci. 9:991-1001 (2000); Campbell et al., Curr. Genet. 16:53-56 (1989), and EP 238 023. The expression of heterologous protein in Trichoderma is described in U.S. Pat. No. 6,022,725; U.S. Pat. No. 6,268,328; Harkki et al. Enzyme Microb. Technol. 13:227-233 (1991); Harkki et al., BioTechnol. 7:596-603 (1989); EP 244,234; EP 215,594; and Nevalainen et al., “The Molecular Biology of Trichoderma and its Application to the Expression of Both Homologous and Heterologous Genes”, in Molecular Industrial Mycology, Eds. Leong and Berka, Marcel Dekker Inc., NY (1992) pp. 129-148). Reference is also made to WO96/00787 and Bajar et al., Proc. Natl. Acad. Sci. USA 88:8202-8212 (1991) for transformation of Fusarium strains.
In one specific embodiment, the preparation of Trichoderma sp. for transformation involves the preparation of protoplasts from fungal mycelia (see, Campbell et al., Curr. Genet. 16:53-56 (1989); Pentilla et al., Gene 61:155-164 (1987)). Agrobacterium tumefaciens-mediated transformation of filamentous fungi is known (see de Groot et al., Nat. Biotechnol. 16:839-842 (1998)). Reference is also made to U.S. Pat. No. 6,022,725 and U.S. Pat. No. 6,268,328 for transformation procedures used with filamentous fungal hosts.
In some embodiments, genetically stable transformants are constructed with vector systems whereby the nucleic acid encoding the variant glucoamylase is stably integrated into a host strain chromosome. Transformants are then purified by known techniques.
In some further embodiments, the host cells are plant cells, such as cells from a monocot plant (e.g., corn, wheat, and sorghum) or cells from a dicot plant (e.g., soybean). Methods for making DNA constructs useful in transformation of plants and methods for plant transformation are known. Some of these methods include Agrobacterium tumefaciens mediated gene transfer; microprojectile bombardment, PEG mediated transformation of protoplasts, electroporation and the like. Reference is made to U.S. Pat. No. 6,803,499, U.S. Pat. No. 6,777,589; Fromm et al., BioTechnol. 8:833-839 (1990); Potrykus et al., Mol. Gen. Genet. 199:169-177 (1985).
7.3. Production of Glucoamylases
The present disclosure further relates to methods of producing the variant glucoamylases, which comprises transforming a host cell with an expression vector comprising a polynucleotide encoding a variant glucoamylase according to the disclosure, culturing the host cell under conditions suitable for expression and production of the variant glucoamylase and optionally recovering the variant glucoamylase. In one aspect, a method of expressing a variant glucoamylase according to the disclosure, the method comprising obtaining a host cell or a cell as disclosed herein and expressing the glucoamylase variant from the cell or host cell, and optionally purifying the glucoamylase variant, is provided. In one aspect, the glucoamylase variant is purified.
In the expression and production methods of the present disclosure the host cells are cultured under suitable conditions in shake flask cultivation, small scale or large scale fermentations (including continuous, batch and fed batch fermentations) in laboratory or industrial fermentors, with suitable medium containing physiological salts and nutrients (see, e.g., Pourquie, J. et al., Biochemistry And Genetics Of Cellulose Degradation, eds. Aubert, J. P. et al., Academic Press, pp. 71-86, 1988 and Ilmen, M. et al., Appl. Environ. Microbiol. 63:1298-1306 (1997)). Common commercially prepared media (e.g., Yeast Malt Extract (YM) broth, Luria Bertani (LB) broth and Sabouraud Dextrose (SD) broth) find use in the present disclosure. Culture conditions for bacterial and filamentous fungal cells are known in the art and may be found in the scientific literature and/or from the source of the fungi such as the American Type Culture Collection and Fungal Genetics Stock Center. In cases where a glucoamylase coding sequence is under the control of an inducible promoter, the inducing agent (e.g., a sugar, metal salt or antimicrobial), is added to the medium at a concentration effective to induce glucoamylase expression.
In some embodiments, the present disclosure relates to methods of producing the variant glucoamylase in a plant host comprising transforming a plant cell with a vector comprising a polynucleotide encoding a glucoamylase variant according to the disclosure and growing the plant cell under conditions suitable for the expression and production of the variant.
In some embodiments, assays are carried out to evaluate the expression of a variant glucoamylase by a cell line that has been transformed with a polynucleotide encoding a variant glucoamylase encompassed by the disclosure. The assays can be carried out at the protein level, the RNA level and/or by use of functional bioassays particular to glucoamylase activity and/or production. Some of these assays include Northern blotting, dot blotting (DNA or RNA analysis), RT-PCR (reverse transcriptase polymerase chain reaction), in situ hybridization using an appropriately labeled probe (based on the nucleic acid coding sequence) and conventional Southern blotting and autoradiography.
In addition, the production and/or expression of a variant glucoamylase may be measured in a sample directly, for example, by assays directly measuring reducing sugars such as glucose in the culture medium and by assays for measuring glucoamylase activity, expression and/or production. In particular, glucoamylase activity may be assayed by the 3,5-dinitrosalicylic acid (DNS) method (see Goto et al., Biosci. Biotechnol. Biochem. 58:49-54 (1994)). In additional embodiments, protein expression, is evaluated by immunological methods, such as immunohistochemical staining of cells, tissue sections or immunoassay of tissue culture medium, (e.g., by Western blot or ELISA). Such immunoassays can be used to qualitatively and quantitatively evaluate expression of a glucoamylase. The details of such methods are known to those of skill in the art and many reagents for practicing such methods are commercially available.
The glucoamylases of the present disclosure may be recovered or purified from culture media by a variety of procedures known in the art including centrifugation, filtration, extraction, precipitation and the like.
8. Compositions and Uses
In one aspect, the use of a glucoamylase variant as described herein for the preparation of an enzymatic composition, is provided.
The variant glucoamylases of the disclosure may be used in enzyme compositions including but not limited to starch hydrolyzing and saccharifying compositions, cleaning and detergent compositions (e.g., laundry detergents, dish washing detergents, and hard surface cleaning compositions), alcohol fermentation compositions, and in animal feed compositions. Further, the variant glucoamylases may be used in, for example, brewing, healthcare, textile, environmental waste conversion processes, biopulp processing, and biomass conversion applications. The variant glucoamylases of the disclosure may be used in enzyme compositions including a starch hydrolyzing composition, a saccharifying composition, a detergent, an alcohol fermentation enzymatic composition, and an animal feed. In one aspect, the composition is a starch hydrolyzing composition.
In some embodiments, an enzyme composition comprising a variant glucoamylase encompassed by the disclosure will be optionally used in combination with any one or combination of the following enzymes—alpha-amylases, proteases, pullulanases, isoamylases, cellulases, hemicellulases, xylanases, cyclodextrin glycotransferases, lipases, phytases, laccases, oxidases, esterases, cutinases, xylanases, granular starch hydrolyzing enzymes and other glucoamylases.
In some embodiments, an enzyme composition comprising a variant glucoamylase encompassed by the disclosure will be optionally used in combination with any one or combination of the following enzymes—amylase, protease, pullulanase, cellulase, glucanase, xylanase, arabinofuranosidase, ferulic acid esterase, xylan acetyl esterase and a further glucoamylase. In some embodiments, an enzyme composition comprising a variant glucoamylase encompassed by the disclosure will be optionally used in combination with any one or combination of the following enzymes—amylase, pullulanase and a further glucoamylase. In some embodiments, an enzyme composition comprising a variant glucoamylase encompassed by the disclosure will be optionally used in combination with any one or combination of the following enzymes—amylase and pullulanase. In a further aspect, the amylase is alpha-amylase and/or isoamylase. In a further aspect, the glucanase is exoglucanase and/or endoglucanase.
In some embodiments, the enzyme composition will include an alpha-amylase such as fungal alpha-amylases (e.g., Aspergillus sp.) or bacterial alpha-amylases (e.g., Bacillus sp. such as B. stearothermophilus, B. amyloliquefaciens and B. licheniformis) and variants and hybrids thereof. In the present context, an alpha-amylase (EC. 3.2.1.1) catalyses the endohydrolysis of (1->4)-alpha-D-glucosidic linkages in oligosaccharides and polysaccharides. An alpha-amylase acts on starch, glycogen and related polysaccharides and oligosaccharides in a random manner; reducing groups are liberated in the alpha-configuration. In some embodiments, the alpha-amylase is an acid stable alpha-amylase. In some embodiments, the alpha-amylase is Aspergillus kawachi alpha-amylase (AkAA), see U.S. Pat. No. 7,037,704. Other alpha-amylases contemplated for use in the compositions of the disclosure include, but are not limited to, bacterial alpha-amylases such as those from Bacillus subtilis (AmyE), and Bacillus licheniformis (AmyL) and Geobacillus stearothermophilus (AmyS) as described by Gray et al. (1986) (Gray G L, Mainzer S E, Rey M W, Lamsa M H, Kindle K L, Carmona C and Requadt C “Structural genes encoding the thermophilic alpha-amylases of Bacillus stearothermophilus and Bacillus licheniformis” Journal of Bacteriology (1986) 166(2) p 635-643) along with variants and combinations, including combinations of variants of the above. Variants of AmyE, AmyL and AmyS are well known and examples are described in US Patent Application 20100015686 A1 (“Variant Alpha-Amylases from Bacillus subtilis and Methods of Uses, Thereof”), US Patent Application 20090314286 A1 (“Geobacillus stearothermophilus Alpha-Amylase (AmyS) Variants with Improved Properties”), WO/2006/066594) (“Alpha-Amylase Variants”), US 20090238923 A1 (“Variants Of Bacillus Licheniformis Alpha-Amylase With Increased Thermostability And/Or Decreased Calcium Dependence”). Commercially available alpha-amylases contemplated for use in the compositions of the disclosure are known and include GZYME G997, SPEZYME® FRED, SPEZYME® XTRA AMYLEX® 4T, AMYLEX® 3T and AMYLEX® XT (Danisco US, Inc, Genencor Division), TERMAMYL® 120-L and SUPRA® (Novozymes, A/S).
In some embodiments, the enzyme composition will include a pullulanase (EC 3.2.1.41). In one aspect, the pullulanases used herein is pullulanase from e.g. Pyrococcus or Bacillus sp, such as Bacillus acidopullulyticus (e.g., the one described in FEMS Microbiol. Letters 115: 97-106) or Bacillus deramificans, or Bacillus naganoencis. In one aspect, the pullulanase is the Bacillus acidopullulyticus PulB enzyme, described in the paper by Kelly et al. FEMS Microbiology Letters 115 (1994) 97-106. The pullulanase may also be an engineered pullulanases from, e.g., a Bacillus strain. Other pullulanases which are preferably used in the processes according to the invention include: Bacillus deramificans (U.S. Pat. No. 5,736,375), or the pullulanase may be derived from Pyrococcus woesei described in PCT/DK91/00219, or the pullulanase may be derived from Fervidobacterium sp. Ven 5 described in PCT/DK92/00079, or the pullulanase may be derived from Thermococcus celer described in PCT/DK95/00097, or the pullulanase may be derived from Pyrodictium abyssei described in PCT/DK95/00211, or the pullulanase may be derived from Fervidobacterium pennavorans described in PCT/DK95/00095, or the pullulanase may be derived from Desulforococcus mucosus described in PCT/DK95/00098. The pullulanase (EC 3.2.1.41) may also be derived from, but not limited to, Klebsiella (Aerobacter) spp. (PulA); for example Klebsiella planticola, Klebsiella (Aerobacter) aerogenes and Klebsiella pneumoniae (see: Katsuragi et al. Journal of Bacteriology (1987) 169(5) p 2301-2306; Fouts et al. PLoS Genetics (2008) 4(7), E1000141). These pullulanases, along with those from, for example, Bacillus acidopullulyticus are members of Glycoside Hydrolase Family 13 In some embodiments, the enzyme composition will include an acid fungal protease. In a further embodiment, the acid fungal protease is derived from a Trichoderma sp. and may be any one of the proteases disclosed in U.S. Pat. No. 7,563,607 (published as US 2006/0154353 Jul. 13, 2006), incorporated herein by reference. In a further embodiment, the enzyme composition will include a phytase from Buttiauxiella spp. (e.g., BP-17, see also variants disclosed in PCT patent publication WO 2006/043178).
In other embodiments, the variant glucoamylases of the disclosure may be combined with other glucoamylases. In some embodiments, the glucoamylases of the disclosure will be combined with one or more glucoamylases derived from strains of Aspergillus or variants thereof, such as A. oryzae, A. niger, A. kawachi, and A. awamori; glucoamylases derived from strains of Humicola or variants thereof, particularly H. grisea, such as the glucoamylase having at least about 90%, about 93%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to SEQ ID NO: 3 disclosed in WO 05/052148; glucoamylases derived from strains of Talaromyces or variants thereof, particularly T. emersonii; glucoamylases derived from strains of Athelia and particularly A. rolfsii; glucoamylases derived from strains of Penicillium, particularly P. chrysogenum. 
In particular, the variant glucoamylases may be used for starch conversion processes, and particularly in the production of dextrose for fructose syrups, specialty sugars and in alcohol and other end-product (e.g., organic acid, ascorbic acid, and amino acids) production from fermentation of starch containing substrates (G. M. A. van Beynum et al., Eds. (1985) Starch Conversion Technology, Marcel Dekker Inc. NY). Dextrins produced using variant glucoamylase compositions of the disclosure may result in glucose yields of at least 80%, at least 85%, at least 90% and at least 95%. Production of alcohol from the fermentation of starch substrates using glucoamylases encompassed by the disclosure may include the production of fuel alcohol or portable alcohol. In some embodiments, the production of alcohol will be greater when the variant glucoamylase is used under the same conditions as the parent glucoamylase. In some embodiments, the production of alcohol will be between about 0.5% and 2.5% better, including but not limited to about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%. about 1.7%, about 1.8%, about 1.9%, about 2.0%, about 2.1%, about 2.2%, about 2.3%, and about 2.4% more alcohol than the parent glucoamylase.
In some embodiments, the variant glucoamylases of the disclosure will find use in the hydrolysis of starch from various plant-based substrates, which are used for alcohol production. In some embodiments, the plant-based substrates will include corn, wheat, barley, rye, milo, rice, sugar cane, potatoes and combinations thereof. In some embodiments, the plant-based substrate will be fractionated plant material, for example a cereal grain such as corn, which is fractionated into components such as fiber, germ, protein and starch (endosperm) (U.S. Pat. No. 6,254,914 and U.S. Pat. No. 6,899,910). Methods of alcohol fermentations are described in The Alcohol Textbook, K. A. Jacques et al., Eds. 2003, Nottingham University Press, UK.
In certain embodiments, the alcohol will be ethanol. In particular, alcohol fermentation production processes are characterized as wet milling or dry milling processes. In some embodiments, the variant glucoamylase will be used in a wet milling fermentation process and in other embodiments the variant glucoamylase will find use in a dry milling process.
Dry grain milling involves a number of basic steps, which generally include: grinding, cooking, liquefaction, saccharification, fermentation and separation of liquid and solids to produce alcohol and other co-products. Plant material and particularly whole cereal grains, such as corn, wheat or rye are ground. In some cases, the grain may be first fractionated into component parts. The ground plant material may be milled to obtain a coarse or fine particle. The ground plant material is mixed with liquid (e.g., water and/or thin stillage) in a slurry tank. The slurry is subjected to high temperatures (e.g., about 90° C. to about 105° C. or higher) in a jet cooker along with liquefying enzymes (e.g., alpha-amylases) to solublize and hydrolyze the starch in the grain to dextrins. The mixture is cooled down and further treated with saccharifying enzymes, such as glucoamylases encompassed by the instant disclosure, to produce glucose. The mash containing glucose may then be fermented for approximately 24 to 120 hours in the presence of fermentation microorganisms, such as ethanol producing microorganism and particularly yeast (Saccharomyces spp). The solids in the mash are separated from the liquid phase and alcohol such as ethanol and useful co-products such as distillers' grains are obtained.
In some embodiments, the saccharification step and fermentation step are combined and the process is referred to as simultaneous saccharification and fermentation or simultaneous saccharification, yeast propagation and fermentation.
In other embodiments, the variant glucoamylase is used in a process for starch hydrolysis wherein the temperature of the process is between about 30° C. and about 75° C., in some embodiments, between about 40° C. and about 65° C. In some embodiments, the variant glucoamylase is used in a process for starch hydrolysis at a pH between about 3.0 and about 6.5. The fermentation processes in some embodiments include milling of a cereal grain or fractionated grain and combining the ground cereal grain with liquid to form a slurry that is then mixed in a single vessel with a variant glucoamylase according to the disclosure and optionally other enzymes such as, but not limited to, alpha-amylases, other glucoamylases, phytases, proteases, pullulanases, isoamylases or other enzymes having granular starch hydrolyzing activity and yeast to produce ethanol and other co-products (see e.g., U.S. Pat. No. 4,514,496, WO 04/081193, and WO 04/080923).
In some embodiments, the disclosure pertains to a method of saccharifying a liquid starch solution, which comprises an enzymatic saccharification step using a variant glucoamylase of the disclosure. The liquid starch solution may be produced by solubilising starch in water or an aqueous buffer and optionally heating to gelatinize the starch. Further partial degradation of the starch by amylases may be applied.
The present invention provides a method of using glucoamylase variants of the invention for producing glucose and the like from starch. Generally, the method includes the steps of partially hydrolyzing precursor starch in the presence of alpha-amylase and then further hydrolyzing the release of D-glucose from the non-reducing ends of the starch or related oligo- and polysaccharide molecules in the presence of glucoamylase by cleaving alpha-(1-4) and alpha-(1-6) glucosidic bonds. The partial hydrolysis of the precursor starch utilizing alpha-amylase provides an initial breakdown of the starch molecules by hydrolyzing internal alpha-(1-4)-linkages. In commercial applications, the initial hydrolysis using alpha-amylase is run at a temperature of approximately 105° C. A very high starch concentration is processed, usually 30% to 40% solids. The initial hydrolysis is usually carried out for five minutes at this elevated temperature. The partially hydrolyzed starch can then be transferred to a second tank and incubated for approximately one hour at a temperature of 85° to 90° C. to derive a dextrose equivalent (D.E.) of 10 to 15. The step of further hydrolyzing the release of D-glucose from the non-reducing ends of the starch or related oligo- and polysaccharides molecules in the presence of glucoamylase is normally carried out in a separate tank at a reduced temperature between 30° and 60° C. Often the temperature of the substrate liquid is dropped to between 55° C. and 60° C. The pH of the solution is dropped from 6 to 6.5 to a range between 3 and 5.5. Often, the pH of the solution is 4 to 4.5. The glucoamylase is added to the solution and the reaction is carried out for 24-72 hours, such as 36-48 hours.
Examples of saccharification processes wherein the glucoamylase variants of the invention may be used include the processes described in JP 3-224493; JP 1-191693; JP 62-272987; and EP 452,238. The glucoamylase variant(s) described herein may be used in combination with an enzyme that hydrolyzes only alpha-(1-6)-glucosidic bonds in molecules with at least four glucosyl residues. Preferentially, the glucoamylase variant can be used in combination with pullulanase or alpha-amylase. The use of alpha-amylase and pullulanase for debranching, the molecular properties of the enzymes, and the potential use of the enzymes with glucoamylase is set forth in G. M. A. van Beynum et al., Starch Conversion Technology, Marcel Dekker, New York, 1985, 101-142.
In one embodiment, the use of a glucoamylase variant as described herein in a starch conversion process, such as in a continuous saccharification step, is provided. The glucoamylase variants described herein may also be used in immobilised form. This is suitable and often used for producing maltodextrins or glucose syrups or specialty syrups, such as maltose syrups and further for the raffinate stream of oligosaccharides in connection with the production of fructose syrups.
When the desired final sugar product is, e.g., high fructose syrup the dextrose syrup may be converted into fructose. After the saccharification process the pH is increased to a value in the range of 6-8, such as pH 7.5, and the calcium is removed by ion exchange. The dextrose syrup is then converted into high fructose syrup using, e.g., an immobilized glucose isomerase (such as SweetzymeTM IT).
In other embodiments, the variant glucoamylase is used in a process for beer brewing. Brewing processes are well-known in the art, and generally involve the steps of malting, mashing, and fermentation. Mashing is the process of converting starch from the milled barley malt and solid adjuncts into fermentable and un-fermentable sugars to produce wort. Traditional mashing involves mixing milled barley malt and adjuncts with water at a set temperature and volume to continue the biochemical changes initiated during the malting process. The mashing process is conducted over a period of time at various temperatures in order to activate the endogenous enzymes responsible for the degradation of proteins and carbohydrates. After mashing, the wort is separated from the solids (spent grains). Following wort separation, the wort may be fermented with brewers' yeast to produce a beer. The short-branched glucose oligomers formed during mashing may be further hydrolyzed by addition of exogenous enzymes like glucoamylases and/or alpha-amylases, beta-amylases and pullulanase, among others. The wort may be used as it is or it may be concentrated and/or dried. The concentrated and/or dried wort may be used as brewing extract, as malt extract flavoring, for non-alcoholic malt beverages, malt vinegar, breakfast cereals, for confectionery etc. The wort is fermented to produce an alcoholic beverage, typically a beer, e.g., ale, strong ale, bitter, stout, porter, lager, export beer, malt liquor, barley wine, happoushu, high-alcohol beer, low-alcohol beer, low-calorie beer, or light beer. In another typical embodiment, the wort is fermented to produce portable ethanol.
In some embodiments, the disclosure pertains to a method of hydrolyzing and saccharifying gelatinised and liquefied (typically) grist starch to be used in brewing, whereby an enzymatic composition comprising one or more glucoamylases as contemplated herein, is used to enhance the amount of brewers' yeast fermentable sugars obtained from the starch. A brewing process is used to produce the portable product, beer, where fermentable sugars are converted to ethanol and CO2 by fermentation with brewers' yeast. The fermentable sugars are traditionally derived from starch in cereal grains, optionally supplemented with fermentable sugar sources such as glucose and maltose syrups and cane sugar. Briefly, beer production, well-known in the art, typically includes the steps of malting, mashing, and fermentation.
Historically the first step in beer production is malting—steeping, germination and drying of cereal grain (e.g. barley). During malting enzymes are produced in the germinating cereal (e.g. barley) kernel and there are certain changes in its chemical constituents (known as modification) including some degradation of starch, proteins and beta-glucans.
The malted cereal is milled to give a grist which may be mixed with a milled adjunct (e.g. non-germinated cereal grain) to give a mixed grist. The grist is mixed with water and subjected to mashing; a previously cooked (gelatinised and liquefied) adjunct may be added to the mash. The mashing process is conducted over a period of time at various temperatures in order to hydrolyse cereal proteins, degrade beta-glucans and solubilise and hydrolyse the starch. The hydrolysis of the grist starch in the malt and adjunct in traditional mashing is catalysed by two main enzymes endogenous to malted barley. Alpha-amylase, randomly cleaves alpha-1,4 bonds in the interior of the starch molecule fragmenting them into smaller dextrins. Beta-amylase sequentially cleaves alpha-1,4 bonds from the non-reducing end of the these dextrins producing mainly maltose. Both alpha- and beta-amylase are unable to hydrolyse the alpha-1,6 bonds which forms the branching points of the starch chains in the starch molecule, which results in the accumulation of limit dextrins in the mash. Malt does contain an enzyme, limit dextrinase, which catalyses the hydrolysis of alpha-1,6 bonds but it only shows weak activity at mashing temperatures due to its thermolability. After mashing, the liquid extract (wort) is separated from the spent grain solids (i.e. the insoluble grain and husk material forming part of grist). The objectives of wort separation include: • to obtain good extract recovery, • to obtain good filterability, and • to produce clear wort. Extract recovery and filterability of the wort are important in the economics of the brewing process.
The composition of the wort depends on the raw materials, mashing process and profiles and other variables. A typical wort comprises 65-80% fermentable sugars (glucose, maltose and maltotriose, and 20-35% non-fermentable limit dextrins (sugars with a higher degree of polymerization than maltotriose). An insufficiency of starch hydrolytic enzymes during mashing can arise when brewing with high levels of adjunct unmalted cereal grists. A source of exogenous enzymes, capable of producing fermentable sugars during the mashing process is thus needed. Furthermore, such exogenous enzymes are also needed to reduce the level of non-fermentable sugars in the wort, with a corresponding increase in fermentable sugars, in order to brew highly attenuated beers with a low carbohydrate content. Herein disclosed is a enzyme composition for hydrolysis of starch comprising at least one glucoamylase as contemplated herein, which can be added to the mash or used in the mashing step of a brewing process, in order to cleave alpha-1,4 bonds and/or alpha-1,6 bonds in starch grist and thereby increase the fermentable sugar content of the wort and reduce the residue of non-fermentable sugars in the finished beer. In addition, the wort, so produced may be dried (by for example spray drying) or concentrated (e.g. boiling and evaporation) to provide a syrup or powder.
The grist, as contemplated herein, may comprise any starch and/or sugar containing plant material derivable from any plant and plant part, including tubers, roots, stems, leaves and seeds. Often the grist comprises grain, such as grain from barley, wheat, rye, oat, corn, rice, milo, millet and sorghum, and more preferably, at least 10%, or more preferably at least 15%, even more preferably at least 25%, or most preferably at least 35%, such as at least 50%, at least 75%, at least 90% or even 100% (w/w) of the grist of the wort is derived from grain. Most preferably the grist comprises malted grain, such as barley malt. Preferably, at least 10%, or more preferably at least 15%, even more preferably at least 25%, or most preferably at least 35%, such as at least 50%, at least 75%, at least 90% or even 100% (w/w) of the grist of the wort is derived from malted grain. Preferably the grist comprises adjunct, such as non-malted grain from barley, wheat, rye, oat, corn, rice, milo, millet and sorghum, and more preferably, at least 10%, or more preferably at least 15%, even more preferably at least 25%, or most preferably at least 35%, such as at least 50%, at least 75%, at least 90% or even 100% (w/w) of the grist of the wort is derived from non-malted grain or other adjunct. Adjunct comprising readily fermentable carbohydrates such as sugars or syrups may be added to the malt mash before, during or after the mashing process of the invention but is preferably added after the mashing process. A part of the adjunct may be treated with an alpha-amylase, and/or endopeptidase (protease) and/or a endoglucanase, and/or heat treated before being added to the mash. The enzyme composition, as contemplated herein, may include additional enzyme(s), preferably an enzyme selected from among an alpha-amylase, protease, pullulanase, isoamylase, cellulase, glucanase such as exoglucanase or endoglucanase, xylanase, arabinofuranosidase, feruloyl esterase, xylan acetyl esterase, phytase and glucoamylase. During the mashing process, starch extracted from the grist is gradually hydrolyzed into fermentable sugars and smaller dextrins. Preferably the mash is starch negative to iodine testing, before wort separation.
In one aspect, a pullulanase (E. C. 3.2.1.41) enzyme activity is exogenously supplied and present in the mash. The pullulanase may be added to the mash ingredients, e.g., the water and/or the grist before, during or after forming the mash.
In another aspect, an alpha-amylase enzyme activity is exogenously supplied and present in the mash. The alpha-amylase may be added to the mash ingredients, e.g., the water and/or the grist before, during or after forming the mash.
In a further aspect, both pullulanase and alpha-amylase enzyme activities are exogenously supplied and present in the mash. The alpha-amylase and pullulanase may be added to the mash ingredients, e.g., the water and/or the grist before, during or after forming the mash.
A further enzyme may be added to the mash, said enzyme being selected from the group consisting of among amylase, protease, pullulanase, isoamylase, cellulase, glucanase, xylanase, arabinofuranosidase, ferulic acid esterase, xylan acetyl esterase, phytase and a further glucoamylase.
Prior to the third step of the brewing process, fermentation, the wort is typically transferred to a brew kettle and boiled vigorously for 50-60 minutes. A number of important processes occur during wort boiling (further information may be found in “Technology Brewing and Malting” by Wolfgang Kunze of the Research and Teaching Institute of Brewing, Berlin (VLB), 3rd completely updated edition, 2004, ISBN 3-921690-49-8) including inactivation of the endogenous malt enzymes and any exogenous enzyme added to the mash or adjunct. The boiled wort is then cooled, pitched with brewers' yeast and fermented at temperatures typically ranging from 8-16° C. to convert the fermentable sugars to ethanol. A low-alcohol beer can be produced from the final beer, by a process of vacuum evaporation that serves to selectively remove alcohol.
In an alternative embodiment, the disclosure pertains to a method of enhancing the amount of fermentable sugars in the wort, using an enzymatic composition comprising one or more glucoamylases as contemplated herein (e.g. thermolabile glucoamylase), whereby the enzymatic composition is added to the wort after it has been boiled, such that the one or more glucoamylases are active during the fermentation step. The enzymatic composition can be added to the boiled wort either before, simultaneously, or after the wort is pitched with the brewers' yeast. At the end of the fermentation and maturation step the beer, which may optionally be subjected to vacuum evaporation to produce a low-alcohol beer, is then pasteurized. An inherent advantage of this method lies in the duration of the fermentation process, which is about 6-15 days (depending on pitching rate, fermentation, temperature, etc), which allows more time for the enzymatic cleavage of non-fermentable sugars, as compared to the short mashing step (2-4 h duration). A further advantage of this method lies in the amount of the enzymatic composition needed to achieve the desired decrease in non-fermentable sugars (and increase in fermentable sugars), which corresponds to a significantly lower number of units of enzymatic activity (e.g. units of glucoamylase activity) than would need to be added to the mash to achieve a similar decrease in non-fermentable sugars. In addition, it removes the difficulties often seen during wort separation, especially by lautering, when high dose rates of glucoamylase are added in the mash.
In one aspect, the disclosure pertains to an enzymatic composition comprising at least one additional enzyme selected among amylase, protease, pullulanase, isoamylase, cellulase, glucanase, xylanase, arabinofuranosidase, ferulic acid esterase, xylan acetyl esterase, phytase and a further glucoamylase.
In a further aspect, the disclosure pertains to an enzymatic composition, wherein the composition comprises at least one additional enzyme selected among alpha-amylase and/or pullulanase.
In a further aspect, the disclosure pertains to an enzymatic composition, wherein the composition further comprises alpha-amylase and pullulanase.
In a further aspect, the disclosure pertains to an enzymatic composition, which enzymatic composition comprises less than 1, less than 0.8, less than 0.6, less than 0.5, less than 0.4, less than 0.2, less than 0.125, less than 0.1, less than 0.05, less than 0.01, or less than 0.005 XU of xylanase activity per GAU of a glucoamylase variant as described herein. If desired, xylanase activity may be reduced by different methods known to the skilled person such as e.g. heat treatment, passing through wheat bran, or other materials, which may selectively adsorb xylanase activity.
In a further aspect, the disclosure pertains to an enzymatic composition, which enzymatic composition comprises less than 400, less than 200, less than 50, less than 20, or less than 2 XU of xylanase activity per gram of the composition.
In a further aspect, the disclosure pertains to an enzymatic composition, which enzymatic composition comprises between 0.1-20, 0.1-10, 0.1-5 or 0.2-3 SSU of alpha-amylase activity per GAU of a glucoamylase variant as described herein.
In a further aspect, the disclosure pertains to an enzymatic composition, which enzymatic composition comprises between 0.30-10, 1-8, 3-10 or 5-9 PU of pullulanase activity per GAU of a glucoamylase variant as described herein.
In a further aspect, the disclosure pertains to an enzymatic composition, which enzymatic composition comprises between 0.95-20 SSU of alpha-amylase activity per GAU of a glucoamylase variant as described herein and between 0.30-10 PU of pullulanase activity per GAU of a glucoamylase variant as described herein.
In a further aspect, the disclosure pertains to an enzymatic composition, which enzymatic composition comprises between 0.95-20 SSU of alpha-amylase activity per GAU of a glucoamylase variant as described herein and between 0.30-10 PU of pullulanase activity per GAU of a glucoamylase variant as described herein and less than 1, less than 0.8, less than 0.6, less than 0.5, less than 0.4, less than 0.2, less than 0.125, less than 0.1, less than 0.05, less than 0.01, or less than 0.005 XU of xylanase activity per GAU of a glucoamylase variant as described herein.
In a further aspect, the disclosure pertains to an enzymatic composition, which enzymatic composition comprises between 0.05-10, 0.1-10, 0.1-8, 0.1-5, 0.1-3, 0.2-3, 0.2-2 PU of pullulanase activity per GAU of a glucoamylase variant as described herein.
In a further aspect, the disclosure pertains to an enzymatic composition, which enzymatic composition comprises between 0.1-20, 1-15, 2-10, 3-10 SSU of alpha-amylase activity per GAU of a glucoamylase variant as described herein.
In a further aspect, the disclosure pertains to an enzymatic composition, which enzymatic composition comprises between 0.05-10 PU of pullulanase activity per GAU of a glucoamylase variant as described herein and between 0.1-20 SSU of alpha-amylase activity per GAU of a glucoamylase variant as described herein.
In a further aspect, the disclosure pertains to an enzymatic composition, which enzymatic composition comprises between 0.1-5 PU of pullulanase activity per GAU of a glucoamylase variant as described herein and between 1-15 SSU of alpha-amylase activity per GAU of a glucoamylase variant as described herein.
In a further aspect, the disclosure pertains to an enzymatic composition, which enzymatic composition comprises between 0.2-2 PU of pullulanase activity per GAU of a glucoamylase variant as described herein and between 2-10 SSU of alpha-amylase activity per GAU of a glucoamylase variant as described herein.
In a further aspect, the disclosure pertains to an enzymatic composition, which enzymatic composition comprises between 0.05-10 PU of pullulanase activity per GAU of a glucoamylase variant as described herein and between 0.1-20 SSU of alpha-amylase activity per GAU of a glucoamylase variant as described herein and less than 1, less than 0.8, less than 0.6, less than 0.5, less than 0.4, less than 0.2, less than 0.125, less than 0.1, less than 0.05, less than 0.01, or less than 0.005 XU of xylanase activity per GAU of a glucoamylase variant as described herein.
In a further aspect, the disclosure pertains to an enzymatic composition, which enzymatic composition comprises between 0.1-5 PU of pullulanase activity per GAU of a glucoamylase variant as described herein and between 1-15 SSU of alpha-amylase activity per GAU of a glucoamylase variant as described herein and less than 1, less than 0.8, less than 0.6, less than 0.5, less than 0.4, less than 0.2, less than 0.125, less than 0.1, less than 0.05, less than 0.01, or less than 0.005 XU of xylanase activity per GAU of a glucoamylase variant as described herein.
In a further aspect, the disclosure pertains to an enzymatic composition, which enzymatic composition comprises between 0.2-2 PU of pullulanase activity per GAU of a glucoamylase variant as described herein and between 2-10 SSU of alpha-amylase activity per GAU of a glucoamylase variant as described herein and less than 1, less than 0.8, less than 0.6, less than 0.5, less than 0.4, less than 0.2, less than 0.125, less than 0.1, less than 0.05, less than 0.01, or less than 0.005 XU of xylanase activity per GAU of a glucoamylase variant as described herein.
In one aspect, the glucoamylase variant as described herein is added in an amount of 500-20000 GAU/kg grist. In another aspect, the glucoamylase variant as described herein is added in an amount of 750-10000 GAU/kg grist. In a further aspect, the glucoamylase variant as described herein is added in an amount of 1000-7500 GAU/kg grist.
The present disclosure also provides an animal feed composition or formulation comprising at least one variant glucoamylase encompassed by the disclosure. Methods of using a glucoamylase enzyme in the production of feeds comprising starch are provided in WO 03/049550 (herein incorporated by reference in its entirety). Briefly, the glucoamylase variant is admixed with a feed comprising starch. The glucoamylase is capable of degrading resistant starch for use by the animal. In some embodiments a glucoamylase variant as described herein is used in processes in the generation of fuels based on starch feed stocks. Other objects and advantages of the present disclosure are apparent from the present specification.
Further embodiments according to the invention:
Embodiment 1
Use of a glucoamylase variant comprising two or more amino acid substitutions relative to interconnecting loop 2′ with the amino acid sequence from position 518 to position 543 of SEQ ID NO:2 or equivalent sequence of residues in a parent glucoamylase, and/or loop 1 with the amino acid sequence from position 21 to position 51 of SEQ ID NO:2 or equivalent sequence of residues in a parent glucoamylase, and/or helix 2 with the amino acid sequence from position 52 to position 68 of SEQ ID NO:2 or equivalent sequence of residues in a parent glucoamylase, and/or loop 11 with the amino acid sequence from position 396 to position 420 of SEQ ID NO:2 or equivalent sequence of residues in a parent glucoamylase, and/or helix 12 with the amino acid sequence from position 421 to position 434 of SEQ ID NO:2 or equivalent sequence of residues in a parent glucoamylase for reducing the synthesis of condensation products during hydrolysis of starch.
Embodiment 2
Use of a glucoamylase variant, which when in its crystal, form has a crystal structure for which the atomic coordinates of the main chain atoms have a root-mean-square deviation from the atomic coordinates of the equivalent main chain atoms of TrGA (as defined in Table 20 in WO2009/067218) of less than 0.13 nm following alignment of equivalent main chain atoms, and which have a linker region, a starch binding domain and a catalytic domain, said variant comprising two or more amino acid substitutions relative to the amino acid sequence of the parent glucoamylase in interconnecting loop 2′ of the starch binding domain, and/or in loop 1, and/or in helix 2, and/or in loop 11, and/or in helix 12 of the catalytic domain for reducing the synthesis of condensation products during hydrolysis of starch.
Embodiment 3
The use of a glucoamylase variant according to any one of the embodiments 1-2, wherein said two or more amino acid substitutions are relative to the interconnecting loop 2′ with the amino acid sequence from position 518 to position 543 of SEQ ID NO:2, and/or loop 1 with the amino acid sequence from position 21 to position 51 of SEQ ID NO:2, and/or helix 2 with the amino acid sequence from position 52 to position 68 of SEQ ID NO:2, and/or loop 11 with the amino acid sequence from position 396 to position 420 of SEQ ID NO:2, and/or helix 12 with the amino acid sequence from position 421 to position 434 of SEQ ID NO:2.
Embodiment 4
The use of a glucoamylase variant according to any one of the embodiments 1-3, wherein the two or more amino acid substitutions are at least one amino acid substitution in the interconnecting loop 2′ and at least one amino acid substitution in loop 1 and/or helix 2 and/or loop 11 and/or helix 12.
Embodiment 5
The use of a glucoamylase variant according to any one of the embodiments 1-4, wherein the two or more amino acid substitutions are 1, 2, 3 or 4 amino acid substitutions in the interconnecting loop 2′ and 1, 2, 3 or 4 amino acid substitutions in loop 1 and/or helix 2 and/or loop 11 and/or helix 12.
Embodiment 6
The use of a glucoamylase variant according to any one of the embodiments 1-5, wherein the two or more amino acid substitutions are at least one amino acid substitution in interconnecting loop 2′ and at least one amino acid substitution in loop 1.
Embodiment 7
The use of a glucoamylase variant according to any one of the embodiments 1-6, wherein the two or more amino acid substitutions are at least one amino acid substitution in interconnecting loop 2′ and at least one amino acid substitution in helix 2.
Embodiment 8
The use of a glucoamylase variant according to any one of the embodiments 1-7, wherein the two or more amino acid substitutions are at least one amino acid substitution in interconnecting loop 2′ and at least one amino acid substitution in loop 11.
Embodiment 9
The use of a glucoamylase variant according to any one of the embodiments 1-8, wherein the two or more amino acid substitutions are at least one amino acid substitution in interconnecting loop 2′ and at least one amino acid substitution in helix 12.
Embodiment 10
The use of a glucoamylase variant according to any one of the embodiments 1-9, wherein the two or more amino acid substitutions are at least one amino acid substitution in interconnecting loop 2′ and at least one amino acid substitution in loop 1 and at least one amino acid substitution in helix 2.
Embodiment 11
The use of a glucoamylase variant according to any one of embodiments 1-10, wherein the glucoamylase variant has at least one amino acid substitution within position 520-543, 530-543, or 534-543 of interconnecting loop 2′, the positions corresponding to the respective position in SEQ ID NO:2 or equivalent positions in a parent glucoamylase.
Embodiment 12
The use of a glucoamylase variant according to any one of embodiments 1-11, wherein the glucoamylase variant has at least one amino acid substitution within the amino acid sequence of position 30-50, 35-48, or 40-46 of loop 1, the positions corresponding to the respective position in SEQ ID NO:2 or equivalent positions in a parent glucoamylase.
Embodiment 13
The use of a glucoamylase variant according to any one of embodiments 1-12, wherein the glucoamylase variant has at least one amino acid substitution within the amino acid sequence of position 50-66, 55-64, or 58-63 of helix 2, the positions corresponding to the respective position in SEQ ID NO:2 or equivalent positions in a parent glucoamylase.
Embodiment 14
The use of a glucoamylase variant according to any one of embodiments 1-13, wherein the glucoamylase variant has at least one amino acid substitution within the amino acid sequence of position 405-420, 410-420, or 415-420 of loop 11, the positions corresponding to the respective position in SEQ ID NO:2 or equivalent positions in a parent glucoamylase.
Embodiment 15
The use of a glucoamylase variant according to any one of embodiments 1-14, wherein the glucoamylase variant has at least one amino acid substitution within the amino acid sequence of position 421-434, 425-434, or 428-434 of helix 12, the positions corresponding to the respective position in SEQ ID NO:2 or equivalent positions in a parent glucoamylase.
Embodiment 16
The use of a glucoamylase variant according to any one of embodiments 1-15, wherein the glucoamylase variant has at least 80%, 85%, 90%, 95%, 98%, or 99.5% sequence identity to the parent glucoamylase.
Embodiment 17
The use of a glucoamylase variant according to any one of embodiments 1-16, wherein the glucoamylase variant has at least 80%, 85%, 90%, 95%, 98%, or 99.5% sequence identity to SEQ ID NO: 1, 2, 3, 5, 6, 7, 8, or 9.
Embodiment 18
The use of a glucoamylase variant to any one of the embodiments 1-17, wherein the glucoamylase variant has a starch binding domain that has at least 96%, 97%, 98%, 99%, or 99.5% sequence identity with the starch binding domain of SEQ ID NO: 1, 2, 11, 385, 386, 387, 388, 389, or 390.
Embodiment 19
The use of a glucoamylase variant according to any one of the embodiments 1-18, wherein the glucoamylase variant has a catalytic domain that has at least 80%, 85%, 90%, 95%, or 99.5% sequence identity with the catalytic domain of SEQ ID NO: 1, 2, 3, 5, 6, 7, 8, or 9.
Embodiment 20
The use of a glucoamylase variant according to any one of embodiments 1-19, wherein the glucoamylase variant has at least 80%, 85%, 90%, 95%, 98%, or 99.5% sequence identity to SEQ ID NO:2.
Embodiment 21
The use of a glucoamylase variant according to any one of embodiments 1-20, wherein the condensation product is isomaltose.
Embodiment 22
The use of a glucoamylase variant according to any one of embodiments 1-21, wherein the hydrolysis of starch is in a brewing process.
Embodiment 23
The use of a glucoamylase variant according to any one of embodiments 1-22, wherein the glucoamylase exhibit an enhanced production of fermentable sugar(s) as compared to the parent glucoamylase, such as TrGA.
Embodiment 24
The use of a glucoamylase variant according to any one of embodiments 1-23, wherein the glucoamylase exhibit an enhanced production of fermentable sugars in a mashing step of the brewing process as compared to the parent glucoamylase, such as TrGA.
Embodiment 25
The use of a glucoamylase variant according to any one of embodiments 1-24, wherein the glucoamylase exhibit an enhanced production of fermentable sugars in a fermentation step of the brewing process as compared to the parent glucoamylase, such as TrGA.
Embodiment 26
The use of a glucoamylase variant according to any one of embodiments 1-25, wherein the fermentable sugar is glucose.
Embodiment 27
The use of a glucoamylase variant according to any one of embodiments 1-26, wherein the hydrolysis of starch is in a process for producing glucose syrup.
Embodiment 28
The use of a glucoamylase variant according to any one of embodiments 1-27, wherein the glucoamylase exhibit a reduced ratio between isomaltose synthesis (IS) and starch hydrolysis activity (SH) as compared to the parent glucoamylase, such as TrGA.
Embodiment 29
The use of a glucoamylase variant according to any one of embodiments 1-28, wherein the glucoamylase exhibit a reduced starch hydrolysis activity, which is not more than 5%, not more than 10% or not more than 15% reduced as compared to the parent glucoamylase, such as TrGA.
Embodiment 30
The use of a glucoamylase variant according to any one of embodiments 1-29, wherein the glucoamylase exhibit an enhanced real degree of fermentation as compared to the parent glucoamylase such as TrGA.
Embodiment 31
The use of a glucoamylase variant according to any one of embodiments 1-30, wherein the glucoamylase forms a lower amount of condensation products than the amount of condensation products formed by the glucoamylase Aspergillus niger (AnGA) (SEQ ID NO: 6) under comparable conditions.
Embodiment 32
The use of a glucoamylase variant according to any one of embodiments 1-31, wherein the glucoamylase forms an amount of condensation products which amount is essentially the same as, not more than 5% higher, not more than 8% higher or not more than 10% higher than the amount of condensation products formed by Aspergillus niger (AnGA) (SEQ ID NO: 6) under comparable conditions.
Embodiment 33
The use of a glucoamylase variant according to any one of embodiments 31-32, wherein dosing of the glucoamylases are the same based on protein concentration.
Embodiment 34
The use of a glucoamylase variant according to any one of embodiments 31-33, wherein dosing of the glucoamylases are the same based on measurement of activity in activity assays.
Embodiment 35
The use of a glucoamylase variant according to any one of embodiments 1-34, which glucoamylase variant has an amino acid substitution in position 539 and one or more amino acid substitutions in a position selected from position 44, 61, 417 and 431, the positions corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase.
Embodiment 36
The use of a glucoamylase variant according to any one of embodiments 1-35, which glucoamylase variant has an amino acid substitution in position 539 and a) an amino acid substitution in position 44 and/or b) amino acid substitutions in both positions 417 and 431, the positions corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase.
Embodiment 37
The use of a glucoamylase variant according to any one of embodiments 1-36, which glucoamylase variant has an amino acid substitution in position 539 and an amino acid substitution in position 44, the positions corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase.
Embodiment 38
The use of a glucoamylase variant according to any one of embodiments 1-37, which glucoamylase variant has an amino acid substitution in position 539 and amino acid substitutions in positions 417 and 431, the positions corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase.
Embodiment 39
The use of a glucoamylase variant according to any one of embodiments 1-38, which glucoamylase variant has an amino acid substitution in position 539 and amino acid substitutions in positions 44 and 61, the positions corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase.
Embodiment 40
The use of a glucoamylase variant according to any one of embodiments 1-39, which glucoamylase variant has an amino acid substitution in position 43, the position corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase.
Embodiment 41
The use of a glucoamylase variant according to any one of embodiments 1-40, which glucoamylase variant has an amino acid substitution in position 61, the position corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase.
Embodiment 42
The use of a glucoamylase variant according to any one of embodiments 1-41, wherein the amino acid substitution in position 539 is 539R, the position corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase.
Embodiment 43
The use of a glucoamylase variant according to any one of embodiments 1-42, wherein the amino acid substitution in position 44 is 44R, the position corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase.
Embodiment 44
The use of a glucoamylase variant according to any one of embodiments 1-43, wherein the amino acid substitution in position 417 is 417R/V, the position corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase.
Embodiment 45
The use of a glucoamylase variant according to any one of embodiments 1-44, wherein the amino acid substitution in position 417 is 417R, the position corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase.
Embodiment 46
The use of a glucoamylase variant according to any one of embodiments 1-45, wherein the amino acid substitution in position 417 is 417V, the position corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase.
Embodiment 47
The use of a glucoamylase variant according to any one of embodiments 1-46, wherein the amino acid substitution in position 431 is 431L, the position corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase.
Embodiment 48
The use of a glucoamylase variant according to any one of embodiments 1-47, wherein the amino acid substitution in position 43 is 43R, the position corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase.
Embodiment 49
The use of a glucoamylase variant according to any one of embodiments 1-48, wherein the amino acid substitution in position 61 is 61I, the position corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase.
Embodiment 50
A glucoamylase variant as defined in any one of embodiments 1-49.
Embodiment 51
A glucoamylase variant comprising two or more amino acid substitutions, wherein an amino acid substitution is in position 539 and an amino acid substitution is in position 44, the positions corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase, and which sequence has at least 80% sequence identity to the parent glucoamylase, and wherein the amino acid substitution in position 44 is not 44C.
Embodiment 52
The glucoamylase variant according to embodiment 51 comprising two or more amino acid substitutions, wherein an amino acid substitution is in position 539 and an amino acid substitution is 44R, the positions corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase.
Embodiment 53
The glucoamylase variant according to any one of embodiments 51-52 comprising an amino acid substitution in position 61, the position corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase.
Embodiment 54
The glucoamylase variant according to any one of embodiments 51-53, wherein the glucoamylase variant has at least 85%, 90%, 95%, 98%, or 99.5% sequence identity to the parent glucoamylase.
Embodiment 55
The glucoamylase variant according to any one of embodiments 51-54, wherein the glucoamylase variant has at least 85%, 90%, 95%, 98%, or 99.5% sequence identity to SEQ ID NO: 1, 2, 3, 5, 6, 7, 8, or 9.
Embodiment 56
The glucoamylase variant according to any one of embodiments 51-55, wherein the glucoamylase variant has at least 85%, 90%, 95%, 98%, or 99.5% sequence identity to SEQ ID NO:2.
Embodiment 57
The glucoamylase variant according to any one of embodiments 51-56, wherein the amino acid substitution in position 539 is 539R, the position corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase.
Embodiment 58
The glucoamylase variant according to any one of embodiments 51-57, wherein the amino acid substitution in position 44 is 44R, the position corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase.
Embodiment 59
The glucoamylase variant according to any one of embodiments 51-58, wherein the amino acid substitution in position 61 is 61I, the position corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase.
Embodiment 60
The glucoamylase variant according to any one of embodiments 51-59 comprising the following amino acid substitutions:

