Source: https://patents.google.com/patent/US20080241901A1/en
Timestamp: 2018-08-19 10:51:26
Document Index: 473771084

Matched Legal Cases: ['application No. 60', 'ARTE1', 'ARTE2', 'ARTE3', 'ARTE1', 'ARTE2', 'ARTE3']

US20080241901A1 - Hybrid Enzymes - Google Patents
US20080241901A1
US20080241901A1 US11953933 US95393307A US2008241901A1 US 20080241901 A1 US20080241901 A1 US 20080241901A1 US 11953933 US11953933 US 11953933 US 95393307 A US95393307 A US 95393307A US 2008241901 A1 US2008241901 A1 US 2008241901A1
US11953933
US7749744B2 (en )
This application is a division of U.S. patent application Ser. No. 11/490,949, filed Jul. 21, 2006 which is a continuation of U.S. patent application Ser. No. 10/974,508, filed on Oct. 27, 2004, which claims the benefit under 35 U.S.C. 119 of U.S. provisional application No. 60/515,017 filed Oct. 28, 2003, the contents of which are incorporated herein by reference.
The present invention relates, inter alia, to a hybrid between at least one carbohydrate-binding module (“CBM”) and at least the catalytic module (CM) of a glucoamylase. The invention also relates to the use of the hybrid enzyme in a starch process in which granular starch is degraded into sugars, e.g., a syrup, or which may be use as nutrient for yeasts in the production of a fermentation product, such as especially ethanol.
Granular starch consists of microscopic granules, which are insoluble in water at room temperature. When an aqueous starch slurry is heated, the granules swell and eventually burst, dispersing the starch molecules into the solution, During this “gelatinzation” process there is a dramatic increase in viscosity. As the solids level is 30-40% in a typical industrial process, the starch has to be thinned or “liquefied” so that it can be handled. This reduction in viscosity is today mostly obtained by enzymatic degradation. During the liquefaction step, the long-chained starch is degraded into smaller branched and linear units (maltodextrins) by an alpha-amylase. The liquefaction process is typically carried out at about 105-110° C. for about 5 to 10 minutes followed by about 1-2 hours at about 95° C. The temperature is then lowered to 60° C., a glucoamylase or a beta-amylase and optionally a debranching enzyme, such as an isoamylase or a pullulanase, are added and the saccharification process proceeds for about 24 to 72 hours.
The term “soluble starch hydrolysate” is understood as the soluble products of the processes of the invention and may comprise mono-, di-, and oligosaccharides, such as glucose, maltose, maltodextrins, cyclodextrins and any mixture of these. Preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%. 98% or at least 99% of the dry solids of the granular starch is converted into a soluble starch hydrolysate.
The term polypeptide “homology” is understood as the degree of identity between two sequences indicating a derivation of the first sequence from the second. The homology may suitably be determined by means of computer programs known in the art such as GAP provided in the GCG program package (Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wis., USA 53711) (Needleman, S. B. and Wunsch, C. D., (1970), Journal of Molecular Biology, 48, 443-453. The following settings for amino acid sequence comparison are used, GAP creation penalty of 3.0 and GAP extension penalty of 0.1.
In the ratter formula, A-CBM is the N-terminal or the C-terminal region of an amino acid sequence comprising at least the carbohydrate-binding module (CBM) per se. MR is the middle region (the “linker”), and X is the sequence of amino acid residues of a polypeptide encoded by a DNA sequence encoding the enzyme (or other protein) to which the CBM is to be linked.
TTTTTTAAATSTSKATTSSSSSSAAATTSSS, (SEQ ID NO: 21)
STGATSPGGSSGS, (SEQ ID NO: 27)
PEPTPEPT. (SEQ ID NO 22)
The tinker may also be fragments of the above linkers.
In another preferred embodiment the hybrid enzymes has a linker sequence which differs from the amino acid sequence shown in SEQ ID NO. 20, SEQ ID NO: 21, SEQ ID NO: 27, or SEQ ID NO: 22 in no more than 10 positions, no more than 9 positions, no more than 8 positions, no more than 7 positions, no more than 6 positions, no more than 5 positions, no more than 4 positions, no more than 3 positions, no more than 2 positions, or even no more than 1 position.
CBMs may be found as integral parts of large polypeptides or proteins consisting of two or more polypeptide amino acid sequence regions, especially in hydrolytic enzymes (hydrolases) which typically comprise a catalytic module containing the active site for substrate hydrolysis and a carbohydrate-binding module (CBM) for binding to the carbohydrate substrate in question. Such enzymes can comprise more than one catalytic module and, e.g., one, two or three CBMs, and optionally further comprise one or more polypeptide amino acid sequence regions inking the CBM(s) with the catalytic module(s), a region of the latter type usually being denoted a “linker”. Examples of hydrolytic enzymes comprising a CBM—some of which have already been mentioned above—are cellulases, alpha-amylases, xylanases, mannanases, arabinofuranosidases, acetylesterases and chitinases. CBMs have also been found in algae, e.g., in the red alga Porphyra purpurea in the form of a non-hydrolytic polysaccharide-binding protein.
