# Patent

## Title
Method for producing glucosidase, enzyme composition, and method for hydrolyzing biomass

## Abstract

A method for producing a mutant glucosidase includes introducing DNA encoding a secretion signal sequence and DNA encoding Asn-X-Ser or Asn-X-Thr into DNA encoding a glucosidase derived from a thermophile, and introducing the resulting DNA into a eukaryotic microorganism and expressing it as a secretory protein. An enzyme composition contains the mutant glucosidase.

## Background
RELATED APPLICATIONS
This application is a continuation of U.S. application Ser. No. 13/581,841, filed Aug. 30, 2012, which is a §371 of International Application No. PCT/JP2011/051406, with an international filing date of Jan. 26, 2011 (WO 2011/108312 A1, published Sep. 9, 2011), which is based on Japanese Patent Application No. 2010-044242, filed Mar. 1, 2010, the subject matter of which is incorporated herein by reference.
This disclosure contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy, created on Jan. 29, 2013, is named HIR12128.txt and is 175,796 bytes in size.


TECHNICAL FIELD
This disclosure relates to a method for producing a glycosylated mutant glucosidase derived from a thermophile, an enzyme composition containing this enzyme, and a method for hydrolyzing biomass using the enzyme composition.
BACKGROUND
Various techniques are available for saccharification of cellulose, among which an enzymatic saccharification method, which uses mild reaction conditions and achieves high sugar yield, has become the mainstream of development.
Cellulase, which is a cellulose degrading enzyme, is roughly classified into cellobiohydrolase, which acts on the crystalline regions of cellulose, and endoglucanase, which reduces the molecular weight by acting on within the cellulose molecular chain. These cellulases are known to be inhibited by cellobiose, which is one of the products of cellulose degradation. Meanwhile, β-glucosidase is an enzyme that acts on a water-soluble oligosaccharide or cellobiose and catalyzes a hydrolysis reaction of the β-glycosidic bond. Particularly, β-glucosidase is an enzyme necessary for the acquisition of plenty of glucose, which is useful as a fermentation raw material. Also, it is known that the reactions mediated by cellobiohydrolase or endoglucanase are inhibited by the accumulation of cellobiose, which is produced by cellulose degradation. That is, β-glucosidase has an effect of greatly improving the cellulose degradation efficiency, owing to its capability of drastically reducing the accumulation of cellobiose produced by cellulose degradation.
Cellulose is contained abundantly in herbaceous plants and woody plants, which are collectively called cellulosic biomass. Cellulosic biomass contains, in addition to cellulose, hemicellulose such as xylan and arabinan, and lignin. Particularly, being an aromatic polymer compound, lignin contained in cellulosic biomass is known to act in an inhibitory manner in the enzymatic saccharification by cellulase derived from filamentous fungi. Although the mechanism of inhibition of cellulase derived from filamentous fungi by lignin has not been entirely elucidated yet, the reduced degradation efficiency caused by adsorption of cellulase to lignin is proposed as one of the causes (P. Hetti et al., Journal of Biotechnology, 107, 65 to 72 (2004)).
A heat-resistant enzyme is highly stable and retains its activity for a long time even under high temperature conditions; therefore, the application of a heat-resistant enzyme as an industrial enzyme is under study. A large number of heat-resistant enzymes have been confirmed among the enzymes possessed by thermophiles or hyperthermophiles.
Also with regard to heat-resistant β-glucosidase, it has been identified from several species of thermophiles or hyperthermophiles. Specifically, heat-resistant β-glucosidase has been identified from organisms such as Pyrococcus furiosus, Pyrococcus horikoshii, Thermotoga maritima, Sulfolobus shibatae, and Clostridium thermocellum. 
Cellulase or β-glucosidase derived from filamentous fungi is known to be glycosylated (P. Christian et al., Trichoderma and Gliocladium: Basic Biology, Taxonomy and Genetics, Vol. 1, 121 to 138 (1998)). As a general function of a sugar chain in such a glycosylated protein, effects such as improving protein solubility, improving physical stability, and improving protease resistance are known (H. Ohba et al., Biosci. Biotech. Biochem., 59, 1581 to 1583 (1995)). As a function conferred by the possession of a sugar chain by a saccharification enzyme such as cellulase, it is disclosed that glycosylation of xylanase with N-linked sugar chains results in an increased expression level of xylanase (WO/2005/093072).
SUMMARY OF INVENTION
Technical Problem
An object of the present invention is to provide a glycosylated mutant glucosidase derived from a thermophile, and further, to provide an enzyme composition exhibiting high degradation efficiency in the process of hydrolysis of cellulose, particularly lignin-containing lignocellulose, by mixing the above glucosidase and cellulase.
Solution to Problem
The present inventors conducted an intensive research to achieve the aforementioned object. As a result, they have found that a glycosylated mutant glucosidase derived from a thermophile can be applied to cellulose degradation.
That is, the present invention is composed of the following technical means.

    (1) A method for producing a mutant glucosidase derived from a thermophile that has selectively attached thereto a sugar chain and also has a glucosidase activity, comprising:
    
        (i) preparing DNA encoding a mutant glucosidase derived from a thermophile by introducing a DNA sequence encoding Asn-X-Ser or Asn-X-Thr (wherein, X is any amino acid except proline) into DNA encoding a glucosidase derived from a thermophile that is originally devoid of a glycosylation sequence, and further, adding a DNA sequence encoding a secretion signal sequence to the DNA encoding the mutant glucosidase,
        (ii) introducing the DNA encoding the mutant glucosidase to which the DNA sequence encoding the secretion signal sequence has been added into an eukaryotic microorganism so that a mutant glucosidase encoded by the DNA of the mutant glucosidase is expressed as a secretory protein, and
        (iii) isolating and purifying the mutant glucosidase thus expressed as a secretory protein.
    