    
    
        a. D44R and A539R; or
        b. D44R, N61I and A539R,

the positions corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase.

    
    


Embodiment 61
The glucoamylase variant according to any one of embodiments 51-60 consisting of SEQ ID NO:2 and having the following amino acid substitutions:

    
    
        a. D44R and A539R; or
        b. D44R, N61I and A539R,

the positions corresponding to the respective position in SEQ ID NO:2.

    
    


Embodiment 62
The glucoamylase variant according to any one of embodiments 51-61, wherein the glucoamylase variant has a starch binding domain that has at least 96%, 97%, 98%, 99%, or 99.5% sequence identity with the starch binding domain of SEQ ID NO: 1, 2, 11, 385, 386, 387, 388, 389, or 390.
Embodiment 63
The glucoamylase variant according to any one of embodiments 51-62, wherein the glucoamylase variant has a catalytic domain that has at least 80%, 85%, 90%, 95%, or 99.5% sequence identity with the catalytic domain of SEQ ID NO: 1, 2, 3, 5, 6, 7, 8, or 9.
Embodiment 64
The glucoamylase variant according to any one of embodiments 50-63, wherein the parent glucoamylase is selected from a glucoamylase obtained from a Trichoderma spp., an Aspergillus spp., a Humicola spp., a Penicillium spp., a Talaromyces spp., or a Schizosaccharmyces spp.
Embodiment 65
The glucoamylase variant according to any one of embodiments 50-64, wherein the parent glucoamylase is obtained from a Trichoderma spp. or an Aspergillus spp.
Embodiment 66
The glucoamylase variant according to any one of embodiments 50-65, which glucoamylase exhibit an enhanced production of fermentable sugar(s) as compared to the parent glucoamylase such as TrGA.
Embodiment 67
The glucoamylase variant according to any one of embodiments 50-66, which glucoamylase exhibit an enhanced production of fermentable sugars in the mashing step of the brewing process as compared to the parent glucoamylase such as TrGA.
Embodiment 68
The glucoamylase variant according to any one of embodiments 50-67, which glucoamylase exhibit an enhanced production of fermentable sugars in the fermentation step of the brewing process as compared to the parent glucoamylase such as TrGA.
Embodiment 69
The glucoamylase variant according to embodiment 68, wherein the fermentable sugar is glucose.
Embodiment 70
The glucoamylase variant according to any one of embodiments 50-69, which glucoamylase exhibit a reduced ratio between isomaltose synthesis and starch hydrolysis activity (IS/SH ratio) as compared to the parent glucoamylase such as TrGA.
Embodiment 71
The glucoamylase variant according to any one of embodiments 50-70, which glucoamylase exhibit a reduced starch hydrolysis activity which is not more than 5%, not more than 10% or not more than 15% reduced as compared to the parent glucoamylase such as TrGA.
Embodiment 72
The glucoamylase variant according to any one of embodiments 50-71, which glucoamylase exhibit an enhanced real degree of fermentation as compared to the parent glucoamylase such as TrGA.
Embodiment 73
The glucoamylase variant according to any one of embodiments 50-72, which glucoamylase forms a lower amount of condensation products than the amount of condensation products formed by Aspergillus niger (AnGA) (SEQ ID NO: 6) under the same conditions.
Embodiment 74
The glucoamylase variant according to any one of embodiments 50-73, which glucoamylase forms an amount of condensation products which amount is essentially the same as, not more than 5%, not more than 8%, or not more than 10% higher than the amount of condensation products formed by Aspergillus niger (AnGA) (SEQ ID NO: 6) under the same conditions.
Embodiment 75
The glucoamylase variant according to any one of embodiments 73-74, wherein the dosing of the glucoamylases are the same based on protein concentration.
Embodiment 76
The glucoamylase variant according to any one of embodiments 73-74, wherein the dosing of the glucoamylases are the same based on measurement of activity in activity assays.
Embodiment 77
The glucoamylase variant according to any one of embodiments 50-76, which glucoamylase has been purified.
Embodiment 78
A polynucleotide encoding a glucoamylase variant according to any of embodiments 50-77.
Embodiment 79
A vector comprising the polynucleotide according to embodiment 78, or capable of expressing a glucoamylase variant according to any of embodiments 50-77.
Embodiment 80
A host cell comprising a vector according to embodiment 79.
Embodiment 81
A host cell which has stably integrated into the chromosome a nucleic acid encoding the variant glucoamylase according to any of embodiments 50-80.
Embodiment 82
A cell capable of expressing a glucoamylase variant according to any one of embodiments 50-76.
Embodiment 83
The host cell according to any one of embodiments 78-81, or the cell according to embodiment 81, which is a bacterial, fungal or yeast cell.
Embodiment 84
The host cell according to embodiment 83, which is Trichoderma spp. such as Trichoderma reesei. 
Embodiment 85
The host cell according to any one of embodiments 83-84, which is a protease deficient and/or xylanase deficient and/or native glucanase deficient host cell.
Embodiment 86
A method of expressing a glucoamylase variant, the method comprising obtaining a host cell or a cell according to any one of embodiments 80-85 and expressing the glucoamylase variant from the cell or host cell, and optionally purifying the glucoamylase variant.
Embodiment 87
The method according to embodiment 86 comprising purifying the glucoamylase variant.
Embodiment 88
Use of a glucoamylase variant according to any one of embodiments 50-76 for the preparation of an enzymatic composition.
Embodiment 89
An enzymatic composition comprising at least one glucoamylase variant according to any one of embodiments 50-77.
Embodiment 90
The enzymatic composition according to embodiment 89 comprising at least one glucoamylase variant according to any one of embodiments 50-77, wherein the composition is selected from among a starch hydrolyzing composition, a saccharifying composition, a detergent, an alcohol fermentation enzymatic composition, and an animal feed.
Embodiment 91
The enzymatic composition according to embodiment 90, which is a starch hydrolyzing composition.
Embodiment 92
The enzymatic composition according to any one of embodiments 89-91 comprising at least one additional enzyme selected among amylase, protease, pullulanase, cellulase, glucanase, xylanase, arabinofuranosidase, ferulic acid esterase, xylan acetyl esterase, and a further glucoamylase.
Embodiment 93
The enzymatic composition according to embodiment 89-92, wherein the at least one additional enzyme is selected among amylase, pullulanase, and a further glucoamylase.
Embodiment 94
The enzymatic composition according to embodiment 89-93, wherein the at least one additional is selected among amylase and pullulanase.
Embodiment 95
The enzymatic composition according to any one of embodiments 89-94, wherein the amylase is selected among alpha-amylase, and isoamylase.
Embodiment 96
A method for converting starch or partially hydrolyzed starch into a syrup containing glucose, said process including saccharifying a liquid starch solution in the presence of at least one glucoamylase variant according to any one of embodiments 50-77 or an enzymatic composition according to any one of embodiments 89-95.
Embodiment 97
The method according to embodiment 96 of saccharifying a liquid starch solution, which comprises an enzymatic saccharification step using a glucoamylase variant according to embodiment 50-77 or an enzymatic composition according to any one of embodiments 89-95.
Embodiment 98
The method according to any one of embodiments 96-97, further comprising contacting the liquid starch solution with at least one additional enzyme.
Embodiment 99
The method according to embodiment 98, wherein the additional enzyme is selected among amylase, protease, pullulanase, cellulase, glucanase, xylanase, arabinofuranosidase, ferulic acid esterase, xylan acetyl esterase, and glucoamylase.
Embodiment 100
The method according to embodiment 96-99, wherein the additional enzyme is amylase and pullulanase.
Embodiment 101
The method according to embodiment any one of embodiments 96-100, wherein the amylase is selected among alpha-amylase, and isoamylase.
Embodiment 102
Use of a glucoamylase variant according to any one of embodiments 50-77 in a starch conversion process, such as a in a continuous starch conversion process.
Embodiment 103
Use of a glucoamylase variant according to any one of embodiments 50-77 in a process for producing oligosaccharides, maltodextrins, or glucose syrups.
Embodiment 104
Use of a glucoamylase variant according to any one of embodiments 50-77 in a process for producing high fructose corn syrup.
Embodiment 105
A method for producing a wort for brewing comprising forming a mash from a grist, and contacting the mash with a glucoamylase variant according to any one of embodiments 50-77 or an enzymatic composition according to any one of embodiments 89-95.
Embodiment 106
The method of embodiment 105, further comprising contacting the mash with one or more additional enzyme(s)
Embodiment 107
The method according to embodiment 106, wherein the one or more enzyme(s) is selected among amylase, protease, pullulanase, cellulase, endoglucanase, xylanase, arabinofuranosidase, ferulic acid esterase, xylan acetyl esterase, and glucoamylase.
Embodiment 108
The method according to embodiment 107, wherein the one or more enzyme(s) is amylase and/or pullulanase.
Embodiment 109
The method according to embodiment any one of embodiments 107-108, wherein the amylase is alpha-amylase and/or isoamylase.
Embodiment 110
The method according to any one of embodiments 105-109, wherein the grist comprises one or more of malted grain, unmalted grain, adjunct, and any combination thereof.
Embodiment 111
The method of any one of embodiments 105-110, further comprising fermenting the wort to obtain a fermented beverage.
Embodiment 112
The method of any one of embodiments 105-111, further comprising fermenting the wort to obtain a beer.
Embodiment 113
A method for production of a beer which comprises:

    
    
        a. preparing a mash,
        b. filtering the mash to obtain a wort, and
        c. fermenting the wort to obtain a beer,

wherein a glucoamylase variant according to any one of embodiments 50-77 is added to: step (a) and/or step (b) and/or step (c).

    
    


Embodiment 114
The method of embodiment 113, wherein the beer is subjected to a pasteurization step.
Embodiment 115
Use of a glucoamylase variant according to any one of embodiments 50-77 to enhance the production of fermentable sugars in either the mashing step or the fermentation step of a brewing process.
Embodiment 116
A beer, wherein the beer is produced by the steps of:

    
    
        a. preparing a mash,
        b. filtering the mash to obtain a wort,
        c. fermenting the wort to obtain a beer, and
    
    


d. pasteurizing the beer,
wherein a glucoamylase variant according to any one of embodiments 50-77 is added to: step (a) and/or step (b) and/or step (c).
Embodiment 117
The beer of embodiment 116, wherein the pasteurized beer is further characterized as being:

    
    
        a. essentially without glucoamylase activity; and/or
        b. a low-calorie beer and/or a low-alcohol beer.
    
    


Embodiment 118
Use of a glucoamylase variant according to any one of embodiments 50-77 in an alcohol fermentation process.
Embodiment 119
A screening method for identification of a glucoamylase variant having a reduced ratio between isomaltose synthesis and starch hydrolysis activity (IS/SH ratio) as compared to the parent glucoamylase.
Embodiment 120
A screening method for identification of a glucoamylase variant having the same or increased starch hydrolysis activity and reduced isomaltose synthesis, which is not more than 5%, not more than 10% or not more than 15% reduced as compared to the parent glucoamylase and having a reduced ratio between isomaltose synthesis and starch hydrolysis activity (IS/SH ratio) as compared to the parent glucoamylase.
Embodiment 121
A screening method for identification of a glucoamylase variant having a reduced synthesis of condensation products during hydrolysis of starch, the method comprising the steps of measuring the isomaltose synthesis and starch hydrolysis activity of glucoamylase variants and selecting the variants having a reduced starch hydrolysis activity which is not more than 5%, not more than 10% or not more than 15% reduced as compared to the parent glucoamylase and having a reduced ratio between isomaltose synthesis and starch hydrolysis activity (IS/SH ratio) as compared to the parent glucoamylase.
Embodiment 122
The glucoamylase variant obtained by the method according to any one of embodiments 119-121.
Further embodiments also part of the invention:
Further Embodiment 1
A glucoamylase variant comprising the following amino acid substitutions:

    
    
        a. D44R and A539R; or
        b. D44R, N61I and A539R,

the positions corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase, wherein the glucoamylase variant has at least 80% sequence identity with SEQ ID NO: 1 or 2, or the parent glucoamylase.

    
    


Further Embodiment 2
The glucoamylase variant according to further embodiment 1 comprising the following amino acid substitutions:

    
    
        a. D44R and A539R; or
        b. D44R, N61I and A539R,

the positions corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase, wherein the glucoamylase variant has at least 80% sequence identity with SEQ ID NO: 1 or 2, or the parent glucoamylase.

    
    


Further Embodiment 3
The glucoamylase variant according to any one of further embodiments 1-2 comprising the following amino acid substitutions:

    
    
        a. D44R, N61I and A539R,

the positions corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase, wherein the glucoamylase variant has at least 80% sequence identity with SEQ ID NO: 1 or 2, or the parent glucoamylase.

    
    


Further Embodiment 4
The glucoamylase variant according to any one of further embodiments 1-2 comprising the following amino acid substitutions:

    
    
        a. D44R and A539R,

the positions corresponding to the respective position in SEQ ID NO:2 or an equivalent position in a parent glucoamylase, wherein the glucoamylase variant has at least 80% sequence identity with SEQ ID NO: 1 or 2, or the parent glucoamylase.

    
    


Further Embodiment 5
The glucoamylase variant of any one of further embodiments 1-4, wherein the glucoamylase variant has at least 85% or 90% sequence identity with SEQ ID NO: 1 or 2.
Further Embodiment 6
The glucoamylase variant of further embodiment 5, wherein the glucoamylase variant has at least 95% sequence identity with SEQ ID NO: 1 or 2.
Further Embodiment 7
The glucoamylase variant of further embodiment 6, wherein the glucoamylase variant has at least 99.5% sequence identity with SEQ ID NO: 1 or 2.
Further Embodiment 8
The glucoamylase variant of any one of further embodiments 1-7, wherein the parent glucoamylase comprises SEQ ID NO: 1 or 2.
Further Embodiment 9
The glucoamylase variant of further embodiment 8, wherein the parent glucoamylase consists of SEQ ID NO: 1 or 2.
Further Embodiment 10
The glucoamylase variant according to any one of further embodiments 1-9, wherein the glucoamylase variant has a starch binding domain that has at least 96%, 97%, 98%, 99%, or 99.5% sequence identity with the starch binding domain of SEQ ID NO: 1, 2, 11, 385, 386, 387, 388, 389, or 390.
Further Embodiment 11
The glucoamylase variant according to any one of further embodiments 1-10, wherein the glucoamylase variant has a catalytic domain that has at least 80%, 85%, 90%, 95%, or 99.5% sequence identity with the catalytic domain of SEQ ID NO: 1, 2, 3, 5, 6, 7, 8, or 9.
Further Embodiment 12
The glucoamylase variant according to any one of further embodiments 1-11, wherein the parent glucoamylase is selected from a glucoamylase obtained from a Trichoderma spp., an Aspergillus spp., a Humicola spp., a Penicillium spp., a Talaromyces spp., or a Schizosaccharmyces spp.
Further Embodiment 13
The glucoamylase variant according to any one of further embodiments 1-12, wherein the parent glucoamylase is obtained from a Trichoderma spp. or an Aspergillus spp.
Further Embodiment 14
The glucoamylase variant according to any one of further embodiments 1-13, which glucoamylase exhibit an enhanced production of fermentable sugar(s) as compared to the parent glucoamylase.
Further Embodiment 15
The glucoamylase variant according to any one of further embodiments 1-14, which glucoamylase exhibit an enhanced production of fermentable sugars in the mashing step of the brewing process as compared to the parent glucoamylase.
Further Embodiment 16
The glucoamylase variant according to any one of further embodiments 1-15, which glucoamylase exhibit an enhanced production of fermentable sugars in the fermentation step of the brewing process as compared to the parent glucoamylase.
Further Embodiment 17
The glucoamylase variant according to further embodiment 16, wherein the fermentable sugar is glucose.
Further Embodiment 18
The glucoamylase variant according to any one of further embodiments 1-17, which glucoamylase exhibit a reduced ratio between isomaltose synthesis and starch hydrolysis activity (IS/SH ratio) as compared to the parent glucoamylase.
Further Embodiment 19
The glucoamylase variant according to any one of further embodiments 1-18, which glucoamylase exhibit a reduced starch hydrolysis activity which is not more than 5%, not more than 10% or not more than 15% reduced as compared to the parent glucoamylase.
Further Embodiment 20
The glucoamylase variant according to any one of further embodiments 1-19, which glucoamylase exhibit an enhanced real degree of fermentation as compared to the parent glucoamylase.
Further Embodiment 21
The glucoamylase variant according to any one of further embodiments 1-20, which glucoamylase forms a lower amount of condensation products than the amount of condensation products formed by Aspergillus niger (AnGA) (SEQ ID NO: 6) under the same conditions.
Further Embodiment 22
The glucoamylase variant according to any one of further embodiments 1-21, which glucoamylase forms an amount of condensation products which amount is essentially the same as, not more than 5%, not more than 8%, or not more than 10% higher than the amount of condensation products formed by Aspergillus niger (AnGA) (SEQ ID NO: 6) under the same conditions.
Further Embodiment 23
The glucoamylase variant according to any one of further embodiments 18-21, wherein the dosing of the glucoamylases are the same based on protein concentration.
Further Embodiment 24
The glucoamylase variant according to any one of further embodiments 18-23, wherein the dosing of the glucoamylases are the same based on measurement of activity in activity assays.
Further Embodiment 25
The glucoamylase variant according to any one of further embodiments 1-24, which glucoamylase has been purified.
Further Embodiment 26
A polynucleotide encoding a glucoamylase variant according to any of further embodiments 1-25.
Further Embodiment 27
A vector comprising the polynucleotide according to further embodiment 26, or capable of expressing a glucoamylase variant according to any of further embodiments 1-25.
Further Embodiment 28
A host cell comprising a vector according to further embodiment 27.
Further Embodiment 29
A host cell which has stably integrated into the chromosome a nucleic acid encoding the variant glucoamylase according to any of further embodiments 1-25.
Further Embodiment 30
A cell capable of expressing a glucoamylase variant according to any one of further embodiments 1-25.
Further Embodiment 31
The host cell according to any one of further embodiments 28-29, or the cell according to further embodiment 30, which is a bacterial, fungal or yeast cell.
Further Embodiment 32
The host cell according to further embodiment 31, which is Trichoderma spp. such as Trichoderma reesei. 
Further Embodiment 33
The host cell according to any one of further embodiments 28-29 and 31-32, which is a protease deficient and/or xylanase deficient and/or glucanase deficient host cell.
Further Embodiment 34
A method of expressing a glucoamylase variant, the method comprising obtaining a host cell or a cell according to any one of further embodiments 28-33 and expressing the glucoamylase variant from the cell or host cell, and optionally purifying the glucoamylase variant.
Further Embodiment 35
The method according to further embodiment 34 comprising purifying the glucoamylase variant.
Further Embodiment 36
Use of a glucoamylase variant according to any one of further embodiments 1-25 for the preparation of an enzymatic composition.
Further Embodiment 37
An enzymatic composition comprising at least one glucoamylase variant according to any one of further embodiments 1-25.
Further Embodiment 38
An enzymatic composition comprising at least one glucoamylase variant according to any one of embodiments 1-25, said enzyme composition comprising one or more further enzymes.
Further Embodiment 39
The enzymatic composition according to any one of further embodiments 37-38 comprising at least one glucoamylase variant according to any one of further embodiments 1-25, wherein the composition is selected from among a starch hydrolyzing composition, a saccharifying composition, a detergent composition, an alcohol fermentation enzymatic composition, and an animal feed composition.
Further Embodiment 40
An enzymatic composition according to any one of further embodiments 36-39 comprising at least one additional enzyme selected among amylase, protease, pullulanase, isoamylase, cellulase, glucanase, xylanase, arabinofuranosidase, ferulic acid esterase, xylan acetyl esterase, phytase and a further glucoamylase.
Further Embodiment 41
The enzymatic composition according to any one of further embodiments 36-40, wherein the composition comprises at least one additional enzyme selected among alpha-amylase and/or pullulanase.
Further Embodiment 42
The enzymatic composition according to any one of further embodiments 36-41, wherein the composition comprises alpha-amylase and pullulanase.
Further Embodiment 43
The enzymatic composition according to any one of further embodiments 36-42, which enzymatic composition comprises less than 1, less than 0.8, less than 0.6, less than 0.5, less than 0.4, less than 0.2, less than 0.125, less than 0.1, less than 0.05, less than 0.01, or less than 0.005 XU of xylanase activity per GAU of a glucoamylase variant according to any one of further embodiments 1-25.
Further Embodiment 44
The enzymatic composition according to any one of further embodiments 36-43, which enzymatic composition comprises less than 400, less than 200, less than 50, less than 20, or less than 2 XU of xylanase activity per gram of the composition.
Further Embodiment 45
The enzymatic composition according to any one of further embodiments 36-44, which enzymatic composition comprises between 0.1-20, 1-15, 2-10, or 3-10 SSU of alpha-amylase activity per GAU of a glucoamylase variant according to any one of further embodiments 1-25.
Further Embodiment 46
The enzymatic composition according to any one of further embodiments 36-45, which enzymatic composition comprises between 0.05-10, 0.1-10, 0.1-8, 0.1-5, 0.1-3, 0.2-3, or 0.2-2 PU of pullulanase activity per GAU of a glucoamylase variant according to any one of further embodiments 1-25.
Further Embodiment 47
The enzymatic composition according to any one of further embodiments 36-46, which enzymatic composition comprises between 0.05-10 PU of pullulanase activity per GAU of a glucoamylase variant according to any one of further embodiments 1-25 and between 0.1-20 SSU of alpha-amylase activity per GAU of a glucoamylase variant according to any one of further embodiments 1-25.
Further Embodiment 48
The enzymatic composition according to any one of further embodiments 36-47, which enzymatic composition comprises between 0.05-10 PU of pullulanase activity per GAU of a glucoamylase variant according to any one of further embodiments 1-25 and between 0.1-20 SSU of alpha-amylase activity per GAU of a glucoamylase variant according to any one of further embodiments 1-25 and less than 1, less than 0.8, less than 0.6, less than 0.5, less than 0.4, less than 0.2, less than 0.125, less than 0.1, less than 0.05, less than 0.01, or less than 0.005 XU of xylanase activity per GAU of a glucoamylase according to any one of further embodiments 1-25.
Further Embodiment 49
A method for producing a wort for brewing comprising forming a mash from a grist, and contacting the mash with a glucoamylase variant according to any one of further embodiments 1-25 or an enzymatic composition according to any one of further embodiments 36-48.
Further Embodiment 50
The method of further embodiment 49, further comprising contacting the mash with one or more additional enzyme(s)
Further Embodiment 51
The method according to further embodiment 50, wherein the one or more enzyme(s) is selected among amylase, protease, pullulanase, isoamylase, cellulase, endoglucanase, xylanase, arabinofuranosidase, ferulic acid esterase, xylan acetyl esterase, phytase and glucoamylase.
Further Embodiment 52
The method according to further embodiment 51, wherein the one or more enzyme(s) is/are alpha-amylase and/or pullulanase.
Further Embodiment 53
The method according to any one of further embodiments 49-52, wherein the grist comprises one or more of malted grain, unmalted grain, adjunct, and any combination thereof.
Further Embodiment 54
The method of any one of further embodiments 49-53, further comprising fermenting the wort to obtain a fermented beverage.
Further Embodiment 55
The method of any one of further embodiments 49-54, further comprising fermenting the wort to obtain a beer.
Further Embodiment 56
A method for production of a beer which comprises:

    
    
        a. preparing a mash,
        b. filtering the mash to obtain a wort, and
        c. fermenting the wort to obtain a beer,

wherein a glucoamylase variant according to any one of further embodiments 1-25 or an enzymatic composition according to any one of further embodiments 36-48 is added to: step (a) and/or step (b) and/or step (c).

    
    


Further Embodiment 57
The method of further embodiment 55, wherein the beer is subjected to a pasteurization step.
Further Embodiment 58
Use of a glucoamylase variant according to any one of further embodiments 1-25 or an enzymatic composition according to any one of further embodiments 36-48 to enhance the production of fermentable sugars in either the mashing step or the fermentation step of a brewing process.
Further Embodiment 59
A beer, wherein the beer is produced by the steps of:

    
    
        a. preparing a mash,
        b. filtering the mash to obtain a wort,
        c. fermenting the wort to obtain a beer, and
        d. pasteurizing the beer,
        e. wherein a glucoamylase variant according to any one of further embodiments 1-25 or an enzymatic composition according to any one of further embodiments 36-48 is added to: step (a) and/or step (b) and/or step (c).
    
    


Further Embodiment 60
The beer of further embodiment 59, wherein the pasteurized beer is further characterized as being:

    
    
        a. essentially without glucoamylase activity; and/or
        b. a low-calorie beer and/or a low-alcohol beer.
    
    


The invention will now be further described by way of the following non-limiting examples.
EXAMPLES
Assays and Methods
The following assays and methods are used in the examples provided below. The methods used to provide variants are described below. However, it should be noted that different methods may be used to provide variants of a parent enzyme and the invention is not limited to the methods used in the examples. It is intended that any suitable means for making variants and selection of variants may be used.
pNPG Glucoamylase Activity Assay for 96-Well Microtiter Plates
The reagent solutions were: NaAc buffer: 200 mM sodium acetate buffer pH 4.5; Substrate: 50 mM p-nitrophenyl-α-D-glucopyranoside (Sigma N-1377) in NaAc buffer (0.3 g/20 ml) and stop solution: 800 mM glycine-NaOH buffer pH 10. 30 μl filtered supernatant was placed in a fresh 96-well flat bottom micro titer plate (MTP). To each well 50 μl NaAc buffer and 120 μl substrate was added and incubated for 30 minutes at 50° C. (Thermolab systems iEMS Incubator/shaker HT). The reaction was terminated by adding 100 μl stop solution. The absorbance was measured at 405 nm in a MTP-reader (Molecular Devices Spectramax 384 plus) and the activity was calculated using a molar extinction coefficient of 0.011 μM/cm.
Thermal Stability Assay 1
With a stock dilution of 150 ppm of purified enzyme (in 50 mM NaAc pH 4.0), a 3 ppm dilution was made by adding 6 μl to 294 μl 50 mM NaAc buffer pH 4.5. The diluted sample was equally divided over 2 MTPs. One MTP (initial plate) was incubated for 1 hr at 4° C. and the other MTP (residual plate) was incubated at 64° C. (Thermolab systems iEMS Incubator/Shaker HT) for 1 hr. The residual plate was chilled for 10 min on ice. 60 μl of both the initial plate and the residual plate was added to 120 μl 4% soluble corn starch pH 3.7 and incubated for 2 hrs at 32° C., 900 rpm in 2 separate MTPs (Thermolabsystems iEMS Incubator/Shaker HT). Activity of both plates was measured in the Hexokinase activity assay, using the ethanol application assay described below.
Thermal stability was calculated as % residual activity as follows:




  
    
      
        
          ABS
          340
        
        ⁢
        
          
        
        ⁢
        residual
      
      -
      blank
    
    
      
        
          ABS
          340
        
        ⁢
        
          
        
        ⁢
        initial
      
      -
      blank
    
  
  ×
  100
  ⁢
  %




Hexokinase Activity Assay

Hexokinase cocktail: 10-15 minutes prior to use, 90 ml water was added to a BoatIL container glucose HK R1 (IL test glucose (HK) kit, Instrument Laboratory #182507-40) and gently mixed. 100 μl of Hexokinase cocktail was added to 85 μl of dH2O. 15 μl of sample was added to the mixtures and incubated for 10 minutes in the dark at room temperature. Absorbance was read at 340 nm in a MTP-reader after 10 minutes. Glucose concentrations were calculated according to a glucose (0-1.6 mg/ml) standard curve.
Ethanol Application—Glucose Release from Corn Starch
8% stock solution: 8 g of soluble corn starch (Sigma #S4180) was suspended in 40 ml dH2O at room temperature. The slurry was added in portions to 50 ml of boiling dH2O in a 250 ml flask and cooked for 5 minutes. The starch solution was cooled to 25° C. while stirring and the volume adjusted with remain 10 ml of dH2O.
Stop solution: 800 mM Glycine-NaOH buffer, pH 10.
4% (m/v) soluble starch working solution: stock solution was diluted (1:1) with 100 mM sodium acetate buffer pH 4.0.
6 μl of 150 ppm purified enzyme was diluted with 294 μl 50 mM NaAc buffer pH 4.0 in a flat bottom 96-well MTP. 60 μl of this dilution was added to 120 μl 4% soluble corn starch pH 4.0 and incubated for 2 hrs at 32° C. 900 rpm (Thermolabsystems iEMS Incubator/Shaker HT). The reaction was stopped by adding 90 μl 4° C.—cold Stop Solution. The sample was placed on ice for 20 minutes. Starch was spun down at 1118×g at 10° C. for 5 minutes (SIGMA 6K15) and 15 μl supernatant was used in the Hexokinase activity assay described above to determine the glucose content.
Data Analysis and Calculation of Performance Index of Ethanol Screening Assay
Protein levels were measured using a microfluidic electrophoresis instrument (Caliper Life Sciences, Hopkinton, Mass., USA). The microfluidic chip and protein samples were prepared according to the manufacturer's instructions (LabChip® HT Protein Express, P/N 760301). Culture supernatants were prepared and stored in 96-well microtiter plates at −20° C. until use, when they were thawed by warming in a 37° C. incubator for 30 minutes. After shaking briefly, 2 μl of each culture sample was transferred to a 96-well PCR plate (Bio-Rad, Hercules, Calif., USA) containing 7 μl samples buffer (Caliper) followed by heating the plate to 90° C. for 5 minutes on a thermostatically controlled plate heater. The plate was allowed to cool before adding 40 μl water to each sample. The plate was then placed in the instrument along with a protein standard supplied and calibrated by the manufacturer. As the proteins move past a focal point in the chip, the fluorescence signal is recorded and the protein concentration is determined by quantitating the signal relative to the signal generated by the calibrated set of protein standards.
After the Caliper protein determination the data is processed in the following way.
The calibration ladders are checked for correctness of the peak pattern. If the calibration ladder that is associated with the run does not suffice, it is replaced by a calibration ladder of an adjacent run. For peak detection, the default settings of the global peak find option of the caliper software are used. The peak of interest is selected at 75 kDA+/−10%. The result is exported to a spreadsheet program and the peak area is related to the corresponding activity (ABS340-blank measurement) in the ethanol screening assay.
With the area and activity numbers of 12 Wild Type samples, a calibration line is made using the “Enzyme Kinetics” equation of the program Grafit Version 5 (Erithacus Software, Horley, UK) in combination with a non-linear fit function. The default settings are used to calculate the Km and Vmax parameters. Based on these two parameters, a Michaelis-Menten reference line is made and the specific activity of each variant is calculated.
Based on the specific activity the performance index (PI) is calculated. The PI of a variant is the quotient “Variant-specific activity/WT-specific activity.” The PI of WT is 1.0 and a variant with a PI>1.0 has a specific activity that is greater than WT.
Purification of TrGA Variants
Culture supernatants of expressed TrGA variants were purified in one step by affinity chromatography using an AKTA explorer 100 FPLC system (Amersham Biosciences, Piscataway, N.J.). β-cyclodextrin (Sigma-Aldrich, Zwijndrecht, The Netherlands; 85.608-8) was coupled to epoxy activated Sepharose beads (GE Healthcare, Diegem, Belgium; 17-0480-01) and employed for purification. The column was equilibrated with 25 mM sodium acetate buffer pH 4.3 followed by application of concentrated enzyme sample. Bound variants were eluted from the column with 25 mM sodium acetate buffer pH 4.3 containing 10 mM α-cyclodextrin (Sigma, 28705). Purified samples were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Protein Quantification of Purified TrGA Variants
The protein concentration of purified TrGA variants was determined by anion exchange chromatography using an AKTA explorer 100 FPLC system. Purified sample was injected onto a ResourceQ_1 ml column (GE Healthcare) and a linear gradient of 0 to 500 mM NaCl in 25 mM sodium acetate buffer pH 4.3 was applied to elute bound protein. The peak area was determined and the protein concentration was calculated relative to a TrGA standard with know concentration.
Liquefact Assay
Glucose release of the variants was determined on corn mash liquefact from a local ethanol producer in a 6-well plate. Each well of the plate was filled with 6 g of 26% DS liquefact pH 4.3. Subsequently, 300 ppm enzyme and 400 ppm urea was added and 250 μl sample was collected after 2, 4 and 6 hr incubation at 32° C. The sample was centrifuged for 5 minutes at 14.000×g and 50 μl of the supernatant was transferred to an eppendorf tube containing 50 μl of kill solution (1.1 N sulfuric acid) and allowed to stand for 5 minutes. 250 μl of water was added to the tube and then filtered with a 0.22 μm filter plate and injected onto an HPX-87H column as described below.
Evaluation of Performance of TrGA Variant in Ethanol Fermentations
A sample of corn mash liquefact from a local ethanol producer was obtained and diluted in some cases to 26% DS using thin stillage. The pH of the slurry was adjusted to pH 4.3 using 4 N sulfuric acid. A 100 g or 50 g aliquot of mash was put into a 125 ml shake flask and placed into a 32° C. incubator and allowed to equilibrate. After addition of 100 μl 400 ppm urea, 1 ml purified variant at intended concentration or purified TrGA at 2 different concentrations was added to the shake flasks. Finally, 333 μl of a solution of Red Star Red yeast (15 g hydrated for 30 minutes in 45 ml DI water; Lesaffre yeast Corp. Milwaukee, Wis.) was added to each sample; Samples were collected at 5, 21, 28, 48 and 52 hours and analyzed by HPLC (Agilent 1200 series) using an Aminex HPX-87H column (Bio-Rad).
Ethanol and Carbohydrate Determinations
A 2 ml eppendorf centrifuge tube was filled with fermentor beer and cooled on ice for 10 minutes. The sample was centrifuged for 3 minutes at 14.000×g and 500 μl of the supernatant was transferred to a test tube containing 50 μl of kill solution (1.1 N sulfuric acid) and allowed to stand for 5 minutes. 5.0 ml of water was added to the test tube and then filtered into a 0.22 μm filter plate (multiscreen, Millipore, Amsterdam, the Netherlands) and run on HPLC. Column Temperature: 60° C.; mobile phase: 0.01 N sulfuric acid; flow rate 0.6 ml/min; detector: R1; injection volume: 20 μl. The column separates molecules based on charge and molecular weight; DP1 (monosaccharides); DP2 (disaccharides); DP3 (trisaccharides); DP>3 (oligosaccharides sugars having a degree of polymerization greater than 3); succinic acid; lactic acid; glycerol; methanol; ethanol.
Determination of GAU Activity
Substrate: p-Nitrophenyl-β-maltoside (4 mM), plus thermostable β-glucosidase (5 U/ml) (from assay R-AMGR305/04; Megazyme International Wicklow, Ireland) was freshly prepared
Buffer: 200 mM Sodium acetate buffer (pH 4.5).
Enzyme samples were diluted by at least a factor 10 in sodium acetate buffer In a 96 well plate: 20 μl substrate was mixed with 20 μL enzyme solution and incubate at 40° C. with agitation for 10 minutes. 300 μL 2% Trizma base was added to terminate reaction and develop the colour. Absorbance at 400 nm was measured against a reagent blank.
Blanks were prepared by adding 300 μL of Trizma base solution (2%) to 20 μL of substrate with vigorous stirring, followed by the enzyme solution (20 μL). Activity was calculated as follows:




  
    Activity
    ⁢
    
      
    
    ⁢
    
      (
      
        GAU
        ⁢
        
          /
        
        ⁢
        ml
      
      )
    
  
  =
  
    
      
        Δ
        ⁢
        
          
        
        ⁢
        
          A
          400
        
      
      10
    
    ·
    
      340
      20
    
    ·
    
      1
      18.1
    
    ·
    
      1
      
        0
        ,
        88
      
    
    ·
    Dilution
  




Where: GAU=International units of enzyme activity. One Unit is the amount of enzyme which release one μmole of p-nitrophenol from the substrate per minute at the defined pH and temperature. ΔA400=absorbance (reaction)−Absorbance (blank). 10=incubation time (min). 340=final reaction volume (μL). 20=volume of enzyme assayed (μL) 18.1=EmM p-nitrophenol in 2% trizma base (pH ̃8.5) at 400 nm (unit: μM−1*cm−1). 0.88=Light path (cm)
Starch Hydrolysis Activity (SH Activity):
Buffer: 0.1 M Citrate buffer pH 5.4 (made from 0.1 M citric acid and 0.1 M Tri-sodium citrate)
Substrate: 30% soluble starch (Merck, v.nr 1.01257.1000) in buffer (heat slightly until all starch is in solution)
Enzyme: Glucoamylase standardised to 3 GAU/ml on basis of above assay.
60 μL 30% starch was transferred to a 96 well PCR plate. 60 μL enzyme sample or standard was added and mixed by pumping a couple of times with the pipette. Following steps until incubation was carried not as fast as possible.
The PCR plate was covered with sealing tape and following PCR programme was run: 6 min at 63° C., 6 min at 99° C. and 10 min at 4° C. Lid was not heated. After the temperature cycle the PCR plate was centrifuged (app 1 min at 300 rpm) to collect all liquid in the bottom of the wells. Plates were stored at 4° C. until further analysis. Glucose concentration was measured according to method below and the hydrolysis activity was calculated as follows:




  
    Starch
    ⁢
    
      
    
    ⁢
    hydrolysis
    ⁢
    
      
    
    ⁢
    activity
    ⁢
    
      
    
    ⁢
    
      (
      
        M
        ⁢
        
          /
        
        ⁢
        min
      
      )
    
  
  =
  
    
      
        [
        glucose
        ]
      
      ⁢
      
        g
        l
      
    
    
      180
      ⁢
      
        g
        mol
      
      ×
      6
      ⁢
      
        
      
      ⁢
      min
    
  




Isomaltose Hydrolysis Activity

Same as for starch hydrolysis activity except that substrate is 2% iso-maltose (Sigma 17253) and the first step in the PCR programme is 10 min at 63° C. instead of 6 min.
Determination of Glucose Concentration
Modified from the Megazyme© D-glucose assay (KGLUC 04/06) and used to determine the amount of glucose released from starch and isomaltose hydrolysis reactions.
The contents of bottle 1 [Reagent Buffer: Potassium phosphate buffer (1.0 M, pH 7.4), p-hydroxybenzoic acid (0.22 M) and sodium azide (0.4% w/v)] was diluted to 1 L with distilled water. The contents of bottle 2 [Reagent Enzymes: Glucose oxidase (>12,000 U) plus peroxidase (>650 U) and 4-aminoantipyrine (80 mg). Freeze-dried powder] was diluted in approx. 20 mL of solution 1 and quantitatively transferred to the bottle containing the remainder of solution 1. This is Glucose Determination Reagent (GOPOD Reagent). It was either used fresh or stored frozen and dark. Before use it was checked that the absorbance (A510) of this solution was less than 0.05 when read against distilled water.
In a 96 well plate, add 250 μL of GOPOD reagent to 10 μL of sample solution. Cover the plate with sealing tape and incubate in an Eppendorf thermomixer at 40° C., 700 rpm for 20 min. Read absorbances at 510 nm. A glucose standard curve is made from solutions of 1.4; 1.2; 0.8; 0.4; 0.2 and 0 mg/ml glucose in milli-q water and used for calculation of the sample glucose concentrations.
Determination of Maltose and Isomaltose Synthesis by TLC
Substrate: 30% glucose in 0.1M citric acid buffer, pH 5.4 (heated slightly to bring all glucose into solution).
Enzyme: Glucoamylase standardized to 3 GAU/ml Aspergillus niger glucoamylase product (AnGA; Diazyme®X4, Danisco, Denmark) and Trichoderma reesei glucoamylase product (TrGA Diazyme TR8 Danisco, Denmark) were always run as references.
Reference: Heat inactivated enzyme sample and/or buffer solution (not used in all cases).
Standards: 1) 0.3% Maltose and 0.1% isomaltose in demineralised water. 2) 0.2% Maltose and 0.05% isomaltose in demineralised water. 3) 0.3% Maltose and 0.1% isomaltose in demineralised water.
Reaction conditions: 60 μl substrate was mixed with 60 μL enzyme solution in wells of a PCR-plate. The plate with was covered with sealing tape and following temperature was run: 120 min at 63° C., 6 min at 99° C., 10 min at 4° C. Lid heated to 70° C. After incubation the plates were given a moderate centrifugation (1 min at 300 rpm), and they were store at 4° C. until further analysis. All samples were run in duplicate.
Quantification of Maltose and Iso-Maltose: Preheat TLC-Plate at 167° C. for 10 min prior to sample application. Dilute all samples and standards 20 times in demineralised water. An automatic TLC sampler (ATS4, CAMAG, Muttenz, Switzerland) was used for accurately transferring 4 μL samples to the TLC plate. Each plate could contain 20 samples, placed in 4 mm wide bands. Plates were heated for 10 min at 40° C. to let bands dry out. The TLC-plates were eluted in AcN, EtAc, 1-propanol, H2O (85:20:50:40), whereafter the plates were heated 5 min at 167° C. to remove excess solvent. The plate was dipped up side down (i.e. by hold the plate on the edge near where the samples were applied) in 5% H2SO4:EtOH (95:5). The dipping solution was made daily. The plates were heated 3 min at 167° C. to visualize spots. Determination of spot intensity was done by scanning in a TLC scanner (CAMAG scanner 3, Muttenz, Switzerland) and quantification was done by drawing a standard-curve based on all maltose and isomaltose concentrations vs. spot intensities. Both maltose and isomaltose concentrations were calculated from this curve, using the fact that the spot intensity vs. concentration is equal for the two compounds.
FIG. 10 depicts an example of a TLC plate with standards containing different concentrations of glucose, maltose and isomaltose and samples containing reaction products from glucose incubated with Aspergillus niger glucoamylase product (AnGA; Diazyme®X4, Danisco, Denmark) and Trichoderma reesei glucoamylase product (TrGA Diazyme TR8 Danisco, Denmark). Blind is glucose incubated without enzyme.
The isomaltose synthesis activity (IS activity) is calculated on basis of the isomaltose concentrations determined by TLC according to the following formula:




  
    Isomaltose
    ⁢
    
      
    
    ⁢
    synthesis
    ⁢
    
      
    
    ⁢
    activity
    ⁢
    
      
    
    ⁢
    
      (
      
        M
        ⁢
        
          /
        
        ⁢
        min
      
      )
    
  
  =
  
    
      
        [
        Isomaltose
        ]
      
      ⁢
      
        
      
      ⁢
      %
      ×
      10
      ⁢
      
        g
        l
      
    
    
      342
      ⁢
      
        g
        mol
      
      ×
      120
      ⁢
      
        
      
      ⁢
      min
    
  




Thermal Stability Assay 2

As a measure of thermostability of the enzymes under the conditions used in the present experiments, the GAU activity was determined according to the above assay before and after incubation of enzymes in 15% glucose, 0.1M citrate buffer, pH 5.4 at 63° C. for 120 min. Data is presented as % activity lost.
Production of GA by Fermentation
400× Trace element solution: Dilute in 1000 ml of demi water: Anhudrous Citric Acid (175 g), FeSO4*7 H2O (200 g), ZnSO4*7 H2O (16 g), CuSO4*5 H2O (3.2 g), MnSO4*H2O (1.4 g), H3BO3 (0.8 g). It may be helpful to acidify this to get all components into solution. The solution was filtered and sterilized.
LD-medium: Add to  ̃800 ml of demi water: Casamino acids (9 g), MgSO4*7H2O (1 g), (NH4)2SO4 (5 g), KH2PO4 (4.5 g), CaCl2*2H2O (1 g), piperazine-1,4-bis(propanesulfonic acid (PIPPS) buffer (33 g), 400× T. reesei trace elements (2.5 ml), Adjust pH to 5.5 with NaOH 4N. Adjust final volume to 920 ml.
2×Amd S Base ager (1 liter): Mix KH2PO4 (30 g), 1M Acetamide (20 ml), 1M CsCl (20 ml), 20% MgSO4.7H2O (6 ml), 20% CaCl2.2H2O (6 ml), T. reesei spore elements 400× (2 ml), 50% glucose.H2O (80 ml). Adjust pH to 4.5 with 4N NaOH Make up to 1 L and filter sterilize. Store at 4° C.
Initial Culture: Strains were grown on AmdS-Base agar plates. To produce agar plates minimal media agar was boiled and after cooling down to app. 50° C. it was diluted with 2× AmdS Base 1:1 and poured on petri dishes. After sporulation (app. 6-7 days) the plates were scraped with 2 ml saline 0.015% Tween 80. Approx 1 ml was added to glycerol tubes containing 500-600 μl 35% glycerol and stored at −80° C. The pre-culture fermentations were started directly from this spore suspension.
Pre Culture: The medium is made by adding 2.5% glucose to the LD-medium, which is subsequently made up to 1 L. To produce biomass 50 μl spore suspension is added to 100 ml medium (sterilised in 500 ml shake flask). The flasks are incubated on a rotary shaker at 30° C., 180 rpm for 2 days, then 10 ml suspension is used to inoculate a new shake flask, which is incubated under similar conditions for 1 day. The content of this flask is used to inoculate a fermentor.
Main Culture: To make 1 L of medium, 40 ml glucose/sophorose mix (Danisco, Jamsa, Finland) was added to the LD-medium and made up to 1 L. 6 L fermentors containing 4 L of medium were inoculated with the pre-culture, and grown at pH 3.5 for approximately 16 hours at 34° C., until CER/OUR (Carbondioxide Excretion Rate/Oxygen Uptake Rate) started falling. Then temperature was lowered to 28° C., pH was raised to 4.0 and the fermentation was continued for approximately 80 hours.
Brew Analysis with Determination of Real Degree of Fermentation (RDF)
Pure Malt Brew Analysis
Analysis was carried out at using the following procedure: 70 g milled pilsner malt (Weyermann, Bamberg, Germany) was mashed with 266 ml water. The temperature cycle after mashing in (mixing malt and water) was: 140 minutes at 63.9° C., increasing to 73.9° C. over 10 minutes, 5 minutes at 73.9° C. At the end of mashing, the mashes were cooled, made up to 350 g and filtered. Filtrate volumes were measured after 30 minutes. The filtrated worts were sampled for specific gravity determination, then heated to 99° C. for 10 minutes in a water bath in order to destroy any residual glucoamylase activity. The heat treatment results in a loss of 1.5 g per 200 ml wort. The worts were fermented at 18° C. and 100 rpm in 500 ml conical flasks after yeast addition for at least 88 hours and no more than 120 hours. Specific gravity was determined on the ferments.
Malt-Adjunct Brew Analysis
A modified decoction mashing, using corn grist as adjunct was employed. The brewing protocol was modified from US 2009014247. 40% of the malt was substituted with corn grist with a moisture content of 12.6% (Benntag Nordic; Nordgetreide GmBH Liibec, Germany). All corn grist was heated to 100° C. at 2° C./min, together with 54% of the water and 5% of the malt (well modified Pilsner malt; Fuglsang Denmark). 5 min rests were held at 72° C. and 80° C. and a 10 min rest was held at 100° C. Hereafter the adjunct was cooled to 64° C. and combined with the main mash, also at 64° C. Enzymes were added at this stage, and the 64° C. rest was extended to 250 min. After fermentation the RDF values were determined.
Real degree of fermentation (RDF) value may be calculated according to the equation below:




  
    RDF
    ⁡
    
      (
      %
      )
    
  
  =
  
    
      (
      
        1
        -
        
          RE
          
            P
            initial
            
            
              
              o
          
        
      
      )
    
    ×
    100
  




Where: RE=real extract=(0.1808×°Pinitial)+(0.8192×°Pfinal), °Pinitial is the specific gravity of the standardised worts before fermentation and °Pfinal is the specific gravity of the fermented worts expressed in degree plato.
In the present context, Real degree of fermentation (RDF) was determined as follows:
After fermentation samples were filtered and degassed. Specific gravity and alcohol concentration was determined on the ferments using a Beer Alcolyzer Plus and a DMA 5000 Density meter (both from Anton Paar, Graz, Austria). Based on these measurements, the real degree of fermentation (RDF) value is calculated, by the instrument, according to the equation below:




  
    RFD
    ⁡
    
      (
      %
      )
    
  
  =
  
    
      
        OE
        -
        
          E
          ⁡
          
            (
            r
            )
          
        
      
      OE
    
    ×
    100
  




Where: E(r) is the real extract in degree Plato (°P) and OE is the original extract in °P.
Xylanase Assay Method
Samples were diluted in citric acid (0.1 M)—di-sodium-hydrogen phosphate (0.2 M) buffer, pH 5.0, to obtain approx. OD590=0.7 in this assay. Three different dilutions of the sample were pre-incubated for 5 minutes at 40° C. At time=5 minutes, 1 Xylazyme tablet (crosslinked, dyed xylan substrate, Megazyme, Bray, Ireland) was added to the enzyme solution in a reaction volume of 1 ml. At time=15 minutes the reaction was terminated by adding 10 ml of 2% TRIS/NaOH, pH 12. Blanks were prepared using 1000 μl buffer instead of enzyme solution. The reaction mixture was centrifuged (1500×g, 10 minutes, 20° C.) and the OD of the supernatant was measured at 590 nm. One xylanase unit (XU) is defined as the xylanase activity increasing OD590 with 0.025 per minute.
Pullulanase Assay Method
Principle:
On incubation of Red-Pullulan (partially depolymerised pullulan, which is dyed with Procion Red MX-5B, from assay kit S-RPUL, Megazyme Int., Bray, Ireland) with pullulanase or limit-dextrinase the substrate is depolymerised by an endo-mechanism to produce low molecular-weight dyed fragments which remain in solution on addition of ethanol to the reaction mixture. High-molecular weight material is removed by centrifugation, and the colour of the supernatant is measured at 510 nm. Pullulanase in the assay solution is determined by reference to a standard curve.
Substrate:
0.5 g of powdered substrate was dissolved in to 25 mL of 0.5 M potassium chloride solution.
Buffer: Sodium Acetate, 200 mM, pH 5.0
Enzyme Preparation:
Enzyme is diluted at least 10 times in buffer. If the resulting A510 reading was above 1.0 the enzyme was further diluted.
Assay Procedure:
In a test tube 1.0 mL of enzyme solution, pre-equilibrated to 40° C., was mixed with 0.5 mL substrate solution, pre-equilibrated to 40° C. The mix was incubated at 40° C. for 10 min. Reaction was terminated and high-molecular weight substrate was precipitated by the adding 2.5 mL ethanol (95% v/v). The tubes were equilibrate to room temperature for 10 min, then stirred for 10 sec on a vortex mixer and centrifuged at 1,000 g for 10 min. Supernatants were transferred to a 96 well plate and the absorbance of blank and reaction solutions was measured at 510 nm against distilled water. The blank reading was subtracted from the sample reading to obtain the A510 used in the formula below.
Activity is determined from the formula: milli-PU/mL=360*A510+11.
The blank is prepared by adding ethanol to the Red-Pullulan substrate before addition of the enzyme.
One Unit of activity is defined as the amount of enzyme required to release one mole of D-glucose reducing sugar equivalents per minute from borohydride reduced pullulan, under the defined assay conditions (see Megazyme method S-RPUL 10/08, Megazyme Int., Bray, Ireland).
Alpha-Amylase Assay (SSU) Method
Principle:
Method is based on the release of reducing groups measured, by reference to a standard curve, as glucose from a 20 minute hydrolysis of 4% potato soluble starch at pH 4.5 and 50° C. One Soluble Starch Unit (SSU) is the activity which liberates 1 milligram of glucose equivalents per minute.
Substrate:
4% (w/v) potato soluble starch (Sigma, S 2630) solution in 0.05 M sodium acetate buffer, pH 4.5. The potato soluble starch is slurried in deionised water then added to vigorously boiling water in a flask. The starch solution is boiled, with stirring, for three minutes then cooled to 25° C. and acetic acid and sodium hydroxide solution added to give a 0.05 M sodium acetate buffer, pH 4.5, when made up to the final concentration.
DNS Solution:
1 L of DNS solution is made up by adding to water: 16 g sodium hydroxide followed by 10 g 3-5 dinitro salicylic acid and then 300 g sodium potassium tartrate, each component being dissolved before the next is added. The solution is made up to volume and stored in the dark.
Assay Procedure:
Add 0.4 ml starch substrate and 0.1 ml 0.5M sodium acetate buffer, pH 4.5 to a test tube which is capped and equilibrated to temperature in a water bath set at 50° C. Add 0.1 ml of diluted enzyme solution and after 20 minutes stop the reaction by adding 0.1 ml 2% (w/v) NaOH. Run each enzyme assay in duplicate along with an enzyme blank.
Color Development:
To each tube (assay and blank) add 1.5 ml water and 2.0 ml DNS solution, mix and place in a boiling water bath for 5 minutes then cool the tubes in an ice bath for 10 minutes. Let the tubes stand at room temperature for 20 minutes and read the absorbance at 543 nm.
Standard Curve:
Construct a glucose standard curve corresponding to 0.0-1.0 mg glucose per 2.2 ml water, then adding 2.0 ml of DNS reagent and proceeding as described.
Calculation Of Enzyme Activity (SSU)
Convert absorbance values (A543) of samples to mg of glucose using the standard curve, and calculate the ΔA543 mg glucose values [sample−blank] (ΔA543 must be between 0.2-0.4). Activity is determined from the formula:

SSU/ml or g=average(ΔA543 mg glucose values[sample−blank])×(1/0.1)×(1/20)×Enzyme Dilution

Example 1
Construction of TrGA Site Evaluation Libraries (SELs) in the pTTT Vector for Expression in Trichoderma reesei 
A Trichoderma reesei cDNA sequence (SEQ ID NO: 4) was cloned into pDONRTM201 via the Gateway® BP recombination reaction (Invitrogen, Carlsbad, Calif., USA) resulting in the entry vector pDONR-TrGA (FIG. 2). The cDNA sequence (SEQ ID NO: 4) encodes the TrGA signal peptide, the pro-sequence, and the mature protein, including the catalytic domain, linker region and starch binding domain (SEQ ID NO: 1). SEQ ID NO: 4 and SEQ ID NO: 1 are shown in FIGS. 1B and 1B. FIG. 1C illustrates the precursor and mature protein TrGA domains.
To express the TrGA protein in Trichoderma reesei, the TrGA coding sequence (SEQ ID NO: 4) was cloned into the Gateway compatible destination vector pTTT-Dest (FIG. 3) via the GATEWAY® LR recombination reaction. The expression vector contained the T. reesei cbhI-derived promoter and terminator regions that allowed for strong inducible expression of a gene of interest. The vector also contained the Aspergillus nidulans amdS selective marker that allowed for growth of the transformants on acetamide as a sole nitrogen source. The expression vector also contained T. reesei telomere regions that allowed for non-chromosomal plasmid maintenance in a fungal cell. On the destination pTTT-Dest plasmid, the cbhI promoter and terminator regions were separated by the chloramphenicol resistance gene, CmR, and the lethal E. coli gene, ccdB, flanked by the bacteriophage lambda-based specific recombination sites attR1, attR2. This configuration allowed for direct selection of recombinants containing the TrGA gene under control of the cbhI regulatory elements in the right orientation via the GATEWAY® LR recombination reaction. The final expression vector pTTT-TrGA is shown in FIG. 4.
SELs were constructed using the pDONR-TrGA entry vector (FIG. 2) as a template and the primers listed in Table 2. All primers used in the mutagenesis experiments contained the triplet NNS (N=A, C, T, G, and S=C or G) at the position that aligns with the codon of the TrGA sequence designed to be mutated (SEQ ID NO: 1), allowing for a random incorporation of nucleotides at the preselected position. Construction of each SEL library started with two independent PCR amplifications on the pDONR-TrGA entry vector: one using the Gateway F (pDONR201-FW) and a specific mutagenesis primer R (Table 2), and the other—the Gateway primer R (pDONR201-RV) and a specific mutagenesis primer F (Table 2). High fidelity PHUSION DNA polymerase (Finnzymes OY, Espoo, Finland) was used in a PCR amplification reaction including 0.2 μM primers. The reactions were carried out for 25 cycles according to the protocol provided by Finnzymes. 1 μl aliquots of the PCR fragments obtained were used as templates for a subsequent fusion PCR reaction together with the Gateway FW and Gateway RV primers (Invitrogen). This PCR amplification, after 22 cycles, produced a population of the full-length linear TrGA DNA fragments randomly mutated at the specific codon position. The fragments were flanked by the Gateway-specific attL1, attL2 recombination sites on both ends. The DNA fragments were purified with a CHARGESWITCH® PCR clean-up kit (Invitrogen, Carlsbad USA) and then recombined with 100 ng of the pTTT-destination vector (FIG. 3) using the LR CLONASETM II enzyme mix according to the protocol supplied by Invitrogen. The recombination products that were generated were transformed into E. coli Max Efficiency DH5α, as described by the supplier (Invitrogen). The final expression constructs pTTT-TrGA with mutations at the desired position were selected by plating bacteria on 2×YT agar plates (16 g/L Bacto Tryptone (Difco), 10 g/L Bacto Yeast Extract (Difco), 5 g/L NaCl, 16 g/L Bacto Agar (Difco)) with 100 μg/ml ampicillin.
96 single colonies from each library were grown for 24 hrs at 37° C. in MTP containing 200 μL 2×YT medium with 100 μg/ml ampicillin. Cultures were used directly to amplify PCR fragments encompassing the region where a specific mutation was introduced. The specific PCR products obtained were sequenced using an ABI3100 sequence analyzer (Applied Biosystems). Each library contained from 15 to 19 different TrGA variants in the final expression vector. These variants were individually transformed into T. reesei, as described below. Libraries are numbered from 1 to 182 referencing the specific amino acid residue in the TrGA sequence that was randomly mutated.







TABLE 2







Primers used to generate TrGA SELs









AA-
F/



position
R
DNA Sequence 5′ to 3′





pDONR201-
F
TCGCGTTAACGCTAGCATGGATCTC (SEQ ID




NO: 13)


 


pDONR201-
R
TCGCGTTAACGCTAGCATGGATCTC (SEQ ID




NO: 14)


 


4
F
CGTCACCAAGAGGTCTGTTGACNNSTTCATCAGCAC




CGAGACGCC (SEQ ID NO: 15)


 


4
R
GTCAACAGACCTCTTGGTGACGTCG (SEQ ID




NO: 16)


 


5
F
CACCAAGAGGTCTGTTGACGACNNSATCAGCACCG




AGACGCCTATTGC (SEQ ID NO: 17)


 


5
R
GTCGTCAACAGACCTCTTGGTGAC (SEQ ID




NO: 18)


 


10
F
TGACGACTTCATCAGCACCGAGNNSCCTATTGCA




CTG (SEQ ID NO: 19)


 


10
R
CTCGGTGCTGATGAAGTCGTC (SEQ ID




NO: 20)


 


12
F
TCATCAGCACCGAGACGCCTNNSGCACTGAACAATC




TTCTTTGCA (SEQ ID NO: 21)


 


12
R
AGGCGTCTCGGTGCTGATGAAGTCG (SEQ ID




NO: 22)


 


14
F
CAGCACCGAGACGCCTATTGCANNSAACAATCTT




CTT (SEQ ID NO: 23)


 


14
R
TGCAATAGGCGTCTCGGTGCT (SEQ ID NO: 24)


 


15
F
CACCGAGACGCCTATTGCACTGNNSAATCTTCTT




TGC (SEQ ID NO: 25)


 


15
R
CAGTGCAATAGGCGTCTCGGT (SEQ ID NO: 26)


 


23
F
CAATCTTCTTTGCAATGTTGGTNNSGATGGATGC




CGT (SEQ ID NO: 27)


 


23
R
ACCAACATTGCAAAGAAGATTG (SEQ ID NO: 28)


 


24
F
TTCTTTGCAATGTTGGTCCTNNSGGATGCCGTGCATT




CGGCACAT (SEQ ID NO: 29)


 


24
R
AGGACCAACATTGCAAAGAAGATTG (SEQ ID




NO: 30)


 


29
F
GTCCTGATGGATGCCGTGCANNSGGCACATCAGCTGG




TGCGGTGA (SEQ ID NO: 31)


 


29
R
TGCACGGCATCCATCAGGACCAACA (SEQ ID




NO: 32)


 


42
F
TGCGGTGATTGCATCTCCCAGCNNSATTGACCC




GGAC (SEQ ID NO: 33)


 


42
R
GCTGGGAGATGCAATCACCGCA (SEQ ID NO: 34)


 


43
F
TGATTGCATCTCCCAGCACANNSGACCCGGACTACTA




TTACATGT (SEQ ID NO: 35)


 


43
R
TGTGCTGGGAGATGCAATCACCGCA (SEQ ID




NO: 36)


 


44
F
TTGCATCTCCCAGCACAATTNNSCCGGACTACTATTA




CATGTGGA (SEQ ID NO: 37)


 


44
R
AATTGTGCTGGGAGATGCAATCACC (SEQ ID




NO: 38)


 


45
F
CATCTCCCAGCACAATTGACNNSGACTACTATTACA




TGTGGACGC (SEQ ID NO: 39)


 


45
R
GTCAATTGTGCTGGGAGATGCAATC (SEQ ID




NO: 40)


 


46
F
CTCCCAGCACAATTGACCCGNNSTACTATTACATGT




GGACGCGAGA (SEQ ID NO: 41)


 


46
R
CGGGTCAATTGTGCTGGGAGATGCA (SEQ ID




NO: 42)


 


47
F
CCAGCACAATTGACCCGGACNNSTATTACATGTGGA




CGCGAGATA (SEQ ID NO: 43)


 


47
R
GTCCGGGTCAATTGTGCTGGGAGAT (SEQ ID




NO: 44)


 


49
F
CAATTGACCCGGACTACTATNNSATGTGGACGCGAG




ATAGCGCTC (SEQ ID NO: 45)


 


49
R
ATAGTAGTCCGGGTCAATTGTGCTG (SEQ ID




NO: 46)


 


51
F
ACCCGGACTACTATTACATGNNSACGCGAGATAGC




GCTCTTGTCT (SEQ ID NO: 47)


 


51
R
CATGTAATAGTAGTCCGGGTCAATT (SEQ ID




NO: 48)


 


59
F
GACGCGAGATAGCGCTCTTGTCNNSAAGAACCTC




ATC (SEQ ID NO: 49)


 


59
R
GACAAGAGCGCTATCTCGCGT (SEQ ID NO: 50)


 


60
F
GCGAGATAGCGCTCTTGTCTTCNNSAACCTCATC




GAC (SEQ ID NO: 51)


 


60
R
GAAGACAAGAGCGCTATCTCG (SEQ ID NO: 52)


 


61
F
AGATAGCGCTCTTGTCTTCAAGNNSCTCATCGAC




CGC (SEQ ID NO: 53)


 


61
R
CTTGAAGACAAGAGCGCTATC (SEQ ID NO: 54)


 


65
F
TGTCTTCAAGAACCTCATCGACNNSTTCACCGAA




ACG (SEQ ID NO: 55)


 


65
R
GTCGATGAGGTTCTTGAAGAC (SEQ ID NO: 56)


 


67
F
CAAGAACCTCATCGACCGCTTCNNSGAAACGTA




CGAT (SEQ ID NO: 57)


 


67
R
GAAGCGGTCGATGAGGTTCTT (SEQ ID NO: 58)


 


68
F
GAACCTCATCGACCGCTTCACCNNSACGTACGA




TGCG (SEQ ID NO: 59)


 


68
R
GGTGAAGCGGTCGATGAGGTT (SEQ ID NO: 60)


 


70
F
TCGACCGCTTCACCGAAACGNNSGATGCGGGCCT




GCAGCGCCGCA (SEQ ID NO: 61)


 


70
R
CGTTTCGGTGAAGCGGTCGATGAGG (SEQ ID




NO: 62)


 


72
F
CCGCTTCACCGAAACGTACGATNNSGGCCTGCA




GCGC (SEQ ID NO: 63)


 


72
R
ATCGTACGTTTCGGTGAAGCGG (SEQ ID NO: 64)


 


73
F
CTTCACCGAAACGTACGATGCGNNSCTGCAGCG




CCGC (SEQ ID NO: 65)


 


73
R
CGCATCGTACGTTTCGGTGAA (SEQ ID NO: 66)


 


75
F
AAACGTACGATGCGGGCCTGNNSCGCCGCATCGAG




CAGTACATTA (SEQ ID NO: 67)


 


75
R
CAGGCCCGCATCGTACGTTTCGGTG (SEQ ID




NO: 68)


 


76
F
CGTACGATGCGGGCCTGCAGNNSCGCATCGAGCAGT




ACATTACTG (SEQ ID NO: 69)


 


76
R
CTGCAGGCCCGCATCGTACGTTTCG (SEQ ID




NO: 70)


 


94
F
CTCTCCAGGGCCTCTCTAACNNSTCGGGCTCCCTCGC




GGACGGCT (SEQ ID NO: 71)


 


94
R
GTTAGAGAGGCCCTGGAGAGTGACC (SEQ ID




NO: 72)


 


97
F
GGGCCTCTCTAACCCCTCGGGCNNSCTCGCGGAC




GGC (SEQ ID NO: 73)


 


97
R
GCCCGAGGGGTTAGAGAGGCC (SEQ ID NO: 74)


 


98
F
CCTCTCTAACCCCTCGGGCTCCNNSGCGGACGG




CTCT (SEQ ID NO: 75)


 


98
R
GGAGCCCGAGGGGTTAGAGAG (SEQ ID NO: 76)


 


99
F
CTCTAACCCCTCGGGCTCCCTCNNSGACGGCTCT




GGT (SEQ ID NO: 77)


 


99
R
GAGGGAGCCCGAGGGGTTAGA (SEQ ID NO: 78)


 


100
F
ACCCCTCGGGCTCCCTCGCGNNSGGCTCTGGTCTC




GGCGAGCCCA (SEQ ID NO: 79)


 


100
R
CGCGAGGGAGCCCGAGGGGTTAGAG (SEQ ID




NO: 80)


 


102
F
CTCGGGCTCCCTCGCGGACGGCNNSGGTCTCGGC




GAG (SEQ ID NO: 81)


 


102
R
GCCGTCCGCGAGGGAGCCCGA (SEQ ID NO: 82)


 


110
F
TGGTCTCGGCGAGCCCAAGTTTNNSTTGACCCTG




AAG (SEQ ID NO: 83)


 


110
R
AAACTTGGGCTCGCCGAGACCA (SEQ ID NO: 84)


 


111
F
TCTCGGCGAGCCCAAGTTTGAGNNSACCCTGAA




GCCT (SEQ ID NO: 85)


 


111
R
CTCAAACTTGGGCTCGCCGAG (SEQ ID NO: 86)


 


113
F
CGAGCCCAAGTTTGAGTTGACCNNSAAGCCTTTC




ACC (SEQ ID NO: 87)


 


113
R
GGTCAACTCAAACTTGGGCTC (SEQ ID NO: 88)


 


114
F
CCAAGTTTGAGTTGACCCTGNNSCCTTTCACCGGC




AACTGGGGTC (SEQ ID NO: 89)


 


114
R
CAGGGTCAACTCAAACTTGGGCTCG (SEQ ID




NO: 90)


 


116
F
TTGAGTTGACCCTGAAGCCTNNSACCGGCAACTGG




GGTCGACCGCA (SEQ ID NO: 91)


 


116
R
AGGCTTCAGGGTCAACTCAAACTTG (SEQ ID




NO: 92)


 


119
F
CCCTGAAGCCTTTCACCGGCNNSTGGGGTCGACCG




CAGCGGGATG (SEQ ID NO: 93)


 


119
R
GCCGGTGAAAGGCTTCAGGGTCAAC (SEQ ID




NO: 94)


 


122
F
CTTTCACCGGCAACTGGGGTNNSCCGCAGCGGGAT




GGCCCAGCTC (SEQ ID NO: 95)


 


122
R
ACCCCAGTTGCCGGTGAAAGGCTTC (SEQ ID




NO: 96)


 


125
F
GCAACTGGGGTCGACCGCAGNNSGATGGCCCAGCT




CTGCGAGCCA (SEQ ID NO: 97)


 


125
R
CTGCGGTCGACCCCAGTTGCCGGTG (SEQ ID




NO: 98)


 


133
F
GGATGGCCCAGCTCTGCGAGCCNNSGCCTTGATT




GGA (SEQ ID NO: 99)


 


133
R
GGCTCGCAGAGCTGGGCCATCC (SEQ ID




NO: 100)


 


137
F
TGCGAGCCATTGCCTTGATTNNSTACTCAAAGTGGC




TCATCAACA (SEQ ID NO: 101)


 


137
R
AATCAAGGCAATGGCTCGCAGAGCT (SEQ ID




NO: 102)


 


140
F
CATTGCCTTGATTGGATACTCANNSTGGCTCAT




CAAC (SEQ ID NO: 103)


 


140
R
TGAGTATCCAATCAAGGCAATG (SEQ ID




NO: 104)


 


144
F
TGGATACTCAAAGTGGCTCATCNNSAACAACTAT




CAG (SEQ ID NO: 105)


 


144
R
GATGAGCCACTTTGAGTATCC (SEQ ID




NO: 106)


 


145
F
ATACTCAAAGTGGCTCATCAACNNSAACTATCAG




TCG (SEQ ID NO: 107)


 


145
R
GTTGATGAGCCACTTTGAGTA (SEQ ID




NO: 108)


 


146
F
CAAAGTGGCTCATCAACAACNNSTATCAGTCGACT




GTGTCCAACG (SEQ ID NO: 109)


 


146
R
GTTGTTGATGAGCCACTTTGAGTAT (SEQ ID




NO: 110)


 


147
F
AAAGTGGCTCATCAACAACAACNNSCAGTCGACT




GTG (SEQ ID NO: 111)


 


147
R
GTTGTTGTTGATGAGCCACTT (SEQ ID




NO: 112)


 


148
F
GGCTCATCAACAACAACTATNNSTCGACTGTGTCCA




ACGTCATCT (SEQ ID NO: 113)


 


148
R
ATAGTTGTTGTTGATGAGCCACTTT (SEQ ID




NO: 114)


 


152
F
CAACAACTATCAGTCGACTGTGNNSAACGTCATC




TGG (SEQ ID NO: 115)


 


152
R
CACAGTCGACTGATAGTTGTT (SEQ ID




NO: 116)


 


153
F
CAACTATCAGTCGACTGTGTCCNNSGTCATCTGG




CCT (SEQ ID NO: 117)


 


153
R
GGACACAGTCGACTGATAGTT (SEQ ID




NO: 118)


 


164
F
GCCTATTGTGCGCAACGACCTCNNSTATGTTGCC




CAGT (SEQ ID NO: 119)


 


164
R
GAGGTCGTTGCGCACAATAGG (SEQ ID




NO: 120)


 


169
F
ACCTCAACTATGTTGCCCAGNNSTGGAACCAAACC




GGCTTTGACC (SEQ ID NO: 121)


 


169
R
CTGGGCAACATAGTTGAGGTCGTTG (SEQ ID




NO: 122)


 


172
F
ATGTTGCCCAGTACTGGAACNNSACCGGCTTTGACC




TCTGGGAAG (SEQ ID NO: 123)


 


172
R
GTTCCAGTACTGGGCAACATAGTTG (SEQ ID




NO: 124)


 


175
F
AGTACTGGAACCAAACCGGCNNSGACCTCTGGGAA




GAAGTCAATG (SEQ ID NO: 125)


 


175
R
GCCGGTTTGGTTCCAGTACTGGGCA (SEQ ID




NO: 126)


 


178
F
ACCAAACCGGCTTTGACCTCNNSGAAGAAGTCAATG




GGAGCTCAT (SEQ ID NO: 127)


 


178
R
GAGGTCAAAGCCGGTTTGGTTCCAG (SEQ ID




NO: 128)


 


180
F
CCGGCTTTGACCTCTGGGAANNSGTCAATGGGAGCT




CATTCTTTA (SEQ ID NO: 129)


 


180
R
TTCCCAGAGGTCAAAGCCGGTTTGG (SEQ ID




NO: 130)


 


181
F
GCTTTGACCTCTGGGAAGAANNSAATGGGAGCTCAT




TCTTTACTG (SEQ ID NO: 131)


 


181
R
TTCTTCCCAGAGGTCAAAGCCGGTT (SEQ ID




NO: 132)


 


182
F
CTTTGACCTCTGGGAAGAAGTCNNSGGGAGCTCATT




C (SEQ ID NO: 133)


 


182
R
GACTTCTTCCCAGAGGTCAAAG (SEQ ID




NO: 134)


 


204
F
TGTCGAGGGCGCCACTCTTGCTNNSACTCTTGGCCA




G (SEQ ID NO: 135)


 


204
R
AGCAAGAGTGGCGCCCTCGAC (SEQ ID




NO: 136)


 


205
F
CGAGGGCGCCACTCTTGCTGCCNNSCTTGGCCAGTC




G (SEQ ID NO: 137)


 


205
R
GGCAGCAAGAGTGGCGCCCTC (SEQ ID




NO: 138)


 


208
F
CTCTTGCTGCCACTCTTGGCNNSTCGGGAAGCGCTTA




TTCATCTG (SEQ ID NO: 139)


 


208
R
GCCAAGAGTGGCAGCAAGAGTGGCG (SEQ ID




NO: 140)


 


211
F
CCACTCTTGGCCAGTCGGGANNSGCTTATTCATCTGT




TGCTCCCC (SEQ ID NO: 141)


 


211
R
TCCCGACTGGCCAAGAGTGGCAGCA (SEQ ID




NO: 142)


 


214
F
TGGCCAGTCGGGAAGCGCTTATNNSTCTGTTGCTCC




C (SEQ ID NO: 143)


 


214
R
ATAAGCGCTTCCCGACTGGCC (SEQ ID




NO: 144)


 


216
F
GTCGGGAAGCGCTTATTCATCTNNSGCTCCCCAGGT




T (SEQ ID NO: 145)


 


216
R
AGATGAATAAGCGCTTCCCGA (SEQ ID




NO: 146)


 


219
F
CGCTTATTCATCTGTTGCTCCCNNSGTTTTGTGCTT




T (SEQ ID NO: 147)


 


219
R
GGGAGCAACAGATGAATAAGC (SEQ ID




NO: 148)


 


228
F
TGTGCTTTCTCCAACGATTCNNSGTGTCGTCTGGTG




GATACGTCG (SEQ ID NO: 149)


 


228
R
GAATCGTTGGAGAAAGCACAAAAC




CT (SEQ ID NO: 150)


 


229
F
GTGCTTTCTCCAACGATTCTGGNNSTCGTCTGGTG




GA (SEQ ID NO: 151)


 


229
R
CCAGAATCGTTGGAGAAAGCA (SEQ ID




NO: 152)


 


230
F
CTTTCTCCAACGATTCTGGGTGNNSTCTGGTGGAT




ACG (SEQ ID NO: 153)


 


230
R
CACCCAGAATCGTTGGAGAAA (SEQ ID




NO: 154)


 


231
F
TCTCCAACGATTCTGGGTGTCGNNSGGTGGATAC




GTC (SEQ ID NO: 155)