The “Carbohydrate-Binding Module of Family 20” or a CBM-20 module is in the context of this invention defined as a sequence of approximately 100 amino acids having at least 45% homology to the Carbohydrate-Binding Module (CBM) of the polypeptide disclosed in FIG. 1 by Joergensen et al (1997) in Biotechnol. Lett. 19:1027-1031. The CBM comprises the least 102 amino acids of the polypeptide, i.e. the subsequence from amino acid 582 to amino acid 683. The numbering of Glycoside Hydrolase Families applied in this disclosure follows the concept of Coutinho, P. M. & Henrissat, B. (1999) CAZy—Carbohydrate-Active Enzymes server at URL: afmb-cnrs-mrs.fr/˜cazy/CAZY/index.html or alternatively Coutinho, P. M. & Henrissat, B. 1999; The modular structure of cellulases and other carbohydrate-active enzymes: an integrated database approach. In “Genetics, Biochemistry and Ecology of Cellulose Degradation”., K. Ohmiya, K. Hayashi, K. Sakka, Y. Kobayashi, S. Karita and T, Kimura eds., Uni Publishers Co., Tokyo, pp. 15-23, and Bourne, Y. & Henrissat, B, 2001; Glycoside hydrolases and glycosyltransferases: families and functional modules, Current Opinion in Structural Biology 11:593-600.
CBMs of Carbohydrate-Binding Module Family 20 suitable for the invention may be derived from glucoamylases of Aspergillus amatory (SWISSPROT Q12537), Aspergillus kawachii (SWISSPROT P23176), Aspergillus niger (SWISSPROT P04064), Aspergillus oryzae (SWISSPROT P36914), from alpha-amylases of Aspergillus kawachii (EMBL:#AB008370), Aspergillus nidulans (NCBI AAF17100.1), from beta-amylases of Bacillus cereus (SWISSPROT P36924), or from CGTases of Bacillus circulans (SWISSPROT P43379). Preferred is a CBM from the alpha-amylase of Aspergillus kawachii (EMBL:#AB008370) as well as CBMs having at least 50%, 60%, 70%, 80%, 90%, 95%, 97% or even at least 99% homology to the CBM of the alpha-amylase of Aspergillus kawachii (EMBL:#AB008370), i.e. a CBM having at least 50%, 60%, 70%, 8:0% 90%, 95%, 97% or even at least 99% homology to the amino acid sequence of SEQ ID NO: 2. Also preferred for the invention are the CBMs of Carbohydrate-Binding Module Family 20 having the amino acid sequences shown in SEQ ID NO: 3 (Bacillus flavorthermus CBM), SEQ ID NO: 4 (Bacillus sp. CBM), and SEQ ID NO: 5 (Alcaliphilic Bacillus CBM). Further preferred CBMs include the CBMs of the glucoamylase from Hormoconis sp. such as from Hormoconis resinae (Syn. Creosote fungus or Amorphotheca resinae) such as the CBM of SWISSPROT:Q03045 (SEQ ID NO: 6), from Lentinula sp. such as from Lentinula edodes (shiitake mushroom) such as the CBM of SPTREMBL:Q9P4C5 (SEQ ID NO: 7), from Neurospora sp. such as from Neurospora crassa such as the CBM of SWISSPROT:P14804 (SEQ ID NO: 8), from Talaromyces sp. such as from Talaromyces byssochiamydioides such as the CBM of NN005220 (SEQ ID NO: 9), from Geosmithia sp. such as from Geosmithia cylindrospora, such as the CBM of NN48286 (SEQ ID NO, 10), from Scorias sp. such as from Scorias spongiosa such as the CBM of NN007096 (SEQ ID NO, 11), from Eupenicillium sp. such as from Eupenicillium ludwigii such as the CBM of NN005968 (SEQ ID NO: 12), from Aspergillus sp. such as from Aspergillus japonicus such as the CBM of NN001136 (SEQ ID NO: 13), from Penicillium sp. such as from Penicillium cf. miczynskii such as the CBM of NN48691 (SEQ ID NO: 14), from Mz1 Penicillium sp, such as the CBM of NN48690 (SEQ ID NO: 15), from Thysanophora sp. such as the CBM of NN48711 (SEQ ID NO: 16), and from Humicola sp. such as from Humicola grisea var. thermoidea such as the CBM of SPTREMBL:Q12623 (SEQ ID NO: 17). Most preferred CBMs include the CBMs of the glucoamylase from Aspergillus sp. such as from Aspergillus niger such as SEQ ID NO: 18, and Athelia sp. such as from Athelia rolfsii, such as SEQ ID NO: 19. Also preferred for the invention is any CBD having at least 50%, 60%, 70%, 80%, 90%, 95%, 97% or even at least 99% homology to any of the afore mentioned CBD amino acid sequences.
Other CBM may be found in a glucoamylase from Mucor circinelloides, Rhizopus oryzae, Arxula adeninivorans.
Glucoamylase which are suitable as the basis for CBM/glucoamylase hybrids of the present invention include, e.g., glucoamylase derived from a fungal organism, bacterium or a plant. Preferred glucoamylases are of fungal or bacterial origin, selected from the group consisting of Aspergillus glucoamylases, in particular A. niger G1 or G2 glucoamylase (Boel et al. (1984), EMBO J. 3 (5), p. 1097-1102) shown in SEQ ID NO: 24), or variants thereof, such as disclosed in WO 92/00381, WO 00/04136 add WO 01/04273 (from Novozymes, Denmark); the A. awamori glucoamylase (WO 84/02921), A. oryzae (Agric. Biol. Chem. (1991), 55 (4), p 941-949), or variants or fragments thereof. Other glucoamylases include Athelia rolfsii glucoamylase (U.S. Pat. No. 4,727,046) shown in SEQ ID NO. 26, Talaromyces glucoamylases, in particular, derived from Talaromyces emersonii (WO 99/28448) shown in SEQ ID NO: 25), Talaromyces leycettanus (U.S. Pat. No. Re. 32,153), Talaromyces duponti, Talaromyces themophilus (U.S. Pat. No. 4,587,215). Bacterial glucoamylases contemplated include glucoamylases from the genus Clostridium, in particular C. thermoamylolyticum (EP 135,138), and C. thermohydrosulfuricum (WO 86/01831). The glucoamylase may be with or without is native CBM, but comprises at least the catalytic module (CM).