    
    (2) The method for producing a mutant glucosidase according to (1), wherein the sugar chain is a high mannose type sugar chain.
    (3) The method for producing a mutant glucosidase according to (1) or (2), wherein the glucosidase derived from a thermophile is a glucosidase derived from a thermophile selected from the group consisting of the genus Sulfolobus, the genus Thermoplasma, the genus Caldivirga, the genus Thermosphaera, the genus Pyrococcus, the genus Picrophilus, the genus Caldivirga, and the genus Fervidobacterium. 
    (4) The method for producing a glucosidase according to any of (1) to (3), wherein the glucosidase derived from a thermophile is a protein comprising:
    
        (i) a same amino acid sequence as any of amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20, or
        (ii) an amino acid sequence having 85% or more identity with any of amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20, and also a β-glucosidase activity.
    
    
    (5) The method for producing a mutant glucosidase according to any of (1) to (4), wherein the eukaryotic microorganism is Pichia pastoris. 
    (6) The method for producing a mutant glucosidase according to any of (1) to (5), wherein the secretion signal sequence is a α factor secretion signal sequence.
    (7) The method for producing a mutant glucosidase according to any of (1) to (6), wherein the mutant glucosidase derived from a thermophile comprises an amino acid sequence shown in any of SEQ ID NO: 6, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, and SEQ ID NO: 56.
    (8) An enzyme composition for saccharification of biomass comprising cellulase and the mutant glucosidase derived from a thermophile obtained by the production method according to any of (1) to (7).
    (9) The enzyme composition for saccharification of biomass according to (8), wherein the cellulase is a mixture of cellulases derived from filamentous fungi.
    (10) The enzyme composition for saccharification of biomass according to (8) or (9), wherein the mixture of cellulases derived from filamentous fungi is a mixture of cellulases derived from the genus Trichoderma. 
    (11) A method for hydrolyzing biomass, comprising using the enzyme composition according to any of (8) to (10).
    (12) The method for hydrolyzing biomass according to (11), comprising filtering a hydrolysate obtained by the aforementioned enzyme composition through an ultrafiltration membrane, and separating and recovering the used enzyme composition.


The present specification encompasses the contents described in the specification and/or drawings of JP Patent Application No. 2010-044242, based on which the present application claims priority.
Advantageous Effects of Invention
Compared to the use of a glucosidase derived from a thermophile with an unglycosylated cellulase mixture, the glycosylated mutant glucosidase derived from a thermophile obtained by the present invention can achieve higher cellulose degradation efficiency in the hydrolysis of cellulosic biomass. This effect is prominent particularly in the hydrolysis of lignocellulose. Also, the glycosylated mutant glucosidase derived from a thermophile according to the present invention has low adsorptivity for cellulosic biomass, particularly for lignocellulose, and for an ultrafiltration membrane, and also for an ultrafiltration membrane used for separation of a sugar solution from the hydrolysate, and thus achieves excellent enzyme recovery from the hydrolysate.



BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 shows an alignment of a β-glucosidase derived from Pyrococcus furiosus (PfuBGL) shown in SEQ ID NO: 4, a β-glucosidase derived from Trichoderma reesei (TriReBGL) shown in SEQ ID NO: 1, and a β-glucosidase derived from Aspergillus niger (AspNgBGL) shown in SEQ ID NO: 2 in Example 1. The glycosylation sequence in SEQ ID NO: 1 and SEQ ID NO: 2 was underlined, and the glycosylation sequence-introduction site (H60, L61, and Y62) in SEQ ID NO: 4 was similarly underlined.
FIG. 2-1 shows an alignment of the amino acid sequence of SEQ ID NO: 6 (gPfuBGL) and the amino acid sequences of SEQ ID NO: 8 (ThAggBGY), SEQ ID NO: 10 (CmGHFP), SEQ ID NO: 12 (SaBGAL), SEQ ID NO: 14 (SsoBGAL), SEQ ID NO: 16 (PtBGAL), SEQ ID NO: 18 (TvBGAL), and SEQ ID NO: 20 (FnGHFP) in Example 2. The glycosylation sequence Asn-Arg-Thr (N-R-T) inserted in the sequence of SEQ ID NO: 6 was underlined. Also, the site corresponding to the glycosylation site Asn-Arg-Thr (N-R-T) in the sequence of SEQ ID NO: 6 in SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 20 was similarly underlined.
FIG. 2-2 is a sequel to FIG. 2-1.
FIG. 3 is a diagram showing the band pattern of polyacrylamide gel electrophoresis for PfuBGL (left) prepared in Comparative Example 1 and the glycosylated mutant PfuBGL prepared in Example 2 without EndoH treatment (right) and with EndoH treatment (middle). A reduction in the molecular weight of the glycosylated mutant PfuBGL by EndoH treatment is confirmed.
FIG. 4 is a graph showing the results of evaluation of enzyme heat stability by measuring the changes in the amount of glucose produced by cellobiose degradation by PfuBGL at a heat retention time of 50 to 90° C. in Example 4.
FIG. 5 is a graph showing the results of evaluation of enzyme heat stability by measuring the changes in the amount of glucose produced by cellobiose degradation by the glycosylated mutant PfuBGL at a heat retention time of 50 to 90° C. in Example 4.
FIG. 6 is a graph showing the results of comparison of the changes in the amount of glucose produced between the case in which an enzyme composition containing a cellulase derived from the genus Trichoderma+the glycosylated mutant PfuBGL was allowed to act on the lignocellulose substrate and the case in which an enzyme composition containing a cellulase derived from the genus Trichoderma+PfuBGL was allowed to act on the lignocellulose substrate in Example 5. As the substrate, 5 wt. % lignocellulose was used, and the reactions were allowed to proceed up to 28 hours at 50° C., and the reaction product was sampled as appropriate and measured for the glucose concentration. Cellulase was added at 0.5 mg/mL and glucosidase was added at 0.005 mg/mL ( 1/100 of the amount of cellulase).
FIG. 7 is a diagram showing the basic structure of an N-linked sugar chain. In the basic structure of an N-linked sugar chain, two N-acetylglucosamine residues, and further, three mannose residues are bound to the Asn side chain of a glucosidase derived from a thermophile.
FIG. 8 is a graph showing the results of evaluation of enzyme heat stability by measuring the changes in the residual activity of AggBGY (relative value to 0 hour of heating) in cellobiose degradation at a heat retention time of 50 to 90° C. in Example 12.
FIG. 9 is a graph showing the results of evaluation of enzyme heat stability by measuring the changes in the residual activity of the glycosylated mutant AggBGY (relative value to 0 hour of heating) in cellobiose degradation at a heat retention time of 50 to 90° C. in Example 12.
FIG. 10 is a graph showing the results of evaluation of enzyme heat stability by measuring the changes in the residual activity of CmGHFP (relative value to 0 hour of heating) in cellobiose degradation at a heat retention time of 50 to 90° C. in Example 12.
FIG. 11 is a graph showing the results of evaluation of enzyme heat stability by measuring the changes in the residual activity of the glycosylated mutant CmGHFP (relative value to 0 hour of heating) in cellobiose degradation at a heat retention time of 50 to 90° C. in Example 12.
FIG. 12 is a graph showing the results of evaluation of enzyme heat stability by measuring the changes in the residual activity of SaBGAL (relative value to 0 hour of heating) in cellobiose degradation at a heat retention time of 50 to 90° C. in Example 12.
FIG. 13 is a graph showing the results of evaluation of enzyme heat stability by measuring the changes in the residual activity of the glycosylated mutant SaBGAL (relative value to 0 hour of heating) in cellobiose degradation at a heat retention time of 50 to 90° C. in Example 12.
FIG. 14 is a graph showing the results of evaluation of enzyme heat stability by measuring the changes in the residual activity of SsoBGAL (relative value to 0 hour of heating) in cellobiose degradation at a heat retention time of 50 to 90° C. in Example 12.
FIG. 15 is a graph showing the results of evaluation of enzyme heat stability by measuring the changes in the residual activity of the glycosylated mutant SsoBGAL (relative value to 0 hour of heating) in cellobiose degradation at a heat retention time of 50 to 90° C. in Example 12.
FIG. 16 is a graph showing the results of evaluation of enzyme heat stability by measuring the changes in the residual activity of PtBGAL (relative value to 0 hour of heating) in cellobiose degradation at a heat retention time of 50 to 90° C. in Example 12.
FIG. 17 is a graph showing the results of evaluation of enzyme heat stability by measuring the changes in the residual activity of the glycosylated mutant PtBGAL (relative value to 0 hour of heating) in cellobiose degradation at a heat retention time of 50 to 90° C. in Example 12.
FIG. 18 is a graph showing the results of evaluation of enzyme heat stability by measuring the changes in the residual activity of TvBGAL (relative value to 0 hour of heating) in cellobiose degradation at a heat retention time of 50 to 90° C. in Example 12.
FIG. 19 is a graph showing the results of evaluation of enzyme heat stability by measuring the changes in the residual activity of the glycosylated mutant TvBGAL (relative value to 0 hour of heating) in cellobiose degradation at a heat retention time of 50 to 90° C. in Example 12.
FIG. 20 is a graph showing the results of evaluation of enzyme heat stability by measuring the changes in the residual activity of FnGHFP (relative value to 0 hour of heating) in cellobiose degradation at a heat retention time of 50 to 90° C. in Example 12.
FIG. 21 is a graph showing the results of evaluation of enzyme heat stability by measuring the changes in the residual activity of the glycosylated mutant FnGHFP (relative value to 0 hour of heating) in cellobiose degradation at a heat retention time of 50 to 90° C. in Example 12.