 


231
R
CGACACCCAGAATCGTTGGAGA (SEQ ID




NO: 156)


 


236
F
GGTGTCGTCTGGTGGATACGTCNNSTCCAACATC




AACAC (SEQ ID NO: 157)


 


236
R
GACGTATCCACCAGACGACAC (SEQ ID




NO: 158)


 


239
F
TGGTGGATACGTCGACTCCAACNNSAACACCAAC




GAG (SEQ ID NO: 159)


 


239
R
GTTGGAGTCGACGTATCCACC (SEQ ID




NO: 160)


 


240
F
TGGATACGTCGACTCCAACATCNNSACCAACGAG




GGCA (SEQ ID NO: 161)


 


240
R
GATGTTGGAGTCGACGTATCCA (SEQ ID




NO: 162)


 


241
F
ATACGTCGACTCCAACATCAACNNSAACGAGGGCAG




GAC (SEQ ID NO: 163)


 


241
R
GTTGATGTTGGAGTCGACGTA (SEQ ID




NO: 164)


 


242
F
TCGACTCCAACATCAACACCNNSGAGGGCAGGACTG




GCAAGGATG (SEQ ID NO: 165)


 


242
R
GGTGTTGATGTTGGAGTCGACGTAT (SEQ ID




NO: 166)


 


243
F
ACTCCAACATCAACACCAACNNSGGCAGGACTGGCA




AGGATGTCA (SEQ ID NO: 167)


 


243
R
GTTGGTGTTGATGTTGGAGTCGACG (SEQ ID




NO: 168)


 


244
F
CTCCAACATCAACACCAACGAGNNSAGGACTGGCA




AG (SEQ ID NO: 169)


 


244
R
CTCGTTGGTGTTGATGTTGGAGT (SEQ ID




NO: 170)


 


245
F
ACATCAACACCAACGAGGGCNNSACTGGCAAGGAT




GTCAACTCCG (SEQ ID NO: 171)


 


245
R
GCCCTCGTTGGTGTTGATGTTGGAGT (SEQ ID




NO: 172)


 


263
F
TTCCATCCACACCTTCGATCCCNNSCTTGGCTGTG




AC (SEQ ID NO: 173)


 


263
R
GGGATCGAAGGTGTGGATGGA (SEQ ID




NO: 174)


 


264
F
CATCCACACCTTCGATCCCAACNNSGGCTGTGACG




CA (SEQ ID NO: 175)


 


264
R
GTTGGGATCGAAGGTGTGGAT (SEQ ID




NO: 176)


 


265
F
CCACACCTTCGATCCCAACCTTNNSTGTGACGCAG




GC (SEQ ID NO: 177)


 


265
R
AAGGTTGGGATCGAAGGTGTG (SEQ ID




NO: 178)


 


268
F
CGATCCCAACCTTGGCTGTGACNNSGGCACCTTCC




AGC (SEQ ID NO: 179)


 


268
R
GTCACAGCCAAGGTTGGGATC (SEQ ID




NO: 180)


 


269
F
TCCCAACCTTGGCTGTGACGCANNSACCTTCCAG




CCA (SEQ ID NO: 181)


 


269
R
TGCGTCACAGCCAAGGTTGGG (SEQ ID




NO: 182)


 


276
F
AGGCACCTTCCAGCCATGCAGTNNSAAAGCGCTC




TCC (SEQ ID NO: 183)


 


276
R
ACTGCATGGCTGGAAGGTGCC (SEQ ID




NO: 184)


 


284
F
CAAAGCGCTCTCCAACCTCAAGNNSGTTGTCGAC




TCCT (SEQ ID NO: 185)


 


284
R
CTTGAGGTTGGAGAGCGCTTT (SEQ ID




NO: 186)


 


291
F
GGTTGTTGTCGACTCCTTCCGCNNSATCTACGGC




GTG (SEQ ID NO: 187)


 


291
R
GCGGAAGGAGTCGACAACAAC (SEQ ID




NO: 188)


 


292
F
TTGTCGACTCCTTCCGCTCCNNSTACGGCGTGAAC




AAGGGCATTC (SEQ ID NO: 189)


 


292
R
GGAGCGGAAGGAGTCGACAACAACC (SEQ ID




NO: 190)


 


294
F
ACTCCTTCCGCTCCATCTACNNSGTGAACAAGGGCA




TTCCTGCCG (SEQ ID NO: 191)


 


294
R
GTAGATGGAGCGGAAGGAGTCGACA (SEQ ID




NO: 192)


 


297
F
GCTCCATCTACGGCGTGAACNNSGGCATTCCTGCCGG




TGCTGCCG (SEQ ID NO: 193)


 


297
R
GTTCACGCCGTAGATGGAGCGGAAG (SEQ ID




NO: 194)


 


300
F
CTACGGCGTGAACAAGGGCATTNNSGCCGGTGCTGC




CG (SEQ ID NO: 195)


 


300
R
AATGCCCTTGTTCACGCCGTA (SEQ ID




NO: 196)


 


301
F
CGGCGTGAACAAGGGCATTCCTNNSGGTGCTGCCG




TC (SEQ ID NO: 197)


 


301
R
AGGAATGCCCTTGTTCACGCC (SEQ ID




NO: 198)


 


303
F
GAACAAGGGCATTCCTGCCGGTNNSGCCGTCGCCA




TT (SEQ ID NO: 199)


 


303
R
ACCGGCAGGAATGCCCTTGTT (SEQ ID




NO: 200)


 


309
F
GTGCTGCCGTCGCCATTGGCNNSTATGCAGAGGAT




GTGTACTACA (SEQ ID NO: 201)


 


309
R
GCCAATGGCGACGGCAGCACCGGCA (SEQ ID




NO: 202)


 


310
F
CTGCCGTCGCCATTGGCCGGNNSGCAGAGGATGTGT




ACTACAACG (SEQ ID NO: 203)


 


310
R
CCGGCCAATGGCGACGGCAGCACCG (SEQ ID




NO: 204)


 


311
F
TGCCGTCGCCATTGGCCGGTATNNSGAGGATGTG




TAC (SEQ ID NO: 205)


 


311
R
ATACCGGCCAATGGCGACGGC (SEQ ID




NO: 206)


 


313
F
CCATTGGCCGGTATGCAGAGNNSGTGTACTACAA




CGGCAACCCTT (SEQ ID NO: 207)


 


313
F
CCATTGGCCGGTATGCAGAGNNSGTGTACTACAAC




GGCAACCCTT (SEQ ID NO: 208)


 


313
R
CTCTGCATACCGGCCAATGGCGACG (SEQ ID




NO: 209)


 


313
R
CTCTGCATACCGGCCAATGGCGACG (SEQ ID




NO: 210)


 


314
F
TTGGCCGGTATGCAGAGGATNNSTACTACAACGGCAA




CCCTTGGT (SEQ ID NO: 211)


 


314
R
ATCCTCTGCATACCGGCCAATGGCG (SEQ ID




NO: 212)


 


315
F
GCCGGTATGCAGAGGATGTGNNSTACAACGGCAACC




CTTGGTATC (SEQ ID NO: 213)


 


315
R
CACATCCTCTGCATACCGGCCAATG (SEQ ID




NO: 214)


 


316
F
GGTATGCAGAGGATGTGTACNNSAACGGCAACCCTT




GGTATCTTG (SEQ ID NO: 215)


 


316
R
GTACACATCCTCTGCATACCGGCCAAT (SEQ ID




NO: 216)


 


317
F
ATGCAGAGGATGTGTACTACNNSGGCAACCCTTGGTA




TCTTGCTA (SEQ ID NO: 217)


 


317
F
ATGCAGAGGATGTGTACTACNNSGGCAACCCTTGGTA




TCTTGCTA (SEQ ID NO: 218)


 


317
R
GTAGTACACATCCTCTGCATACCGGC (SEQ ID




NO: 219)


 


317
R
GTAGTACACATCCTCTGCATACCGGC (SEQ ID




NO: 220)


 


321
F
TGTACTACAACGGCAACCCTNNSTATCTTGCTACATT




TGCTGCTG (SEQ ID NO: 221)


 


321
F
TGTACTACAACGGCAACCCTNNSTATCTTGCTACATT




TGCTGCTG (SEQ ID NO: 222)


 


321
R
AGGGTTGCCGTTGTAGTACACATCC (SEQ ID




NO: 223)


 


321
R
AGGGTTGCCGTTGTAGTACACATCC (SEQ ID




NO: 224)


 


338
F
GCAGCTGTACGATGCCATCTACNNSTGGAAGAAGA




CG (SEQ ID NO: 225)


 


338
R
GTAGATGGCATCGTACAGCTG (SEQ ID




NO: 226)


 


340
F
ACGATGCCATCTACGTCTGGNNSAAGACGGGCTCC




ATCACGGTGA (SEQ ID NO: 227)


 


340
R
CCAGACGTAGATGGCATCGTACAGC (SEQ ID




NO: 228)


 


341
F
ATGCCATCTACGTCTGGAAGNNSACGGGCTCCATCA




CGGTGACCG (SEQ ID NO: 229)


 


341
R
CTTCCAGACGTAGATGGCATCGTAC




AGC (SEQ ID NO: 230)


 


342
F
ATGCCATCTACGTCTGGAAGAAGNNSGGCTCCA




TCACG (SEQ ID NO: 231)


 


342
R
CTTCTTCCAGACGTAGATGGC (SEQ ID




NO: 232)


 


344
F
CTACGTCTGGAAGAAGACGGGCNNSATCACGG




TGACC (SEQ ID NO: 233)


 


344
R
GCCCGTCTTCTTCCAGACGTAG (SEQ ID




NO: 234)


 


346
F
CTGGAAGAAGACGGGCTCCATCNNSGTGACCGCC




ACCTC (SEQ ID NO: 235)


 


346
R
GATGGAGCCCGTCTTCTTCCA (SEQ ID




NO: 236)


 


349
F
GACGGGCTCCATCACGGTGACCNNSACCTCCCTG




GCC (SEQ ID NO: 237)


 


349
R
GGTCACCGTGATGGAGCCCGT (SEQ ID




NO: 238)


 


350
F
GCTCCATCACGGTGACCGCCNNSTCCCTGGCCTTC




TTCCAGGAGC (SEQ ID NO: 239)


 


350
R
GGCGGTCACCGTGATGGAGCCCGTC (SEQ ID




NO: 240)


 


356
F
CCACCTCCCTGGCCTTCTTCNNSGAGCTTGTTCCTGG




CGTGACGG (SEQ ID NO: 241)


 


356
R
GAAGAAGGCCAGGGAGGTGGCGGTC (SEQ ID




NO: 242)


 


359
F
CCTGGCCTTCTTCCAGGAGCTTNNSCCTGGCGTGA




CG (SEQ ID NO: 243)


 


359
R
AAGCTCCTGGAAGAAGGCCAG (SEQ ID




NO: 244)


 


361
F
CTTCTTCCAGGAGCTTGTTCCTNNSGTGACGGCC




GGG (SEQ ID NO: 245)


 


361
R
AGGAACAAGCTCCTGGAAGAA (SEQ ID




NO: 246)


 


363
F
AGGAGCTTGTTCCTGGCGTGNNSGCCGGGACCTA




CTCCAGCAGCT (SEQ ID NO: 247)


 


363
R
CACGCCAGGAACAAGCTCCTGGAAG (SEQ ID




NO: 248)


 


364
F
GGAGCTTGTTCCTGGCGTGACGNNSGGGACCTACT




CC (SEQ ID NO: 249)


 


364
R
CGTCACGCCAGGAACAAGCTC (SEQ ID




NO: 250)


 


368
F
GCGTGACGGCCGGGACCTACNNSAGCAGCTCTTCG




ACCTTTACCA (SEQ ID NO: 251)


 


368
R
GTAGGTCCCGGCCGTCACGCCAGGA (SEQ ID




NO: 252)


 


369
F
TGACGGCCGGGACCTACTCCNNSAGCTCTTCGACC




TTTACCAACA (SEQ ID NO: 253)


 


369
R
GGAGTAGGTCCCGGCCGTCACGCCA (SEQ ID




NO: 254)


 


375
F
CTCCAGCAGCTCTTCGACCTTTNNSAACATCATC




AACG (SEQ ID NO: 255)


 


375
R
AAAGGTCGAAGAGCTGCTGGA (SEQ ID




NO: 256)


 


376
F
GCAGCTCTTCGACCTTTACCNNSATCATCAACGCC




GTCTCGACAT (SEQ ID NO: 257)


 


376
R
GGTAAAGGTCGAAGAGCTGCTGGAG (SEQ ID




NO: 258)


 


379
F
TTCGACCTTTACCAACATCATCNNSGCCGTCTCG




ACA (SEQ ID NO: 259)


 


379
R
GATGATGTTGGTAAAGGTCGA (SEQ ID




NO: 260)


 


382
F
TACCAACATCATCAACGCCGTCNNSACATACGC




CGAT (SEQ ID NO: 261)


 


382
R
GACGGCGTTGATGATGTTGGT (SEQ ID




NO: 262)


 


390
F
GACATACGCCGATGGCTTCCTCNNSGAGGCTGCC




AAG (SEQ ID NO: 263)


 


390
R
GAGGAAGCCATCGGCGTATGT (SEQ ID




NO: 264)


 


391
F
ATACGCCGATGGCTTCCTCAGCNNSGCTGCCAAG




TAC (SEQ ID NO: 265)


 


391
R
GCTGAGGAAGCCATCGGCGTA (SEQ ID




NO: 266)


 


393
F
CGATGGCTTCCTCAGCGAGGCTNNSAAGTACGTC




CCC (SEQ ID NO: 267)


 


393
R
AGCCTCGCTGAGGAAGCCATC (SEQ ID




NO: 268)


 


394
F
TGGCTTCCTCAGCGAGGCTGCCNNSTACGTCCCC




GCC (SEQ ID NO: 269)


 


394
R
GGCAGCCTCGCTGAGGAAGCC (SEQ ID




NO: 270)


 


395
F
TCCTCAGCGAGGCTGCCAAGNNSGTCCCCGCCGACG




GTTCGCTGG (SEQ ID NO: 271)


 


395
R
CTTGGCAGCCTCGCTGAGGAAGCCA (SEQ ID




NO: 272)


 


398
F
AGGCTGCCAAGTACGTCCCCNNSGACGGTTCGCTGGC




CGAGCAGTT (SEQ ID NO: 273)


 


398
R
GGGGACGTACTTGGCAGCCTCGCTG (SEQ ID




NO: 274)


 


401
F
AGTACGTCCCCGCCGACGGTNNSCTGGCCGAGCAG




TTTGACCGCA (SEQ ID NO: 275)


 


401
R
ACCGTCGGCGGGGACGTACTTGGCAG (SEQ ID




NO: 276)


 


408
F
CGCTGGCCGAGCAGTTTGACNNSAACAGCGGCACTCC




GCTGTCTG (SEQ ID NO: 277)


 


408
R
GTCAAACTGCTCGGCCAGCGAACCG (SEQ ID




NO: 278)


 


409
F
TGGCCGAGCAGTTTGACCGCNNSAGCGGCACTCCGCT




GTCTGCGC (SEQ ID NO: 279)


 


409
R
GCGGTCAAACTGCTCGGCCAGCGAA (SEQ ID




NO: 280)


 


410
F
GGCCGAGCAGTTTGACCGCAACNNSGGCACTCCG




CTG (SEQ ID NO: 281)


 


410
R
GTTGCGGTCAAACTGCTCGGC (SEQ ID




NO: 282)


 


412
F
AGTTTGACCGCAACAGCGGCNNSCCGCTGTCTGCGC




TTCACCTGA (SEQ ID NO: 283)


 


412
R
GCCGCTGTTGCGGTCAAACTGCTCG (SEQ ID




NO: 284)


 


415
F
GCAACAGCGGCACTCCGCTGNNSGCGCTTCACCTGA




CGTGGTCGT (SEQ ID NO: 285)


 


415
R
CAGCGGAGTGCCGCTGTTGCGGTCA (SEQ ID




NO: 286)


 


417
F
CAGCGGCACTCCGCTGTCTGCGNNSCACCTGACGT




GGT (SEQ ID NO: 287)


 


417
R
CGCAGACAGCGGAGTGCCGCT (SEQ ID




NO: 288)


 


418
F
GCACTCCGCTGTCTGCGCTTNNSCTGACGTGGTCGT




ACGCCTCGT (SEQ ID NO: 289)


 


418
R
AAGCGCAGACAGCGGAGTGCCGCTG (SEQ ID




NO: 290)


 


421
F
TGTCTGCGCTTCACCTGACGNNSTCGTACGCCTCGTT




CTTGACAG (SEQ ID NO: 291)


 


421
R
CGTCAGGTGAAGCGCAGACAGCGGA (SEQ ID




NO: 292)


 


430
F
GTACGCCTCGTTCTTGACAGCCNNSGCCCGTCGG




GCT (SEQ ID NO: 293)


 


430
R
GGCTGTCAAGAACGAGGCGTA (SEQ ID




NO: 294)


 


431
F
CGCCTCGTTCTTGACAGCCACGNNSCGTCGGGC




TGGC (SEQ ID NO: 295)


 


431
R
CGTGGCTGTCAAGAACGAGGC (SEQ ID




NO: 296)


 


433
F
TCTTGACAGCCACGGCCCGTNNSGCTGGCATCGTG




CCCCCCTCGT (SEQ ID NO: 297)


 


433
R
ACGGGCCGTGGCTGTCAAGAACGAG (SEQ ID




NO: 298)


 


436
F
CCACGGCCCGTCGGGCTGGCNNSGTGCCCCCCTCGT




GGGCCAACA (SEQ ID NO: 299)


 


436
R
GCCAGCCCGACGGGCCGTGGCTGTC (SEQ ID




NO: 300)


 


442
F
TGGCATCGTGCCCCCCTCGTGGNNSAACAGCAG




CGCT (SEQ ID NO: 301)


 


442
R
CCACGAGGGGGGCACGATGCC (SEQ ID




NO: 302)


 


443
F
CATCGTGCCCCCCTCGTGGGCCNNSAGCAGCGCT




AGC (SEQ ID NO: 303)


 


443
R
GGCCCACGAGGGGGGCACGAT (SEQ ID




NO: 304)


 


444
F
CGTGCCCCCCTCGTGGGCCAACNNSAGCGCTAGC




ACG (SEQ ID NO: 305)


 


444
R
GTTGGCCCACGAGGGGGGCAC (SEQ ID




NO: 306)


 


448
F
GTGGGCCAACAGCAGCGCTAGCNNSATCCCCT




CGACG (SEQ ID NO: 307)


 


451
F
GCAGCGCTAGCACGATCCCCNNSACGTGCTC




CGGCGCGTCCGTGG (SEQ ID NO: 308)


 


451
R
GGGGATCGTGCTAGCGCTGCTGTTG (SEQ ID




NO: 309)


 


493
F
CTACACGCCCCTGCCCTGCGCGNNSCCAACCTCC




GTG (SEQ ID NO: 310)


 


493
R
CGCGCAGGGCAGGGGCGTGTA (SEQ ID




NO: 311)


 


494
F
CACGCCCCTGCCCTGCGCGACCNNSACCTCCGTG




GCC (SEQ ID NO: 312)


 


494
R
GGTCGCGCAGGGCAGGGGCGT (SEQ ID




NO: 313)


 


495
F
GCCCCTGCCCTGCGCGACCCCANNSTCCGTGGCC




GTC (SEQ ID NO: 314)


 


495
R
TGGGGTCGCGCAGGGCAGGGG (SEQ ID




NO: 315)


 


501
F
CCCAACCTCCGTGGCCGTCACCNNSCACGAGCTC




GTGT (SEQ ID NO: 316)


 


501
R
GGTGACGGCCACGGAGGTTGG (SEQ ID




NO: 317)


 


502
F
AACCTCCGTGGCCGTCACCTTCNNSGAGCTCGTG




TCG (SEQ ID NO: 318)


 


502
R
GAAGGTGACGGCCACGGAGGT (SEQ ID




NO: 319)


 


503
F
CTCCGTGGCCGTCACCTTCCACNNSCTCGTGTCGA




CACA (SEQ ID NO: 320)


 


503
R
GTGGAAGGTGACGGCCACGGA (SEQ ID




NO: 321)


 


508
F
CTTCCACGAGCTCGTGTCGACANNSTTTGGCCAG




ACG (SEQ ID NO: 322)


 


508
R
TGTCGACACGAGCTCGTGGAA (SEQ ID




NO: 323)


 


511
F
GCTCGTGTCGACACAGTTTGGCNNSACGGTCAAGGTG




(SEQ ID NO: 324)


 


511
R
GCCAAACTGTGTCGACACGAG (SEQ ID




NO: 325)


 


514
F
CACAGTTTGGCCAGACGGTCNNSGTGGCGGGCAACG




CCGCGGCCC (SEQ ID NO: 326)


 


514
R
GACCGTCTGGCCAAACTGTGTCGAC (SEQ ID




NO: 327)


 


517
F
TGGCCAGACGGTCAAGGTGGCGNNSAACGCCGCGGC




CCTGGG (SEQ ID NO: 328)


 


517
R
CGCCACCTTGACCGTCTGGCCAAACTG (SEQ ID




NO: 329)


 


518
F
CCAGACGGTCAAGGTGGCGGGCNNSGCCGCGGCCCT




GGGCAACT (SEQ ID NO: 330)


 


518
R
GCCCGCCACCTTGACCGTCTGGCCAAA (SEQ ID




NO: 331)


 


519
F
GACGGTCAAGGTGGCGGGCAACNNSGCGGCCCTGGG




CAACT (SEQ ID NO: 332)


 


519
R
GTTGCCCGCCACCTTGACCGTCTGGCC (SEQ ID




NO: 333)


 


520
F
GGTCAAGGTGGCGGGCAACGCCNNSGCCCTGGGCAA




CTGGA (SEQ ID NO: 334)


 


520
R
GGCGTTGCCCGCCACCTTGACCGTCTG (SEQ ID




NO: 335)


 


525
F
CAACGCCGCGGCCCTGGGCAACNNSAGCACGAGCGCC




GCCG (SEQ ID NO: 336)


 


525
R
GTTGCCCAGGGCCGCGGCGTTGCCCGC (SEQ ID




NO: 337)


 


527
F
CGCGGCCCTGGGCAACTGGAGCNNSAGCGCCGCCGT




GGCTC (SEQ ID NO: 338)


 


527
R
GCTCCAGTTGCCCAGGGCCGCGGCGTT (SEQ ID




NO: 339)


 


531
F
CAACTGGAGCACGAGCGCCGCCNNSGCTCTGGACGC




CGTCA (SEQ ID NO: 340)


 


531
R
GGCGGCGCTCGTGCTCCAGTTGCCCAG (SEQ ID




NO: 341)


 


533
F
GAGCACGAGCGCCGCCGTGGCTNNSGACGCCGTCAAC




TATGC (SEQ ID NO: 342)


 


533
R
AGCCACGGCGGCGCTCGTGCTCCAGTT (SEQ ID




NO: 343)


 


535
F
GAGCGCCGCCGTGGCTCTGGACNNSGTCAACTATGCC




GATA (SEQ ID NO: 344)


 


535
R
GTCCAGAGCCACGGCGGCGCTCGTGCT (SEQ ID




NO: 345)


 


536
F
CGCCGCCGTGGCTCTGGACGCCNNSAACTATGCCGAT




AACC (SEQ ID NO: 346)


 


536
R
GGCGTCCAGAGCCACGGCGGCGCTCGT (SEQ ID




NO: 347)


 


537
F
CGCCGTGGCTCTGGACGCCGTCNNSTATGCCGAT




AAC (SEQ ID NO: 348)


 


537
F
CGCCGTGGCTCTGGACGCCGTCNNSTATGCCGATAAC




CACCCC (SEQ ID NO: 349)


 


537
R
GACGGCGTCCAGAGCCACGGCGGCGCT (SEQ ID




NO: 350)


 


537
R
GACGGCGTCCAGAGCCACGGCGGCGCT (SEQ ID




NO: 351)


 


538
F
CGTGGCTCTGGACGCCGTCAACNNSGCCGATAACCAC




CCCC (SEQ ID NO: 352)


 


538
R
GTTGACGGCGTCCAGAGCCACGGCGGCG (SEQ ID




NO: 353)


 


539
F
GGCTCTGGACGCCGTCAACTATNNSGATAACCACCCC




CTGT (SEQ ID NO: 354)


 


539
R
ATAGTTGACGGCGTCCAGAGCCACGGC (SEQ ID




NO: 355)


 


540
F
TCTGGACGCCGTCAACTATGCCNNSAACCACCCCCTG




TGGATT (SEQ ID NO: 356)


 


540
R
GGCATAGTTGACGGCGTCCAGAGCCAC (SEQ ID




NO: 357)


 


541
F
GGACGCCGTCAACTATGCCGATNNSCACCCCCTGTGGA




TTGGG (SEQ ID NO: 358)


 


541
R
ATCGGCATAGTTGACGGCGTCCAGAGC (SEQ ID




NO: 359)


 


545
F
CTATGCCGATAACCACCCCCTGNNSATTGGGACGGTC




AACCTC (SEQ ID NO: 360)


 


545
R
CAGGGGGTGGTTATCGGCATAGTTGAC (SEQ ID




NO: 361)


 


546
F
TGCCGATAACCACCCCCTGTGGNNSGGGACGGTCAA




CCTCGAG (SEQ ID NO: 362)


 


546
R
CCACAGGGGGTGGTTATCGGCATAGTT (SEQ ID




NO: 363)


 


547
F
CGATAACCACCCCCTGTGGATTNNSACGGTCAACCTC




GAGGCT (SEQ ID NO: 364)


 


547
R
AATCCACAGGGGGTGGTTATCGGCATA (SEQ ID




NO: 365)


 


549
F
CCACCCCCTGTGGATTGGGACGNNSAACCTCGAGGC




TGGAGAC (SEQ ID NO: 366)


 


549
R
CGTCCCAATCCACAGGGGGTGGTTATC (SEQ ID




NO: 367)


 


551
F
CCTGTGGATTGGGACGGTCAACNNSGAGGCTGGAGA




CGTCGTG (SEQ ID NO: 368)


 


551
R
GTTGACCGTCCCAATCCACAGGGGGTG (SEQ ID




NO: 369)


 


561
F
TGGAGACGTCGTGGAGTACAAGNNSATCAATGTGGG




CCAAGAT (SEQ ID NO: 370)


 


561
R
CTTGTACTCCACGACGTCTCCAGCCTC (SEQ ID




NO: 371)


 


563
F
CGTCGTGGAGTACAAGTACATCNNSGTGGGCCAAG




ATGGCTCC (SEQ ID NO: 372)


 


563
R
GATGTACTTGTACTCCACGACGTCTCC (SEQ ID




NO: 373)


 


567
F
CAAGTACATCAATGTGGGCCAANNSGGCTCCGTGAC




CTGGGAG (SEQ ID NO: 374)


 


567
R
TTGGCCCACATTGATGTACTTGTACTC (SEQ ID




NO: 375)


 


569
F
CATCAATGTGGGCCAAGATGGCNNSGTGACCTGGGA




GAGTGAT (SEQ ID NO: 376)


 


569
R
GCCATCTTGGCCCACATTGATGTACTTG (SEQ ID




NO: 377)


 


577
F
CGTGACCTGGGAGAGTGATCCCNNSCACACTTACAC




GGTTCCT (SEQ ID NO: 378)


 


577
R
GGGATCACTCTCCCAGGTCACGGAGCC (SEQ ID




NO: 379)


 


579
F
CTGGGAGAGTGATCCCAACCACNNSTACACGGTTCC




TGCGGTG (SEQ ID NO: 380)


 


579
R
GTGGTTGGGATCACTCTCCCAGGTCAC (SEQ ID




NO: 381)


 


583
F
TCCCAACCACACTTACACGGTTNNSGCGGTGGCTTG




TGTGACG (SEQ ID NO: 382)


 


583
R
AACCGTGTAAGTGTGGTTGGGATCACT (SEQ ID




NO: 383)









Example 2
Transformation of TrGA SELs into Trichoderma reesei 
The SELs were transformed into T. reesei using the PEG protoplast method. The E. coli clones of the SELs confirmed by sequence analysis were grown overnight at 37° C. in deep well microtiter plates (Greiner Art. No. 780271) containing 1200 μl of 2×YT medium with ampicillin (100 μg/ml) and kanamycin (50 μg/ml). Plasmid DNAs were isolated from the cultures using CHEMAGIC® Plasmid Mini Kit (Chemagen—Biopolymer Technologie AG, Baesweiler, Germany) and were transformed individually into a T. reesei host strain derived from RL-P37 bearing four gene deletions (Δcbh1, Δcbh2, Δegl1, Δegl2, i.e., “quad-deleted;” see U.S. Pat. No. 5,847,276, WO 92/06184, and WO 05/001036) using the PEG-Protoplast method (Penttilä et al. (1987) Gene 61:155-164) with the following modifications.
For protoplast preparation, spores were grown for 16-24 hours at 24° C. in Trichoderma Minimal Medium (MM) (20 g/L glucose, 15 g/L KH2PO4, pH 4.5, 5 g/L (NH4)2SO4, 0.6 g/L MgSO4.7H2O, 0.6 g/L CaCl2.2H2O, 1 ml of 1000× T. reesei Trace elements solution {5 g/L FeSO4.7H2O, 1.4 g/L ZnSO4.7H2O, 1.6 g/L MnSO4.H2O, 3.7 g/L CoCl2.6H2O}) with shaking at 150 rpm. Germinating spores were harvested by centrifugation and treated with 15 mg/ml of β-D-glucanase-G (Interspex—Art. No. 0439-1) solution to lyse the fungal cell walls. Further preparation of protoplasts was performed by a standard method, as described by Penttila et al. (1987 supra).
The transformation method was scaled down 10 fold. In general, transformation mixtures containing up to 600 ng of DNA and 1-5×105 protoplasts in a total volume of 25 μl were treated with 200 ml of 25% PEG solution, diluted with 2 volumes of 1.2 M sorbitol solution, mixed with 3% selective top agarose MM with acetamide (the same Minimal Medium as mentioned above but (NH4)2SO4 was substituted with 20 mM acetamide) and poured onto 2% selective agarose with acetamide either in 24 well microtiter plates or in a 20×20 cm Q-tray divided in 48 wells. The plates were incubated at 28° C. for 5 to 8 days. Spores from the total population of transformants regenerated on each individual well were harvested from the plates using a solution of 0.85% NaCl, 0.015% Tween 80. Spore suspensions were used to inoculate fermentations in 96 wells MTPs. In the case of 24 well MTPs, an additional plating step on a fresh 24 well MTP with selective acetamide MM was introduced in order to enrich the spore numbers.
Example 3
Fermentation of T. reesei Transformants Expressing TrGA Variants in a MTP Format
The transformants were fermented and the supernatants containing the expressed variant TrGA proteins were tested for various properties. In brief, 96-well filter plates (Corning Art. No. 3505) containing in each well 200 μl of LD-GSM medium (5.0 g/L (NH4)2SO4, 33 g/L 1,4-Piperazinebis(propanesulfonic acid), pH 5.5, 9.0 g/L Casamino acids, 1.0 g/L KH2PO4, 1.0 g/L CaCl2.2H2O, 1.0 g/L MgSO4.7H2O, 2.5 ml/L of 1000× T. reesei trace elements, 20 g/L Glucose, 10 g/L Sophorose) were inoculated in quadruplicate with spore suspensions of T. reesei transformants expressing TrGA variants (more than 104 sporesper well). The plates were incubated at 28° C. with 230 rpm shaking and 80% humidity for 6 days. Culture supernatants were harvested by vacuum filtration. The supernatants were used in different assays for screening of variants with improved properties.
Example 4
Preparation of the Whole Broth Samples from GA-Producing Transformants
TrGA producing transformants were initially pre-grown in 250 ml shake flasks containing 30 ml of ProFlo medium. Proflo medium contained: 30 g/L α-lactose, 6.5 g/L (NH4)2SO4, 2 g/L KH2PO4, 0.3 g/L MgSO4.7H2O, 0.2 g/L CaCl2.2H2O, 1 ml/L 1000× trace element salt solution as mentioned above, 2 ml/L 10% Tween 80, 22.5 g/L ProFlo cottonseed flour (Traders protein, Memphis, Tenn.), 0.72 g/L CaCO3. After two days of growth at 28° C. and 140 rpm, 10% of the Proflo culture was transferred into a 250 ml shake flask containing 30 ml of Lactose Defined Medium. The composition of the Lactose Defined Medium was as follows: 5 g/L (NH4)2SO4, 33 g/L 1,4-Piperazinebis (propanesulfonic acid) buffer, pH 5.5, 9 g/L casamino acids, 4.5 g/L KH2PO4, 1.0 g/L MgSO4.7H2O, 5 ml/L Mazu DF60-P antifoam (Mazur Chemicals, IL), 1 ml/L of 1000× trace element solution. 40 ml/L of 40% (w/v) lactose solution was added to the medium after sterilization. Shake flasks with the Lactose Defined Medium were incubated at 28° C., 140 rpm for 4-5 days.
Mycelium was removed from the culture samples by centrifugation and the supernatant was analyzed for total protein content (BCA Protein Assay Kit, Pierce Cat. No. 23225) and GA activity, as described above in the Assays and Methods section.
The protein profile of the whole broth samples was determined by SDS-PAGE electrophoresis. Samples of the culture supernatant were mixed with an equal volume of 2× sample loading buffer with reducing agent and separated on NUPAGE® Novex 10% Bis-Tris Gel with MES SDS Running Buffer (Invitrogen, Carlsbad, Calif., USA). Polypeptide bands were visualized in the SDS gel with SIMPLYBLUE SafeStain (Invitrogen, Carlsbad, Calif., USA).
Example 5
Thermal Stability of the Variants
The thermal stability was measured according to above assay “Thermal stability assay 2”.
The parent molecule under the conditions described had a residual activity of 87.2%, Table 3 shows the residual activity for the variants, which were selected from an initial screen for fermentation in large scale and further analysis. The material used was crude fermentation broth from shake flasks. Residual activity was calculated on basis of GAU activity before and after 120 min incubation at 63° C. in 0.1M citrate buffer pH 5.4, containing 15% glucose.







TABLE 3







Thermostability for selected TrGA variants, shown as residual


activity after incubation for 120 min at 63° C. in


0.1M citrate buffer pH 5.4, containing 15% glucose.










Variant
Residual activity













D44R/N61I/A539R (BRW
84.2



11)




L417R/A431L/A539R
88.5



(VAR16)




D44R/A539R (BRW 1)
99.0



N61I/L417V/A431L/A539R
86.2



(VAR3)




I43R/L417V/A431L/A539R
89.8



(VAR13)




BRW 2
92.4



I43Q/D44C/N61I/L417V/
86.6



E503A/Q511H/A539R (ALL3)




I43Q/D44C/L417V/E503A/
90.1



Q511H (ALL1)




I43R/N61I/L417R/E503A/
84.6



Q511H/A539R (ALL8)




I43Q/N61I/T430M/Q511H/
83.0



A539R (RB19)




I43Q/N61I/T430A/A431L/
82.3



Q511H/A539R (RB10)




Diazyme X4 (AnGA reference
72.3



product)




Diazyme TR8 (TrGA reference
87.2



product)




I43R/G73F/T430A (C2
88.3



Negative Control)




I43Q/L417V/Q511H (RB13
74.1



Negative Control)









Example 6
Determination of Isomaltose Synthesis and Starch Hydrolysis and Ratio Thereof
Variants were tested according to above assays: “Starch hydrolysis activity” and “Determination of maltose and isomaltose synthesis by TLC”. The IS/SH ratio was calculated from the results of these analysis as described. Table 4 summarises the data for the variants selected for fermentation in large scale and further analysis. The material used was crude fermentation broth from shake flasks.







TABLE 4







Isomaltose synthesis activity (IS), starch hydrolysis


activity (SH) and IS/SH ratio of selected TrGA variants











Variant
Iso-

Iso-



(Parent
maltose
Starch
maltose



glucaoamylase
synthesis
hydro-
synthesis



TrGA, SEQ ID
M/min
lysis
Relative to
(IS/SH)


No: 2)
*10E−05
M/min
DiazymeX4
*10E−04














D44R/N61I/A539R
2.7
0.078
1.1
3.4


(BRW 11)






L417R/A431L/A539R
3.4
0.078
1.4
4.4


(VAR16)






D44R/A539R (BRW 1)
3.2
0.071
1.4
4.5


N61I/L417V/A431L/
4.3
0.089
1.5
4.8


A539R (VAR3)






I43R/L417V/A431L/
4.1
0.082
1.6
5.0


A539R (VAR13)






BRW 2
3.9
0.079
1.6
5.0


I43Q/D44C/N61I/
3.8
0.074
1.6
5.1


L417V/E503A/Q511H/






A539R (ALL3)






I43Q/D44C/L417V/
3.7
0.072
1.6
5.2


E503A/Q511H (ALL1)






I43R/N61I/L417R/
4.3
0.079
1.7
5.4


E503A/Q511H/A539R






(ALL8)






I43Q/N61I/T430M/
4.7
0.085
1.8
5.6


Q511H/A539R (RB19)






I43Q/N61I/T430A/
5.3
0.089
1.8
5.9


A431L/Q511H/A539R






(RB10)






Diazyme X4 (AnGA


1.0
3.2


reference product)






Diazyme TR8 (TrGA


2.5
7.9


reference product)






I43R/G73F/T430A (C2
8.7
0.0789
5.0
16


Negative Control)






I43Q/L417V/Q511H
8.1
0.0508
3.4
11


(RB13 Negative






Control)









Example 7
Brew Analysis with Determination of Real Degree of Fermentation (RDF)
All the variants shown in table 3 and 4 were grown in fermentors and GA enzyme was collected and purified (as described above under “Purification of TrGA variants”). The purified enzymes were reanalysed for IS/SH ratio as described above in Example 6 and thermostability was measured as described in Example 5. Brew analysis with determination of RDF value was carried out on the four variants which showed the best combination of IS/SH ratio and thermostability (Brew11, Brew1, Var16 and Var13) as described above under “Brew analysis with determination of real degree of fermentation (RDF)”. RDF values are listed in Table 5.







TABLE 5







RDF values of selected purified TrGA-variants,


purified wild type TrGA and purified AnGA.


Below values were obtained using the above


described “Pure malt brew analysis”-method.











Purified GA
RDF
STDEV














AnGA (purified from DIAZYME ®
82.5
0.064



X4)





TrGA (purified from DIAZYME ®
82.0
0.028



TR8)





D44R/N61I/A539R (BRW 11)
82.50
0.005



D44R/A539R (BRW 1)
82.33
0.050



L417R/A431L/A539R (VAR16)
81.93
0.113



I43R/L417V/A431L/A539R
81.86
0.085



(VAR13)









Example 8
Construction and Characterization of Combinatorial Variants
Based on data a selected set of variants with single substitutions were further characterized. These variants have single substitution at positions: 43, 44, 61, 73, 294, 417, 430, 431, 503, 511, 535, 539, and 563. Among these sites, 43, 44, and 294 were identified in a previous screening experiment in Schizosaccharomyces pombe. See WO 08/045,489, which is incorporated herein by reference. Variants were purified from large-scale fermentation, and PIs of thermal stability and specific activities were determined. Specifically, specific activities were determined using various substrates, including DP7, cornstarch, and liquefact. The results are shown in Table 6.







TABLE 6







PIs of a selected set of single site variants, each


of which is obtained from a 500 ml fermentation.














P.I.
P.I.
P.I.
P.I.




DP7-
CornStarch-
Thermal
Liquefact-



Variants
FPLC
FPLC
Stability
FPLC
















N61I
1.16
1.35
1.00
1.66



A431L
1.15
1.38
1.18
1.51



L417V
1.18
1.32
1.02
1.40



A431Q
1.06
1.20
0.92
1.24



G294C
1.01
0.84
0.94
1.23



N563K
1.07
1.12
1.97
1.15



Q511H
1.05
1.09
1.52
1.13



T430M
1.05
1.15
0.89
1.09



E503A
1.08
1.16
1.40
1.09



I43Q
1.11
1.24
0.94
1.08



A539R
1.15
1.37
1.43
1.08



I43R
1.03
1.07
1.41
1.07



L417R
1.23
1.27
1.51
1.04



T430A
1.13
1.35
1.23
1.04



G73F
1.06
1.06
1.45
1.03



D44R
0.97
1.06
1.46
0.98



N563I
1.09
1.22
2.06
0.92



D44C
0.80
0.82
0.96
0.91



E503V
1.17
1.07
1.66
0.88



A535R
1.09
1.44
1.47
0.85









Additionally, combinatorial variants were constructed using the PCR method with substitutions among: 43, 44, 61, 73, 294, 417, 430, 431, 503, 511, 535, 539, and 563. Briefly, the combinatorial variants were constructed by using plasmid pDONR-TrGA (FIG. 2) as the backbone. The methodology to construct combinatorial variants is based on the Gateway technology (Invitrogen, Carlsbad, Calif.). The primers used to create the combinatorial variants are shown in Tables 2 and 7. The following synthetic construct approach was chosen for the construction of all combinatorial variants.
CTCTCT [XbaI site] [MF] GAGAGGGG [attB1] [GAP combinatorial variant][attB2 sites] CCCCAGAG [MR][HindIII] AGAGAG
This construct was treated with restriction enzymes Xba-I and HindIII. The digested fragments were ligated into Xba-I/HindIII treated pBC (a pUC19 derived vector). The ligation mixture was transformed to E. coli DH10B (Invitrogen, Carlsbad, Calif.) and plated onto selective agar supplemented with 100 μg/ml ampicillin. The plates were incubated for 16 h at 37° C. Colonies from the selective plates were isolated and inoculated into selective liquid medium. After 16 h incubation at 37° C. and 250 rpm the plasmids were isolated using a standard plasmid isolation kit and combined with pDONR 2.21 (Invitrogen, Carlsbad, Calif.) to create a Gateway entry vector with the specific combinatorial variants. The reaction mixture was transformed into E. coli Max efficiency DH5α (Invitrogen, Carlsbad, Calif.) and plated on selective agar (2×TY supplemented with 50 μg kanamycin/ml). After overnight incubation at 37° C., single colonies were picked for sequence analysis (BaseClear B.V., Leiden, Netherlands). The combinatorial variants were subcloned in pTrexTrTel and expressed in a T. reesei host strain as described in WO 06/060062.