A preferred glucoamylase is the A. niger glucoamylase disclosed in SEQ ID NO: 24, or a glucoamylase that has more than 50%, such as 60%, 70%, 80%, 90%. 95%, 96%, 97%, 98% or 99% homology(identity) to the amino acid sequence shown in SEQ ID NO: 24.
Another preferred glucoamylase is the Athelia rolfsii glucoamylase shown in SEQ ID NO: 26, or a glucoamylase that has more than 50%, such as 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% homology(identity) to the amino acid sequence shown in SEQ ID NO: 26.
A third preferred glucoamylase is the Talaromyces emersonii glucoamylase shown in SEQ ID NO: 25, or a glucoamylase that has more than 50%, such as 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% homology(identity) to the amino acid sequence shown in SEQ ID NO: 25.
In an aspect the invention relates to a hybrid enzyme which comprises an amino acid sequence of a catalytic module having glulcoamylase activity and an amino acid sequence of a carbohydrate-binding module. In a preferred embodiment the catalytic module is of fungal origin. In a more preferred embodiment the catalytic module is derived from a strain of Talaromyces, preferably Talaromyces emersonii, a strain of Aspergillus, preferably Aspergillus niger or a strain of Athelia, preferably Athelia rolfsii.
In another preferred embodiment the hybrid enzyme of the invention comprises a catalytic module having glucoamylase activity derived from Athelia rolfsii and a carbohydrate-binding module from Aspergillus niger or Talaromyces emersonii. The hybrid may in one embodiment include a linker sequence, preferably from Aspergillus niger, Athelia rolfsii or Talaromyces emersonii between the catalytic module and the carbohydrate-binding module.
Preferably the hybrid enzyme comprises a CBM sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 97% or even at least 99% homology to any of the amino acid sequences shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SE ID NO: 17, SE ID NO: 18, or SEQ ID NO: 19.
Even more preferred the hybrid enzyme comprises a CBM sequence having an amino acid sequence shown in SEQ ID NO: 28. In yet another preferred embodiment the CBM sequence has an amino acid sequence which differs from the amino acid sequence amino acid sequence shown in SEQ ID NO: 28, or any one of the other CBM sequences, in no more than 10 amino acid positions, no more than 9 positions, no more than 8 positions, no more than 7 positions, no more than 6 positions, no more than 5 positions, no more than 4 positions, no more than 3 positions, no more than 2 positions, or even no more than 1 position. In a most preferred embodiment the hybrid enzyme comprises a CBM derived from a glucoamylase from Athelia rolfsii, such as the AMG from Athelia rolfsii AHU 9627 described in U.S. Pat. No. 4,727,026 or the COM from Aspergillus niger.
Fusion Name CM SBM (start of SBM- underlined)
SEQ ID NO: 29 ANTE1 AN TE SSVPGTCSATSATGPYSTATNTVWPSSGSGSST
SEQ ID NO: 30 ANTE2 AN TE SSVPGTCAATSAIGTYSTATNTVWPSSGSGSST
SEQ ID NO: 31 ANTE3 AN TE SSVPGTCAATSAIGTYSSVTVTSWPSSGSGSST
SEQ ID NO: 32 ANAR1 AN AR SSVPGTCSTGATSPGGSSGSVEVTFDVYATTVY
SEQ ID NO: 33 ANAR2 AN AR SSVPGTCAATSAIGTGSSGSVEVTFDVYATTVY
SEQ ID NO: 34 ANAR3 AN AR SSVPGTCAATSAIGTYSSVTVTSWFDVYATTVY
SEQ ID NO: 35 ARTE1 AR TE GVSTSCSATSATGPYSTATNTVWPSSGSGSSTT
SEQ ID NO: 36 ARTE2 AR TE GVSTSCSTGATSPGYSTATNTVWPSSGSGSSTT
SEQ ID NO: 37 ARTE3 AR TE GVSTSCSTGATSPGGSSGSVEVTPSSGSGSSTT
SEQ ID NO: 38 ARAN1 AR AN GVSTSCAATSAIGTYSSVTVTSWPSIVATGGTT
SEQ ID NO: 39 ARAN2 AR AN GVSTSCSTGATSPGYSSVTVTSWPSIVATGGTT
SEQ ID NO: 40 ARAN3 AR AN GVSTSCSTGATSPGGSSGSVEVTPSIVATGGTT
SEQ ID NO: 41 TEAN1 TE AN SVPAVCAATSAIGTYSSVTVTSWPSIVATGGTT
SEQ ID NO: 42 TEAN2 TE AN SVPAVCSATSATGPYSSVTVTSWPSIVATGGTT
SEQ ID NO: 43 TEAN3 TE AN SVPAVCSATSATGPYSTATNTVWPSIVATGGTT
SEQ ID NO: 44 TEAR1 TE AR SSVPAVCSTGATSPGGSSGSVEVTFDVYATTVY
SEQ ID NO: 45 TEAR2 TE AR SSVPAVCSATSATGPYSSGSVEVTFDVYATTVY
SEQ ID NO: 46 TEAR3 TE AR SSVPAVCSATSATGPYSTATNTVWFDVYATTVY
CM: catalytic module; SBM: starch binding module; AN: Aspergillus niger; TE: Talaromyces emersonii; AR: Athelia rolfsii.