DESCRIPTION OF EMBODIMENTS
Hereinbelow, the present invention will be described in detail.
The “glucosidase” in the present invention refers to an enzyme having an activity of hydrolyzing a disaccharide having a β-glycosidic bond (i.e., the β-glucosidase activity). Although a group of enzymes belonging to β-glucosidase is listed under the Enzyme Commission (EC) No: EC 3.2.1.21, a protein not belong to β-glucosidase in terms of EC number but having the aforementioned β-glucosidase activity is also encompassed by glucosidase in the present invention. Examples of the glucosidase include galactosidase, mannosidase, and a glucosidic bond hydrolase family protein.
In the present invention, a thermophile is a generic term for a group of microorganisms that can live at 50° C. or higher, and particularly, a hyperthermophile refers to a group of microorganisms that can live at 80° C. or higher. Examples of the thermophile include the genus Sulfolobus, the genus Thermoplasma, the genus Caldivirga, the genus Thermosphaera, the genus Pyrococcus, the genus Picrophilus, the genus Caldivirga, and the genus Fervidobacterium. 
The glucosidase derived from a thermophile is publicly known, and for example, it is registered at GenBank under NP—577802, which can be used in the present invention. Preferably, the glucosidase derived from a thermophile has the amino acid sequence shown in SEQ ID NOs: 4, 8, 10, 12, 14, 16, 18, and 20. More preferably, the glucosidase derived from a thermophile consists of the amino acid sequence shown in SEQ ID NOs: 4, 8, 10, 12, 14, 16, 18, and 20. In the present invention, the glucosidase derived from a thermophile also encompasses a protein having the amino acid sequence of SEQ ID NOs: 4, 8, 10, 12, 14, 16, 18 and 20 that has been subjected to one or multiple deletion, substitution, addition, or insertion, or deletion, substitution, addition, or insertion of one or a plurality of amino acids, and having a β-glucosidase activity. Here, the range of “one or a plurality” is not particularly limited; however, it is preferably 10 or less, more preferably five or less, particularly preferably four or less, or one or two. Also, in the present invention, the glucosidase derived from a thermophile also encompasses a protein having an amino acid sequence with 85% or more, more preferably 90% or more, and most preferably 95% or more identity with the amino acid sequence shown in SEQ ID NOs: 4, 8, 10, 12, 14, 16, 18, and 20 according to calculation using The Basic Local Alignment Search Tool (BLAST) at the National Center for Biological Information and the like (for example, default parameter(s), i.e., the initially set parameter(s)), preferably consisting of the above amino acid sequence, and having the β-glucosidase activity. Here, the term “identity” refers to the percentage of the identical amino acid and homologous amino acid residues relative to the overlapping total amino acid residues in the optimal alignment when two amino acid sequences are aligned either with or without a gap introduced between the amino acid sequences. The identity can be obtained by using a method commonly known to those skilled in the art, sequence analysis software (a publicly known algorithm such as BLAST and FASTA), and the like. The “β-glucosidase activity” is as defined above, and this activity can be measured by, for example, adding an enzyme solution to a cellobiose substrate solution obtained by dissolving cellobiose in a 50 mM acetic acid-sodium acetate buffer (pH 5.0), allowing the reaction to proceed at 30 to 85° C. for 30 minutes, terminating the reaction by changing pH as needed, and quantitating the glucose concentration in the resulting reaction solution using a glucose quantitation kit.
In the present invention, the “glucosidase derived from a thermophile” does not encompass a glucosidase naturally having a glycosylation sequence in its amino acid sequence, and it is limited to a glucosidase that is naturally devoid of a glycosylation sequence.
A “sugar chain” as used to in the present invention has a structure in which monosaccharides are linked via a glycosidic bond, and it is terminally bonded to the amino acid side chain of the peptide sequence of the glucosidase derived from a thermophile by covalent bonding. The presence or absence of “sugar chain” can be confirmed by staining glucosidase separated by SDS electrophoresis by a generally known periodic acid-Schiff base (PAS) reaction.
A sugar chain is mainly classified into an N-linked sugar chain, which binds to the asparagine side chain, and an O-linked sugar chain, which binds to the serine and threonine side chains, and the sugar chain is preferably an N-linked sugar chain. For an N-linked sugar chain, a structure having a basic skeleton having two N-acetylglucosamine residues and three mannose residues with respect to the asparagine side chain is given as an example (FIG. 7). Sugar molecules are additionally bound to this basic structure by the enzymatic action, whereby various sugar chain structures are composed. The sugar chain structure varies depending on the kind of a microorganism used as a host, the culture condition of the host, and the like. The glycosylated glucosidase derived from a thermophile refers to a compound to which various sugar chain structures are attached.
Whether the sugar chain bonded to the glucosidase derived from a thermophile is either N-linked or O-linked can be confirmed by, for example, allowing each of N-linked glycanase that specifically hydrolyzes the terminal portion of an N-linked sugar chain and O-linked glycanase that specifically hydrolyzes the terminal portion of an O-linked sugar chain to act on the glucosidase, performing SDS electrophoresis, and then comparing the changes in the molecular weight of the glucosidase. As the N-linked glycanase employed here, N-glycosidase F derived from Flavobacterium meningosepticum (PNGaseF), endo-β-N-acetylglucosaminidase derived from Streptomyces plicatus, and the like can be used. Also, as the O-linked glycanase, endo-α-N-acetylgalactosaminidase derived from Streptococcus and the like can be used.
The sugar chain in the mutant glucosidase of the present invention is preferably a high mannose type. Here, the term “high mannose type” refers to an N-linked sugar chain in which four or more mannose residues are linked per two N-acetylglucosamine residues or glucosamine residues that compose the sugar chain. As the sugar other than mannose, other monosaccharides such as glucose may be contained. When glucose is contained, it is normally bound to the non-reducing end of mannose of a high mannose type sugar chain.
Examples of an N-linked sugar chain other than a high mannose type include a complex type sugar chain. A complex type is characterized by containing, as a sugar other than mannose and N-acetylglucosamine, various kinds of sugars such as fructose and sialic acid as its components. Compared to a high mannose type, the ratio of N-acetylglucosamine in a sugar chain is increased, and the ratio of mannose to two N-acetylglucosamine residues is three or less.
Whether or not a given N-linked sugar chain is a high mannose type or a hybrid type can be confirmed by, for example, transferring glucosidase having been subjected to electrophoresis to a PVDF membrane, reacting it with sugar chain-specific lectin, and examining the color development. Examples of the sugar chain-specific lectin used here include Concanavalin A (ConA), lectin from Ricinus communis (RCA12), lectin from Ulex europaeus (UEA-1), and peanut lectin (PNA). If the glucosidase is stained with ConA, it can be confirmed as a high mannose type N-linked sugar chain, while if it is stained with RCA120, it can be confirmed as a hybrid type N-linked sugar chain. Also, as another judgment technique, the sugar chain structure can be confirmed by separating the sugar chain-composing sugar from sufficiently purified glucosidase and quantitating the monosaccharide component of the sugar thus separated by analyzing it with MALDI-TOF/MS or HPLC.
The “glycosylation sequence” refers to the amino acid sequence of the part that is subjected to glycosylation in the process of expression and translation in a eukaryotic organism.
Examples of the glycosylation sequence include the consensus sequence of an N-linked sugar chain, which is Asn-X-Ser or Asn-X-Thr (wherein, X is any amino acid except proline), and the consensus sequence of an O-linked sugar chain, which is Cys-X-Ser-X-Pro-Cys (wherein, X is any amino acid except proline) (SEQ ID NO: 63); however, the glycosylation sequence is not limited thereto. Preferably, the glycosylation sequence is the consensus sequence of an N-linked sugar chain. Here, examples of amino acid except proline include Gly, Ala, Val, Leu, Ile, Phe, Tyr, Trp, Met, Cys, Ser, Thr, Asp, Glu, His, Lys, Arg, Asn, and Gln.
The glycosylated mutant glucosidase derived from a thermophile according to the present invention (hereinbelow, referred to as a “mutant glucosidase”) refers to a mutant glucosidase resulting from introduction of the amino acid sequence composing the aforementioned glycosylation sequence into the aforementioned amino acid sequence of a glucosidase derived from a thermophile, wherein the amino acid sequence is selectively glycosylated. One or two or more glycosylation sequences may be introduced, and these sequences may be all the same or contain multiple kinds of glycosylation sequences.
As the glycosylation sequence-introduction site, such a position that does not inactivate the original enzyme activity by the introduction is preferably selected. The method for determining such a glycosylation sequence-introduction site can be carried out by the following step 1) and step 2).