TABLE 7







Primers used to construct combinatorial variants











SEQ ID


Primer
DNA sequence
NO:












Var1 Basic-1
GAGAGAGTGCGGGCCTCTTCGCTATTTCTAGA
391


 


Var1 Basic-2
CAAAATAAAATCATTATTTGTCTAGAAATAGCGAAGAGGC
392


 


Var1 Basic-3
CAAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGT
393


 


Var1 Basic-4
TTGCTCATCAATGTGTTGCAACGAACAGGTCACTATCAGT
394


 


Var1 Basic-5
TGCAACACATTGATGAGCAATGCTTTTTTATAATGCCAAC
395


 


Var1 Basic-6
AGCCTGCTTTTTTGTACAAAGTTGGCATTATAAAAAAGCA
396


 


Var1 Basic-7
TTTGTACAAAAAAGCAGGCTATGCACGTCCTGTCGACTGC
397


 


Var1 Basic-8
CAACGGAGCCGAGCAGCACCGCAGTCGACAGGACGTGCAT
398


 


Var1 Basic-9
GGTGCTGCTCGGCTCCGTTGCCGTTCAAAAGGTCCTGGGA
399


 


Var1 Basic-10
AGACCGCTTGATCCTGGTCTTCCCAGGACCTTTTGAACGG
400


 


Var1 Basic-11
AGACCAGGATCAAGCGGTCTGTCCGACGTCACCAAGAGGT
401


 


Var1 Basic-12
GCTGATGAAGTCGTCAACAGACCTCTTGGTGACGTCGGAC
402


 


Var1 Basic-13
CTGTTGACGACTTCATCAGCACCGAGACGCCTATTGCACT
403


 


Var1 Basic-14
CATTGCAAAGAAGATTGTTCAGTGCAATAGGCGTCTCGGT
404


 


Var1 Basic-15
GAACAATCTTCTTTGCAATGTTGGTCCTGATGGATGCCGT
405


 


Var1 Basic-16
CCAGCTGATGTGCCGAATGCACGGCATCCATCAGGACCAA
406


 


Var1 Basic-17
GCATTCGGCACATCAGCTGGTGCGGTGATTGCATCTCCCA
407


 


Var1 Basic-18
GTAGTCCGGGTCAATTGTGCTGGGAGATGCAATCACCGCA
408


 


Var1 Basic-19
GCACAATTGACCCGGACTACTATTACATGTGGACGCGAGA
409


 


Var1 Basic-20
TCTTGAAGACAAGAGCGCTATCTCGCGTCCACATGTAATA
410


 


Var1 Basic-21
TAGCGCTCTTGTCTTCAAGAACCTCATCGACCGCTTCACC
411


 


Var1 Basic-22
AGGCCCGCATCGTACGTTTCGGTGAAGCGGTCGATGAGGT
412


 


Var1 Basic-23
GAAACGTACGATGCGGGCCTGCAGCGCCGCATCGAGCAGT
413


 


Var1 Basic-24
AGTGACCTGGGCAGTAATGTACTGCTCGATGCGGCGCTGC
414


 


Var1 Basic-25
ACATTACTGCCCAGGTCACTCTCCAGGGCCTCTCTAACCC
415


 


Var1 Basic-26
CGTCCGCGAGGGAGCCCGAGGGGTTAGAGAGGCCCTGGAG
416


 


Var1 Basic-27
CTCGGGCTCCCTCGCGGACGGCTCTGGTCTCGGCGAGCCC
417


 


Var1 Basic-28
TTCAGGGTCAACTCAAACTTGGGCTCGCCGAGACCAGAGC
418


 


Var1 Basic-29
AAGTTTGAGTTGACCCTGAAGCCTTTCACCGGCAACTGGG
419


 


Var1 Basic-30
GCCATCCCGCTGCGGTCGACCCCAGTTGCCGGTGAAAGGC
420


 


Var1 Basic-31
GTCGACCGCAGCGGGATGGCCCAGCTCTGCGAGCCATTGC
421


 


Var1 Basic-32
ACTTTGAGTATCCAATCAAGGCAATGGCTCGCAGAGCTGG
422


 


Var1 Basic-33
CTTGATTGGATACTCAAAGTGGCTCATCAACAACAACTAT
423


 


Var1 Basic-34
ACGTTGGACACAGTCGACTGATAGTTGTTGTTGATGAGCC
424


 


Var1 Basic-35
CAGTCGACTGTGTCCAACGTCATCTGGCCTATTGTGCGCA
425


 


Var1 Basic-36
GGCAACATAGTTGAGGTCGTTGCGCACAATAGGCCAGATG
426


 


Var1 Basic-37
ACGACCTCAACTATGTTGCCCAGTACTGGAACCAAACCGG
427


 


Var1 Basic-38
CTTCTTCCCAGAGGTCAAAGCCGGTTTGGTTCCAGTACTG
428


 


Var1 Basic-39
CTTTGACCTCTGGGAAGAAGTCAATGGGAGCTCATTCTTT
429


 


Var1 Basic-40
CGGTGCTGGTTGGCAACAGTAAAGAATGAGCTCCCATTGA
430


 


Var1 Basic-41
ACTGTTGCCAACCAGCACCGAGCACTTGTCGAGGGCGCCA
431


 


Var1 Basic-42
GCCAAGAGTGGCAGCAAGAGTGGCGCCCTCGACAAGTGCT
432


 


Var1 Basic-43
CTCTTGCTGCCACTCTTGGCCAGTCGGGAAGCGCTTATTC
433


 


Var1 Basic-44
AAACCTGGGGAGCAACAGATGAATAAGCGCTTCCCGACTG
434


 


Var1 Basic-45
ATCTGTTGCTCCCCAGGTTTTGTGCTTTCTCCAACGATTC
435


 


Var1 Basic-46
TATCCACCAGACGACACCCAGAATCGTTGGAGAAAGCACA
436


 


Var1 Basic-47
TGGGTGTCGTCTGGTGGATACGTCGACTCCAACATCAACA
437


 


Var1 Basic-48
GCCAGTCCTGCCCTCGTTGGTGTTGATGTTGGAGTCGACG
438


 


Var1 Basic-49
CCAACGAGGGCAGGACTGGCAAGGATGTCAACTCCGTCCT
439


 


Var1 Basic-50
CGAAGGTGTGGATGGAAGTCAGGACGGAGTTGACATCCTT
440


 


Var1 Basic-51
GACTTCCATCCACACCTTCGATCCCAACCTTGGCTGTGAC
441


 


Var1 Basic-52
CATGGCTGGAAGGTGCCTGCGTCACAGCCAAGGTTGGGAT
442


 


Var1 Basic-53
GCAGGCACCTTCCAGCCATGCAGTGACAAAGCGCTCTCCA
443


 


Var1 Basic-54
GTCGACAACAACCTTGAGGTTGGAGAGCGCTTTGTCACTG
444


 


Var1 Basic-55
ACCTCAAGGTTGTTGTCGACTCCTTCCGCTCCATCTACGG
445


 


Var1 Basic-56
CAGGAATGCCCTTGTTCACGCCGTAGATGGAGCGGAAGGA
446


 


Var1 Basic-57
CGTGAACAAGGGCATTCCTGCCGGTGCTGCCGTCGCCATT
447


 


Var1 Basic-58
ACATCCTCTGCATACCGGCCAATGGCGACGGCAGCACCGG
448


 


Var1 Basic-59
GGCCGGTATGCAGAGGATGTGTACTACAACGGCAACCCTT
449


 


Var1 Basic-60
AGCAAATGTAGCAAGATACCAAGGGTTGCCGTTGTAGTAC
450


 


Var1 Basic-61
GGTATCTTGCTACATTTGCTGCTGCCGAGCAGCTGTACGA
451


 


Var1 Basic-62
TCTTCCAGACGTAGATGGCATCGTACAGCTGCTCGGCAGC
452


 


Var1 Basic-63
TGCCATCTACGTCTGGAAGAAGACGGGCTCCATCACGGTG
453


 


Var1 Basic-64
AAGGCCAGGGAGGTGGCGGTCACCGTGATGGAGCCCGTCT
454


 


Var1 Basic-65
ACCGCCACCTCCCTGGCCTTCTTCCAGGAGCTTGTTCCTG
455


 


Var1 Basic-66
GTAGGTCCCGGCCGTCACGCCAGGAACAAGCTCCTGGAAG
456


 


Var1 Basic-67
GCGTGACGGCCGGGACCTACTCCAGCAGCTCTTCGACCTT
457


 


Var1 Basic-68
CGGCGTTGATGATGTTGGTAAAGGTCGAAGAGCTGCTGGA
458


 


Var1 Basic-69
TACCAACATCATCAACGCCGTCTCGACATACGCCGATGGC
459


 


Var1 Basic-70
TTGGCAGCCTCGCTGAGGAAGCCATCGGCGTATGTCGAGA
460


 


Var1 Basic-71
TTCCTCAGCGAGGCTGCCAAGTACGTCCCCGCCGACGGTT
461


 


Var1 Basic-72
GTCAAACTGCTCGGCCAGCGAACCGTCGGCGGGGACGTAC
462


 


Var1 Basic-73
CGCTGGCCGAGCAGTTTGACCGCAACAGCGGCACTCCGCT
463


 


Var1 Basic-74
ACGTCAGGTGAACCGCAGACAGCGGAGTGCCGCTGTTGCG
464


 


Var1 Basic-75
GTCTGCGGTTCACCTGACGTGGTCGTACGCCTCGTTCTTG
465


 


Var1 Basic-76
GCCCGACGAAGCGTGGCTGTCAAGAACGAGGCGTACGACC
466


 


Var1 Basic-77
ACAGCCACGCTTCGTCGGGCTGGCATCGTGCCCCCCTCGT
467


 


Var1 Basic-78
GCTAGCGCTGCTGTTGGCCCACGAGGGGGGCACGATGCCA
468


 


Var1 Basic-79
GGGCCAACAGCAGCGCTAGCACGATCCCCTCGACGTGCTC
469


 


Var1 Basic-80
ATCCGACCACGGACGCGCCGGAGCACGTCGAGGGGATCGT
470


 


Var1 Basic-81
CGGCGCGTCCGTGGTCGGATCCTACTCGCGTCCCACCGCC
471


 


Var1 Basic-82
TGCGACGGAGGGAATGACGTGGCGGTGGGACGCGAGTAGG
472


 


Var1 Basic-83
ACGTCATTCCCTCCGTCGCAGACGCCCAAGCCTGGCGTGC
473


 


Var1 Basic-84
CGTGTAGGGAGTACCGGAAGGCACGCCAGGCTTGGGCGTC
474


 


Var1 Basic-85
CTTCCGGTACTCCCTACACGCCCCTGCCCTGCGCGACCCC
475


 


Var1 Basic-86
AGGTGACGGCCACGGAGGTTGGGGTCGCGCAGGGCAGGGG
476


 


Var1 Basic-87
AACCTCCGTGGCCGTCACCTTCCACGAGCTCGTGTCGACA
477


 


Var1 Basic-88
TTGACCGTCTGGCCAAACTGTGTCGACACGAGCTCGTGGA
478


 


Var1 Basic-89
CAGTTTGGCCAGACGGTCAAGGTGGCGGGCAACGCCGCGG
479


 


Var1 Basic-90
CGTGCTCCAGTTGCCCAGGGCCGCGGCGTTGCCCGCCACC
480


 


Var1 Basic-91
CCCTGGGCAACTGGAGCACGAGCGCCGCCGTGGCTCTGGA
481


 


Var1 Basic-92
TATCACGATAGTTGACGGCGTCCAGAGCCACGGCGGCGCT
482


 


Var1 Basic-93
CGCCGTCAACTATCGTGATAACCACCCCCTGTGGATTGGG
483


 


Var1 Basic-94
CCAGCCTCGAGGTTGACCGTCCCAATCCACAGGGGGTGGT
484


 


Var1 Basic-95
ACGGTCAACCTCGAGGCTGGAGACGTCGTGGAGTACAAGT
485


 


Var1 Basic-96
ATCTTGGCCCACATTGATGTACTTGTACTCCACGACGTCT
486


 


Var1 Basic-97
ACATCAATGTGGGCCAAGATGGCTCCGTGACCTGGGAGAG
487


 


Var1 Basic-98
TGTAAGTGTGGTTGGGATCACTCTCCCAGGTCACGGAGCC
488


 


Var1 Basic-99
TGATCCCAACCACACTTACACGGTTCCTGCGGTGGCTTGT
489


 


Var1 Basic-100
TCCTTGACAACCTGCGTCACACAAGCCACCGCAGGAACCG
490


 


Var1 Basic-101
GTGACGCAGGTTGTCAAGGAGGACACCTGGCAGTCGTAAA
491


 


Var1 Basic-102
CTTTGTACAAGAAAGCTGGGTTTACGACTGCCAGGTGTCC
492


 


Var1 Basic-103
CCCAGCTTTCTTGTACAAAGTTGGCATTATAAGAAAGCAT
493


 


Var1 Basic-104
TTGCAACAAATTGATAAGCAATGCTTTCTTATAATGCCAA
494


 


Var1 Basic-105
TGCTTATCAATTTGTTGCAACGAACAGGTCACTATCAGTC
495


 


Var1 Basic-106
TCAAATAATGATTTTATTTTGACTGATAGTGACCTGTTCG
496


 


Var1 Basic-107
AAAATAAAATCATTATTTGAAGCTTAAGCCTGGGGTGCCT
497


 


Var1 Basic-108
AGAGAGTCATTAGGCACCCCAGGCTTAAGCT
498


 


Var2-18
GTAGTCCGGGTCTTGTGTGCTGGGAGATGCAATCACCGCA
499


 


Var2-19
GCACACAAGACCCGGACTACTATTACATGTGGACGCGAGA
500


 


Var3-21
TAGCGCTCTTGTCTTCAAGATTCTCATCGACCGCTTCACC
501


 


Var3-22
AGGCCCGCATCGTACGTTTCGGTGAAGCGGTCGATGAGAA
502


 


Var4-92
TATCACGATAGTTGACACGGTCCAGAGCCACGGCGGCGCT
503


 


Var4-93
CCGTGTCAACTATCGTGATAACCACCCCCTGTGGATTGGG
504


 


Var5-18
GTAGTCCGGGTCTTGTGTGCTGGGAGATGCAATCACCGCA
505


 


Var5-19
GCACACAAGACCCGGACTACTATTACATGTGGACGCGAGA
506


 


Var5-21
TAGCGCTCTTGTCTTCAAGATTCTCATCGACCGCTTCACC
507


 


Var5-22
AGGCCCGCATCGTACGTTTCGGTGAAGCGGTCGATGAGAA
508


 


Var6-21
TAGCGCTCTTGTCTTCAAGATTCTCATCGACCGCTTCACC
509


 


Var6-22
AGGCCCGCATCGTACGTTTCGGTGAAGCGGTCGATGAGAA
510


 


Var6-92
TATCACGATAGTTGACACGGTCCAGAGCCACGGCGGCGCT
511


 


Var6-93
CCGTGTCAACTATCGTGATAACCACCCCCTGTGGATTGGG
512


 


Var7-92
TATCACGATAGTTGACACGGTCCAGAGCCACGGCGGCGCT
513


 


Var7-93
CCGTGTCAACTATCGTGATAACCACCCCCTGTGGATTGGG
514


 


Var7-18
GTAGTCCGGGTCTTGTGTGCTGGGAGATGCAATCACCGCA
515


 


Var7-19
GCACACAAGACCCGGACTACTATTACATGTGGACGCGAGA
516


 


Var8-18
GTAGTCCGGGTCTTGTGTGCTGGGAGATGCAATCACCGCA
517


 


Var8-19
GCACACAAGACCCGGACTACTATTACATGTGGACGCGAGA
518


 


Var8-21
TAGCGCTCTTGTCTTCAAGATTCTCATCGACCGCTTCACC
519


 


Var8-22
AGGCCCGCATCGTACGTTTCGGTGAAGCGGTCGATGAGAA
520


 


Var8-92
TATCACGATAGTTGACACGGTCCAGAGCCACGGCGGCGCT
521


 


Var8-93
CCGTGTCAACTATCGTGATAACCACCCCCTGTGGATTGGG
522


 


Var9-18
GTAGTCCGGGTCTTGTGTGCTGGGAGATGCAATCACCGCA
523


 


Var9-19
GCACACAAGACCCGGACTACTATTACATGTGGACGCGAGA
524


 


Var9-21
TAGCGCTCTTGTCTTCAAGATTCTCATCGACCGCTTCACC
525


 


Var9-22
AGGCCCGCATCGTACGTTTCGGTGAAGCGGTCGATGAGAA
526


 


Var9-92
TATCACGATAGTTGACACGGTCCAGAGCCACGGCGGCGCT
527


 


Var9-93
CCGTGTCAACTATCGTGATAACCACCCCCTGTGGATTGGG
528


 


Var9-76
GCCCGACGAAGAGCGGCTGTCAAGAACGAGGCGTACGACC
529


 


Var9-77
ACAGCCGCTCTTCGTCGGGCTGGCATCGTGCCCCCCTCGT
530


 


Var10-76
GCCCGACGAAGAGCGGCTGTCAAGAACGAGGCGTACGACC
531


 


Var10-77
ACAGCCGCTCTTCGTCGGGCTGGCATCGTGCCCCCCTCGT
532


 


Var10-88
TTGACCGTATGGCCAAACTGTGTCGACACGAGCTCGTGGA
533


 


Var10-89
CAGTTTGGCCATACGGTCAAGGTGGCGGGCAACGCCGCGG
534


 


Var10-92
TATCACGATAGTTGACACGGTCCAGAGCCACGGCGGCGCT
535


 


Var10-93
CCGTGTCAACTATCGTGATAACCACCCCCTGTGGATTGGG
536


 


Var10-96
ATCTTGGCCCACAATGATGTACTTGTACTCCACGACGTCT
537


 


Var10-97
ACATCATTGTGGGCCAAGATGGCTCCGTGACCTGGGAGAG
538


 


Var10-18
GTAGTCCGGGTCTTGTGTGCTGGGAGATGCAATCACCGCA
539


 


Var10-19
GCACACAAGACCCGGACTACTATTACATGTGGACGCGAGA
540


 


Var11-76
GCCCGACGAAGAGCGGCTGTCAAGAACGAGGCGTACGACC
541


 


Var11-77
ACAGCCGCTCTTCGTCGGGCTGGCATCGTGCCCCCCTCGT
542


 


Var11-88
TTGACCGTATGGCCAAACTGTGTCGACACGAGCTCGTGGA
543


 


Var1l-89
CAGTTTGGCCATACGGTCAAGGTGGCGGGCAACGCCGCGG
544


 


Var1l-92
TATCACGATAGTTGACACGGTCCAGAGCCACGGCGGCGCT
545


 


Var1l-93
CCGTGTCAACTATCGTGATAACCACCCCCTGTGGATTGGG
546


 


Var1l-96
ATCTTGGCCCACAATGATGTACTTGTACTCCACGACGTCT
547


 


Var11-97
ACATCATTGTGGGCCAAGATGGCTCCGTGACCTGGGAGAG
548


 


Var11-21
TAGCGCTCTTGTCTTCAAGATTCTCATCGACCGCTTCACC
549


 


Var1l-22
AGGCCCGCATCGTACGTTTCGGTGAAGCGGTCGATGAGAA
550


 


Var12-76
GCCCGACGAAGAGCGGCTGTCAAGAACGAGGCGTACGACC
551


 


Var12-77
ACAGCCGCTCTTCGTCGGGCTGGCATCGTGCCCCCCTCGT
552


 


Var12-88
TTGACCGTATGGCCAAACTGTGTCGACACGAGCTCGTGGA
553


 


Var12-89
CAGTTTGGCCATACGGTCAAGGTGGCGGGCAACGCCGCGG
554


 


Var12-92
TATCACGATAGTTGACACGGTCCAGAGCCACGGCGGCGCT
555


 


Var12-93
CCGTGTCAACTATCGTGATAACCACCCCCTGTGGATTGGG
556


 


Var12-96
ATCTTGGCCCACAATGATGTACTTGTACTCCACGACGTCT
557


 


Var12-97
ACATCATTGTGGGCCAAGATGGCTCCGTGACCTGGGAGAG
558


 


Var12-18
GTAGTCCGGGTCTTGTGTGCTGGGAGATGCAATCACCGCA
559


 


Var12-19
GCACACAAGACCCGGACTACTATTACATGTGGACGCGAGA
560


 


Var12-21
TAGCGCTCTTGTCTTCAAGATTCTCATCGACCGCTTCACC
561


 


Var12-22
AGGCCCGCATCGTACGTTTCGGTGAAGCGGTCGATGAGAA
562


 


Var13-18
GTAGTCCGGGTCACGTGTGCTGGGAGATGCAATCACCGCA
563


 


Var13-19
GCACACGTGACCCGGACTACTATTACATGTGGACGCGAGA
564


 


Var14-18
GTAGTCCGGGTCACGTGTGCTGGGAGATGCAATCACCGCA
565


 


Var14-19
GCACACGTGACCCGGACTACTATTACATGTGGACGCGAGA
566


 


Var14-21
TAGCGCTCTTGTCTTCAAGATTCTCATCGACCGCTTCACC
567


 


Var14-22
AGGCCCGCATCGTACGTTTCGGTGAAGCGGTCGATGAGAA
568


 


Var15-18
GTAGTCCGGGTCACGTGTGCTGGGAGATGCAATCACCGCA
569


 


Var15-19
GCACACGTGACCCGGACTACTATTACATGTGGACGCGAGA
570


 


Var15-21
TAGCGCTCTTGTCTTCAAGATTCTCATCGACCGCTTCACC
571


 


Var15-22
AGGCCCGCATCGTACGTTTCGGTGAAGCGGTCGATGAGAA
572


 


Var15-92
TATCACGATAGTTGACACGGTCCAGAGCCACGGCGGCGCT
573


 


Var15-93
CCGTGTCAACTATCGTGATAACCACCCCCTGTGGATTGGG
574


 


Var15-76
GCCCGACGAAGAGCGGCTGTCAAGAACGAGGCGTACGACC
575


 


Var15-77
ACAGCCGCTCTTCGTCGGGCTGGCATCGTGCCCCCCTCGT
576


 


Var16-74
ACGTCAGGTGACGCGCAGACAGCGGAGTGCCGCTGTTGCG
577


 


Var16-75
GTCTGCGCGTCACCTGACGTGGTCGTACGCCTCGTTCTTG
578


 


Var17-74
ACGTCAGGTGACCCGCAGACAGCGGAGTGCCGCTG1TGCG
579


 


Var17-75
GTCTGCGGGTCACCTGACGTGGTCGTACGCCTCGTTCTTG
580


 


Var18-22
AGAAACGCATCGTACGTTTCGGTGAAGCGGTCGATGAGGT
581


 


Var18-23
GAAACGTACGATGCGTTTCTGCAGCGCCGCATCGAGCAGT
582


 


Var18-74
ACGTCAGGTGACGCGCAGACAGCGGAGTGCCGCTGTTGCG
583


 


Var18-75
GTCTGCGCGTCACCTGACGTGGTCGTACGCCTCGTTCTTG
584


 


Var18-76
GCCCGACGGGCCGTGGCTGTCAAGAACGAGGCGTACGACC
585


 


Var18-77
ACAGCCACGGCCCGTCGGGCTGGCATCGTGCCCCCCTCGT
586


 


Var18-87
AACCTCCGTGGCCGTCACCTTCCACGTTCTCGTGTCGACA
587


 


Var18-88
TTGACCGTCTGGCCAAACTGTGTCGACACGAGAACGTGGA
588


 


Var18-96
ATCTTGGCCCACTTTGATGTACTTGTACTCCACGACGTCT
589


 


Var18-97
ACATCAAAGTGGGCCAAGATGGCTCCGTGACCTGGGAGAG
590


 


Var19-18
GTAGTCCGGGTCACGTGTGCTGGGAGATGCAATCACCGCA
591


 


Var19-19
GCACACGTGACCCGGACTACTATTACATGTGGACGCGAGA
592


 


Var19-22
AGAAACGCATCGTACGTTTCGGTGAAGCGGTCGATGAGGT
593


 


Var19-23
GAAACGTACGATGCGTTTCTGCAGCGCCGCATCGAGCAGT
594


 


Var19-74
ACGTCAGGTGACGCGCAGACAGCGGAGTGCCGCTGTTGCG
595


 


Var19-75
GTCTGCGCGTCACCTGACGTGGTCGTACGCCTCGTTCTTG
596


 


Var19-76
GCCCGACGGGCCGTGGCTGTCAAGAACGAGGCGTACGACC
597


 


Var19-77
ACAGCCACGGCCCGTCGGGCTGGCATCGTGCCCCCCTCGT
598


 


Var19-87
AACCTCCGTGGCCGTCACCTTCCACGTTCTCGTGTCGACA
599


 


Var19-88
TTGACCGTCTGGCCAAACTGTGTCGACACGAGAACGTGGA
600


 


Var19-96
ATCTTGGCCCACTTTGATGTACTTGTACTCCACGACGTCT
601


 


Var19-97
ACATCAAAGTGGGCCAAGATGGCTCCGTGACCTGGGAGAG
602


 


Var20-18
GTAGTCCGGGTCACGTGTGCTGGGAGATGCAATCACCGCA
603


 


Var20-19
GCACACGTGACCCGGACTACTATTACATGTGGACGCGAGA
604


 


Var20-22
AGAAACGCATCGTACGTTTCGGTGAAGCGGTCGATGAGGT
605


 


Var20-23
GAAACGTACGATGCGTTTCTGCAGCGCCGCATCGAGCAGT
606


 


Var20-74
ACGTCAGGTGAAGCGCAGACAGCGGAGTGCCGCTGTTGCG
607


 


Var20-75
GTCTGCGCTTCACCTGACGTGGTCGTACGCCTCGTTCTTG
608


 


Var20-76
GCCCGACGGGCCGTGGCTGTCAAGAACGAGGCGTACGACC
609


 


Var20-77
ACAGCCACGGCCCGTCGGGCTGGCATCGTGCCCCCCTCGT
610


 


Var20-87
AACCTCCGTGGCCGTCACCTTCCACGTTCTCGTGTCGACA
611


 


Var20-88
TTGACCGTATGGCCAAACTGTGTCGACACGAGAACGTGGA
612


 


Var20-89
CAGTTTGGCCATACGGTCAAGGTGGCGGGCAACGCCGCGG
613


 


Var20-93
TATCGGCATAGTTGACGGCGTCCAGAGCCACGGCGGCGCT
614


 


Var20-94
CGCCGTCAACTATGCCGATAACCACCCCCTGTGGATTGGG
615


 


Var20-96
ATCTTGGCCCACTTTGATGTACTTGTACTCCACGACGTCT
616


 


Var20-97
ACATCAAAGTGGGCCAAGATGGCTCCGTGACCTGGGAGAG
617


 


GAV Basic-1
ACAAGTTTGTACAAAAAAGCAGGCT
618


 


GAV Basic-2
GCAGTCGACAGGACGTGCATAGCCTGCTTTTTTGTACAAA
619


 


GAV Basic-3
ATGCACGTCCTGTCGACTGCGGTGCTGCTCGGCTCCGTTG
620


 


GAV Basic-4
TCCCAGGACCTTTTGAACGGCAACGGAGCCGAGCAGCACC
621


 


GAV Basic-5
CCGTTCAAAAGGTCCTGGGAAGACCAGGATCAAGCGGTCT
622


 


GAV Basic-6
ACCTCTTGGTGACGTCGGACAGACCGCTTGATCCTGGTCT
623


 


GAV Basic-7
GTCCGACGTCACCAAGAGGTCTGTTGACGACTTCATCAGC
624


 


GAV Basic-8
AGTGCAATAGGCGTCTCGGTGCTGATGAAGTCGTCAACAG
625


 


GAV Basic-9
ACCGAGACGCCTATTGCACTGAACAATCTTCTTTGCAATG
626


 


GAV Basic-10
ACGGCATCCATCAGGACCAACATTGCAAAGAAGATTGTTC
627


 


GAV Basic-11
TTGGTCCTGATGGATGCCGTGCATTCGGCACATCAGCTGG
628


 


GAV Basic-12
TGGGAGATGCAATCACCGCACCAGCTGATGTGCCGAATGC
629


 


GAV Basic-13
TGCGGTGATTGCATCTCCCAGCACAATTGACCCGGACTAC
630


 


GAV Basic-14
TCTCGCGTCCACATGTAATAGTAGTCCGGGTCAATTGTGC
631


 


GAV Basic-15
TATTACATGTGGACGCGAGATAGCGCTCTTGTCTTCAAGA
632


 


GAV Basic-16
GGTGAAGCGGTCGATGAGGTTCTTGAAGACAAGAGCGCTA
633


 


GAV Basic-17
ACCTCATCGACCGCTTCACCGAAACGTACGATGCGGGCCT
634


 


GAV Basic-18
ACTGCTCGATGCGGCGCTGCAGGCCCGCATCGTACGTTTC
635


 


GAV Basic-19
GCAGCGCCGCATCGAGCAGTACATTACTGCCCAGGTCACT
636


 


GAV Basic-20
GGGTTAGAGAGGCCCTGGAGAGTGACCTGGGCAGTAATGT
637


 


GAV Basic-21
CTCCAGGGCCTCTCTAACCCCTCGGGCTCCCTCGCGGACG
638


 


GAV Basic-22
GGGCTCGCCGAGACCAGAGCCGTCCGCGAGGGAGCCCGAG
639


 


GAV Basic-23
GCTCTGGTCTCGGCGAGCCCAAGTTTGAGTTGACCCTGAA
640


 


GAV Basic-24
CCCAGTTGCCGGTGAAAGGCTTCAGGGTCAACTCAAACTT
641


 


GAV Basic-25
GCCTTTCACCGGCAACTGGGGTCGACCGCAGCGGGATGGC
642


 


GAV Basic-26
GCAATGGCTCGCAGAGCTGGGCCATCCCGCTGCGGTCGAC
643


 


GAV Basic-27
CCAGCTCTGCGAGCCATTGCCTTGATTGGATACTCAAAGT
644


 


GAV Basic-28
ATAGTTGTTGTTGATGAGCCACTTTGAGTATCCAATCAAG
645


 


GAV Basic-29
GGCTCATCAACAACAACTATCAGTCGACTGTGTCCAACGT
646


 


GAV Basic-30
TGCGCACAATAGGCCAGATGACGTTGGACACAGTCGACTG
647


 


GAV Basic-31
CATCTGGCCTATTGTGCGCAACGACCTCAACTATGTTGCC
648


 


GAV Basic-32
CCGGTTTGGTTCCAGTACTGGGCAACATAGTTGAGGTCGT
649


 


GAV Basic-33
CAGTACTGGAACCAAACCGGCTTTGACCTCTGGGAAGAAG
650


 


GAV Basic-34
AAAGAATGAGCTCCCATTGACTTCTTCCCAGAGGTCAAAG
651


 


GAV Basic-35
TCAATGGGAGCTCATTCTTTACTGTTGCCAACCAGCACCG
652


 


GAV Basic-36
TGGCGCCCTCGACAAGTGCTCGGTGCTGGTTGGCAACAGT
653


 


GAV Basic-37
AGCACTTGTCGAGGGCGCCACTCTTGCTGCCACTCTTGGC
654


 


GAV Basic-38
GAATAAGCGCTTCCCGACTGGCCAAGAGTGGCAGCAAGAG
655


 


GAV Basic-39
CAGTCGGGAAGCGCTTATTCATCTGTTGCTCCCCAGGTTT
656


 


GAV Basic-40
GAATCGTTGGAGAAAGCACAAAACCTGGGGAGCAACAGAT
657


 


GAV Basic-41
TGTGCTTTCTCCAACGATTCTGGGTGTCGTCTGGTGGATA
658


 


GAV Basic-42
TGTTGATGTTGGAGTCGACGTATCCACCAGACGACACCCA
659


 


GAV Basic-43
CGTCGACTCCAACATCAACACCAACGAGGGCAGGACTGGC
660


 


GAV Basic-44
AGGACGGAGTTGACATCCTTGCCAGTCCTGCCCTCGTTGG
661


 


GAV Basic-45
AAGGATGTCAACTCCGTCCTGACTTCCATCCACACCTTCG
662


 


GAV Basic-46
GTCACAGCCAAGGTTGGGATCGAAGGTGTGGATGGAAGTC
663


 


GAV Basic-47
ATCCCAACCTTGGCTGTGACGCAGGCACCTTCCAGCCATG
664


 


GAV Basic-48
TGGAGAGCGCTTTGTCACTGCATGGCTGGAAGGTGCCTGC
665


 


GAV Basic-49
CAGTGACAAAGCGCTCTCCAACCTCAAGGTTGTTGTCGAC
666


 


GAV Basic-50
CCGTAGATGGAGCGGAAGGAGTCGACAACAACCTTGAGGT
667


 


GAV Basic-51
TCCTTCCGCTCCATCTACGGCGTGAACAAGGGCATTCCTG
668


 


GAV Basic-52
AATGGCGACGGCAGCACCGGCAGGAATGCCCTTGTTCACG
669


 


GAV Basic-53
CCGGTGCTGCCGTCGCCATTGGCCGGTATGCAGAGGATGT
670


 


GAV Basic-54
AAGGGTTGCCGTTGTAGTACACATCCTCTGCATACCGGCC
671


 


GAV Basic-55
GTACTACAACGGCAACCCTTGGTATCTTGCTACATTTGCT
672


 


GAV Basic-56
TCGTACAGCTGCTCGGCAGCAGCAAATGTAGCAAGATACC
673


 


GAV Basic-57
GCTGCCGAGCAGCTGTACGATGCCATCTACGTCTGGAAGA
674


 


GAV Basic-58
CACCGTGATGGAGCCCGTCTTCTTCCAGACGTAGATGGCA
675


 


GAV Basic-59
AGACGGGCTCCATCACGGTGACCGCCACCTCCCTGGCCTT
676


 


GAV Basic-60
CAGGAACAAGCTCCTGGAAGAAGGCCAGGGAGGTGGCGGT
677


 


GAV Basic-61
CTTCCAGGAGCTTGTTCCTGGCGTGACGGCCGGGACCTAC
678


 


GAV Basic-62
AAGGTCGAAGAGCTGCTGGAGTAGGTCCCGGCCGTCACGC
679


 


GAV Basic-63
TCCAGCAGCTCTTCGACCTTTACCAACATCATCAACGCCG
680


 


GAV Basic-64
GCCATCGGCGTATGTCGAGACGGCGTTGATGATGTTGGTA
681


 


GAV Basic-65
TCTCGACATACGCCGATGGCTTCCTCAGCGAGGCTGCCAA
682


 


GAV Basic-66
AACCGTCGGCGGGGACGTACTTGGCAGCCTCGCTGAGGAA
683


 


GAV Basic-67
GTACGTCCCCGCCGACGGTTCGCTGGCCGAGCAGTTTGAC
684


 


GAV Basic-68
AGCGGAGTGCCGCTGTTGCGGTCAAACTGCTCGGCCAGCG
685


 


GAV Basic-69
CGCAACAGCGGCACTCCGCTGTCTGCGCTTCACCTGACGT
686


 


GAV Basic-70
CAAGAACGAGGCGTACGACCACGTCAGGTGAAGCGCAGAC
687


 


GAV Basic-71
GGTCGTACGCCTCGTTCTTGACAGCCACGGCCCGTCGGGC
688


 


GAV Basic-72
ACGAGGGGGGCACGATGCCAGCCCGACGGGCCGTGGCTGT
689


 


GAV Basic-73
TGGCATCGTGCCCCCCTCGTGGGCCAACAGCAGCGCTAGC
690


 


GAV Basic-74
GAGCACGTCGAGGGGATCGTGCTAGCGCTGCTGTTGGCCC
691


 


GAV Basic-75
ACGATCCCCTCGACGTGCTCCGGCGCGTCCGTGGTCGGAT
692


 


GAV Basic-76
GGCGGTGGGACGCGAGTAGGATCCGACCACGGACGCGCCG
693


 


GAV Basic-77
CCTACTCGCGTCCCACCGCCACGTCATTCCCTCCGTCGCA
694


 


GAV Basic-78
GCACGCCAGGCTTGGGCGTCTGCGACGGAGGGAATGACGT
695


 


GAV Basic-79
GACGCCCAAGCCTGGCGTGCCTTCCGGTACTCCCTACACG
696


 


GAV Basic-80
GGGGTCGCGCAGGGCAGGGGCGTGTAGGGAGTACCGGAAG
697


 


GAV Basic-81
CCCCTGCCCTGCGCGACCCCAACCTCCGTGGCCGTCACCT
698


 


GAV Basic-82
TGTCGACACGAGCTCGTGGAAGGTGACGGCCACGGAGGTT
699


 


GAV Basic-83
TCCACGAGCTCGTGTCGACACAGTTTGGCCAGACGGTCAA
700


 


GAV Basic-84
CCGCGGCGTTGCCCGCCACCTTGACCGTCTGGCCAAACTG
701


 


GAV Basic-85
GGTGGCGGGCAACGCCGCGGCCCTGGGCAACTGGAGCACG
702


 


GAV Basic-86
TCCAGAGCCACGGCGGCGCTCGTGCTCCAGTTGCCCAGGG
703


 


GAV Basic-87
AGCGCCGCCGTGGCTCTGGACGCCGTCAACTATGCCGATA
704


 


GAV Basic-88
CCCAATCCACAGGGGGTGGTTATCGGCATAGTTGACGGCG
705


 


GAV Basic-89
ACCACCCCCTGTGGATTGGGACGGTCAACCTCGAGGCTGG
706


 


GAV Basic-90
ACTTGTACTCCACGACGTCTCCAGCCTCGAGGTTGACCGT
707


 


GAV Basic-91
AGACGTCGTGGAGTACAAGTACATCAATGTGGGCCAAGAT
708


 


GAV Basic-92
CTCTCCCAGGTCACGGAGCCATCTTGGCCCACATTGATGT
709


 


GAV Basic-93
GGCTCCGTGACCTGGGAGAGTGATCCCAACCACACTTACA
710


 


GAV Basic-94
ACAAGCCACCGCAGGAACCGTGTAAGTGTGGTTGGGATCA
711


 


GAV Basic-95
CGGTTCCTGCGGTGGCTTGTGTGACGCAGGTTGTCAAGGA
712


 


GAV Basic-96
TTTACGACTGCCAGGTGTCCTCCTTGACAACCTGCGTCAC
713


 


GAV Basic-97
GGACACCTGGCAGTCGTAAACCCAGCTTTCTTGTACAAAG
714


 


GAV Basic-98
ACCACTTTGTACAAGAAAGCTGGG
715


 


All1-13
TGCGGTGATTGCATCTCCCAGCACACTTTGCCCGGACTAC
716


 


All1-14
TCTCGCGTCCACATGTAATAGTAGTCCGGGCAAAGTGTGC
717


 


All1-69
CGCAACAGCGGCACTCCGCTGTCTGCGGTTCACCTGACGT
718


 


All1-70
CAAGAACGAGGCGTACGACCACGTCAGGTGAACCGCAGAC
719


 


All1-82
TGTCGACACGAGCGCGTGGAAGGTGACGGCCACGGAGGTT
720


 


All1-83
TCCACGCGCTCGTGTCGACACAGTTTGGCCACACGGTCAA
721


 


All1-84
CCGCGGCGTTGCCCGCCACCTTGACCGTGTGGCCAAACTG
722


 


All1-87
AGCGCCGCCGTGGCTCTGGACGCCGTCAACTATAGAGATA
723


 


All1-88
CCCAATCCACAGGGGGTGGTTATCTCTATAGTTGACGGCG
724


 


All2-13
TGCGGTGATTGCATCTCCCAGCACACTTGACCCGGACTAC
725


 


All2-14
TCTCGCGTCCACATGTAATAGTAGTCCGGGTCAAGTGTGC
726


 


All2-69
CGCAACAGCGGCACTCCGCTGTCTGCGGTTCACCTGACGT
727


 


All2-70
CAAGAACGAGGCGTACGACCACGTCAGGTGAACCGCAGAC
728


 


All2-82
TGTCGACACGAGCGCGTGGAAGGTGACGGCCACGGAGGTT
729


 


All2-83
TCCACGCGCTCGTGTCGACACAGTTTGGCCACACGGTCAA
730


 


All2-84
CCGCGGCGTTGCCCGCCACCTTGACCGTGTGGCCAAACTG
731


 


All2-87
AGCGCCGCCGTGGCTCTGGACGCCGTCAACTATAGAGATA
732


 


All2-88
CCCAATCCACAGGGGGTGGTTATCTCTATAGTTGACGGCG
733


 


All3-13
TGCGGTGATTGCATCTCCCAGCACACTTTGCCCGGACTAC
734


 


All3-14
TCTCGCGTCCACATGTAATAGTAGTCCGGGCAAAGTGTGC
735


 


All3-16
GGTGAAGCGGTCGATGAGGATCTTGAAGACAAGAGCGCTA
736


 


All3-17
TCCTCATCGACCGCTTCACCGAAACGTACGATGCGGGCCT
737


 


All3-69
CGCAACAGCGGCACTCCGCTGTCTGCGGTTCACCTGACGT
738


 