CM: catalytic module; SBM: starch bending module, AN: Aspergillus niger, TE: Talaromyces emersonii; AR: Athelia rolfsii,
The vectors of the present invention preferably contain one or more selectable markers, which permit easy selection of transformed cents. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.
Examples of selectable markers for use in a filamentous fungus host cell may be selected from the group including, but not limited to, amdS (acetamidase), argB (omithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hygB (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase), trpC (anthranilate synthase), and glufosinate resistance markers, as well as equivalents from other species. Preferred for use in an Aspergillus cell are the amdS and pyrG markers of Aspergillus nidulans or Aspergillus oryzae and the bar marker of Streptomyces hygroscopicus. Furthermore, selection may be accomplished by co-transformation, e.g., as described in WO 91/17243, where the selectable marker is on a separate vector.
In an even more preferred embodiment, the filamentous fungus host cell is a cell of a species of, but not limited to a cell selected from the group consisting of a strain belonging to a species of Aspergillus, preferably Aspergillus oryzae, Aspergillus niger, Aspergillus awamori, Aspergillus kawachii, or a strain of Fusarium, such as a strain of Fusarium oxysporium, Fusarium graminearum (in the perfect state named Gibberella zeae, previously Sphaeria zeae, synonym with Gibberella roseum and Gibberella roseum f. sp. cerealis), or Fusarium sulphureum (in the prefect state named Gibberella puricaris, synonym with Fusarium trichothecioides, Fusarium bactridoides, Fusarium sambucium, Fusarium roseum, and Fusarium roseum var. graminearum), Fusarium cerealis (synonym with Fusarium crookwellense), or Fusarium venenatum.
Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se, Suitable procedures for transformation of Aspergillus host cells are described in EP 238 023 and Yelton et al., 1984, Proceedings of the National Academy of Sciences USA 81: 1470-1474. Suitable methods for transforming Fusarium species are described by Malardier et al., 1989, Gene 78: 147-156 and WO 96/00787.
Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New York, Ito et al., 1983, Journal of Bacteriology 153: 163, and Hinnen et al., 1978, Proceedings of the National Academy of Sciences USA 75; 1920.
The techniques used to isolate or done a DNA sequence encoding a polypeptide of interest are known in the art and include isolation from genomic DNA, preparation from cDNA, or a combination thereof. The cloning of the DNA sequences of the present invention from such genomic DNA can be effected, e.g., by using the welt known polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments with shared structural features. See, e.g., Innis et al., 1990, PCR: A Guide to Methods and Application, Academic Press, New York. Other DNA amplification procedures such as ligase chain reaction (LCR), ligated activated transcription (LAT) and DNA sequence-based amplification (NASBA) may be used.
The alpha-amylase may according to the invention be of any origin. Preferred are alpha-amylases of fungal or bacterial origin. The alpha-amylase may be a Bacillus alpha-amylase, such as, derived from a strain of B. licheniformis, B. amyloliquefaciens, B. stearothermophilus, and B. subtilis. Other alpha-amylases include alpha-amylase derived from a strain of the Bacillus sp. NCIB 12289, NCIB 12512, NCIB 12513 or DSM 9375, all of which are described in detail in WO 95/26397, and the alpha-amylase described by Tsukamoto et al. Biochemical and Biophysical Research Communications, 151 (1988), pp. 25-31. Other alpha-amylase variants and hybrids are described in WO 96/23874, WO 97/41213, and WO 99/19467.
Other alpha-amylase includes alpha-amylases derived from a strain of Aspergillus, such as, Aspergillus oryzae and Aspergillus niger alpha-amylases. In a preferred embodiment the alpha-amylase is an acid alpha-amylase. In a more preferred embodiment the acid alpha-amylase is an acid fungal alpha-amylase or an acid bacterial alpha-amylase. More preferably, the acid alpha-amylase is an acid fungal alpha-amylase derived from the genus Aspergillus. A commercially available acid fungal amylase is SP288 (available from Novozymes A/S, Denmark). In a preferred embodiment, the alpha-amylase is an acid alpha-amylase. The term “acid alpha-amylase” means an alpha-amylase (E.C. 3.2.1.1) which added in an effective amount has activity at a pH in the range of 3.0 to 7.0, preferably from 3.5 to 6.0, or more preferably from 4.0-5.0. A preferred acid fungal alpha-amylase is a Fungamyl-like alpha-amylase. In the present disclosure, the term “Fungamyl-like alpha-amylase” indicates an alpha-amylase which exhibits a high homology, i e. more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% 90%, 95%, 96%, 97%, 98%, 99% or even 100% homology(identity) to the amino acid sequence shown in SEQ ID NO: 10 in WO 96/23874.
Preferred commercial compositions comprising alpha-amylase include MYCOLASE from DSM (Gist Brocades), BAN™, TERMAMYL™ SC, FUNGAMYL™, LIQUOZYME™ X and SAN™ SUPER, SAN™ EXTRA L (Novozymes A/S) and CLARASE™ L-40,000, DEX-LO™. SPEYME FRED, SPEZYME™ AA, and SPEZYME™ DELTA AA (Genencor Int,), and the acid fungal alpha-amylase sold under the trade name SP288 (available from Novozymes A/S, Denmark).