    Step 1) Perform the amino acid sequence alignment analysis between a glucosidase derived from filamentous fungi naturally possessing a glycosylation sequence and a glucosidase derived from a thermophile that is naturally devoid of a glycosylation sequence to clarify a relative positional relationship of the glycosylation sequence of the glucosidase derived from filamentous fungi in the glucosidase derived from a thermophile to specify the glycosylation sequence-introduction site. As the alignment tool, a plenty of well-known software such as ClustalW can be used. The glucosidase derived from filamentous fungi naturally possessing a glycosylation sequence is preferably a glucosidase derived from the genus Trichoderma or a glucosidase derived from the genus Aspergillosis. The amino acid sequences of these glucosidases derived from filamentous fungi are publicly known, and preferably, a β-glucosidase derived from Trichoderma reesei having the amino acid sequence of SEQ ID NO: 1 or a β-glucosidase derived from Aspergillus niger having the amino acid sequence of SEQ ID NO: 2 is used.
    Step 2) Next, confirm if the glycosylation sequence-introduction site in the glucosidase derived from a thermophile that has been specified by the aforementioned alignment analysis is present on the surface of the enzyme. Whether or not it is present on the surface of the enzyme can be found out by using the crystal structure of the objective glucosidase derived from a thermophile. Such a crystal structure can be retrieved from a database such as Protein Data Bank if it is known. Also, a crystal structure may be obtained by actually performing X-ray crystallography, etc.


In the present invention, the glycosylation sequence-introduction site is preferably selected in accordance with the aforementioned step 1) and step 2); however, as another technique, it can be selected in accordance with the following step 3) and step 4).

    Step 3) Obtain the Accessible Surface Area (ASA) (Å2) of the amino acid residue by analytical software, and based on the value thus obtained, select the amino acid residue that is exposed near the surface of the glucosidase derived from a thermophile. The ASA of each amino acid residue can be calculated using ASA analytical software such as AREAIMOL (ccp4 package) (Collaborative Computing Project Number 4 (CCP4) of UK Science and Engineering Research Council), SURFace (Barry Honig's group, the Department of Biochemistry and Molecular Biophysics and Center of Computational Biology and Bioinformatics of Columbia), and ASAP (Institute for Molecular Bioscience, University of Queensland and the ARC Centre in Bioinformatics), all of which are obtainable from websites. If an amino acid residue is calculated as having an ASA of 1 Å2 or greater, then it is assumed to be exposed to the surface, and as the glycosylation sequence-introduction site, an amino acid residue with an ASA of 2 Å2 or greater is preferably selected. Particularly, in order to introduce the three amino acid residues (Asn-X-Ser or Asn-X-Thr: X is any amino acid except proline), which form the consensus sequence for an N-linked sugar chain, it is preferable to select a part containing three or more consecutive amino acids with an ASA of 2 Å2 or greater.
    Step 4) Select, from among the glycosylation sequence-introduction sites in a glucosidase derived from a thermophile selected by the step 3) above, a position that is too far from the enzyme active site of the glucosidase derived from a thermophile to cause a reduction in the enzyme activity by introduction of the glycosylation sequence. The distance from the enzyme active site can be found out by using the crystal structure of the objective glucosidase derived from a thermophile in a similar manner to the above. Specifically, after excluding the amino acid residues within a distance of 3.5 Å from the enzyme active site, the amino acid residue that is further apart than this distance is preferably selected.


For example, application of the aforementioned step 3) and step 4) to PfuBGL represented by the amino acid sequence of SEQ ID NO: 4 will be as follows. First of all, using AREAIMOL (ccp4 package), ASA is calculated by assuming that the solvent molecule has 1.4 Å, and the amino acid residue with an ASA of 2 Å2 or greater is extracted (Table 1 (i)). Subsequently, a part containing three or more consecutive amino acids with an ASA of 2 Å2 or greater is extracted (Table 1 (ii)). Further, based on the information of the crystal structure of PfuBGL, the amino acid residues within a distance of 3.5 Å from the enzyme active site are specified (23 residues of S13, R78, N207, I263, I303, G304, V305, N306, Y307, S343, D344, L367, I370, I371, T372, D378, R384, Y387, H391, D404, V405, R406, N407, Y408, L409, H410, W411, and F427) and then excluded. As a result, the glycosylation sequence-introduction site can be selected (Table 1 (iii)).