All3-70
CAAGAACGAGGCGTACGACCACGTCAGGTGAACCGCAGAC
739


 


All3-82
TGTCGACACGAGCGCGTGGAAGGTGACGGCCACGGAGGTT
740


 


All3-83
TCCACGCGCTCGTGTCGACACAGTTTGGCCACACGGTCAA
741


 


All3-84
CCGCGGCGTTGCCCGCCACCTTGACCGTGTGGCCAAACTG
742


 


All3-87
AGCGCCGCCGTGGCTCTGGACGCCGTCAACTATAGAGATA
743


 


All3-88
CCCAATCCACAGGGGGTGGTTATCTCTATAGTTGACGGCG
744


 


All4-13
TGCGGTGATTGCATCTCCCAGCACACTTGACCCGGACTAC
745


 


All4-14
TCTCGCGTCCACATGTAATAGTAGTCCGGGTCAAGTGTGC
746


 


All4-16
GGTGAAGCGGTCGATGAGGATCTTGAAGACAAGAGCGCTA
747


 


All4-17
TCCTCATCGACCGCTTCACCGAAACGTACGATGCGGGCCT
748


 


All4-69
CGCAACAGCGGCACTCCGCTGTCTGCGGTTCACCTGACGT
749


 


All4-70
CAAGAACGAGGCGTACGACCACGTCAGGTGAACCGCAGAC
750


 


All4-82
TGTCGACACGAGCGCGTGGAAGGTGACGGCCACGGAGGTT
751


 


All4-83
TCCACGCGCTCGTGTCGACACAGTTTGGCCACACGGTCAA
752


 


All4-84
CCGCGGCGTTGCCCGCCACCTTGACCGTGTGGCCAAACTG
753


 


All4-87
AGCGCCGCCGTGGCTCTGGACGCCGTCAACTATAGAGATA
754


 


All4-88
CCCAATCCACAGGGGGTGGTTATCTCTATAGTTGACGGCG
755


 


All5-13
TGCGGTGATTGCATCTCCCAGCACAAGAGACCCGGACTAC
756


 


All5-14
TCTCGCGTCCACATGTAATAGTAGTCCGGGTCTCTTGTGC
757


 


All5-69
CGCAACAGCGGCACTCCGCTGTCTGCGGTTCACCTGACGT
758


 


All5-70
CAAGAACGAGGCGTACGACCACGTCAGGTGAACCGCAGAC
759


 


All5-82
TGTCGACACGAGCGCGTGGAAGGTGACGGCCACGGAGGTT
760


 


All5-83
TCCACGCGCTCGTGTCGACACAGTTTGGCCACACGGTCAA
761


 


All5-84
CCGCGGCGTTGCCCGCCACCTTGACCGTGTGGCCAAACTG
762


 


All5-87
AGCGCCGCCGTGGCTCTGGACGCCGTCAACTATAGAGATA
763


 


All5-88
CCCAATCCACAGGGGGTGGTTATCTCTATAGTTGACGGCG
764


 


All6-13
TGCGGTGATTGCATCTCCCAGCACAAGAGACCCGGACTAC
765


 


All6-14
TCTCGCGTCCACATGTAATAGTAGTCCGGGTCTCTTGTGC
766


 


All6-16
GGTGAAGCGGTCGATGAGGATCTTGAAGACAAGAGCGCTA
767


 


All6-17
TCCTCATCGACCGCTTCACCGAAACGTACGATGCGGGCCT
768


 


All6-69
CGCAACAGCGGCACTCCGCTGTCTGCGGTTCACCTGACGT
769


 


All6-70
CAAGAACGAGGCGTACGACCACGTCAGGTGAACCGCAGAC
770


 


All6-82
TGTCGACACGAGCGCGTGGAAGGTGACGGCCACGGAGGTT
771


 


All6-83
TCCACGCGCTCGTGTCGACACAGTTTGGCCACACGGTCAA
772


 


All6-84
CCGCGGCGTTGCCCGCCACCTTGACCGTGTGGCCAAACTG
773


 


All6-87
AGCGCCGCCGTGGCTCTGGACGCCGTCAACTATAGAGATA
774


 


All6-88
CCCAATCCACAGGGGGTGGTTATCTCTATAGTTGACGGCG
775


 


All7-13
TGCGGTGATTGCATCTCCCAGCACAAGAGACCCGGACTAC
776


 


All7-14
TCTCGCGTCCACATGTAATAGTAGTCCGGGTCTCTTGTGC
777


 


All7-69
CGCAACAGCGGCACTCCGCTGTCTGCGAGACACCTGACGT
778


 


All7-70
CAAGAACGAGGCGTACGACCACGTCAGGTGTCTCGCAGAC
779


 


All7-82
TGTCGACACGAGCGCGTGGAAGGTGACGGCCACGGAGGTT
780


 


All7-83
TCCACGCGCTCGTGTCGACACAGTTTGGCCACACGGTCAA
781


 


All7-84
CCGCGGCGTTGCCCGCCACCTTGACCGTGTGGCCAAACTG
782


 


All7-87
AGCGCCGCCGTGGCTCTGGACGCCGTCAACTATAGAGATA
783


 


All7-88
CCCAATCCACAGGGGGTGGTTATCTCTATAGTTGACGGCG
784


 


All8-13
TGCGGTGATTGCATCTCCCAGCACAAGAGACCCGGACTAC
785


 


All8-14
TCTCGCGTCCACATGTAATAGTAGTCCGGGTCTCTTGTGC
786


 


All8-16
GGTGAAGCGGTCGATGAGGATCTTGAAGACAAGAGCGCTA
787


 


All8-17
TCCTCATCGACCGCTTCACCGAAACGTACGATGCGGGCCT
788


 


All8-69
CGCAACAGCGGCACTCCGCTGTCTGCGAGACACCTGACGT
789


 


All8-70
CAAGAACGAGGCGTACGACCACGTCAGGTGTCTCGCAGAC
790


 


All8-82
TGTCGACACGAGCGCGTGGAAGGTGACGGCCACGGAGGTT
791


 


All8-83
TCCACGCGCTCGTGTCGACACAGTTTGGCCACACGGTCAA
792


 


All8-84
CCGCGGCGTTGCCCGCCACCTTGACCGTGTGGCCAAACTG
793


 


All8-87
AGCGCCGCCGTGGCTCTGGACGCCGTCAACTATAGAGATA
794


 


All8-88
CCCAATCCACAGGGGGTGGTTATCTCTATAGTTGACGGCG
795


 


CS1-69
CGCAACAGCGGCACTCCGCTGTCTGCGGTTCACCTGACGT
796


 


CS1-70
CAAGAACGAGGCGTACGACCACGTCAGGTGAACCGCAGAC
797


 


CS1-71
GGTCGTACGCCTCGTTCTTGACAGCCGCGCTCCGTCGGGC
798


 


CS1-72
ACGAGGGGGGCACGATGCCAGCCCGACGGAGCGCGGCTGT
799


 


CS1-83
TCCACGAGCTCGTGTCGACACAGTTTGGCCACACGGTCAA
800


 


CS1-84
CCGCGGCGTTGCCCGCCACCTTGACCGTGTGGCCAAACTG
801


 


CS1-87
AGCGCCGCCGTGGCTCTGGACCGCGTCAACTATCGCGATA
802


 


CS1-88
CCCAATCCACAGGGGGTGGTTATCGCGATAGTTGACGCGG
803


 


CS1-91
AGACGTCGTGGAGTACAAGTACATCATTGTGGGCCAAGAT
804


 


CS1-92
CTCTCCCAGGTCACGGAGCCATCTTGGCCCACAATGATGT
805


 


CS2-69
CGCAACAGCGGCACTCCGCTGTCTGCGGTTCACCTGACGT
806


 


CS2-70
CAAGAACGAGGCGTACGACCACGTCAGGTGAACCGCAGAC
807


 


CS2-71
GGTCGTACGCCTCGTTCTTGACAGCCGCGCAACGTCGGGC
808


 


CS2-72
ACGAGGGGGGCACGATGCCAGCCCGACGTTGCGCGGCTGT
809


 


CS2-83
TCCACGAGCTCGTGTCGACACAGTTTGGCCACACGGTCAA
810


 


CS2-84
CCGCGGCGTTGCCCGCCACCTTGACCGTGTGGCCAAACTG
811


 


CS2-87
AGCGCCGCCGTGGCTCTGGACCGCGTCAACTATCGCGATA
812


 


CS2-88
CCCAATCCACAGGGGGTGGTTATCGCGATAGTTGACGCGG
813


 


CS2-91
AGACGTCGTGGAGTACAAGTACATCATTGTGGGCCAAGAT
814


 


CS2-92
CTCTCCCAGGTCACGGAGCCATCTTGGCCCACAATGATGT
815


 


CS3-69
CGCAACAGCGGCACTCCGCTGTCTGCGGTTCACCTGACGT
816


 


CS3-70
CAAGAACGAGGCGTACGACCACGTCAGGTGAACCGCAGAC
817


 


CS3-71
GGTCGTACGCCTCGTTCTTGACAGCCGCGGCCCGTCGGGC
818


 


CS3-72
ACGAGGGGGGCACGATGCCAGCCCGACGGGCCGCGGCTGT
819


 


CS3-83
TCCACGAGCTCGTGTCGACACAGTTTGGCCACACGGTCAA
820


 


CS3-84
CCGCGGCGTTGCCCGCCACCTTGACCGTGTGGCCAAACTG
821


 


CS3-87
AGCGCCGCCGTGGCTCTGGACCGCGTCAACTATGCCGATA
822


 


CS3-88
CCCAATCCACAGGGGGTGGTTATCGGCATAGTTGACGCGG
823


 


CS3-91
AGACGTCGTGGAGTACAAGTACATCATTGTGGGCCAAGAT
824


 


CS3-92
CTCTCCCAGGTCACGGAGCCATCTTGGCCCACAATGATGT
825


 


CS4-69
CGCAACAGCGGCACTCCGCTGTCTGCGGTTCACCTGACGT
826


 


CS4-70
CAAGAACGAGGCGTACGACCACGTCAGGTGAACCGCAGAC
827


 


CS4-71
GGTCGTACGCCTCGTTCTTGACAGCCGCGGCCCGTCGGGC
828


 


CS4-72
ACGAGGGGGGCACGATGCCAGCCCGACGGGCCGCGGCTGT
829


 


CS4-83
TCCACGAGCTCGTGTCGACACAGTTTGGCCACACGGTCAA
830


 


CS4-84
CCGCGGCGTTGCCCGCCACCTTGACCGTGTGGCCAAACTG
831


 


CS4-87
AGCGCCGCCGTGGCTCTGGACGCCGTCAACTATCGCGATA
832


 


CS4-88
CCCAATCCACAGGGGGTGGTTATCGCGATAGTTGACGGCG
833


 


CS4-91
AGACGTCGTGGAGTACAAGTACATCATTGTGGGCCAAGAT
834


 


CS4-92
CTCTCCCAGGTCACGGAGCCATCTTGGCCCACAATGATGT
835


 


LQ1-50
CAGTAGATGGAGCGGAAGGAGTCGACAACAACCTTGAGGT
836


 


LQ1-51
TCCTTCCGCTCCATCTACTGCGTGAACAAGGGCATTCCTG
837


 


LQ1-69
CGCAACAGCGGCACTCCGCTGTCTGCGAGACACCTGACGT
838


 


LQ1-70
CAAGAACGAGGCGTACGACCACGTCAGGTGTCTCGCAGAC
839


 


LQ1-71
GGTCGTACGCCTCGTTCTTGACAGCCACGCTCCGTCGGGC
840


 


LQ1-72
ACGAGGGGGGCACGATGCCAGCCCGACGGAGCGTGGCTGT
841


 


LQ2-50
CAGTAGATGGAGCGGAAGGAGTCGACAACAACCTTGAGGT
842


 


LQ2-51
TCCTTCCGCTCCATCTACTGCGTGAACAAGGGCATTCCTG
843


 


LQ2-69
CGCAACAGCGGCACTCCGCTGTCTGCGGTACACCTGACGT
844


 


LQ2-70
CAAGAACGAGGCGTACGACCACGTCAGGTGTACCGCAGAC
845


 


LQ2-71
GGTCGTACGCCTCGTTCTTGACAGCCACGCAGCGTCGGGC
846


 


LQ2-72
ACGAGGGGGGCACGATGCCAGCCCGACGCTGCGTGGCTGT
847


 


LQ3-50
CAGTAGATGGAGCGGAAGGAGTCGACAACAACCTTGAGGT
848


 


LQ3-51
TCCTTCCGCTCCATCTACTGCGTGAACAAGGGCATTCCTG
849


 


LQ3-69
CGCAACAGCGGCACTCCGCTGTCTGCGGTACACCTGACGT
850


 


LQ3-70
CAAGAACGAGGCGTACGACCACGTCAGGTGTACCGCAGAC
851


 


LQ3-71
GGTCGTACGCCTCGTTCTTGACAGCCACGTTACGTCGGGC
852


 


LQ3-72
ACGAGGGGGGCACGATGCCAGCCCGACGTAACGTGGCTGT
853


 


LQ3-83
TCCACGAGCTCGTGTCGACACAGTTTGGCCACACGGTCAA
854


 


LQ3-84
CCGCGGCGTTGCCCGCCACCTTGACCGTGTGGCCAAACTG
855


 


LQ4-50
CAGTAGATGGAGCGGAAGGAGTCGACAACAACCTTGAGGT
856


 


LQ4-51
TCCTTCCGCTCCATCTACTGCGTGAACAAGGGCATTCCTG
857


 


LQ4-69
CGCAACAGCGGCACTCCGCTGTCTGCGAGACACCTGACGT
858


 


LQ4-70
CAAGAACGAGGCGTACGACCACGTCAGGTGTCTCGCAGAC
859


 


LQ4-71
GGTCGTACGCCTCGTTCTTGACAGCCACGCAGCGTCGGGC
860


 


LQ4-72
ACGAGGGGGGCACGATGCCAGCCCGACGCTGCGTGGCTGT
861


 


LQ4-83
TCCACGAGCTCGTGTCGACACAGTTTGGCCACACGGTCAA
862


 


LQ4-84
CCGCGGCGTTGCCCGCCACCTTGACCGTGTGGCCAAACTG
863


 


LQ5-69
CGCAACAGCGGCACTCCGCTGTCTGCGCGTCACCTGACGT
864


 


LQ5-70
CAAGAACGAGGCGTACGACCACGTCAGGTGACGCGCAGAC
865


 


LQ5-71
GGTCGTACGCCTCGTTCTTGACAGCCACGCTCCGTCGGGC
866


 


LQ5-72
ACGAGGGGGGCACGATGCCAGCCCGACGGAGCGTGGCTGT
867


 


LQ5-83
TCCACGAGCTCGTGTCGACACAGTTTGGCCACACGGTCAA
868


 


LQ5-84
CCGCGGCGTTGCCCGCCACCTTGACCGTGTGGCCAAACTG
869


 


LQ6-69
CGCAACAGCGGCACTCCGCTGTCTGCGGTTCACCTGACGT
870


 


LQ6-70
CAAGAACGAGGCGTACGACCACGTCAGGTGAACCGCAGAC
871


 


LQ6-71
GGTCGTACGCCTCGTTCTTGACAGCCACGCTTCGTCGGGC
872


 


LQ6-72
ACGAGGGGGGCACGATGCCAGCCCGACGAAGCGTGGCTGT
873


 


LQ6-83
TCCACGAGCTCGTGTCGACACAGTTTGGCCATACGGTCAA
874


 


LQ6-84
CCGCGGCGTTGCCCGCCACCTTGACCGTATGGCCAAACTG
875


 


TS1-13
TGCGGTGATTGCATCTCCCAGCACAAGAGACCCGGACTAC
876


 


TS1-14
TCTCGCGTCCACATGTAATAGTAGTCCGGGTCTCTTGTGC
877


 


TS1-71
GGTCGTACGCCTCGTTCTTGACAGCCGCAGCCCGTCGGGC
878


 


TS1-72
ACGAGGGGGGCACGATGCCAGCCCGACGGGCTGCGGCTGT
879


 


TS1-82
TGTCGACACGAGCACGTGGAAGGTGACGGCCACGGAGGTT
880


 


TS1-83
TCCACGTGCTCGTGTCGACACAGTTTGGCCAGACGGTCAA
881


 


TS1-87
AGCGCCGCCGTGGCTCTGGACCGCGTCAACTATGCCGATA
882


 


TS1-88
CCCAATCCACAGGGGGTGGTTATCGGCATAGTTGACGCGG
883


 


TS1-91
AGACGTCGTGGAGTACAAGTACATCAAAGTGGGCCAAGAT
884


 


TS1-92
CTCTCCCAGGTCACGGAGCCATCTTGGCCCACTTTGATGT
885


 


TS2-13
TGCGGTGATTGCATCTCCCAGCACAATTAGACCGGACTAC
886


 


TS2-14
TCTCGCGTCCACATGTAATAGTAGTCCGGTCTAATTGTGC
887


 


TS2-82
TGTCGACACGAGCGCGTGGAAGGTGACGGCCACGGAGGTT
888


 


TS2-83
TCCACGCGCTCGTGTCGACACAGTTTGGCCACACGGTCAA
889


 


TS2-84
CCGCGGCGTTGCCCGCCACCTTGACCGTGTGGCCAAACTG
890


 


TS2-91
AGACGTCGTGGAGTACAAGTACATCATTGTGGGCCAAGAT
891


 


TS2-92
CTCTCCCAGGTCACGGAGCCATCTTGGCCCACAATGATGT
892


 


TS3-82
TGTCGACACGAGCGCGTGGAAGGTGACGGCCACGGAGGTT
893


 


TS3-83
TCCACGCGCTCGTGTCGACACAGTTTGGCCAGACGGTCAA
894


 


TS3-91
AGACGTCGTGGAGTACAAGTACATCATTGTGGGCCAAGAT
895


 


TS3-92
CTCTCCCAGGTCACGGAGCCATCTTGGCCCACAATGATGT
896


 


TS4-13
TGCGGTGATTGCATCTCCCAGCACAAGAGACCCGGACTAC
897


 


TS4-14
TCTCGCGTCCACATGTAATAGTAGTCCGGGTCTCTTGTGC
898


 


TS4-71
GGTCGTACGCCTCGTTCTTGACAGCCGCGGCCCGTCGGGC
899


 


TS4-72
ACGAGGGGGGCACGATGCCAGCCCGACGGGCCGCGGCTGT
900


 


TS4-82
TGTCGACACGAGCGCGTGGAAGGTGACGGCCACGGAGGTT
901


 


TS4-83
TCCACGCGCTCGTGTCGACACAGTTTGGCCACACGGTCAA
902


 


TS4-84
CCGCGGCGTTGCCCGCCACCTTGACCGTGTGGCCAAACTG
903


 


TS4-91
AGACGTCGTGGAGTACAAGTACATCATTGTGGGCCAAGAT
904


 


TS4-92
CTCTCCCAGGTCACGGAGCCATCTTGGCCCACAATGATGT
905


 


TS5-13
TGCGGTGATTGCATCTCCCAGCACAATTCGCCCGGACTAC
906


 


TS5-14
TCTCGCGTCCACATGTAATAGTAGTCCGGGCGAATTGTGC
907


 


TS5-71
GGTCGTACGCCTCGTTCTTGACAGCCGCGGCCCGTCGGGC
908


 


TS5-72
ACGAGGGGGGCACGATGCCAGCCCGACGGGCCGCGGCTGT
909


 


TS5-83
TCCACGAGCTCGTGTCGACACAGTTTGGCCACACGGTCAA
910


 


TS5-84
CCGCGGCGTTGCCCGCCACCTTGACCGTGTGGCCAAACTG
911


 


TS5-87
AGCGCCGCCGTGGCTCTGGACGCGGTCAACTATGCCGATA
912


 


TS5-88
CCCAATCCACAGGGGGTGGTTATCGGCATAGTTGACCGCG
913


 


GAV st-1
GAGAGGGGACAAGTTTGTACAAAAAAGCAGGCT
914


 


GAV st-2
GCAGTCGACAGGACGTGCATAGCCTGLTTTTTTGTACAAA
915


 


GAV st-3
ATGCACGTCCTGTCGACTGCGGTGCTGCTCGGCTCCGTTG
916


 


GAV st-4
TCCCAGGACCTTTTGAACGGCAACGGAGCCGAGCAGCACC
917


 


GAV st-5
CCGTTCAAAAGGTCCTGGGAAGACCAGGATCAAGCGGTCT
918


 


GAV st-6
ACCTCTTGGTGACGTCGGACAGACCGCTTGATCCTGGTCT
919


 


GAV st-7
GTCCGACGTCACCAAGAGGTCTGTTGACGACTTCATCAGC
920


 


GAV st-8
AGTGCAATAGGCGTCTCGGTGCTGATGAAGTCGTCAACAG
921


 


GAV st-9
ACCGAGACGCCTATTGCACTGAACAATCTTCTTTGCAATG
922


 


GAV st-10
ACGGCATCCATCAGGACCAACATTGCAAAGAAGATTGTTC
923


 


GAV st-11
TTGGTCCTGATGGATGCCGTGCATTCGGCACATCAGCTGG
924


 


GAV st-12
TGGGAGATGCAATCACCGCACCAGCTGATGTGCCGAATGC
925


 


GAV st-13
TGCGGTGATTGCATCTCCCAGCACACAAGACCCGGACTAC
926


 


GAV st-14
TCTCGCGTCCACATGTAATAGTAGTCCGGGTCTTGTGTGC
927


 


GAV st-15
TATTACATGTGGACGCGAGATAGCGCTCTTGTCTTCAAGA
928


 


GAV st-16
GGTGAAGCGGTCGATGAGGTTCTTGAAGACAAGAGCGCTA
929


 


GAV st-17
ACCTCATCGACCGCTTCACCGAAACGTACGATGCGGGCCT
930


 


GAV st-18
ACTGCTCGATGCGGCGCTGCAGGCCCGCATCGTACGTTTC
931


 


GAV st-19
GCAGCGCCGCATCGAGCAGTACATTACTGCCCAGGTCACT
932


 


GAV st-20
GGGTTAGAGAGGCCCTGGAGAGTGACCTGGGCAGTAATGT
933


 


GAV st-21
CTCCAGGGCCTCTCTAACCCCTCGGGCTCCCTCGCGGACG
934


 


GAV st-22
GGGCTCGCCGAGACCAGAGCCGTCCGCGAGGGAGCCCGAG
935


 


GAV st-23
GCTCTGGTCTCGGCGAGCCCAAGTTTGAGTTGACCCTGAA
936


 


GAV st-24
CCCAGTTGCCGGTGAAAGGCTTCAGGGTCAACTCAAACTT
937


 


GAV st-25
GCCTTTCACCGGCAACTGGGGTCGACCGCAGCGGGATGGC
938


 


GAV st-26
GCAATGGCTCGCAGAGCTGGGCCATCCCGCTGCGGTCGAC
939


 


GAV st-27
CCAGCTCTGCGAGCCATTGCCTTGATTGGATACTCAAAGT
940


 


GAV st-28
ATAGTTGTTGTTGATGAGCCACTTTGAGTATCCAATCAAG
941


 


GAV st-29
GGCTCATCAACAACAACTATCAGTCGACTGTGTCCAACGT
942


 


GAV st-30
TGCGCACAATAGGCCAGATGACGTTGGACACAGTCGACTG
943


 


GAV st-31
CATCTGGCCTATTGTGCGCAACGACCTCAACTATGTTGCC
944


 


GAV st-32
CCGGTTTGGTTCCAGTACTGGGCAACATAGTTGAGGTCGT
945


 


GAV st-33
CAGTACTGGAACCAAACCGGCTTTGACCTCTGGGAAGAAG
946


 


GAV st-34
AAAGAATGAGCTCCCATTGACTTCTTCCCAGAGGTCAAAG
947


 


GAV st-35
TCAATGGGAGCTCATTCTTTACTGTTGCCAACCAGCACCG
948


 


GAV st-36
TGGCGCCCTCGACAAGTGCTCGGTGCTGGTTGGCAACAGT
949


 


GAV st-37
AGCACTTGTCGAGGGCGCCACTCTTGCTGCCACTCTTGGC
950


 


GAV st-38
GAATAAGCGCTTCCCGACTGGCCAAGAGTGGCAGCAAGAG
951


 


GAV st-39
CAGTCGGGAAGCGCTTATTCATCTGTTGCTCCCCAGGTTT
952


 


GAV st-40
GAATCGTTGGAGAAAGCACAAAACCTGGGGAGCAACAGAT
953


 


GAV st-41
TGTGCTTTCTCCAACGATTCTGGGTGTCGTCTGGTGGATA
954


 


GAV st-42
TGTTGATGTTGGAGTCGACGTATCCACCAGACGACACCCA
955


 


GAV st-43
CGTCGACTCCAACATCAACACCAACGAGGGCAGGACTGGC
956


 


GAV st-44
AGGACGGAGTTGACATCCTTGCCAGTCCTGCCCTCGTTGG
957


 


GAV st-45
AAGGATGTCAACTCCGTCCTGACTTCCATCCACACCTTCG
958


 


GAV st-46
GTCACAGCCAAGGTTGGGATCGAAGGTGTGGATGGAAGTC
959


 


GAV st-47
ATCCCAACCTTGGCTGTGACGCAGGCACCTTCCAGCCATG
960


 


GAV st-48
TGGAGAGCGCTTTGTCACTGCATGGCTGGAAGGTGCCTGC
961


 


GAV st-49
CAGTGACAAAGCGCTCTCCAACCTCAAGGTTGTTGTCGAC
962


 


GAV st-50
CCGTAGATGGAGCGGAAGGAGTCGACAACAACCTTGAGGT
963


 


GAV st-51
TCCTTCCGCTCCATCTACGGCGTGAACAAGGGCATTCCTG
964


 


GAV st-52
AATGGCGACGGCAGCACCGGCAGGAATGCCCTTGTTCACG
965


 


GAV st-53
CCGGTGCTGCCGTCGCCATTGGCCGGTATGCAGAGGATGT
966


 


GAV st-54
AAGGGTTGCCGTTGTAGTACACATCCTCTGCATACCGGCC
967


 


GAV st-55
GTACTACAACGGCAACCCTTGGTATCTTGCTACATTTGCT
968


 


GAV st-56
TCGTACAGCTGCTCGGCAGCAGCAAATGTAGCAAGATACC
969


 


GAV st-57
GCTGCCGAGCAGCTGTACGATGCCATCTACGTCTGGAAGA
970


 


GAV st-58
CACCGTGATGGAGCCCGTCTTCTTCCAGACGTAGATGGCA
971


 


GAV st-59
AGACGGGCTCCATCACGGTGACCGCCACCTCCCTGGCCTT
972


 


GAV st-60
CAGGAACAAGCTCCTGGAAGAAGGCCAGGGAGGTGGCGGT
973


 


GAV st-61
CTTCCAGGAGCTTGTTCCTGGCGTGACGGCCGGGACCTAC
974


 


GAV st-62
AAGGTCGAAGAGCTGCTGGAGTAGGTCCCGGCCGTCACGC
975


 


GAV st-63
TCCAGCAGCTCTTCGACCTTTACCAACATCATCAACGCCG
976


 


GAV st-64
GCCATCGGCGTATGTCGAGACGGCGTTGATGATGTTGGTA
977


 


GAV st-65
TCTCGACATACGCCGATGGCTTCCTCAGCGAGGCTGCCAA
978


 


GAV st-66
AACCGTCGGCGGGGACGTACTTGGCAGCCTCGCTGAGGAA
979


 


GAV st-67
GTACGTCCCCGCCGACGGTTCGCTGGCCGAGCAGTTTGAC
980


 


GAV st-68
AGCGGAGTGCCGCTGTTGCGGTCAAACTGCTCGGCCAGCG
981


 


GAV st-69
CGCAACAGCGGCACTCCGCTGTCTGCGCTTCACCTGACGT
982


 


GAV st-70
CAAGAACGAGGCGTACGACCACGTCAGGTGAAGCGCAGAC
983


 


GAV st-71
GGTCGTACGCCTCGTTCTTGACAGCCACGGCCCGTCGGGC
984


 


GAV st-72
ACGAGGGGGGCACGATGCCAGCCCGACGGGCCGTGGCTGT
985


 


GAV st-73
TGGCATCGTGCCCCCCTCGTGGGCCAACAGCAGCGCTAGC
986


 


GAV st-74
GAGCACGTCGAGGGGATCGTGCTAGCGCTGCTGTTGGCCC
987


 


GAV st-75
ACGATCCCCTCGACGTGCTCCGGCGCGTCCGTGGTCGGAT
988


 


GAV st-76
GGCGGTGGGACGCGAGTAGGATCCGACCACGGACGCGCCG
989


 


GAV st-77
CCTACTCGCGTCCCACCGCCACGTCATTCCCTCCGTCGCA
990


 


GAV st-78
GCACGCCAGGCTTGGGCGTCTGCGACGGAGGGAATGACGT
991


 


GAV st-79
GACGCCCAAGCCTGGCGTGCCTTCCGGTACTCCCTACACG
992


 


GAV st-80
GGGGTCGCGCAGGGCAGGGGCGTGTAGGGAGTACCGGAAG
993


 


GAV st-81
CCCCTGCCCTGCGCGACCCCAACCTCCGTGGCCGTCACCT
994


 


GAV st-82
TGTCGACACGAGCTCGTGGAAGGTGACGGCCACGGAGGTT
995


 


GAV st-83
TCCACGAGCTCGTGTCGACACAGTTTGGCCACACGGTCAA
996


 


GAV st-84
CCGCGGCGTTGCCCGCCACCTTGACCGTGTGGCCAAACTG
997


 


GAV st-85
GGTGGCGGGCAACGCCGCGGCCCTGGGCAACTGGAGCACG
998


 


GAV st-86
TCCAGAGCCACGGCGGCGCTCGTGCTCCAGTTGCCCAGGG
999


 


GAV st-87
AGCGCCGCCGTGGCTCTGGACGCCGTCAACTATGCCGATA
1000


 


GAV st-88
CCCAATCCACAGGGGGTGGTTATCGGCATAGTTGACGGCG
1001


 


GAV st-89
ACCACCCCCTGTGGATTGGGACGGTCAACCTCGAGGCTGG
1002


 


GAV st-90
ACTTGTACTCCACGACGTCTCCAGCCTCGAGGTTGACCGT
1003


 


GAV st-91
AGACGTCGTGGAGTACAAGTACATCAATGTGGGCCAAGAT
1004


 


GAV st-92
CTCTCCCAGGTCACGGAGCCATCTTGGCCCACATTGATGT
1005


 


GAV st-93
GGCTCCGTGACCTGGGAGAGTGATCCCAACCACACTTACA
1006


 


GAV st-94
ACAAGCCACCGCAGGAACCGTGTAAGTGTGGTTGGGATCA
1007


 


GAV st-95
CGGTTCCTGCGGTGGCTTGTGTGACGCAGGTTGTCAAGGA
1008


 


GAV st-96
TTTACGACTGCCAGGTGTCCTCCTTGACAACCTGCGTCAC
1009


 


GAV st-97
GGACACCTGGCAGTCGTAAACCCAGCTTTCTTGTACAAAG
1010


 


GAV st-98
CTCTGGGGACCACTTTGTACAAGAAAGCTGGG
1011


 


RB1-16
GGTGAAGCGGTCGATGAGGATCTTGAAGACAAGAGCGCTA
1012


 


RB1-17
TCCTCATCGACCGCTTCACCGAAACGTACGATGCGGGCCT
1013


 


RB1-71
GGTCGTACGCCTCGTTCTTGACAGCCGCGGCCCGTCGGGC
1014


 


RB1-72
ACGAGGGGGGCACGATGCCAGCCCGACGGGCCGCGGCTGT
1015


 


RB2-69
CGCAACAGCGGCACTCCGCTGTCTGCGGTTCACCTGACGT
1016


 


RB2-70
CAAGAACGAGGCGTACGACCACGTCAGGTGAACCGCAGAC
1017


 


RB2-71
GGTCGTACGCCTCGTTCTTGACAGCCGCGGCCCGTCGGGC
1018


 


RB2-72
ACGAGGGGGGCACGATGCCAGCCCGACGGGCCGCGGCTGT
1019


 


RB3-71
GGTCGTACGCCTCGTTCTTGACAGCCGCGCTCCGTCGGGC
1020


 


RB3-72
ACGAGGGGGGCACGATGCCAGCCCGACGGAGCGCGGCTGT
1021


 


RB1-71
GGTCGTACGCCTCGTTCTTGACAGCCGCGGCCCGTCGGGC
1022


 


RB1-72
ACGAGGGGGGCACGATGCCAGCCCGACGGGCCGCGGCTGT
1023


 


RB4-82
TGTCGACACGAGCGCGTGGAAGGTGACGGCCACGGAGGTT
1024


 


RB4-83
TCCACGCGCTCGTGTCGACACAGTTTGGCCACACGGTCAA
1025


 


RB4-71
GGTCGTACGCCTCGTTCTTGACAGCCGCGGCCCGTCGGGC
1026


 


RB4-72
ACGAGGGGGGCACGATGCCAGCCCGACGGGCCGCGGCTGT
1027


 


RB5-71
GGTCGTACGCCTCGTTCTTGACAGCCGCGGCCCGTCGGGC
1028


 


RB5-72
ACGAGGGGGGCACGATGCCAGCCCGACGGGCCGCGGCTGT
1029


 


RB5-87
AGCGCCGCCGTGGCTCTGGACGCCGTCAACTATCGCGATA
1030


 


RB5-88
CCCAATCCACAGGGGGTGGTTATCGCGATAGTTGACGGCG
1031


 


RB6-16
GGTGAAGCGGTCGATGAGGATCTTGAAGACAAGAGCGCTA
1032


 


RB6-17
TCCTCATCGACCGCTTCACCGAAACGTACGATGCGGGCCT
1033


 


RB6-71
GGTCGTACGCCTCGTTCTTGACAGCCGCGGCCCGTCGGGC
1034


 


RB6-72
ACGAGGGGGGCACGATGCCAGCCCGACGGGCCGCGGCTGT
1035


 


RB6-87
AGCGCCGCCGTGGCTCTGGACGCCGTCAACTATCGCGATA
1036


 


RB6-88
CCCAATCCACAGGGGGTGGTTATCGCGATAGTTGACGGCG
1037


 


RB7-69
CGCAACAGCGGCACTCCGCTGTCTGCGGTTCACCTGACGT
1038


 


RB7-70
CAAGAACGAGGCGTACGACCACGTCAGGTGAACCGCAGAC
1039


 


RB7-71
GGTCGTACGCCTCGTTCTTGACAGCCGCGGCCCGTCGGGC
1040


 


RB7-72
ACGAGGGGGGCACGATGCCAGCCCGACGGGCCGCGGCTGT
1041


 


RB7-87
AGCGCCGCCGTGGCTCTGGACGCCGTCAACTATCGCGATA
1042


 


RB7-88
CCCAATCCACAGGGGGTGGTTATCGCGATAGTTGACGGCG
1043


 


RB8-71
GGTCGTACGCCTCGTTCTTGACAGCCGCGCTCCGTCGGGC
1044


 


RB8-72
ACGAGGGGGGCACGATGCCAGCCCGACGGAGCGCGGCTGT
1045


 


RB8-87
AGCGCCGCCGTGGCTCTGGACGCCGTCAACTATCGCGATA
1046


 


RB8-88
CCCAATCCACAGGGGGTGGTTATCGCGATAGTTGACGGCG
1047


 


RB9-71
GGTCGTACGCCTCGTTCTTGACAGCCGCGCTCCGTCGGGC
1048


 


RB9-72
ACGAGGGGGGCACGATGCCAGCCCGACGGAGCGCGGCTGT
1049


 


RB9-82
TGTCGACACGAGCGCGTGGAAGGTGACGGCCACGGAGGTT
1050


 


RB9-83
TCCACGCGCTCGTGTCGACACAGTTTGGCCACACGGTCAA
1051


 


RB10-16
GGTGAAGCGGTCGATGAGGATCTTGAAGACAAGAGCGCTA
1052


 


RB10-17
TCCTCATCGACCGCTTCACCGAAACGTACGATGCGGGCCT
1053


 


RB10-71
GGTCGTACGCCTCGTTCTTGACAGCCGCGCTCCGTCGGGC
1054


 


RB10-72
ACGAGGGGGGCACGATGCCAGCCCGACGGAGCGCGGCTGT
1055


 


RB10-87
AGCGCCGCCGTGGCTCTGGACGCCGTCAACTATCGCGATA
1056


 


RB10-88
CCCAATCCACAGGGGGTGGTTATCGCGATAGTTGACGGCG
1057


 


RB11-69
CGCAACAGCGGCACTCCGCTGTCTGCGGTTCACCTGACGT
1058


 


RB11-70
CAAGAACGAGGCGTACGACCACGTCAGGTGAACCGCAGAC
1059


 


RB11-71
GGTCGTACGCCTCGTTCTTGACAGCCGCGCTCCGTCGGGC
1060


 


RB11-72
ACGAGGGGGGCACGATGCCAGCCCGACGGAGCGCGGCTGT
1061


 


RB11-87
AGCGCCGCCGTGGCTCTGGACGCCGTCAACTATCGCGATA
1062


 


RB11-88
CCCAATCCACAGGGGGTGGTTATCGCGATAGTTGACGGCG
1063


 


RB12-16
GGTGAAGCGGTCGATGAGGATCTTGAAGACAAGAGCGCTA
1064


 


RB12-17
TCCTCATCGACCGCTTCACCGAAACGTACGATGCGGGCCT
1065


 


RB13-69
CGCAACAGCGGCACTCCGCTGTCTGCGGTTCACCTGACGT
1066


 


RB13-70
CAAGAACGAGGCGTACGACCACGTCAGGTGAACCGCAGAC
1067


 


RB14-71
GGTCGTACGCCTCGTTCTTGACAGCCACGCTCCGTCGGGC
1068


 


RB14-72
ACGAGGGGGGCACGATGCCAGCCCGACGGAGCGTGGCTGT
1069


 


RB15-87
AGCGCCGCCGTGGCTCTGGACGCCGTCAACTATCGCGATA
1070


 


RB15-88
CCCAATCCACAGGGGGTGGTTATCGCGATAGTTGACGGCG
1071


 


RB16-16
GGTGAAGCGGTCGATGAGGATCTTGAAGACAAGAGCGCTA
1072


 


RB16-17
TCCTCATCGACCGCTTCACCGAAACGTACGATGCGGGCCT
1073


 


RB16-87
AGCGCCGCCGTGGCTCTGGACGCCGTCAACTATCGCGATA
1074


 


RB16-88
CCCAATCCACAGGGGGTGGTTATCGCGATAGTTGACGGCG
1075


 


RB17-82
TGTCGACACGAGCGCGTGGAAGGTGACGGCCACGGAGGTT
1076


 


RB17-83
TCCACGCGCTCGTGTCGACACAGTTTGGCCACACGGTCAA
1077


 


RB17-87
AGCGCCGCCGTGGCTCTGGACGCCGTCAACTATCGCGATA
1078


 


RB17-88
CCCAATCCACAGGGGGTGGTTATCGCGATAGTTGACGGCG
1079


 


RB18-71
GGTCGTACGCCTCGTTCTTGACAGCCATGGCCCGTCGGGC
1080


 


RB18-72
ACGAGGGGGGCACGATGCCAGCCCGACGGGCCATGGCTGT
1081


 


RB18-87
AGCGCCGCCGTGGCTCTGGACGCCGTCAACTATCGCGATA
1082


 


RB18-88
CCCAATCCACAGGGGGTGGTTATCGCGATAGTTGACGGCG
1083


 


RB19-16
GGTGAAGCGGTCGATGAGGATCTTGAAGACAAGAGCGCTA
1084


 


RB19-17
TCCTCATCGACCGCTTCACCGAAACGTACGATGCGGGCCT
1085


 


RB19-71
GGTCGTACGCCTCGTTCTTGACAGCCATGGCCCGTCGGGC
1086


 


RB19-72
ACGAGGGGGGCACGATGCCAGCCCGACGGGCCATGGCTGT
1087


 


RB19-87
AGCGCCGCCGTGGCTCTGGACGCCGTCAACTATCGCGATA
1088


 


RB19-88
CCCAATCCACAGGGGGTGGTTATCGCGATAGTTGACGGCG
1089


 


RB20-16
GGTGAAGCGGTCGATGAGGATCTTGAAGACAAGAGCGCTA
1090


 


RB20-17
TCCTCATCGACCGCTTCACCGAAACGTACGATGCGGGCCT
1091


 


RB20-69
CGCAACAGCGGCACTCCGCTGTCTGCGGTTCACCTGACGT
1092


 


RB20-70
CAAGAACGAGGCGTACGACCACGTCAGGTGAACCGCAGAC
1093


 


RB20-71
GGTCGTACGCCTCGTTCTTGACAGCCATGGCCCGTCGGGC
1094


 


RB20-72
ACGAGGGGGGCACGATGCCAGCCCGACGGGCCATGGCTGT
1095


 


RB20-87
AGCGCCGCCGTGGCTCTGGACGCCGTCAACTATCGCGATA
1096


 


RB20-88
CCCAATCCACAGGGGGTGGTTATCGCGATAGTTGACGGCG
1097









Variants were purified from large-scale fermentation, i.e., 100 ml or 500 ml fermentation, and PIs of thermal stability (Ts) and specific activities were determined. Specifically, specific activities were determined using different substrates, including DP2, DP3, DP4, DP5, DP6, DP7, cornstarch (CS), and liquefact (Liq). PIs are presented in Table 8. “N/D” in Table 8 stands for “not done.”







TABLE 8







PIs of representative combinatorial variants

















Variant
Mutations
DP2
DP3
DP4
DP5
DP6
DP7
CS
Ts
Liq.