According to the invention the process is conducted at a temperature below the initial gelatinization temperature. Preferably the temperature at which the processes are conducted is between 30-60° C. such as at least 30° C., at least 31° C., at least 32° C., at least 33° C., at least 34° C., at least 35° C., at least 36° C., at least 37° C., at least 38° C., at least 39° C., at least 40° C., at least 41° C., at least 42° C., at least 43° C., at least 44° C., at least 45° C., at least 46° C., at least 47° C., at least 48° C., at least 49° C., at least 50° C., at least 51° C., at least 52° C., at least 53° C., at least 54° C., at least 55° C., at least 56° C., at least 57°0 C., at least 58° C., at least 59° C., or preferably at least 60° C.
The pH at which the process of the seventh aspect of the invention is conducted may in be in the range of 3.0 to 7.0, preferably from 3,5 to 6.0, or more preferably from 4.0-5.0.
The granular starch to be processed in the process of the invention may in particular be obtained from tubers, roots, stems, legumes, cereals or whole grain. More specifically the granular starch may be obtained from corns, cobs, wheat, barley, rye, milo, sago, cassava, tapioca, sorghum, rice, peas, bean, banana or potatoes. Specially contemplated are both waxy and non-waxy types of corn and barley. The granular starch to be processed may be a highly refined starch quality, preferably at least 90%, at least 95%, at least 97% or at least 99.5% pure or it may be a more crude starch containing material comprising milled whole grain including non-starch fractions such as germ residues and fibers. The raw material, such as whole grain, is milled in order to open up the structure and allowing for further processing. Two milling processes are preferred according to the invention: wet and dry milling. In dry milling the whole kernel is milled and used. Wet milling gives a good separation of germ and meal (starch granules and protein) and is with a few exceptions applied at locations where the starch hydrolysate is used in production of syrups. Both dry and wet milling is well known in the art of starch processing and is equally contemplated for the process of the invention.
In a final aspect the invention relates to the use of the hybrid enzyme having glucoamylase activity in a process for production of a fermentation product especially ethanol. The process comprises subjecting granular starch in aqueous medium to an alpha-amylase and a hybrid enzyme of the invention in the presence of a fermenting organism. The alpha-amylase may be any of the alpha-amylase, preferably one mentioned above. Preferred are acid fungal alpha-amylases, especially of Aspergillus origin.
“Fermenting organism” refers to any microorganism suitable for use in a desired fermentation process. Suitable fermenting organisms according to the invention are able to ferment, ire., convert, sugars, such as glucose and/or maltose, directly or indirectly into the desired fermentation product. Examples of fermenting organisms include fungal organisms, such as yeast Preferred yeast includes strains of the Sacchromyces spp. and in particular, Sacchromyces cerevisiae. Commercially available yeast include, e.g., RED STAR®/Lesaffre Ethanol Red (available from Red Star/Lesaffre, USA), FALI (available from Fleischmann's Yeast, a division of Burns Philp Food Inc., USA), SUPERSTART (available from Alltech), GERT STRAND (available from Gert Strand AB, Sweden) and FERMIOL (available from DSM Specialties).
Various references and a Sequence Listing are cited herein the disclosures of which are incorporated by reference in their entireties.
Acid stable alpha-amylase, an endo-alpha-amylase (1,4-alpha-D-glucan-glucano-hydrolase, E.C. 3.2.1.1) hydrolyzes alpha-1,4-glucosidic bonds in the inner regions of the starch molecule to form dextrin's and oligosaccharides with different chain lengths. The intensity of color formed with iodine is directly proportional to the concentration of starch. Amylase activity is determined using reverse colorimetry as a reduction in the concentration of starch under the specified analytical conditions.
ALPHA  -  AMYLASE STARCH + IODINE λ = 590   nm   40  ° , pft   2.5  DEXTRINS + OLIGOSACCHARIDES blue / violet   t = 23   sec .  decoloration
Glucoamylase activity may be measured in AmyloGlucosidase Units (AGU). The AGU is defined as the amount of enzyme, which hydrolyzes 1 micromole maltose per minute under the standard conditions 37° C., pH 4.3, substrate, maltose 23.2 mM, buffer: acetate 0.1 M, reaction time 5 minutes.
A folder (EB-SM-0131.02/01) describing this analytical method in more detail is available on request from Novozymes A/S, Denmark, which folder is hereby included by reference,
Unless otherwise stated, DNA manipulations and transformations were performed using standard methods of molecular biology as described in Sambrook et al. (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor lab., Cold Spring Harbor, N.Y. Ausubel, F. M. et al. (eds.) “Current protocols in Molecular Biology”, John Wiley and Sons, 1995; Harwood, C. R., and Cutting, S. M. (eds.).