TABLE 1





Example of selection of the glycosylation sequence-introduction


site in PfuBGL















(i) Amino acid residues with an ASA of 2 Å2 or greater


M1-M9, F17-F19, G22-G25, E27-V34, H37-E40, I42-L51, E53, N54,


Y58-Q64, D67, I68, E70, I68, E70, K71, G73, D75, E82-R85, P88-P90,


F92-D110, P112-115, K117, E118, E120-A127, E129, H130, R132,


K133, S136, D137, K139-F145, Y150-W152, P155-D160, I162, A163,


R165-L167, P169-P173, W176-T181, V183, E184, K187, Y194, H195,


D197-L199, D201, M202, E208, N210-Q215, Y217-S222, F224, P225,


G227-K236, K238-N240, I242, I246, Y249, D250, K253-S259, A265-


E279, E281-D287, E289, T292, I293, H295-W302, Y308, R310-Y313,


A315-Y326, F328-R340, D344, F345, W374, Y350-E352, E355, N356,


K359, Y360, N362-P368, I370, E373, M376, A379-Y383, P385-Y387,


S390, K363, A394, Y396, N397, M399-R406, W411, E418-Q421,


F423-F427, Y431, D433, P443, L446, R449, E450, T453-E460,


A462-K473


(ii) Among (i), a part containing three or more consecutive amino


acid residues


M1-M9, F17-F19, G22-G25, E27-V34, H37-E40, I42-L51, Y58-Q64,


E82-R85, P88-P90, F92-D110, P112-115, E120-A127, K139-F145,


Y150-W152, P155-D160, R165-L167, P169-P173, W176-T181, D197-


L199, N210-Q215, Y217-S222, G227-K236, K238-N240, K253-S259,


A265-E279, E281-D287, H295-W302, R310-Y313, A315-Y326, F328-


R340, Y350-E352, N362-P368, A379-Y383, P385-Y387, M399-R406,


E418-Q421, F423-F427, T453-E460, A462-K473


(iii) Among (ii), an amino acid residue away from the enzyme active


site by 3.5 Å or more


M1-M9, F17-F19, G22-G25, E27-V34, H37-E40, I42-L51, Y58-Q64,


E82-R85, P88-P90, F92-D110, P112-115, E120-A127, K139-F145,


Y150-W152, P155-D160, R165-L167, P169-P173, W176-T181, D197-


L199, N210-Q215, Y217-S222, G227-K236, K238-N240, K253-S259,


A265-E279, E281-D287, H295-W302, R310-Y313, A315-Y326, F328-


R340, Y350-E352, N362-E366, A379-Y383, M399-A403, E418-Q421,


F423-R426, T453-E460, A462-K473









The glycosylation sequence-introduction site is present at one site or preferably at two to five sites.
In the present invention, the term “introduction” indicates that the aforementioned glycosylation sequence is translated into a polypeptide. That is, in the original amino acid sequence of a glucosidase derived from a thermophile, the aforementioned glycosylation sequence may substitute for the existing amino acid sequence, or it may be inserted into the existing amino acid sequence. That is, in the case of substitution, the length of polypeptide remains unchanged relative to before mutation, while in the case of insertion, the length of polypeptide becomes longer by the length of the inserted glycosylation sequence. However, from the viewpoint of retention of the enzyme activity, the introduction of the glycosylation sequence preferably takes place by substitution of the existing amino acid sequence.
Also, as the glycosylation sequence to be introduced, any of the aforementioned glycosylation sequences may be adopted, and as X, any amino acid except proline can be used; however, a glycosylation sequence and an amino acid that are expected to bring little impact when introduced as a mutation should be selected. For example, in the case of an amino acid substitution, a conservative amino acid substitution is desirable. The conservative amino acid substitution refers to substitution that takes place between the amino acids having similar electrical properties, structural properties, polarity or hydrophobicity, etc., and the substitution between these similar amino acids is expected not to alter the phenotype of protein. Examples include a basic amino acid (Lys, Arg, and His), an acidic amino acid (Glu and Asp), an aromatic amino acid (Trp, Phe, Tyr, and His), a branched amino acid (Val, Ile, and Thr), a polar amino acid (Ser, Thr, Tyr, Cys, Met, Gln, Asn, and Gly), and a hydrophobic amino acid (Ala, Val, Leu, and Ile).
In one embodiment, using the aforementioned technique, the glycosylation sequence-introduction site in the amino acid sequence (SEQ ID NO: 4) of the β-glucosidase derived from Pyrococcus furiosus can be specified as H60-L61-Y62 (FIG. 1).
Also, according to the present invention, using the amino acid sequence of the glucosidase derived from a thermophile in which the glycosylation sequence-introduction site is determined in the aforementioned technique, the glycosylation sequence-introduction site can be determined also in another glucosidase derived from a thermophile having the amino acid sequence that is highly identical to the above amino acid sequence. For example, after determining the glycosylation sequence-introduction site in the β-glucosidase derived from Pyrococcus furiosus shown in SEQ ID NO: 4 by the aforementioned technique, an alignment analysis is performed with respect to another enzyme having the amino acid sequence having high identity with the β-glucosidase shown in the SEQ ID NO: 4, and the position in the amino acid sequence of this enzyme that corresponds to the determined introduction site in SEQ ID NO: 4 (for example, H60-L61-Y62) can be determined as the glycosylation sequence-introduction site. Examples of another enzyme having the amino acid sequence having high identity with the β-glucosidase shown in the SEQ ID NO: 4 include the enzymes having the amino sequences shown in SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20.
In one embodiment, using the aforementioned technique, the glycosylation sequence-introduction site in the amino acid sequences shown in SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20 can be specified as N60-L61-N62, D60-L61-Y62, G61-N62-Y63, G61-N62-Y63, D58-L59-Y60, N63-N64-Y65, and K63-Q64-Y65, respectively (FIG. 2-1 and FIG. 2-2).
In one embodiment, the mutant glucosidase according to the present invention comprises, for example, the amino acid sequence shown in SEQ ID NO: 6. Preferably, the mutant glucosidase according to the present invention consists of, for example, the amino acid sequence shown in SEQ ID NO: 6.
In the present invention, the glucosidase activity refers to the cellobiose degradation activity. That is, it is an activity of catalyzing the reaction for producing glucose by hydrolysis of cellobiose, when it is used as a substrate. The mutant glucosidase of the present invention retains preferably 40% or more, more preferably 50% or more, even more preferably 60% or more, and particularly preferably 70% or more activity relative to the cellobiose degradation activity of the wild-type glucosidase. The cellobiose degradation activity of the mutant glucosidase of the present invention relative to the wild-type can be evaluated by, for example, adding the mutant glucosidase or the wild-type glucosidase to a 10 mM cellobiose/50 mM acetate buffer solution, carrying out the enzymatic reaction at 50° C., and then measuring the amount of glucose produced. Here, the amount of glucose produced can be quantitated in accordance with a publicly known technique such as the enzymic method and HPLC.