ALL1
I43Q/D44C/L417V/E503A/Q511H/A539R
0.89
1.22
1.08
1.10
1.17
1.02
1.27
1.95
1.59


ALL2
I43Q/L417V/E503A/Q511H/A539R
1.13
1.50
1.24
1.19
1.32
1.24
1.52
1.84
1.96


ALL3
I43Q/D44C/N61I/L417V/E503A/Q511H/
0.69
1.09
0.91
0.89
0.96
0.99
1.30
1.43
1.64



A539R











ALL4
I43Q/N61I/L417V/E503A/Q511H/A539R
0.89
1.40
1.15
1.15
1.25
1.12
1.52
1.79
1.73


ALL5
I43R/L417V/E503A/Q511H/A539R
1.10
1.50
N/D
N/D
N/D
1.04
1.17
2.37
1.87


ALL6
I43R/N61I/L417V/E503A/Q511H/A539R
0.93
1.52
N/D
N/D
N/D
1.37
1.53
1.84
2.24


ALL7
I43R/L417R/E503A/A539R
1.19
1.38
1.29
1.23
1.33
1.20
1.54
1.67
1.62


ALL8
I43R/N61I/L417R/E503A/Q511H/A539R
1.07
1.56
N/D
N/D
N/D
1.08
1.14
1.84
2.11


C1
G73F/T430A/Q511H
0.82
0.80
0.95
0.95
1.06
0.94
1.07
1.84
0.96


C2
I43R/G73F/T430A
0.96
0.97
1.14
1.17
1.28
1.07
1.25
1.91
1.18


C4
G73F/T430A/E503V/Q511H
0.97
0.80
1.10
1.08
1.17
1.04
1.06
2.57
1.01


C5
D44C/G73F/N563K
0.77
0.77
0.86
0.87
0.95
0.77
0.71
2.16
0.97


C7
D44C/G73F/E503V/Q511H
0.74
0.71
0.82
0.84
0.93
0.78
0.79
2.10
0.89


C8
D44C/G73F/N563K
0.78
0.76
0.81
0.91
0.94
0.79
1.05
2.28
N/D


C9
D44C/G73F/L417R/N563K
0.82
0.86
0.96
1.07
1.08
0.96
1.00
2.57
1.22


C11
D44C/G73F/N563K
0.74
0.68
0.81
0.92
0.90
0.76
1.04
2.42
N/D


C12
I43R/T430A
1.02
1.06
1.21
1.21
1.32
1.03
1.35
1.60
1.26


C13
I43Q/T430A
0.96
0.94
1.13
1.14
1.24
1.04
1.29
1.10
1.45


C14
I43Q/T430A/Q511H
1.08
1.11
1.13
1.14
1.23
1.13
1.34
1.33
1.46


C15
D44C/L417R/N563K
0.84
0.88
0.95
1.07
1.08
0.85
1.09
2.30
0.99


CS1
L417V/T430A/A431L/Q511H/A535R/
0.78
1.19
1.35
1.29
1.42
1.28
1.44
1.56 
2.35



A539R/N563I











CS2
L417V/T430A/A431Q/Q511H/A535R/
0.96
1.39
1.46
1.42
1.52
1.60
1.77
1.56
2.32



A539R/N563I











CS3
L417V/T430A/Q511H/A535R/N563I
1.00
1.17
N/D
N/D
N/D
0.98
1.40
1.69
1.78


CS4
L417V/T430A/Q511H/A539R/N563I
1.11
1.36
N/D
N/D
N/D
1.21
1.53
1.95
1.73


LQ1
G294C/L417R/A431L
1.10
1.47
1.18
1.18
1.35
1.10
0.98
1.04
1.77


LQ2
G294C/L417V/A431Q
1.15
1.56
1.32
1.30
1.46
1.22
1.08
1.02
2.08


LQ3
G294C/L417V/A431L/Q511H
0.99
1.41
N/D
N/D
N/D
N/D
1.21
1.29
2.07


LQ4
G294C/L417R/A431Q/Q511H
1.20
1.49
1.11
1.13
1.27
1.08
1.19
1.43
1.65


LQ5
L417R/A431L/Q511H
N/D
N/D
N/D
N/D
N/D
1.02
1.21
1.56
1.88


LQ6
L417V/A431Q/Q511H
0.94
1.30
1.20
1.18
1.28
1.25
1.33
1.35
2.05


RB1
I43Q/T430A/Q511H/N61I
0.90
1.13
1.00
1.03
1.13
1.08
1.15
1.27
1.47


RB2
I43Q/T430A/Q511H/L417V
1.12
1.34
N/D
N/D
N/D
1.09
1.38
1.42
1.87


RB3
I43Q/T430A/Q511H/A431L
0.87
1.05
1.03
1.05
1.15
1.10
1.25
1.29
1.70


RB4
I43Q/T430A/Q511H/E503A
0.99
1.00
1.05
1.04
1.13
1.09
1.29
1.47
1.50


RB5
I43Q/T430A/Q511H/A539R
0.98
1.22
1.06
1.02
1.11
1.10
1.45
1.47
1.59


RB6
I43Q/T430A/Q511H/N61I/A539R
0.75
1.09
1.09
1.05
1.14
1.23
N/D
0.94
N/D


RB7
I43Q/T430A/Q511H/L417V/A539R
1.20
1.62
N/D
N/D
N/D
1.29
1.44
1.85
2.17


RB8
I43Q/T430A/Q511H/A431L/A539R
0.90
1.38
N/D
N/D
N/D
1.25
1.38
1.59
2.18


RB9
I43Q/T430A/Q511H/A431L/E503A
0.79
0.82
0.95
0.92
1.02
1.03
1.39
1.69
1.55


RB10
I43Q/T430A/Q511H/N61I/A539R/A431L
0.68
1.27
N/D
N/D
N/D
0.87
1.47
1.29
1.73


RB11
I43Q/T430A/Q511H/L417V/A539R/A431L
0.89
1.50
N/D
N/D
N/D
1.05
1.36
1.58
2.03


RB12
I43Q/Q511H/N61I
0.75
0.80
0.91
0.91
1.00
0.82
1.25
1.37
1.56


RB13
I43Q/Q511H/L417V
0.97
0.94
0.96
0.93
1.05
0.94
1.38
1.50
1.50


RB14
I43Q/Q511H/A431L
0.73
0.75
0.89
0.88
0.94
0.89
1.31
1.37
1.55


RB15
I43Q/Q511H/A539R
0.90
0.88
1.03
0.99
1.07
1.05
1.42
1.52
1.69


RB16
I43Q/Q511H/A539R/N61I
0.78
0.96
1.22
1.18
1.26
0.94
1.43
1.60
1.34


RB17
I43Q/Q511H/A539R/E503A
1.02
1.15
1.16
1.12
1.21
1.20
1.38
1.68
1.40


RB18
I43Q/Q511H/A539R/T430M
0.81
0.84
1.03
0.99
1.08
0.54
1.44
1.64
1.40


RB19
I43Q/Q511H/A539R/T430M/N61I
0.77
1.21
N/D
N/D
N/D
1.07
1.50
1.47
1.88


RB20
I43Q/Q511H/A539R/T430M/N61I/L417V
0.73
1.35
N/D
N/D
N/D
0.96
1.55
1.79
1.95


TS1
I43R/T430A/E503V/A535R/N563K
1.09
0.95
1.13
1.09
1.17
1.19
N/D
2.13
N/D


TS2
D44R/E503A/Q511H/N563I
0.80
0.70
0.88
0.89
0.96
0.85
1.14
1.53
1.23


TS3
E503A/N563I
0.93
0.73
0.91
0.88
0.96
0.92
1.17
1.39
1.04


TS4
I43R/T430A/E503A/Q511H/N563K
0.92
0.77
0.95
0.89
1.02
1.06
1.33
1.66
1.33


TS5
D44R/T430A/Q511H/A535R
0.74
0.68
0.83
0.83
0.91
0.91
0.95
1.59
1.21


Var1
L417V/A431L/A539R
0.69
1.06
N/D
1.12
N/D
1.16
1.26
1.18
1.40


Var2
L417V/A431L/A539R/I43Q
0.69
1.13
N/D
1.15
N/D
1.18
1.40
1.12
1.53


Var3
L417V/A431L/A539R/N61I
0.43
0.91
N/D
0.99
N/D
1.06
1.29
1.14
1.38


Var4
L417V/A431L/A539R/A535R
0.64
0.97
N/D
1.03
N/D
1.07
1.22
1.49
1.22


Var5
L417V/A431L/A539R/I43Q/N61I
0.38
0.86
N/D
0.94
N/D
1.04
1.25
1.07
1.24


Var6
L417V/A431L/A539R/N61I/A535R
0.32
0.72
N/D
0.82
N/D
0.90
1.16
1.86
1.15


Var7
L417V/A431L/A539R/A535R/I43Q
0.65
0.99
N/D
1.03
N/D
1.10
1.22
1.29
1.26


Var8
L417V/A431L/A539R/I43Q/N61I/A535R
0.32
0.73
N/D
0.80
N/D
0.94
1.11
1.08
1.12


Var9
L417V/A431L/A539R/143Q/N61I/
0.25
0.70
N/D
0.71
N/D
0.84
0.99
1.13
1.14



A535R/T430A











Var10
L417V/T430A/A431L/Q511H/A535R/
0.53
0.98
N/D
0.97
N/D
1.06
1.08
1.86
1.63



A539R/N563I/I43Q











Var11
L417V/T430A/A431L/Q511H/A535R/
0.38
0.80
N/D
0.86
N/D
0.95
1.16
1.51
1.15



A539R/N563I/N61I











Var12
L417V/T430A/A431L/Q511H/A535R/
0.31
0.78
N/D
0.84
N/D
0.94
1.17
1.81
1.30



A539R/N563I/I43Q/N61I











Var13
L417V/A431L/A539R/I43R
0.67
1.08
N/D
1.12
N/D
1.18
1.32 
1.83
1.55


Var14
L417V/A431L/A539R/I43R/N61I
0.43
0.99
N/D
1.01
N/D
1.09
1.24
1.70
1.48


Var15
L417V/A431L/A539R/I43R/N61I/
0.39
0.94
N/D
0.96
N/D
1.09
1.34
1.96
1.59



A535R/T430A











Var16
L417R/A431L/A539R
0.70
1.01
N/D
1.03
N/D
1.11
1.23
1.33
1.31


Var17
L417G/A431L/A539R
0.70
1.15
N/D
1.14
N/D
1.19
1.20
1.23
1.42


Var18
G73F/E503V/N563K/L417R/A539R
0.93
1.14
N/D
1.12
N/D
1.11
1.08
2.71
0.98


Var19
G73F/E503V/N563K/I43R/L417R/A539R
1.24
1.47
N/D
1.43
N/D
1.33
1.38
2.58
1.21


Var20
G73F/E503V/N563K/I43R/Q511H
0.94
0.95
N/D
0.95
N/D
0.97
1.02
2.47
0.84









Example 9
Homology Between TrGA and AaGA
The crystal structure of the TrGA identified in Example 11 in WO2009/067218 (Danisco US Inc., Genencor Division) page 89-93 incorporated herein by reference was superposed on the previously identified crystal structure of the Aspergillus awamori GA (AaGA). The AaGA crystal structure was obtained from the protein database (PDB) and the form of AaGA that was crystallized was the form containing only a catalytic domain (PDB entry number: 1GLM). The structure of the TrGA with all three regions intact was determined to 1.8 Angstrom resolution herein (see Table 20 in WO2009/067218 (Danisco US Inc., Genencor Division) page 94-216 incorporated herein by reference and Example 11 in WO2009/067218 (Danisco US Inc., Genencor Division) page 89-93 incorporated herein by reference). Using the coordinates (see Table 20 in WO2009/067218 (Danisco US Inc., Genencor Division) page 94-216 incorporated herein by reference), the structure was aligned with the coordinates of the catalytic domain from Aspergillus awamori strain X100 that was determined previously (Aleshin et al., J. Mol. Biol. 238: 575-591 (1994)). As seen in FIGS. 6-7, the structure of the catalytic domain overlapped very closely and allowed the identification of equivalent residues based on the structural superposition.
Based on this analysis, sites were identified that could be mutated in TrGA and result in increased thermostability and/or specific activity. There sites include 108, 124, 175, and 316 at the active site. Also identified were specific pairwise variants Y47W/Y315F and Y47F/Y315W. Other sites identified were 143, D44, P45, D46, R122, R125, V181, E242, Y310, D313, V314, N317, R408, and N409. Because of the high structural homology, it is expected that beneficial variants found at sites in the TrGA would have similar consequence in Aspergillus awamori and other homologous glucoamylases.
The TrGA linker, residues 454-490 is defined as the segment spanning the region between two disulfide bridges, one between residues 222 and 453 and one between residues 491 and 587. Nine of the residues in the linker are prolines. From the crystal structure, the linker extends from the back of the molecule in a wide arc followed by an abrupt turn after the lysine 477 residue on the surface near the substrate binding surface. The linker extends as a random coil that is anchored by interactions of the side chains of Tyr 452, Pro 465, Phe 470, Gln 474, Pro 475, Lys 477, Val 480 and Tyr 486 to regions on the surface of the catalytic domain.
The starch binding domain is composed of a beta-sandwich of two twisted beta sheets, tethered at one end by a disulfide bridge between Cys 491 and Cys 587 and at the other end, having a series of loops that comprise a binding site for starch connected by long loops. The structure of the TrGA SBD is quite similar to the averaged structure of the AnGA SBD determined by NMR (Sorimachi et al., Structure 5: 647-661 (1997)) and the SBD of beta amylase from Bacillus cereus (Mikami, B. et al., Biochemistry 38: 7050-61 (1999)). FIG. 9 shows an alignment of the AnGA and TrGA crystal structures including the SBD. When aligned with one or both of these SBD's, one loop stands out as being highly variable, corresponding to residues 537-543 (in A. niger the loop is 554-560 and in B. cereus the loop is 462-465). In the NMR structure of beta-cyclodextrin, a starch analog complexed to the SBD of AnGA (Sorimachi et al. (1997) supra), the loop shifts substantially upon binding to cyclodextrin. Thus, this loop is designated the “flexible loop.” This flexible loop forms part of the “binding site 2” (see FIG. 9 for this binding site in TrGA). A second binding site was also identified in AnGA (binding site 1), a primary site that shares similarities with other carbohydrate binding proteins. Overall, conservation of residues and even side conformations in the binding site 1 of these SBDs is very high. The figures demonstrate the interactions in these binding sites between the SBD and the catalytic domain that serve to bind to the starch.
Taken together, there appears to be a common pattern for the interactions between the linker and SBD with the catalytic domain. The interaction is in the form of an anchoring side chain that interacts with the surface area of the neighboring domain. In general, the anchor residue is found on the linker segment. In the case of interactions between the CD and SBD, the anchor residues can be contributed from either domain as in the case of residues Ile 43 and Phe 29 that come from the CD or residue 592, which comes from the SBD.
Example 10
Model of Acarbose Binding to TrGA
The crystal structure of the TrGA complexed with the inhibitor acarbose has been determined. Crystals of the complex were obtained by soaking pre-grown native TrGA crystals in acarbose. After soaking for 3 days the crystals were mounted in a seal glass capillary tube and x-ray diffraction was collected with a Rigaku Raxis IV++ image plate detector to a resolution of 2.0 Å. The coordinates were fitted to a difference electron density map. The model was refined to an R-factor of 0.154 with an R-free of 0.201 for a total of 41276 reflection representing all data collected between 27 and 2.0 Å resolution. The model of the resulting refined structure is shown in FIG. 9.
Based on the knowledge that the presence of the SBD has an impact on hydrolysis of insoluble starch, it followed that there should be an interaction of the SBD with larger starch molecules. Thus, the structure of the TrGA was compared with known structures of (1) an acarbose bound CD of AaGA and (2) an SBD from A. niger complexed with beta-cyclodextrin. This showed that the beta-cyclodextrin bound at binding site 2 was close to the substrate location as indicated by the location of acarbose bound to the A. awamori CD. Thus, the coordinates of acarbose from the structure model of the AaGA (pdb enty1GAI, Aleshin, et al. 1994 supra) were aligned into the TrGA active site. Further, the AnGA SBD structure bound to cyclodextrin (pdb entry 1AC0: Sorimachi, et al 1997 supra) was aligned. From this, a model was made for acarbose binding to TrGA (see FIG. 9). The model showed that the SBD would localize the TrGA CD near disrupted starch, and also prevent the enzyme from diffusing away from the substrate while releasing the product from the active site after hydrolysis. The SBD of TrGA would bind to starch along site 1, and favor localization where a disrupted fragment could bind to site 2 within a loose end that points into the catalytic site (the active side for the catalytic domain). This model shows how the proposed function of the enzyme is contributed by the structure of the SBD and linker. The amino acid side chains involved in the specific interaction between the CD, the linker and the SBD are specific for Trichoderma reesei GA, however, in other glucoamylases, complementary sequence changes would enable similar overall interactions and domain juxtaposition.
Based on this model, sites were identified for which substitutions could be made in the TrGA SBD to result in increased stability and/or specific activity. Thus, two loops that are part of binding site 1 are likely candidates for alterations to increase or decrease binding to the larger starch molecule. These are loop 1 (aa 560-570) and loop 2 (aa 523-527). Because the two Trp (tryptophan) residues at amino acids 525 and 572 are likely involved directly in starch binding, they would not be as conducive to change. However, the underlying residues, including 516-518 would be conducive, as would the underlying residues 558-562. The loop from residues 570-578 is also a good candidate for alterations. Residues 534-541 are part of the binding site 2 that interacts with the catalytic site on the CD. Thus, these might be a good candidate for alterations that may increase or decrease specific activity.
Because of the high structural homology of the TrGA SBD, it is expected that beneficial variants found at sites in Trichoderma reesei GA would have similar consequences in Aspergillus awamori and other homologous glucoamylases. Thus, the structure of the TrGA SBD provides a basis for engineering this and related enzymes for altered properties as compared to a parent glucoamylase. These altered properties may be advantageous for processes in the generation of fuels based on starch feed stocks.
Example 11
Enzymes Used
Purified variant of the Trichoderma reesei glucoamulase (TrGA) with mutations D44R and A539R. The variant is expressed in Trichoderma reesei and is hereafter called BRW 1. Glucoamylase product from fermentation of Aspergillus niger, sold under the name DIAZYME® X4. Pullulanase product from Bacillus deramnificans expressed in Bacillus licheniformis, sold under the name DIAZYME® P10. Acid α-amylase product from Aspergillus kawachi expressed in Trichoderma reesei, sold under the name GC626.
Results:
Table 9 below shows the RDF values obtained with different combinations of glucoamylase, pullulanase and alpha-amylase using the above described “Malt-adjunct brew analysis”-method. The glucoamylase, pullulanase and alpha-amylase activity were measured as described above. Three replicates were made for each dose. The average RDF and standard deviation are listed. For glucoamylases the amount of glucoamylase protein added/kg of grist is listed. Additionally the corresponding activity in GAU/kg of grist is listed. For alpha-amylase and pullulanase the number of enzyme units added/kg of grist is listed together with the corresponding amount of enzyme product (GC626 and DIAZYME® P10 respectively) added/kg of grist. *DIAZYME® X4 also contains some alpha-amylase activity. The number of units of alpha-amylase added when dosing this product is also listed in the table.

















Glucoamylase
α-Amylase
Pullulanase 





added
added
added
RDF


















mg
GAU/
SSU/
g prod./
PU/
g. prod./





Enzyme
Protein
kg
kg
kg
kg
kg
Average



Dose
composition
/kg grist
grist
grist
grist
grist
grist
(n = 3)
StDev



















1
DiazymeX4
1022
2871
 1768*
—


83.3
0.047


2
TrGA
1022
4088




82.3
0.054


3
BRW1
1022
3781




83.2
0.050


4
BRW1
2044
7563




84.8
0.001


5
BRW1 +
1022
3781
28172
2.46
1961
2.06
84.1
0.098



GC626 +











Diazyme











P10










6
BRW1 +
2044
7563
56344
4.93
3907
4.11
85.1
0.169



GC626 +











Diazyme











P10









It is seen from Table 9, dose 2 and 3 that BRW1 performs better in terms of RDF obtained than TrGA. This correlates well with the fact that the BRW1 variant has a lower level of reversion activity. When dosed at 1022 mg glucoamylase protein/kg of grist BRW1 performs on level with DIAZYME® X4 (compare dose 1 and 3). Note that the glucoamylase in DIAZYME® X4 (Aspergillus niger glucoamylase) and the BRW1 glucoamylase have similar levels of reversion activity. The alpha-amylase activity present in DIAZYME® X4 probably means that the RDF value obtained is slightly higher than what pure Aspergillus niger glucoamylase would give. This only emphasizes that the BRW1 molecule performs as well if not better than Aspergillus niger glucoamylase.
When the dose of BRW1 is doubled from 1022 to 2044 mg/kg of grist, the RDF value increases from 83.2 to 84.8 (compare doses 3 and 4). RDF values can also be increased by adding auxiliary enzymes. When BRW1 is combined with alpha-amylase at 28172 SSU/kg of grist and pullulanase at 1961 PU/kg of grist the RDF value increases from 83.2 to 84.1 (compare doses 3 and 5). At high dose of BRW1 (2044 mg/kg of grist) there is also a benefit of adding auxiliary enzymes, but not as pronounced as with the low dose of BRW1 (compare differences in RDF between dose 3 and 5 and dose 4 and 6).
Sequences
Following are sequences, which are herein incorporated by reference in their entirety.








SEQ ID NO: 1: Trichoderma reesei glucoamylase, full length; with signal peptide



<210>    1 


<211>  632


<212> PRT


<213> Trichoderma reesei


 


<400>    1 


Met His Val Leu Ser Thr Ala Val Leu Leu Gly Ser Val Ala Val Gln


1               5                   10                  15 


 


Lys Val Leu Gly Arg Pro Gly Ser Ser Gly Leu Ser Asp Val Thr Lys


            20                  25                  30 


 


Arg Ser Val Asp Asp Phe Ile Ser Thr Glu Thr Pro Ile Ala Leu Asn


        35                  40                  45 


 


Asn Leu Leu Cys Asn Val Gly Pro Asp Gly Cys Arg Ala Phe Gly Thr


    50                  55                  60 


 


Ser Ala Gly Ala Val Ile Ala Ser Pro Ser Thr Ile Asp Pro Asp Tyr


65                  70                  75                  80 


 


Tyr Tyr Met Trp Thr Arg Asp Ser Ala Leu Val Phe Lys Asn Leu Ile


                85                  90                  95 


 


Asp Arg Phe Thr Glu Thr Tyr Asp Ala Gly Leu Gln Arg Arg Ile Glu


            100                 105                 110 


 


Gln Tyr Ile Thr Ala Gln Val Thr Leu Gln Gly Leu Ser Asn Pro Ser


        115                 120                 125 


 


Gly Ser Leu Ala Asp Gly Ser Gly Leu Gly Glu Pro Lys Phe Glu Leu


    130                 135                 140 


 


Thr Leu Lys Pro Phe Thr Gly Asn Trp Gly Arg Pro Gln Arg Asp Gly


145                 150                 155                 160 


 


Pro Ala Leu Arg Ala Ile Ala Leu Ile Gly Tyr Ser Lys Trp Leu Ile


                165                 170                 175 


 


Asn Asn Asn Tyr Gln Ser Thr Val Ser Asn Val Ile Trp Pro Ile Val


            180                 185                 190 


 


Arg Asn Asp Leu Asn Tyr Val Ala Gln Tyr Trp Asn Gln Thr Gly Phe


        195                 200                 205 


 


Asp Leu Trp Glu Glu Val Asn Gly Ser Ser Phe Phe Thr Val Ala Asn


    210                 215                 220 


 


Gln His Arg Ala Leu Val Glu Gly Ala Thr Leu Ala Ala Thr Leu Gly


225                 230                 235                 240 


 


Gln Ser Gly Ser Ala Tyr Ser Ser Val Ala Pro Gln Val Leu Cys Phe


                245                 250                 255 


 


Leu Gln Arg Phe Trp Val Ser Ser Gly Gly Tyr Val Asp Ser Asn Ile


            260                 265                 270 


 


Asn Thr Asn Glu Gly Arg Thr Gly Lys Asp Val Asn Ser Val Leu Thr


        275                 280                 285 


 


Ser Ile His Thr Phe Asp Pro Asn Leu Gly Cys Asp Ala Gly Thr Phe


    290                 295                 300 


 


Gln Pro Cys Ser Asp Lys Ala Leu Ser Asn Leu Lys Val Val Val Asp


305                 310                 315                 320 


 


Ser Phe Arg Ser Ile Tyr Gly Val Asn Lys Gly Ile Pro Ala Gly Ala


                325                 330                 335 


 


Ala Val Ala Ile Gly Arg Tyr Ala Glu Asp Val Tyr Tyr Asn Gly Asn


            340                 345                 350 


 


Pro Trp Tyr Leu Ala Thr Phe Ala Ala Ala Glu Gln Leu Tyr Asp Ala


        355                 360                 365 


 


Ile Tyr Val Trp Lys Lys Thr Gly Ser Ile Thr Val Thr Ala Thr Ser


    370                 375                 380 


 


Leu Ala Phe Phe Gln Glu Leu Val Pro Gly Val Thr Ala Gly Thr Tyr


385                 390                 395                 400 


 


Ser Ser Ser Ser Ser Thr Phe Thr Asn Ile Ile Asn Ala Val Ser Thr


                405                 410                 415 


 


Tyr Ala Asp Gly Phe Leu Ser Glu Ala Ala Lys Tyr Val Pro Ala Asp


            420                 425                 430 


 


Gly Ser Leu Ala Glu Gln Phe Asp Arg Asn Ser Gly Thr Pro Leu Ser


        435                 440                 445 


 


Ala Leu His Leu Thr Trp Ser Tyr Ala Ser Phe Leu Thr Ala Thr Ala


    450                 455                 460 


 


Arg Arg Ala Gly Ile Val Pro Pro Ser Trp Ala Asn Ser Ser Ala Ser


465                 470                 475                 480 


 


Thr Ile Pro Ser Thr Cys Ser Gly Ala Ser Val Val Gly Ser Tyr Ser


                485                 490                 495 


 


Arg Pro Thr Ala Thr Ser Phe Pro Pro Ser Gln Thr Pro Lys Pro Gly


            500                 505                 510 


 


Val Pro Ser Gly Thr Pro Tyr Thr Pro Leu Pro Cys Ala Thr Pro Thr


        515                 520                 525 


 


Ser Val Ala Val Thr Phe His Glu Leu Val Ser Thr Gln Phe Gly Gln


    530                 535                 540 


 


Thr Val Lys Val Ala Gly Asn Ala Ala Ala Leu Gly Asn Trp Ser Thr


545                 550                 555                 560 


 


Ser Ala Ala Val Ala Leu Asp Ala Val Asn Tyr Ala Asp Asn His Pro


                565                 570                 575 


 


Leu Trp Ile Gly Thr Val Asn Leu Glu Ala Gly Asp Val Val Glu Tyr


            580                 585                 590 


 


Lys Tyr Ile Asn Val Gly Gln Asp Gly Ser Val Thr Trp Glu Ser Asp


        595                 600                 605 


 


Pro Asn His Thr Tyr Thr Val Pro Ala Val Ala Cys Val Thr Gln Val


    610                 615                 620 


 


Val Lys Glu Asp Thr Trp Gln Ser


625                 630 


 


SEQ ID NO: 2: Trichoderma reesei glucoamylase, mature protein; without signal


peptide


<210>    2


<211>  599


<212> PRT


<213> Trichoderma reesei


 


<400>    2


Ser Val Asp Asp Phe Ile Ser Thr Glu Thr Pro Ile Ala Leu Asn Asn


1               5                   10                  15 


 


Leu Leu Cys Asn Val Gly Pro Asp Gly Cys Arg Ala Phe Gly Thr Ser


            20                  25                  30 


 


Ala Gly Ala Val Ile Ala Ser Pro Ser Thr Ile Asp Pro Asp Tyr Tyr


        35                  40                  45 


 


Tyr Met Trp Thr Arg Asp Ser Ala Leu Val Phe Lys Asn Leu Ile Asp


    50                  55                  60 


 


Arg Phe Thr Glu Thr Tyr Asp Ala Gly Leu Gln Arg Arg Ile Glu Gln


65                  70                  75                  80 


 


Tyr Ile Thr Ala Gln Val Thr Leu Gln Gly Leu Ser Asn Pro Ser Gly


                85                  90                  95 


 


Ser Leu Ala Asp Gly Ser Gly Leu Gly Glu Pro Lys Phe Glu Leu Thr


            100                 105                 110 


 


Leu Lys Pro Phe Thr Gly Asn Trp Gly Arg Pro Gln Arg Asp Gly Pro


        115                 120                 125 


 


Ala Leu Arg Ala Ile Ala Leu Ile Gly Tyr Ser Lys Trp Leu Ile Asn


    130                 135                 140 


 


Asn Asn Tyr Gln Ser Thr Val Ser Asn Val Ile Trp Pro Ile Val Arg


145                 150                 155                 160 


 


Asn Asp Leu Asn Tyr Val Ala Gln Tyr Trp Asn Gln Thr Gly Phe Asp


                165                 170                 175 


 


Leu Trp Glu Glu Val Asn Gly Ser Ser Phe Phe Thr Val Ala Asn Gln


            180                 185                 190 


 


His Arg Ala Leu Val Glu Gly Ala Thr Leu Ala Ala Thr Leu Gly Gln


        195                 200                 205 


 


Ser Gly Ser Ala Tyr Ser Ser Val Ala Pro Gln Val Leu Cys Phe Leu


    210                 215                 220 


 


Gln Arg Phe Trp Val Ser Ser Gly Gly Tyr Val Asp Ser Asn Ile Asn


225                 230                 235                 240 


 


Thr Asn Glu Gly Arg Thr Gly Lys Asp Val Asn Ser Val Leu Thr Ser


                245                 250                 255 


 


Ile His Thr Phe Asp Pro Asn Leu Gly Cys Asp Ala Gly Thr Phe Gln


            260                 265                 270 


 


Pro Cys Ser Asp Lys Ala Leu Ser Asn Leu Lys Val Val Val Asp Ser


        275                 280                 285 


 


Phe Arg Ser Ile Tyr Gly Val Asn Lys Gly Ile Pro Ala Gly Ala Ala


    290                 295                 300 


 


Val Ala Ile Gly Arg Tyr Ala Glu Asp Val Tyr Tyr Asn Gly Asn Pro


305                 310                 315                 320 


 


Trp Tyr Leu Ala Thr Phe Ala Ala Ala Glu Gln Leu Tyr Asp Ala Ile


                325                 330                 335 


 


Tyr Val Trp Lys Lys Thr Gly Ser Ile Thr Val Thr Ala Thr Ser Leu


            340                 345                 350 


 


Ala Phe Phe Gln Glu Leu Val Pro Gly Val Thr Ala Gly Thr Tyr Ser


        355                 360                 365 


 


Ser Ser Ser Ser Thr Phe Thr Asn Ile Ile Asn Ala Val Ser Thr Tyr


    370                 375                 380 


 


Ala Asp Gly Phe Leu Ser Glu Ala Ala Lys Tyr Val Pro Ala Asp Gly


385                 390                 395                 400 


 


Ser Leu Ala Glu Gln Phe Asp Arg Asn Ser Gly Thr Pro Leu Ser Ala


                405                 410                 415 


 


Leu His Leu Thr Trp Ser Tyr Ala Ser Phe Leu Thr Ala Thr Ala Arg


            420                 425                 430 


 


Arg Ala Gly Ile Val Pro Pro Ser Trp Ala Asn Ser Ser Ala Ser Thr


        435                 440                 445 


 


Ile Pro Ser Thr Cys Ser Gly Ala Ser Val Val Gly Ser Tyr Ser Arg


    450                 455                 460 


 


Pro Thr Ala Thr Ser Phe Pro Pro Ser Gln Thr Pro Lys Pro Gly Val


465                 470                 475                 480 


 


Pro Ser Gly Thr Pro Tyr Thr Pro Leu Pro Cys Ala Thr Pro Thr Ser


                485                 490                 495 


 


Val Ala Val Thr Phe His Glu Leu Val Ser Thr Gln Phe Gly Gln Thr


            500                 505                 510 


 


Val Lys Val Ala Gly Asn Ala Ala Ala Leu Gly Asn Trp Ser Thr Ser


        515                 520                 525 


 


Ala Ala Val Ala Leu Asp Ala Val Asn Tyr Ala Asp Asn His Pro Leu


    530                 535                 540 


 


Trp Ile Gly Thr Val Asn Leu Glu Ala Gly Asp Val Val Glu Tyr Lys


545                 550                 555                 560 


 


Tyr Ile Asn Val Gly Gln Asp Gly Ser Val Thr Trp Glu Ser Asp Pro


                565                 570                 575 


 


Asn His Thr Tyr Thr Val Pro Ala Val Ala Cys Val Thr Gln Val Val


            580                 585                 590 


 


Lys Glu Asp Thr Trp Gln Ser


        595 


 


SEQ ID NO: 3: Trichoderma reesei glucoamylase catalytic domain, 1-453 of mature


TrGA, CD


<210>    3


<211>  453


<212> PRT


<213> Trichoderma reesei


 


<400>    3


Ser Val Asp Asp Phe Ile Ser Thr Glu Thr Pro Ile Ala Leu Asn Asn


1               5                   10                  15 


 


Leu Leu Cys Asn Val Gly Pro Asp Gly Cys Arg Ala Phe Gly Thr Ser


            20                  25                  30 


 


Ala Gly Ala Val Ile Ala Ser Pro Ser Thr Ile Asp Pro Asp Tyr Tyr


        35                  40                  45 


 


Tyr Met Trp Thr Arg Asp Ser Ala Leu Val Phe Lys Asn Leu Ile Asp


    50                  55                  60 


 


Arg Phe Thr Glu Thr Tyr Asp Ala Gly Leu Gln Arg Arg Ile Glu Gln


65                  70                  75                  80 


 


Tyr Ile Thr Ala Gln Val Thr Leu Gln Gly Leu Ser Asn Pro Ser Gly


                85                  90                  95 


 


Ser Leu Ala Asp Gly Ser Gly Leu Gly Glu Pro Lys Phe Glu Leu Thr


            100                 105                 110 


 


Leu Lys Pro Phe Thr Gly Asn Trp Gly Arg Pro Gln Arg Asp Gly Pro


        115                 120                 125 


 


Ala Leu Arg Ala Ile Ala Leu Ile Gly Tyr Ser Lys Trp Leu Ile Asn


    130                 135                 140 


 


Asn Asn Tyr Gln Ser Thr Val Ser Asn Val Ile Trp Pro Ile Val Arg


145                 150                 155                 160 


 


Asn Asp Leu Asn Tyr Val Ala Gln Tyr Trp Asn Gln Thr Gly Phe Asp


                165                 170                 175 


 


Leu Trp Glu Glu Val Asn Gly Ser Ser Phe Phe Thr Val Ala Asn Gln


            180                 185                 190 


 


His Arg Ala Leu Val Glu Gly Ala Thr Leu Ala Ala Thr Leu Gly Gln


        195                 200                 205 


 


Ser Gly Ser Ala Tyr Ser Ser Val Ala Pro Gln Val Leu Cys Phe Leu


    210                 215                 220 


 


Gln Arg Phe Trp Val Ser Ser Gly Gly Tyr Val Asp Ser Asn Ile Asn


225                 230                 235                 240 


 


Thr Asn Glu Gly Arg Thr Gly Lys Asp Val Asn Ser Val Leu Thr Ser


                245                 250                 255 


 


Ile His Thr Phe Asp Pro Asn Leu Gly Cys Asp Ala Gly Thr Phe Gln


            260                 265                 270 


 


Pro Cys Ser Asp Lys Ala Leu Ser Asn Leu Lys Val Val Val Asp Ser


        275                 280                 285 


 


Phe Arg Ser Ile Tyr Gly Val Asn Lys Gly Ile Pro Ala Gly Ala Ala


    290                 295                 300 


 


Val Ala Ile Gly Arg Tyr Ala Glu Asp Val Tyr Tyr Asn Gly Asn Pro


305                 310                 315                 320 


 


Trp Tyr Leu Ala Thr Phe Ala Ala Ala Glu Gln Leu Tyr Asp Ala Ile


                325                 330                 335 


 


Tyr Val Trp Lys Lys Thr Gly Ser Ile Thr Val Thr Ala Thr Ser Leu


            340                 345                 350 


 


Ala Phe Phe Gln Glu Leu Val Pro Gly Val Thr Ala Gly Thr Tyr Ser


        355                 360                 365 


 


Ser Ser Ser Ser Thr Phe Thr Asn Ile Ile Asn Ala Val Ser Thr Tyr


    370                 375                 380 


 


Ala Asp Gly Phe Leu Ser Glu Ala Ala Lys Tyr Val Pro Ala Asp Gly


385                 390                 395                 400 


 


Ser Leu Ala Glu Gln Phe Asp Arg Asn Ser Gly Thr Pro Leu Ser Ala


                405                 410                 415 


 


Leu His Leu Thr Trp Ser Tyr Ala Ser Phe Leu Thr Ala Thr Ala Arg


            420                 425                 430 


 


Arg Ala Gly Ile Val Pro Pro Ser Trp Ala Asn Ser Ser Ala Ser Thr


        435                 440                 445 


 


Ile Pro Ser Thr Cys


    450 


 


SEQ ID NO: 4: Trichoderma reesei glucoamylase cDNA


<210>    4


<211> 1899


<212> DNA


<213> Trichoderma reesei


 


<400>    4









atgcacgtcc tgtcgactgc ggtgctgctc ggctccgttg ccgttcaaaa ggtcctggga
60 



 


agaccaggat caagcggtct gtccgacgtc accaagaggt ctgttgacga cttcatcagc
120 


 


accgagacgc ctattgcact gaacaatctt ctttgcaatg ttggtcctga tggatgccgt
180 


 


gcattcggca catcagctgg tgcggtgatt gcatctccca gcacaattga cccggactac
240 


 


tattacatgt ggacgcgaga tagcgctctt gtcttcaaga acctcatcga ccgcttcacc
300 


 


gaaacgtacg atgcgggcct gcagcgccgc atcgagcagt acattactgc ccaggtcact
360 


 


ctccagggcc tctctaaccc ctcgggctcc ctcgcggacg gctctggtct cggcgagccc
420 


 


aagtttgagt tgaccctgaa gcctttcacc ggcaactggg gtcgaccgca gcgggatggc
480 


 


ccagctctgc gagccattgc cttgattgga tactcaaagt ggctcatcaa caacaactat
540 


 


cagtcgactg tgtccaacgt catctggcct attgtgcgca acgacctcaa ctatgttgcc
600 


 


cagtactgga accaaaccgg ctttgacctc tgggaagaag tcaatgggag ctcattcttt
660 


 


actgttgcca accagcaccg agcacttgtc gagggcgcca ctcttgctgc cactcttggc
720 


 


cagtcgggaa gcgcttattc atctgttgct ccccaggttt tgtgctttct ccaacgattc
780 


 


tgggtgtcgt ctggtggata cgtcgactcc aacatcaaca ccaacgaggg caggactggc
840 


 


aaggatgtca actccgtcct gacttccatc cacaccttcg atcccaacct tggctgtgac
900 


 


gcaggcacct tccagccatg cagtgacaaa gcgctctcca acctcaaggt tgttgtcgac
960 


 


tccttccgct ccatctacgg cgtgaacaag ggcattcctg ccggtgctgc cgtcgccatt
1020 


 


ggccggtatg cagaggatgt gtactacaac ggcaaccctt ggtatcttgc tacatttgct
1080 


 


gctgccgagc agctgtacga tgccatctac gtctggaaga agacgggctc catcacggtg
1140 


 


accgccacct ccctggcctt cttccaggag cttgttcctg gcgtgacggc cgggacctac
1200 


 


tccagcagct cttcgacctt taccaacatc atcaacgccg tctcgacata cgccgatggc
1260 


 


ttcctcagcg aggctgccaa gtacgtcccc gccgacggtt cgctggccga gcagtttgac
1320 


 


cgcaacagcg gcactccgct gtctgcgctt cacctgacgt ggtcgtacgc ctcgttcttg
1380 


 


acagccacgg cccgtcgggc tggcatcgtg cccccctcgt gggccaacag cagcgctagc
1440 


 


acgatcccct cgacgtgctc cggcgcgtcc gtggtcggat cctactcgcg tcccaccgcc
1500 


 


acgtcattcc ctccgtcgca gacgcccaag cctggcgtgc cttccggtac tccctacacg
1560 


 


cccctgccct gcgcgacccc aacctccgtg gccgtcacct tccacgagct cgtgtcgaca
1620 


 


cagtttggcc agacggtcaa ggtggcgggc aacgccgcgg ccctgggcaa ctggagcacg
1680 


 


agcgccgccg tggctctgga cgccgtcaac tatgccgata accaccccct gtggattggg
1740 


 


acggtcaacc tcgaggctgg agacgtcgtg gagtacaagt acatcaatgt gggccaagat
1800 


 


ggctccgtga cctgggagag tgatcccaac cacacttaca cggttcctgc ggtggcttgt
1860 


 


gtgacgcagg ttgtcaagga ggacacctgg cagtcgtaa
1899



 








SEQ ID NO: 5: Aspergillus awamori GA (AaGA); CD



<210>    5 


<211>  448


<212> PRT


<213> Aspergillus awamori


 


<400>    5 


Ala Thr Leu Asp Ser Trp Leu Ser Asn Glu Ala Thr Val Ala Arg Thr


1               5                   10                  15 


 


Ala Ile Leu Asn Asn Ile Gly Ala Asp Gly Ala Trp Val Ser Gly Ala


            20                  25                  30 


 