EXAMPLES Example 1 Construction and Expression of Glucoamylase Catalytic Domain-Starch Binding Domain Hybrids
SEQ ID NO: 29 pANTE1 AN TE SSVPGTCSATSATGPYSTATNTVWPSSGSGSST
SEQ ID NO: 30 pANTE2 AN TE SSVPGTCAATSAIGTYSTATNTVWPSSGSGSST
SEQ ID NO: 31 pANTE3 AN TE SSVPGTCAATSAIGTYSSVTVTSWPSSGSGSST
SEQ ID NO: 32 pANAR1 AN AR SSVPGTCSTGATSPGGSSGSVEVTFDVYATTVY
SEQ ID NO: 33 pANAR2 AN AR SSVPGTCAATSAIGTGSSGSVEVTFDVYATTVY
SEQ ID NO: 34 pANAR3 AN AR SSVPGTCAATSAIGTYSSVTVTSWFDVYATTVY
SEQ ID NO: 35 pARTE1 AR TE GVSTSCSATSATGPYSTATNTVWPSSGSGSSTT
SEQ ID NO: 36 pARTE2 AR TE GVSTSCSTGATSPGYSTATNTVWPSSGSGSSTT
SEQ ID NO: 37 pARTE3 AR TE GVSTSCSTGATSPGGSSGSVEVTPSSGSGSSTT
SEQ ID NO: 38 pARAN1 AR AN GVSTSCAATSAIGTYSSVTVTSWPSIVATGGTT
SEQ ID NO: 39 pARAN2 AR AN GVSTSCSTGATSPGYSSVTVTSWPSIVATGGTT
SEQ ID NO: 40 pARAN3 AR AN GVSTSCSTGATSPGGSSGSVEVTPSIVATGGTT
SEQ ID NO: 41 pTEAN1 TE AN SVPAVCAATSAIGTYSSVTVTSWPSIVATGGTT
SEQ ID NO: 42 pTEAN2 TE AN SVPAVCSATSATGPYSSVTVTSWPSIVATGGTT
SEQ ID NO: 43 pTEAN3 TE AN SVPAVCSATSATGPYSTATNTVWPSIVATGGTT
SEQ ID NO: 44 pTEAR1 TE AR SSVPAVCSTGATSPGGSSGSVEVTFDVYATTVY
SEQ ID NO: 45 pTEAR2 TE AR SSVPAVCSATSATGPYSSGSVEVTFDVYATTVY
SEQ ID NO: 46 pTEAR3 TE AR SSVPAVCSATSATGPYSTATNTVWFDVYATTVY
E. coil DH10B (mcrA (mrr-hsdRMS-mcrBC) 80 dlacZM15 lacX74 deoR recA 1endA1 araD139 (ara, leu)7697 galU galK, rpsL nupG), Saccharomyces cerevisiae INVSc1 (MATa, his3D1, leu2, trp1-289, ura3-52) and the E. coli/yeast plasmid shuttle vector pYES2 were purchased from Invitrogen Inc. (San Diego, Calif.).
DNA manipulations were performed essentially as described in Sambrook et al., (1989) Maniatis, T, 1989. Molecular Cloning: A Laboratory Manual (2nd Edition ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., and in Dan Burke, Dean Dawson, Tim Stearns (2000) Methods in Yeast Genetics' A Cold Spring Harbor Laboratory Course Manual. Cold Spring Harbor Laboratory. Restriction endonucleases and T4 DNA ligase were from New England Biolabs. Pwo DNA polymerase (Boehringer Mannheim) was used essentially as prescribed by supplier. For SOE pcr reactions app. 100 ng of each desired pcr fragment was gelpurified, mixed together and submitted to 25 cycles of pcr without adding any primers. Reactions were separated by agarose gel electrophoresis and DNA bands migrating at the expected sizes were cut out from the gel, purified by spin columns and used as template in a new pcr reaction using the flanking primers. Plasmid pSteD226 was constructed in two steps; first the coding sequence of the Talaromyces emersonii glucoamylase (SEQ ID NO: 80) was cloned as a HindIII-XbaI fragment into pYES2 to create pSteD212. Thereafter the AgeI-HindIII fragment of pSteD212 containing the galactose inducible promoter was replaced with an AgeI-HindIII fragment containing the constitutive TPI promoter (Alber and Kawasaki (1982) Nucleotide sequence of the triose phosphate isomerase gene of Saccharomyces cerevisiae. J. Mol. Appl. Gen., 1;419-434) to create pSteD226. Plasmid pLac102 was constructed by cloning the coding sequence of the G1 form of Aspergillus niger glucoamylase (SEQ ID NO: 81) into the yeast/E. coli shuttle vector pMT742 as an EcoRI-HinDIII fragment. For amplification of CD and SBD's the following DNA templates were employed: plasmid pLAc102 carrying the cDNA encoding the G1-form of A. niger G1 glucoamylase; plasmid pSteD226 carrying the cDNA encoding the G1-form of T. emersonii glucoamylase; cDNA synthetized from A. rolfsii containing the G1-form of A. rolfsii glucoamylase.
Oligos utilized to amplify the CD and SBD pcr products are listed in table 2 and Table 3 respectively. SOE PCR products were purified by gel etectrophoresis and Qiagen spincolums, digested with HindIII/XbaI and ligated into HindIII/XbaI cut and gel-purified pSteD226. Following ligation overnight reactions were electrophorated into DH10B and plated onto LB agar plates supplemented with 100 micro g/ml ampicillin (Sigma). Transformants were plate purified and plasmids extracted for sequencing. Integrity of the entire cloned HindIII-XbaI fragment was verified by restriction analysis and DNA sequencing. Plasmids chosen were then transformed into competent yeast InvScI and plated on selective media. Yeast transformants were purified to single colonies and aliquots stored at −80° C. in 15% glycerol.
Oligos used to amplify the glucoamylase Catalytic
Domains; HinDIII site located in fwd primer
underlined; initiator ATG shown in bold.