The mutant glucosidase according to the present invention may be one obtained by any method; however, in order for the introduced glycosylation sequence to be selectively glycosylated, the mutant glucosidase is desirably provided by culturing a eukaryotic cell containing DNA encoding the mutant glucosidase.
DNA encoding the mutant glucosidase can be produced by the following method. That is, it can be produced by determining the glycosylation sequence-introduction site in the amino acid sequence of a glucosidase derived from a thermophile by the aforementioned technique, and then introducing DNA encoding the glycosylation sequence into the part of the nucleotide sequence in DNA encoding the glucosidase that corresponds to the introduction site. Here, the term “DNA” encompasses any nucleic acid that encodes the glucosidase or the glycosylation sequence, which can be cDNA, genomic DNA, a gene, and the like.
Examples of DNA encoding a glucosidase derived from a thermophile include DNA encoding the aforementioned glucosidase derived from a thermophile, for example, DNA comprising the nucleotide sequence shown in SEQ ID NOs: 3, 7, 9, 11, 13, 15, 17, and 19, preferably DNA consisting of the above nucleotide sequences. Also, DNA encoding a glucosidase derived from a thermophile encompasses DNA comprising a nucleotide sequence capable of hybridizing with DNA consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NOs: 3, 7, 9, 11, 13, 15, 17, and 19 under stringent conditions, preferably DNA consisting of the above nucleotide sequence, and encoding a protein having the β-glucosidase activity. The stringent condition may be a low stringent condition, a medium stringent condition, or a high stringent condition. The stringent condition includes, for example, carrying out hybridization in 2 to 5×SSC and 0.2% SDS (wherein, 1×SSC indicates 150 mM sodium chloride, 15 mM sodium citrate, and pH 7.0) at 45 to 70° C., followed by washing with 0.1 to 1×SSC and 0.1 to 0.2% SDS at 45 to 65° C. The stringent condition is described in, for example, Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd edition, 1989, Cold Spring Harbor Laboratory Press. Further, DNA encoding a glucosidase derived from a thermophile encompasses DNA comprising the nucleotide sequence shown in SEQ ID NOs: 3, 7, 9, 11, 13, 15, 17 and 19 that has been subjected to one or multiple deletion, substitution, addition, or insertion, or deletion, substitution, addition, or insertion of one or a plurality of nucleotides and encoding a protein having the β-glucosidase activity. Here, the range of “one or a plurality” is not particularly limited; however, it is preferably 10 or less, more preferably five or less, particularly preferably four or less, or one or two. Furthermore, DNA encoding a glucosidase derived from a thermophile encompasses DNA comprising a nucleotide sequence having 85% or more, more preferably 90% or more, and most preferably 95% or more identity with the nucleotide sequence shown in SEQ ID NOs: 3, 7, 9, 11, 13, 15, 17, and 19 according to calculation using BLAST and the like (for example, default parameter(s), i.e., the initially set parameter(s)), preferably DNA consisting of the above nucleotide sequence, and encoding a protein having the β-glucosidase activity.
Examples of DNA encoding the glycosylation sequence include DNA encoding the aforementioned glycosylation sequence, and it can be appropriately determined in consideration of the host organisms and the degeneracy of the genetic code.
DNA encoding the glycosylation sequence can be introduced into DNA encoding a glucosidase by a publicly known technique such as site-directed mutagenesis and specific mutation introduction by PCR (Sambrook et al., described above).
In one embodiment, examples of DNA encoding the mutant glucosidase include DNA comprising the nucleotide sequence shown in SEQ ID NO: 5. Preferably, examples of DNA encoding the mutant glucosidase include DNA consisting of the nucleotide sequence shown in SEQ ID NO: 5. In SEQ ID NO: 5, DNA encoding the glycosylation sequence is introduced as -aaccgcact- in the positions 178 to 186.
Further, DNA encoding a secretion signal sequence that is suitable for the host may be added to DNA encoding the aforementioned mutant glucosidase. The secretion signal sequence can be appropriately added to the 5′ terminus or 3′ terminus of DNA encoding the mutant glucosidase, and preferably, it is added to the 5′ terminus. DNA encoding the secretion signal sequence may also be incorporated in an expression vector in advance. For example, when the host is yeast, the α factor signal sequence, the α-amylase signal sequence, the glucoamylase signal sequence, the serum albumin signal sequence, the inulinase-derived signal sequence, the invertase signal sequence, the killer protein signal sequence, the lysozyme signal sequence, and the like are used. Particularly in Pichia pastoris, the α factor secretion signal sequence is preferred. The α factor secretion signal sequence is publicly known, and for example, it is registered at GenBank under NP—015137, which can be used in the present invention.
When the genus Trichoderma is used as the host, the cellulase-related signal sequence can be used. The genus Trichoderma has a characteristic of secreting, as cellulase, cellobiohydrolase, xylanase, endoglucanase, xylosidase, and xyloglucanase outside the cell, and these enzymes each have a secretion signal sequence. These signal sequences are publicly known, and peptide sequences containing these signal sequences can be used by functionally linking them to the mutant glucosidase.
DNA encoding the mutant glucosidase having one or more glycosylation sequences that can be subjected to glycosylation by a eukaryotic cell prepared as above is linked to the downstream of the promoter in an appropriate expression vector using a restriction enzyme and a DNA ligase, whereby an expression vector carrying the DNA can be produced.
Examples of the expression vector include a bacterial plasmid, a yeast plasmid, DNA of a phage (such as lambda phage), DNA of a virus such as a retrovirus, a baculovirus, a vaccinia virus, and an adenovirus, a derivative of SV40, and an agrobacterium as a vector for a plant cell, and any other vector can be used as long as it is replicable and viable, and glycosylation is feasible in the host cell. Examples of the expression vector include, when the host is yeast, pPink-HC, pPink-LC, pPinkα-HC, pPCIZ, pPCIZα, pPCI6, pPCI6α, pFLD1, pFLD1α, pGAPZ, pGAPZα, pPIC9K, and pPIC9.
As the promoter, any promoter may be used as long as it is a suitable promoter corresponding to the host to be used for gene expression, and it may be either a constitutive promoter or an inducible promoter. Examples of the promoter include, when the host is yeast, the AOX1 promoter, the TEF1 promoter, the ADE2 promoter, the CYC1 promoter, the GAL-L1 promoter, the AOX2 promoter, the YPT1 promoter, the GAP promoter, and the FLD promoter.