Asp Ser Gly Ile Val Val Ala Ser Pro Ser Thr Asp Asn Pro Asp Tyr


        35                  40                  45 


 


Phe Tyr Thr Trp Thr Arg Asp Ser Gly Leu Val Ile Lys Thr Leu Val


    50                  55                  60 


 


Asp Leu Phe Arg Asn Gly Asp Thr Asp Leu Leu Ser Thr Ile Glu Asn


65                  70                  75                  80 


 


Tyr Ile Ser Ser Gln Ala Ile Val Gln Gly Ile Ser Asn Pro Ser Gly


                85                  90                  95 


 


Asp Leu Ser Ser Gly Gly Leu Gly Glu Pro Lys Phe Asn Val Asp Glu


            100                 105                 110 


 


Thr Ala Tyr Thr Gly Ser Trp Gly Arg Pro Gln Arg Asp Gly Pro Ala


        115                 120                 125 


 


Leu Arg Ala Thr Ala Met Ile Gly Phe Arg Gln Trp Leu Leu Asp Asn


    130                 135                 140 


 


Gly Tyr Thr Ser Ala Ala Thr Glu Ile Val Trp Pro Leu Val Arg Asn


145                 150                 155                 160 


 


Asp Leu Ser Tyr Val Ala Gln Tyr Trp Asn Gln Thr Gly Tyr Asp Leu


                165                 170                 175 


 


Trp Glu Glu Val Asn Gly Ser Ser Phe Phe Thr Ile Ala Val Gln His


            180                 185                 190 


 


Arg Ala Leu Val Glu Gly Ser Ala Phe Ala Thr Ala Val Gly Ser Ser


        195                 200                 205 


 


Cys Ser Trp Cys Asp Ser Gln Ala Pro Gln Ile Leu Cys Tyr Leu Gln


    210                 215                 220 


 


Ser Phe Trp Thr Gly Glu Tyr Ile Leu Ala Asn Phe Asp Ser Ser Arg


225                 230                 235                 240 


 


Ser Gly Lys Asp Thr Asn Thr Leu Leu Gly Ser Ile His Thr Phe Asp


                245                 250                 255 


 


Pro Glu Ala Gly Cys Asp Asp Ser Thr Phe Gln Pro Cys Ser Pro Arg


            260                 265                 270 


 


Ala Leu Ala Asn His Lys Glu Val Val Asp Ser Phe Arg Ser Ile Tyr


        275                 280                 285 


 


Thr Leu Asn Asp Gly Leu Ser Asp Ser Glu Ala Val Ala Val Gly Arg


    290                 295                 300 


 


Tyr Pro Lys Asp Ser Tyr Tyr Asn Gly Asn Pro Trp Phe Leu Cys Thr


305                 310                 315                 320 


 


Leu Ala Ala Ala Glu Gln Leu Tyr Asp Ala Leu Tyr Gln Trp Asp Lys


                325                 330                 335 


 


Gln Gly Ser Leu Glu Ile Thr Asp Val Ser Leu Asp Phe Phe Gln Ala


            340                 345                 350 


 


Leu Tyr Ser Asp Ala Ala Thr Gly Thr Tyr Ser Ser Ser Ser Ser Thr


        355                 360                 365 


 


Tyr Ser Ser Ile Val Asp Ala Val Lys Thr Phe Ala Asp Gly Phe Val


    370                 375                 380 


 


Ser Ile Val Glu Thr His Ala Ala Ser Asn Gly Ser Leu Ser Glu Gln


385                 390                 395                 400 


 


Tyr Asp Lys Ser Asp Gly Asp Glu Leu Ser Ala Arg Asp Leu Thr Trp


                405                 410                 415 


 


Ser Tyr Ala Ala Leu Leu Thr Ala Asn Asn Arg Arg Asn Ser Val Met


            420                 425                 430 


 


Pro Pro Ser Trp Gly Glu Thr Ser Ala Ser Ser Val Pro Gly Thr Cys


        435                 440                 445 


 


SEQ ID NO: 6: Aspergillus niger (AnGA), CD


<210>    6


<211>  449


<212> PRT


<213> Aspergillus niger


 


<400>    6


Ala Thr Leu Asp Ser Trp Leu Ser Asn Glu Ala Thr Val Ala Arg Thr


1               5                   10                  15 


 


Ala Ile Leu Asn Asn Ile Gly Ala Asp Gly Ala Trp Val Ser Gly Ala


            20                  25                  30 


 


Asp Ser Gly Ile Val Val Ala Ser Pro Ser Thr Asp Asn Pro Asp Tyr


        35                  40                  45 


 


Phe Tyr Thr Trp Thr Arg Asp Ser Gly Leu Val Leu Lys Thr Leu Val


    50                  55                  60 


 


Asp Leu Phe Arg Asn Gly Asp Thr Ser Leu Leu Ser Thr Ile Glu Asn


65                  70                  75                  80 


 


Tyr Ile Ser Ala Gln Ala Ile Val Gln Gly Ile Ser Asn Pro Ser Gly


                85                  90                  95 


 


Asp Leu Ser Ser Gly Ala Gly Leu Gly Glu Pro Lys Phe Asn Val Asp


            100                 105                 110 


 


Glu Thr Ala Tyr Thr Gly Ser Trp Gly Arg Pro Gln Arg Asp Gly Pro


        115                 120                 125 


 


Ala Leu Arg Ala Thr Ala Met Ile Gly Phe Gly Gln Trp Leu Leu Asp


    130                 135                 140 


 


Asn Gly Tyr Thr Ser Thr Ala Thr Asp Ile Val Trp Pro Leu Val Arg


145                 150                 155                 160 


 


Asn Asp Leu Ser Tyr Val Ala Gln Tyr Trp Asn Gln Thr Gly Tyr Asp


                165                 170                 175 


 


Leu Trp Glu Glu Val Asn Gly Ser Ser Phe Phe Thr Ile Ala Val Gln


            180                 185                 190 


 


His Arg Ala Leu Val Glu Gly Ser Ala Phe Ala Thr Ala Val Gly Ser


        195                 200                 205 


 


Ser Cys Ser Trp Cys Asp Ser Gln Ala Pro Glu Ile Leu Cys Tyr Leu


    210                 215                 220 


 


Gln Ser Phe Trp Thr Gly Ser Phe Ile Leu Ala Asn Phe Asp Ser Ser


225                 230                 235                 240 


 


Arg Ser Gly Lys Asp Ala Asn Thr Leu Leu Gly Ser Ile His Thr Phe


                245                 250                 255 


 


Asp Pro Glu Ala Ala Cys Asp Asp Ser Thr Phe Gln Pro Cys Ser Pro


            260                 265                 270 


 


Arg Ala Leu Ala Asn His Lys Glu Val Val Asp Ser Phe Arg Ser Ile


        275                 280                 285 


 


Tyr Thr Leu Asn Asp Gly Leu Ser Asp Ser Glu Ala Val Ala Val Gly


    290                 295                 300 


 


Arg Tyr Pro Glu Asp Thr Tyr Tyr Asn Gly Asn Pro Trp Phe Leu Cys


305                 310                 315                 320 


 


Thr Leu Ala Ala Ala Glu Gln Leu Tyr Asp Ala Leu Tyr Gln Trp Asp


                325                 330                 335 


 


Lys Gln Gly Ser Leu Glu Val Thr Asp Val Ser Leu Asp Phe Phe Lys


            340                 345                 350 


 


Ala Leu Tyr Ser Asp Ala Ala Thr Gly Thr Tyr Ser Ser Ser Ser Ser


        355                 360                 365 


 


Thr Tyr Ser Ser Ile Val Asp Ala Val Lys Thr Phe Ala Asp Gly Phe


    370                 375                 380 


 


Val Ser Ile Val Glu Thr His Ala Ala Ser Asn Gly Ser Met Ser Glu


385                 390                 395                 400 


 


Gln Tyr Asp Lys Ser Asp Gly Glu Gln Leu Ser Ala Arg Asp Leu Thr


                405                 410                 415 


 


Trp Ser Tyr Ala Ala Leu Leu Thr Ala Asn Asn Arg Arg Asn Ser Val


            420                 425                 430 


 


Val Pro Ala Ser Trp Gly Glu Thr Ser Ala Ser Ser Val Pro Gly Thr


        435                 440                 445 


 


Cys


 


SEQ ID NO: 7: Aspergillus oryzae (AoGA), CD


<210>    7


<211>  450 


<212> PRT


<213> Aspergillus oryzae


 


<400>    7


Gln Ser Asp Leu Asn Ala Phe Ile Glu Ala Gln Thr Pro Ile Ala Lys


1               5                   10                  15 


 


Gln Gly Tyr Leu Asn Asn Ile Gly Ala Asp Gly Lys Leu Val Glu Gly


            20                  25                  30 


 


Ala Ala Ala Gly Ile Val Tyr Ala Ser Pro Ser Lys Ser Asn Pro Asp


        35                  40                  45 


 


Tyr Phe Tyr Thr Trp Thr Arg Asp Ala Gly Leu Thr Met Glu Glu Tyr


    50                  55                  60 


 


Ile Glu Gln Phe Ile Gly Gly Asp Ala Thr Leu Glu Ser Thr Ile Gln


65                  70                  75                  80 


 


Asn Tyr Val Asp Ser Gln Ala Asn Glu Gln Ala Val Ser Asn Pro Ser


                85                  90                  95 


 


Gly Gly Leu Ser Asp Gly Ser Gly Leu Ala Glu Pro Lys Phe Tyr Tyr


            100                 105                 110 


 


Asn Ile Ser Gln Phe Thr Asp Ser Trp Gly Arg Pro Gln Arg Asp Gly


        115                 120                 125 


 


Pro Ala Leu Arg Ala Ser Ala Leu Ile Ala Tyr Gly Asn Ser Leu Ile


    130                 135                 140 


 


Ser Ser Asp Lys Gln Ser Val Val Lys Ala Asn Ile Trp Pro Ile Tyr


145                 150                 155                 160 


 


Gln Asn Asp Leu Ser Tyr Val Gly Gln Tyr Trp Asn Gln Thr Gly Phe


                165                 170                 175 


 


Asp Leu Trp Glu Glu Val Gln Gly Ser Ser Phe Phe Thr Val Ala Val


            180                 185                 190 


 


Gln His Lys Ala Leu Val Glu Gly Asp Ala Phe Ala Lys Ala Leu Gly


        195                 200                 205 


 


Glu Glu Cys Gln Ala Cys Ser Val Ala Pro Gln Ile Leu Cys His Leu


    210                 215                 220 


 


Gln Asp Phe Trp Asn Gly Ser Ala Val Leu Ser Asn Leu Pro Thr Asn


225                 230                 235                 240 


 


Gly Arg Ser Gly Leu Asp Thr Asn Ser Leu Leu Gly Ser Ile His Thr


                245                 250                 255 


 


Phe Asp Pro Ala Ala Ala Cys Asp Asp Thr Thr Phe Gln Pro Cys Ser


            260                 265                 270 


 


Ser Arg Ala Leu Ser Asn His Lys Leu Val Val Asp Ser Phe Arg Ser


        275                 280                 285 


 


Val Tyr Gly Ile Asn Asn Gly Arg Gly Ala Gly Lys Ala Ala Ala Val


    290                 295                 300 


 


Gly Pro Tyr Ala Glu Asp Thr Tyr Gln Gly Gly Asn Pro Trp Tyr Leu


305                 310                 315                 320 


 


Thr Thr Leu Val Ala Ala Glu Leu Leu Tyr Asp Ala Leu Tyr Gln Trp


                325                 330                 335 


 


Asp Lys Gln Gly Gln Val Asn Val Thr Glu Thr Ser Leu Pro Phe Phe


            340                 345                 350 


 


Lys Asp Leu Ser Ser Asn Val Thr Thr Gly Ser Tyr Ala Lys Ser Ser


        355                 360                 365 


 


Ser Ala Tyr Glu Ser Leu Thr Ser Ala Val Lys Thr Tyr Ala Asp Gly


    370                 375                 380 


 


Phe Ile Ser Val Val Gln Glu Tyr Thr Pro Asp Gly Gly Ala Leu Ala


385                 390                 395                 400 


 


Glu Gln Tyr Ser Arg Asp Gln Gly Thr Pro Val Ser Ala Ser Asp Leu


                405                 410                 415 


 


Thr Trp Ser Tyr Ala Ala Phe Leu Ser Ala Val Gly Arg Arg Asn Gly


            420                 425                 430 


 


Thr Val Pro Ala Ser Trp Gly Ser Ser Thr Ala Asn Ala Val Pro Ser


        435                 440                 445 


 


Gln Cys


    450 


 


SEQ ID NO: 8: Humicola grisea glucoamylase (HgGA); CD


<210>    8


<211>  441 


<212> PRT


<213> Humicola grisea


 


<400>    8


Ala Ala Val Asp Thr Phe Ile Asn Thr Glu Lys Pro Ile Ala Trp Asn


1               5                   10                  15 


 


Lys Leu Leu Ala Asn Ile Gly Pro Asn Gly Lys Ala Ala Pro Gly Ala


            20                  25                  30 


 


Ala Ala Gly Val Val Ile Ala Ser Pro Ser Arg Thr Asp Pro Pro Tyr


        35                  40                  45 


 


Phe Phe Thr Trp Thr Pro Asp Ala Ala Leu Val Leu Thr Gly Ile Ile


    50                  55                  60 


 


Glu Ser Leu Gly His Asn Tyr Asn Thr Thr Leu Gln Gln Val Ser Asn


65                  70                  75                  80 


 


Pro Ser Gly Thr Phe Ala Asp Gly Ser Gly Leu Gly Glu Ala Lys Phe


                85                  90                  95 


 


Asn Val Asp Leu Thr Ala Phe Thr Gly Glu Trp Gly Arg Pro Gln Arg


            100                 105                 110 


 


Asp Gly Pro Pro Leu Arg Ala Ile Ala Leu Ile Gln Tyr Ala Lys Trp


        115                 120                 125 


 


Leu Ile Ala Asn Gly Tyr Lys Ser Thr Ala Lys Ser Val Val Trp Pro


    130                 135                 140 


 


Val Val Lys Asn Asp Leu Ala Tyr Thr Ala Gln Tyr Trp Asn Glu Thr


145                 150                 155                 160 


 


Gly Phe Asp Leu Trp Glu Glu Val Pro Gly Ser Ser Phe Phe Thr Ile


                165                 170                 175 


 


Ala Ser Ser His Arg Ala Leu Thr Glu Gly Ala Tyr Leu Ala Ala Gln


            180                 185                 190 


 


Leu Asp Thr Glu Cys Pro Pro Cys Thr Thr Val Ala Pro Gln Val Leu


        195                 200                 205 


 


Cys Phe Gln Gln Ala Phe Trp Asn Ser Lys Gly Asn Tyr Val Val Ser


    210                 215                 220 


 


Thr Ser Thr Ala Gly Glu Tyr Arg Ser Gly Lys Asp Ala Asn Ser Ile


225                 230                 235                 240 


 


Leu Ala Ser Ile His Asn Phe Asp Pro Glu Ala Gly Cys Asp Asn Leu


                245                 250                 255 


 


Thr Phe Gln Pro Cys Ser Glu Arg Ala Leu Ala Asn His Lys Ala Tyr


            260                 265                 270 


 


Val Asp Ser Phe Arg Asn Leu Tyr Ala Ile Asn Lys Gly Ile Ala Gln


        275                 280                 285 


 


Gly Lys Ala Val Ala Val Gly Arg Tyr Ser Glu Asp Val Tyr Tyr Asn


    290                 295                 300 


 


Gly Asn Pro Trp Tyr Leu Ala Asn Phe Ala Ala Ala Glu Gln Leu Tyr


305                 310                 315                 320 


 


Asp Ala Ile Tyr Val Trp Asn Lys Gln Gly Ser Ile Thr Val Thr Ser


                325                 330                 335 


 


Val Ser Leu Pro Phe Phe Arg Asp Leu Val Ser Ser Val Ser Thr Gly


            340                 345                 350 


 


Thr Tyr Ser Lys Ser Ser Ser Thr Phe Thr Asn Ile Val Asn Ala Val


        355                 360                 365 


 


Lys Ala Tyr Ala Asp Gly Phe Ile Glu Val Ala Ala Lys Tyr Thr Pro


    370                 375                 380 


 


Ser Asn Gly Ala Leu Ala Glu Gln Tyr Asp Arg Asn Thr Gly Lys Pro


385                 390                 395                 400 


 


Asp Ser Ala Ala Asp Leu Thr Trp Ser Tyr Ser Ala Phe Leu Ser Ala


                405                 410                 415 


 


Ile Asp Arg Arg Ala Gly Leu Val Pro Pro Ser Trp Arg Ala Ser Val


            420                 425                 430 


 


Ala Lys Ser Gln Leu Pro Ser Thr Cys


        435                 440 


 


SEQ ID NO: 9: Hypocrea vinosa glucoamylase (HvGA); CD


<210>    9


<211>  452


<212> PRT


<213> Hypocrea vinosa


 


<400>    9


Ser Val Asp Asp Phe Ile Asn Thr Gln Thr Pro Ile Ala Leu Asn Asn


1               5                   10                  15 


 


Leu Leu Cys Asn Val Gly Pro Asp Gly Cys Arg Ala Phe Gly Thr Ser


            20                  25                  30 


 


Ala Gly Ala Val Ile Ala Ser Pro Ser Thr Thr Asp Pro Asp Tyr Tyr


        35                  40                  45 


 


Tyr Met Trp Thr Arg Asp Ser Ala Leu Val Phe Lys Asn Ile Val Asp


    50                  55                  60 


 


Arg Phe Thr Gln Gln Tyr Asp Ala Gly Leu Gln Arg Arg Ile Glu Gln


65                  70                  75                  80 


 


Tyr Ile Ser Ala Gln Val Thr Leu Gln Gly Ile Ser Asn Pro Ser Gly


                85                  90                  95 


 


Ser Leu Ser Asp Gly Ser Gly Leu Gly Glu Pro Lys Phe Glu Leu Thr


            100                 105                 110 


 


Leu Ser Gln Phe Thr Gly Asn Trp Gly Arg Pro Gln Arg Asp Gly Pro


        115                 120                 125 


 


Ala Leu Arg Ala Ile Ala Leu Ile Gly Tyr Ser Lys Trp Leu Ile Asn


    130                 135                 140 


 


Asn Asn Tyr Gln Ser Thr Val Ser Asn Ile Ile Trp Pro Ile Val Arg


145                 150                 155                 160 


 


Asn Asp Leu Asn Tyr Val Ala Gln Tyr Trp Asn Gln Thr Gly Phe Asp


                165                 170                 175 


 


Leu Trp Glu Glu Val Asn Gly Ser Ser Phe Phe Thr Val Ala Asn Gln


            180                 185                 190 


 


His Arg Ala Leu Val Glu Gly Ala Thr Leu Ala Ala Thr Leu Gly Gln


        195                 200                 205 


 


Ser Gly Ser Thr Tyr Ser Ser Val Ala Pro Gln Ile Leu Cys Phe Leu


    210                 215                 220 


 


Gln Arg Phe Trp Val Ser Gly Gly Tyr Ile Asp Ser Asn Ile Asn Thr


225                 230                 235                 240 


 


Asn Glu Gly Arg Thr Gly Lys Asp Ala Asn Ser Leu Leu Ala Ser Ile


                245                 250                 255 


 


His Thr Phe Asp Pro Ser Leu Gly Cys Asp Ala Ser Thr Phe Gln Pro


            260                 265                 270 


 


Cys Ser Asp Lys Ala Leu Ser Asn Leu Lys Val Val Val Asp Her Phe


        275                 280                 285 


 


Arg Ser Ile Tyr Gly Val Asn Lys Gly Ile Pro Ala Gly Ser Ala Val


    290                 295                 300 


 


Ala Ile Gly Arg Tyr Pro Glu Asp Val Tyr Phe Asn Gly Asn Pro Trp


305                 310                 315                 320 


 


Tyr Leu Ala Thr Phe Ala Ala Ala Glu Gln Leu Tyr Asp Ser Val Tyr


                325                 330                 335 


 


Val Trp Lys Lys Thr Gly Ser Ile Thr Val Thr Ser Thr Ser Ser Ala


            340                 345                 350 


 


Phe Phe Gln Glu Leu Val Pro Gly Val Ala Ala Gly Thr Tyr Ser Ser


        355                 360                 365 


 


Ser Gln Ser Thr Phe Thr Ser Ile Ile Asn Ala Ile Ser Thr Tyr Ala


    370                 375                 380 


 


Asp Gly Phe Leu Ser Glu Ala Ala Lys Tyr Val Pro Ala Asp Gly Ser


385                 390                 395                 400 


 


Leu Ala Glu Gln Phe Asp Arg Asn Thr Gly Thr Pro Leu Ser Ala Val


                405                 410                 415 


 


His Leu Thr Trp Ser Tyr Ala Ser Phe Leu Thr Ala Ala Ala Arg Arg


            420                 425                 430 


 


Ala Gly Val Val Pro Pro Ser Trp Ala Ser Ser Gly Ala Asn Thr Val


        435                 440                 445 


 


Pro Ser Ser Cys


    450


 


SEQ ID NO: 10: TrGA, linker region


<210>   10 


<211>   37


<212> PRT


<213> Trichoderma reesei


 


<400>   10 


Ser Gly Ala Ser Val Val Gly Ser Tyr Ser Arg Pro Thr Ala Thr Ser


1               5                   10                  15 


 


Phe Pro Pro Ser Gln Thr Pro Lys Pro Gly Val Pro Ser Gly Thr Pro


            20                  25                  30 


 


Tyr Thr Pro Leu Pro


        35 


 


SEQ ID NO: 11: TrGA, SBD


<210>   11 


<211>  109


<212> PRT


 


<213> Trichoderma reesei


<400>   11 


Cys Ala Thr Pro Thr Ser Val Ala Val Thr Phe His Glu Leu Val Ser


1               5                   10                  15 


 


Thr Gln Phe Gly Gln Thr Val Lys Val Ala Gly Asn Ala Ala Ala Leu


            20                  25                  30 


 


Gly Asn Trp Ser Thr Ser Ala Ala Val Ala Leu Asp Ala Val Asn Tyr


        35                  40                  45 


 


Ala Asp Asn His Pro Leu Trp Ile Gly Thr Val Asn Leu Glu Ala Gly


    50                  55                  60 


 


Asp Val Val Glu Tyr Lys Tyr Ile Asn Val Gly Gln Asp Gly Ser Val


65                  70                  75                  80 


 


Thr Trp Glu Ser Asp Pro Asn His Thr Tyr Thr Val Pro Ala Val Ala


                85                  90                  95 


 


Cys Val Thr Gln Val Val Lys Glu Asp Thr Trp Gln Ser


            100                 105 


 


SEQ ID NO: 12 SVDDFI: start of the TrGA mature protein


<210>   12


<211>    6


<212> PRT


<213> Trichoderma reesei


 


<400>   12


Ser Val Asp Asp Phe Ile


1               5 


 


SEQ ID NO: 384 Talaromyces GA mature protein


<210>  384


<211>  588


<212> PRT


<213> Talaromyces sp.


 


<400>  384


Gly Ser Leu Asp Ser Phe Leu Ala Thr Glu Thr Pro Ile Ala Leu Gln


1               5                   10                  15 


 


Gly Val Leu Asn Asn Ile Gly Pro Asn Gly Ala Asp Val Ala Gly Ala


            20                  25                  30 


 


Ser Ala Gly Ile Val Val Ala Ser Pro Ser Arg Ser Asp Pro Asp Tyr


        35                  40                  45 


 


Phe Tyr Ser Trp Thr Arg Asp Ala Ala Leu Thr Ala Lys Tyr Leu Val


    50                  55                  60 


 


Asp Ala Phe Ile Ala Gly Asn Lys Asp Leu Glu Gln Thr Ile Gln Glu


65                  70                  75                  80 


 


Tyr Ile Ser Ala Gln Ala Gln Val Gln Thr Ile Ser Asn Pro Ser Gly


                85                  90                  95 


 


Asp Leu Ser Thr Gly Gly Leu Gly Glu Pro Lys Phe Asn Val Asn Glu


            100                 105                 110 


 


Thr Ala Phe Thr Gly Pro Trp Gly Arg Pro Gln Arg Asp Gly Pro Ala


        115                 120                 125 


 


Leu Arg Ala Thr Ala Leu Ile Ala Tyr Ala Asn Tyr Leu Ile Asp Asn


    130                 135                 140 


 


Gly Gln Ala Ser Thr Ala Asp Glu Ile Ile Trp Pro Ile Val Gln Asn


145                 150                 155                 160 


 


Asp Leu Ser Tyr Val Thr Gln Tyr Trp Asn Ser Ser Thr Phe Asp Leu


                165                 170                 175 


 


Trp Glu Glu Val Glu Gly Ser Ser Phe Phe Thr Thr Ala Val Gln His


            180                 185                 190 


 


Arg Ala Leu Val Glu Gly Asn Ala Leu Ala Thr Arg Leu Asn His Thr


        195                 200                 205 


 


Cys Pro Asn Cys Val Ser Gln Ala Pro Gln Val Leu Cys Phe Leu Gln


    210                 215                 220 


 


Ser Tyr Trp Thr Gly Ser Tyr Val Leu Ala Asn Phe Gly Gly Ser Gly


225                 230                 235                 240 


 


Arg Ser Gly Lys Asp Val Asn Ser Ile Leu Gly Ser Ile His Thr Phe


                245                 250                 255 


 


Asp Pro Ala Gly Gly Cys Asp Asp Ser Thr Phe Gln Pro Cys Ser Ala


            260                 265                 270 


 


Arg Ala Leu Ala Asn His Lys Val Val Thr Asp Ser Phe Arg Ser Val


        275                 280                 285 


 


Tyr Ala Val Asn Ser Gly Ile Ala Glu Gly Ser Ala Val Ala Val Gly


    290                 295                 300 


 


Arg Tyr Pro Glu Asp Val Tyr Gln Gly Gly Asn Pro Trp Tyr Leu Ala


305                 310                 315                 320 


 


Thr Ala Ala Ala Ala Glu Gln Leu Tyr Asp Ala Ile Tyr Gln Trp Asn


                325                 330                 335 


 


Lys Ile Gly Ser Ile Ser Ile Thr Asp Val Ser Leu Ala Phe Phe Gln


            340                 345                 350 


 


Asp Ile Tyr Pro Ser Ala Ala Val Gly Thr Tyr Asn Ser Gly Ser Ser


        355                 360                 365 


 


Thr Phe Asn Asp Ile Ile Ser Ala Val Gln Thr Tyr Ala Asp Gly Tyr


    370                 375                380 


 


Leu Ser Ile Ile Glu Lys Tyr Thr Pro Ser Asp Gly Ser Leu Thr Glu


385                 390                 395                 400 


 


Gln Phe Ser Arg Ser Asp Gly Thr Pro Leu Ser Ala Ser Gly Leu Thr


                405                 410                 415 


 


Trp Ser Tyr Ala Ser Leu Leu Thr Ala Ala Ala Arg Arg Gln Ser Ile


            420                 425                 430 


 


Val Pro Ala Ser Trp Gly Glu Ser Ser Ala Ser Ser Val Pro Ala Val


        435                 440                 445 


 


Cys Ser Ala Thr Ser Ala Thr Gly Pro Tyr Ser Thr Ala Thr Asn Thr


    450                 455                 460 


 


Ala Trp Pro Ser Ser Gly Ser Gly Pro Ser Thr Thr Thr Ser Val Pro


465                 470                 475                 480 


 


Cys Thr Thr Pro Thr Ser Val Ala Val Thr Phe Asp Glu Ile Val Ser


                485                 490                 495 


 


Thr Thr Tyr Gly Glu Thr Ile Tyr Leu Ala Gly Ser Ile Pro Glu Leu


            500                 505                 510 


 


Gly Asn Trp Ser Pro Ser Ser Ala Ile Pro Leu Arg Ala Asp Ala Tyr


        515                 520                 525 


 


Thr Ser Ser Asn Pro Leu Trp Tyr Val Thr Leu Asn Leu Pro Ala Gly


    530                 535                 540 


 


Thr Ser Phe Glu Tyr Lys Phe Phe Lys Lys Glu Thr Asp Gly Thr Ile


545                 550                 555                 560 


 


Val Trp Glu Asp Asp Pro Asn Arg Ser Tyr Thr Val Pro Ala Tyr Cys


                565                 570                 575 


 


Gly Gln Thr Thr Ala Ile Leu Asp Asp Ser Trp Gln


            580                 585 


 


SEQ ID NO: 385 Humicola grisea GA SBD


<210>  385 


<211>  112


<212> PRT


<213> Humicola grisea


 


<400>  385 


Cys Ala Asp Ala Ser Glu Val Tyr Val Thr Phe Asn Glu Arg Val Ser


1               5                   10                  15 


 


Thr Ala Trp Gly Glu Thr Ile Lys Val Val Gly Asn Val Pro Ala Leu


            20                  25                  30 


 


Gly Asn Trp Asp Thr Ser Lys Ala Val Thr Leu Ser Ala Ser Gly Tyr


        35                  40                  45 


 


Lys Ser Asn Asp Pro Leu Trp Ser Ile Thr Val Pro Ile Lys Ala Thr


    50                  55                  60 


 


Gly Ser Ala Val Gln Tyr Lys Tyr Ile Lys Val Gly Thr Asn Gly Lys


65                  70                  75                  80 


 


Ile Thr Trp Glu Ser Asp Pro Asn Arg Ser Ile Thr Leu Gln Thr Ala


                85                  90                  95 


 


Ser Ser Ala Gly Lys Cys Ala Ala Gln Thr Val Asn Asp Ser Trp Arg


            100                 105                 110 


 


SEQ ID NO: 386 Thermomyces lanuginosus GA SBD


<210>  386


<211>  109


<212> PRT


<213> Thermomyces lanuginosus


 


<400>  386


Cys Thr Pro Pro Ser Glu Val Thr Leu Thr Phe Asn Ala Leu Val Asp


1               5                   10                  15 


 


Thr Ala Phe Gly Gln Asn Ile Tyr Leu Val Gly Ser Ile Pro Glu Leu


            20                  25                  30 


 


Gly Ser Trp Asp Pro Ala Asn Ala Leu Leu Met Ser Ala Lys Ser Trp


        35                  40                  45 


 


Thr Ser Gly Asn Pro Val Trp Thr Leu Ser Ile Ser Leu Pro Ala Gly


    50                  55                  60 


 


Thr Ser Phe Glu Tyr Lys Phe Ile Arg Lys Asp Asp Gly Ser Ser Asp


65                  70                  75                  80 


 


Val Val Trp Glu Ser Asp Pro Asn Arg Ser Tyr Asn Val Pro Lys Asp


                85                  90                  95 


 


Cys Gly Ala Asn Thr Ala Thr Val Asn Ser Trp Trp Arg


            100                 105 


 


SEQ ID NO: 387 Talaromyces emersonii GA SBD


<210>  387


<211>  108


<212> PRT


<213> Talaromyces emersonii


 


<400>  387


Cys Thr Thr Pro Thr Ser Val Ala Val Thr Phe Asp Glu Ile Val Ser


1               5                   10                  15 


 


Thr Ser Tyr Gly Glu Thr Ile Tyr Leu Ala Gly Ser Ile Pro Glu Leu


            20                  25                  30 


 


Gly Asn Trp Ser Thr Ala Ser Ala Ile Pro Leu Arg Ala Asp Ala Tyr


        35                  40                  45 


 


Thr Asn Ser Asn Pro Leu Trp Tyr Val Thr Val Asn Leu Pro Pro Gly


    50                  55                  60 


 


Thr Ser Phe Glu Tyr Lys Phe Phe Lys Asn Gln Thr Asp Gly Thr Ile


65                  70                  75                  80 


 


Val Trp Glu Asp Asp Pro Asn Arg Ser Tyr Thr Val Pro Ala Tyr Cys


                85                  90                  95 


 


Gly Gln Thr Thr Ala Ile Leu Asp Asp Ser Trp Gln


            100                 105 


 


SEQ ID NO: 388 Aspergillus niger GA SBD


<210>  388


<211>  108


<212> PRT


<213> Aspergillus niger


 


<400>  388


Cys Thr Thr Pro Thr Ala Val Ala Val Thr Phe Asp Leu Thr Ala Thr


1               5                   10                  15 


 


Thr Thr Tyr Gly Glu Asn Ile Tyr Leu Val Gly Ser Ile Ser Gln Leu


            20                  25                  30 


 


Gly Asp Trp Glu Thr Ser Asp Gly Ile Ala Leu Ser Ala Asp Lys Tyr


        35                  40                  45 


 


Thr Ser Ser Asp Pro Leu Trp Tyr Val Thr Val Thr Leu Pro Ala Gly


    50                  55                  60 


 


Glu Ser Phe Glu Tyr Lys Phe Ile Arg Ile Glu Ser Asp Asp Ser Val


65                  70                  75                  80 


 


Glu Trp Glu Ser Asp Pro Asn Arg Glu Tyr Thr Val Pro Gln Ala Cys


                85                  90                  95 


 


Gly Thr Ser Thr Ala Thr Val Thr Asp Thr Trp Arg


            100                 105 


 


SEQ ID NO: 389 Aspergillus awamori GA SBD


<210>  389


<211>  108


<212> PRT


<213> Aspergillus awamori


 


<400>  389


Cys Thr Thr Pro Thr Ala Val Ala Val Thr Phe Asp Leu Thr Ala Thr


1               5                   10                  15 


 


Thr Thr Tyr Gly Glu Asn Ile Tyr Leu Val Gly Ser Ile Ser Gln Leu


            20                  25                  30 


 


Gly Asp Trp Asp Thr Ser Asp Gly Ile Ala Leu Ser Ala Asp Lys Tyr


        35                  40                  45 


 


Thr Ser Ser Asn Pro Leu Trp Tyr Val Thr Val Thr Leu Pro Ala Gly


    50                  55                  60 


 


Glu Ser Phe Glu Tyr Lys Phe Ile Arg Ile Glu Ser Asp Asp Ser Val


65                  70                  75                  80 


 


Glu Trp Glu Ser Asp Pro Asn Arg Glu Tyr Thr Val Pro Gln Ala Cys


                85                  90                  95 


 


Gly Glu Ser Thr Ala Thr Val Thr Asp Thr Trp Arg


            100                 105 


 


SEQ ID NO: 390 Thielavia terrestris GA SBD


<210>  390 


<211>  108


<212> PRT


<213> Thielavia terrestris


 


<400>  390 


Cys Ser Thr Pro Thr Ala Val Ala Val Thr Phe Asn Glu Arg Val Thr


1               5                   10                  15 


 


Thr Gln Trp Gly Gln Thr Ile Lys Val Val Gly Asp Ala Ala Ala Leu


            20                  25                  30 


 


Gly Gly Trp Asp Thr Ser Lys Ala Val Pro Leu Ser Ala Ala Gly Tyr


        35                  40                  45 


 


Thr Ala Ser Asp Pro Leu Trp Ser Gly Thr Val Asp Leu Pro Ala Gly


    50                  55                  60 


 


Leu Ala Val Gln Tyr Lys Tyr Ile Asn Val Ala Ala Asp Gly Gly Val


65                  70                  75                  80 


 


Thr Trp Glu Ala Asp Pro Asn His Ser Phe Thr Val Pro Ala Ala Cys


                85                  90                  95 


 


Gly Thr Thr Ala Val Thr Arg Asp Asp Thr Trp Gln


            100                 105 


 


SEQ ID NO: 1098 Trichoderma reesei glucoamylase variant


SVDDFISTETPIALNNLLCNVGPDGCRAFGTSAGAVIASPSTIRPDYYYMWTRDSALVFKNLIDRFTETYDAGLQRRIE


 


QYITAQVTLQGLSNPSGSLADGSGLGEPKFELTLKPFTGNWGRPQRDGPALRAIALIGYSKWLINNNYQSTVSNVIWPI


 


VRNDLNYVAQYWNQTGFDLWEEVNGSSFFTVANQHRALVEGATLAATLGQSGSAYSSVAPQVLCFLQRFWVSSGGYVDS


 


NINTNEGRTGKDVNSVLTSIHTFDPNLGCDAGTFQPCSDKALSNLKVVVDSFRSIYGVNKGIPAGAAVAIGRYAEDVYY


 


NGNPWYLATFAAAEQLYDAIYVWKKTGSITVTATSLAFFQELVPGVTAGTYSSSSSTFTNIINAVSTYADGFLSEAAKY


 


VPADGSLAEQFDRNSGTPLSALHLTWSYASFLTATARRAGIVPPSWANSSASTIPSTCSGASVVGSYSRPTATSFPPSQ


 


TPKPGVPSGTPYTPLPCATPTSVAVTFHELVSTQFGQTVKVAGNAAALGNWSTSAAVALDAVNYRDNHPLWIGTVNLEA


 


GDVVEYKYINVGQDGSVTWESDPNHTYTVPAVACVTQVVKEDTWQS


 


SEQ ID NO: 1099 Trichoderma reesei glucoamylase variant


SVDDFISTETPIALNNLLCNVGPDGCRAFGTSAGAVIASPSTIRPDYYYMWTRDSALVFKILIDRFTETYDAGLQRRIE


 


QYITAQVTLQGLSNPSGSLADGSGLGEPKFELTLKPFTGNWGRPQRDGPALRAIALIGYSKWLINNNYQSTVSNVIWPI


 


VRNDLNYVAQYWNQTGFDLWEEVNGSSFFTVANQHRALVEGATLAATLGQSGSAYSSVAPQVLCFLQRFWVSSGGYVDS


 


NINTNEGRTGKDVNSVLTSIHTFDPNLGCDAGTFQPCSDKALSNLKVVVDSFRSIYGVNKGIPAGAAVAIGRYAEDVYY


 


NGNPWYLATFAAAEQLYDAIYVWKKTGSITVTATSLAFFQELVPGVTAGTYSSSSSTFTNIINAVSTYADGFLSEAAKY


 


VPADGSLAEQFDRNSGTPLSALHLTWSYASFLTATARRAGIVPPSWANSSASTIPSTCSGASVVGSYSRPTATSFPPSQ


 


TPKPGVPSGTPYTPLPCATPTSVAVTFHELVSTQFGQTVKVAGNAAALGNWSTSAAVALDAVNYRDNHPLWIGTVNLEA


 


GDVVEYKYINVGQDGSVTWESDPNHTYTVPAVACVTQVVKEDTWQS






The foregoing applications, and all documents cited therein or during their prosecution (“appln cited documents”) and all documents cited or referenced in the appln cited documents, and all documents cited or referenced herein (“herein cited documents”), and all documents cited or referenced in herein cited documents, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention.
Various modifications and variations of the described methods and system of the disclosure will be apparent to those skilled in the art without departing from the scope and spirit of the disclosure. Although the disclosure has been described in connection with specific representative embodiments, it should be understood that the subject matters as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the disclosure that are obvious to those skilled in the art are intended to be within the scope of the following claims.

## Claims

1. An enzymatic composition comprising:
a) at least one glucoamylase variant comprising the following amino acid substitutions:
i. D44R and A539R; or
ii. D44R, N61I and A539R,

the positions corresponding to the respective position in SEQ ID NO: 2, wherein the glucoamylase variant has at least 90% sequence identity to the entire length of the amino acid sequence set forth in SEQ ID NO: 2;
b) an alpha-amylase; and
c) a pullulanase;

wherein said enzymatic composition comprises between 0.05-10 PU of pullulanase activity per GAU of said glucoamylase variant and between 0.1-20 SSU of alpha-amylase activity per GAU of said glucoamylase variant.

2. A method for producing a wort for brewing comprising forming a mash from a grist, and contacting the mash with an enzymatic composition according to claim 1.

3. The enzymatic composition of claim 1, wherein said composition comprises between 0.1-5 PU of pullulanase activity per GAU of said glucoamylase variant and between 1-15 SSU of alpha-amylase activity per GAU of said glucoamylase variant.

4. The enzymatic composition of claim 1, wherein said composition comprises between 0.2-2 PU of pullulanase activity per GAU of said glucoamylase variant and between 2-10 SSU of alpha-amylase activity per GAU of said glucoamylase variant.

5. The enzymatic composition of claim 1, wherein said glucoamylase variant has at least 95% sequence identity to SEQ ID NO: 2.

6. The enzymatic composition of claim 1, wherein said glucoamylase comprises SEQ ID NO: 1098 or SEQ ID NO: 1099.

7. The enzymatic composition of claim 1, wherein said glucoamylase variant exhibits a reduced ratio between isomaltose synthesis and starch hydrolysis activity (IS/SH ratio) as compared to the glucoamylase of SEQ ID NO: 2.

8. The enzymatic composition of claim 1, wherein said glucoamylase variant exhibits an enhanced real degree of fermentation as compared to the glucoamylase of SEQ ID NO: 2.

9. The method of claim 2, wherein said enzymatic composition comprises between 0.1-5 PU of pullulanase activity per GAU of said glucoamylase variant and between 1-15 SSU of alpha-amylase activity per GAU of said glucoamylase variant.

10. The method of claim 2, wherein said enzymatic composition comprises between 0.2-2 PU of pullulanase activity per GAU of said glucoamylase variant and between 2-10 SSU of alpha-amylase activity per GAU of said glucoamylase variant.

11. The method of claim 2, wherein said glucoamylase variant has at least 95% sequence identity to SEQ ID NO: 2.

12. The method of claim 2, wherein said glucoamylase comprises SEQ ID NO: 1098 or SEQ ID NO: 1099.

13. The method of claim 2, wherein said glucoamylase variant exhibits a reduced ratio between isomaltose synthesis and starch hydrolysis activity (IS/SH ratio) as compared to the glucoamylase of SEQ ID NO: 2.

14. The method of claim 2, wherein said glucoamylase variant exhibits an enhanced real degree of fermentation as compared to the glucoamylase of SEQ ID NO: 2.