Template Fwd primer (listed 5′-3′) Rev. primer product
pLac102 TCGTAAGCTTCACCATGTCGTTCC ACAGGTGCCGGGCACGCT AN1
GATCTCTACTCGCC GCTGGC
pLac102 TCGTAAGCTTCACCATGTCGTTCC GGTACCAATGGCAGATGT AN2
GATCTCTACTCGCC GGCCGC
pLac102 TCGTAAGCTTCACCATGTCGTTCC CCACGAGGTGACAGTCAC AN3
GATCTCTACTCGCC ACTGCTG
(SEQ ID NO: 48) (SEQ ID NO: 50)
pSteD226 TGCAAAGCTTCACCATGGCGTCCC GCAGACGGCAGGGACGCT TE1
TCGTTGCTGG GCTTGC
pSteD226 TGCAAAGCTTCACCATGGCGTCCC TGGGCCCGTGGCAGAGGT TE2
TCGTTGCTGG GGCAGAG
(SEQ ID NO: 51) (SEQ ID NO: 53)
pSteD226 TGCAAAGCTTCACCATGGCGTCCC CCAGACGGTGTTGGTAGC TE3
TCGTTGCTGG CGTGCT
(SEQ ID NO: 51) (SEQ ID NO: 54)
AR cDNA AAGAAAGCTTCACCATGTTTCGTT GCAGGAGGTAGAGACTCC AR1
CACTCCTGGCCTTGGC CTTAGCA
AR cDNA AAGAAAGCTTCACCATGTTTCGTT ACCCGGGCTTGTAGCACC AR2
CACTCCTGGCCTTGGC AGTCGAG
(SEQ ID NO: 55) (SEQ ID NO: 57)
AR cDNA AAGAAAGCTTCACCATGTTTCGTT AGTGACCTCGACACTACC AR3
CACTCCTGGCCTTGGC CGAGGAG
(SEQ ID NO: 55) (SEQ ID NO: 58)
Domains. The XbaI site located at 5′end of the reverse
Template (listed 5′-3′) (listed 5′-3′) product
pLac102 GCAAGCAGCGTCCCTGCCGTCT TAGTATCTAGATCACCGC TEANsbd1
GCGCGGCCACATCTGCCATTGG CAGGTGTCAGTCACCG
TACC (SEQ ID NO: 60)
pLac102 CTCTGCCACCTCTGCCACGGGC TAGTATCTAGATCACCGC TEANsbd2
CCATACAGCAGTGTGACTGTCA CAGGTGTCAGTCACCG
CCTCG (SEQ ID NO: 60)
pLac102 AGCACGGCTACCAACACCGTCT TAGTATCTAGATCACCGC TEANsbd3
GGCCGAGTATCGTGGCTACTGG CAGGTGTCAGTCACCG
CGGC (SEQ ID NO: 60)
pLac102 TGCTAAGGGAGTCTCTACCTCC TAGTATCTAGATCACCGC ARANsbd1
TGCGCGGCCACATCTGCCATTG CAGGTGTCAGTCACCG
GTACC (SEQ ID NO: 60)
pLac102 CTCGACTGGTGCTACAAGCCCG TAGTATCTAGATCACCGC ARANsbd2
GGTTACAGCAGTGTGACTGTCA CAGGTGTCAGTCACCG
pLac102 CTCGACTGGTGCTACAAGCCCG TAGTATCTAGATCACCGC ARANsbd3
pSteD226 TGCTAAGGGAGTCTCTACCTCC TACCTCTAGAATCGTCAC ARTEsbd1
TGCTCTGCCACCTCTGCCACGG TGCCAACTATCGTCAAGA
GCCCAT AGTT
pSteD226 CTCGACTGGTGCTACAAGCCCG TACCTCTAGAATCGTCAC ARTEsbd2
GGTTACAGCACGGCTACCAACA TGCCAACTATCGTCAAGA
CCGTC AGTT
(SEQ ID NO: 68) (SEQ ID NO: 67)
pSteD226 CTCCTCGGGTAGTGTCGAGGTC TACCTCTAGAATCGTCAC ARTEsbd3
ACTCCAAGCTCTGGCTCTGGCA TGCCAACTATCGTCAAGA
GCTCA AGTT
(SEQ ID NO: 69) (SEQ ID NO: 67)
pSteD226 GCCAGCAGCGTGCCCGGCACCT TACCTCTAGAATCGTCAC ANTEsbd1
GTTCTGCCACCTCTGCCACGGG TGCCAACTATCGTCAAGA
C AGTT
(SEQ ID NO: 70) (SEQ ID NO: 67)
pSteD226 GCGGCCACATCTGCCATTGGTA TACCTCTAGAATCGTCAC ANTEsbd2
CCTACAGCACGGCTACCAACAC TGCCAACTATCGTCAAGA
CGTC AGTT
SEQ ID NO: 71) (SEQ ID NO: 67)
pSteD226 CAGCAGTGTGACTGTCACCTCG TACCTCTAGAATCGTCAC ANTEsbd3
TGGCCAAGCTCTGGCTCTGGCA TGCCAACTATCGTCAAGA
GCTC AGTT
SEQ ID NO: 72) (SEQ ID NO: 67)
AR cDNA GCAAGCAGCGTCCCTGCCGTCT CGGCCCTCTAGAATCGTC TEARsbd1
GCTCGACTGGTGCTACAAGCCC ATTAAGATTCATCCCAAG
GGGTG TGTCTTTTTCGG
AR cDNA CTCTGCCACCTCTGCCACGGGC CGGCCCTCTAGAATCGTC TEARsbd2
CCAGGCTCCTCGGGTAGTGTCG ATTAAGATTCATCCCAAG
AGGTC TGTCTTTTTCGG
(SEQ ID NO: 75) (SEQ ID NO: 67)
AR cDNA AGCACGGCTACCAACACCGTCT CGGCCCTCTAGAATCGTC TEARsbd3
GGTTCGACGTTTACGCTACCAC ATTAAGATTCATCCCAAG
AGTAT TGTCTTTTTCGG
(SEQ ID NO: 76) (SEQ ID NO: 67)
AR cDNA GCCAGCAGCGTGCCCGGCACCT CGGCCCTCTAGAATCGTC ANARsbd1
GTTCGACTGGTGCTACAAGCCC ATTAAGATTCATCCCAAG
(SEQ ID NO: 77) (SEQ ID NO: 67)
AR cDNA GCGGCCACATCTGCCATTGGTA CGGCCCTCTAGAATCGTC ANARsbd2
CCGGCTCCTCGGGTAGTGTCGA ATTAAGATTCATCCCAAG
GGTC TGTCTTTTTCGG
(SEQ ID NO: 78) (SEQ ID NO: 67)
AR cDNA CAGCAGTGTGACTGTCACCTCG CGGCCCTCTAGAATCGTC ANARsbd3
TGGTTCGACGTTTACGCTACCA ATTAAGATTCATCCCAAG
CAGTATA TGTCTTTTTCGG
(SEQ ID NO: 79) (SEQ ID NO: 67)
SOE PCR, as described in experimental procedures, was employed to generate the desired CD-SBD fusions. PCR products combinations used in the SOS reactions and the resulting SOE hybrids are listed in Table 4.
The relative performance of glucoamylase-SBM hybrids (TEAN-1, TEAN-3) to pure Talaromyces emersonii glucoamylase was evaluated via mini-scale fermentations. About 380 g of ground corn (ground in a pilot scale hammer mill through a 1.65 mm screen) was added to about 620 g tap water, This mixture was supplemented with 3 mL 1 g/L penicillin. The pH of this slurry was adjusted to 5.0 with 40% H2SO4. The dry solid (DS) level was determined in triplicate to be 32%. Approximately 5 9 of this slurry was added to 15 mL tubes. Enzymes used in this study are detailed below:
TEAR-1 (T. emersonii catalytic module and 250%
A. rolfii SBM hybrid)
TEAR-2 (T. emersonii catalytic module and 215%
21. A hybrid enzyme which comprises an amino acid sequence of a catalytic module having glucoamylase activity and an amino acid sequence of a carbohydrate-binding module.
22. The hybrid enzyme of claim 21, wherein the amino acid sequence of the carbohydrate-binding module is of fungal origin.
23. The hybrid enzyme of claim 21, wherein the amino acid sequence of the carbohydrate-binding module is from Aspergillus sp, Athelia sp. or Talaromyces sp.
24. The hybrid enzyme of claim 21, wherein the amino acid sequence of the carbohydrate-binding module is derived from Aspergillus kawachii.
25. The hybrid enzyme of claim 21, wherein the amino acid sequence of the carbohydrate-binding module is the carbohydrate-binding module having the amino acid sequence shown in SEQ ID NO: 2.
26. The hybrid enzyme of claim 21, wherein the amino acid sequence of the carbohydrate-binding module is derived from Aspergillus niger.
27. The hybrid enzyme of claim 21, wherein the amino acid sequence of the carbohydrate-binding module is the carbohydrate-binding module having the amino acid sequence shown in SEQ ID NO: 18.
28. The hybrid enzyme of claim 21, wherein the amino acid sequence of the carbohydrate-binding module is derived from Athelia sp.
29. The hybrid enzyme of claim 21, wherein the amino acid sequence of the carbohydrate-binding module is the carbohydrate-binding module having the amino acid sequence shown in SEQ ID NO: 28.
30. The hybrid enzyme of claim 21, wherein the catalytic module is a glucoamylase of fungal origin.
31. The hybrid enzyme of claim 21, wherein the catalytic module is derived from a strain of Aspergillus.
32. The hybrid enzyme of claim 21, wherein the catalytic module is derived from a strain of Aspergillus niger or Aspergillus oryzae.
33. The hybrid enzyme of claim 21, wherein the catalytic module is the sequence shown in SEQ ID NO: 24.
34. The hybrid enzyme of claim 21, wherein the catalytic module is derived from a strain of Athelia.
35. The hybrid enzyme of claim 21, wherein the catalytic module is the sequence shown in SEQ ID NO: 26.
36. The hybrid enzyme of claim 21, wherein the catalytic module is derived from a strain of Talaromyces.
37. The hybrid enzyme of claim 21, wherein the catalytic module is the sequence shown in SEQ ID NO: 25.
38. A process of producing ethanol, comprising subjecting granular starch with a hybrid enzyme of claim 1 in aqueous medium in the presence of a fermenting organism.
39. The process of claim 38, wherein the granular starch is further subjected to an alpha-amylase treatment,
40. The process of claim 38, wherein the alpha-amylase is an acid alpha-amylase.
US11953933 2003-10-28 2007-12-11 Hybrid enzymes Active US7749744B2 (en)
US11490949 Division US7312055B2 (en) 2003-10-28 2006-07-21 Hybrid enzymes
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US11953933 Active US7749744B2 (en) 2003-10-28 2007-12-11 Hybrid enzymes
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