The host cell to be used in the present invention may be any host cell as long as it has the glycosylation mechanism. Preferred examples of the host cell include a yeast cell, a fungal cell, an insect cell, a plant cell, and an animal cell. Examples of the yeast cell include the genus Pichia, the genus Saccharomyces, and the genus Schizosaccharomyces. Examples of the fungal cell include the genus Aspergillus and the genus Trichoderma. Examples of the insect cell include Sf9. Examples of the plant cell include the dicotyledons. Examples of the animal cell include CHO, HeLa, and HEK293. More preferably, the host cell is a yeast cell, and even more preferably, it is Pichia pastoris. 
Transformation or transfection can be carried out by a publicly known method such as the calcium phosphate method and electroporation. The mutant glucosidase can be obtained by expressing it in the host cell that has been transformed or transfected as described above under the control of the promoter, and then collecting the expression product. For expression of the mutant glucosidase, the host cell is allowed to proliferate or grow to an appropriate cell density, and the promoter is induced by a temperature shift or chemical induction means such as the addition of isopropyl-1-thio-β-D-galactoside (IPTG), and then the cell is further cultured for a certain period of time.
When the mutant glucosidase is excreted out of the cell, it is directly purified from the medium. When the mutant glucosidase is present outside the cell, the cell is disrupted by physical means such as ultrasonic disintegration and mechanical disintegration or by chemical means such as a cell lysis agent, and then the mutant glucosidase is purified. The mutant glucosidase can be partially or completely purified from the medium of the recombinant cell by a combination of techniques such as ammonium sulfate precipitation or ethanol precipitation, acid extraction, anion or cation exchange chromatography, reverse-phase high-performance chromatography, affinity chromatography, gel filtration chromatography, and electrophoresis.
In the hydrolysis of cellulosic biomass, the glycosylated mutant glucosidase of the present invention has higher heat resistance and can achieve a cellulose degradation efficiency that is 1.2 times, 1.3 time, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, and twice or more as high as the cellulose degradation efficiency achieved when a glucosidase derived from a thermophile is used with an unglycosylated cellulase mixture. The mutant glucosidase of the present invention can be applied to cellulosic biomass such as cellulosic biomass containing a lignin component, Avicel containing almost no lignin component, Solka Floc, and industrial pulp, and it can achieve profound cellulose degradation efficiency particularly in cellulosic biomass containing a lignin component.
The mutant glucosidase obtained as above can be used as an enzyme composition for saccharification of biomass by mixing with cellulase. The “cellulose” as used herein is not particularly limited as long as it is an enzyme having a cellulose degradation activity, and it may be a mixture of one or more kinds of cellulases. Examples of such an enzyme include cellulase, hemicellulase, cellobiohydrolase, endoglucanase, exoglucanase, xylanase, and mannanase.
The cellulase used in the present invention is preferably a mixture of cellulases derived from filamentous fungi. The mixture of cellulases derived from filamentous fungi is a mixture containing at least both endoglucanase and cellobiohydrolase. In order to carry out more efficient saccharification of cellulose, a mixture of cellulases derived from filamentous fungi containing two or more kinds of endoglucanases and/or two or more kinds of cellobiohydrolases is preferred. Examples of the microorganism producing the aforementioned mixture of cellulases derived from filamentous fungi include the genus Trichoderma, the genus Aspergillus, the genus Cellulomonas, the genus Clostridium, the genus Streptomyces, the genus Humicola, the genus Acremonium, the genus Irpex, the genus Mucor, and the genus Talaromyces. Because these microorganisms produce a cellulase in the liquid culture medium, the liquid culture medium may be directly used as an unpurified mixture of cellulases derived from filamentous fungi, or a preparation obtained from a purified product of the liquid culture medium may be used as the mixture of cellulases derived from filamentous fungi. The mixture of cellulases derived from filamentous fungi may simultaneously contain a β-glucosidase produced by the microorganism; however, considering that it does not exist in an adequate amount for cellulose degradation, and also, it is clearly distinguishable from the β-glucosidase derived from the genus Pyrococcus as will be described later, the β-glucosidase produced by the cellulase-producing microorganism is also encompassed by cellulase in the present invention. When a preparation obtained from a purified product of the aforementioned liquid culture medium is used, a substance other than an enzyme such as a protease inhibitor, a dispersant, a solubilizing agent, and a stabilizer can be added and the resulting product may be used as a cellulase preparation.
The mixture of cellulases derived from filamentous fungi used in the present invention is preferably a mixture of cellulases produced by the genus Trichoderma. The genus Trichoderma produces a mixture of cellulases containing at least two kinds of endoglucanases and at least two kinds of cellobiohydrolases into the liquid culture medium, and a mixture of cellulases prepared from such a liquid culture medium is preferably used in the present invention. Among the organisms belonging to the genus Trichoderma, a mixture of cellulases derived from Trichoderma reesei is more preferred. Examples of the mixture of cellulases derived from Trichoderma reesei include a mixture of cellulases derived from Trichoderma reesei QM9414, Trichoderma reesei QM9123, Trichoderma reesei RutC-30, Trichoderma reesei PC3-7, Trichoderma reesei ATCC66589, Trichoderma reesei CL-847, Trichoderma reesei MCG77, Trichoderma reesei MCG80, and Trichoderma viride QM9123. Also, a mutant strain derived from the aforementioned genus Trichoderma that has achieved improved cellulose productivity through mutagenic treatment using a mutagen, ultraviolet ray irradiation, or the like may also be used.
The mixture of cellulases derived from the genus Trichoderma used in the present invention is an enzyme composition containing a plurality of enzyme components such as cellobiohydrolase, endoglucanase, exoglucanase, β-glucosidase, xylanase, and xylosidase. The mixture of cellulases derived from the genus Trichoderma can carry out effective hydrolysis of cellulose owing to the concerted effect or the complementary effect of a plurality of enzyme components in cellulose hydrolysis.
Cellobiohydrolase is a generic term for cellulase that is characterized by starting hydrolysis from the terminal portion of cellulose, and a group of enzymes belonging to cellobiohydrolase is listed under EC No: EC3.2.1.91.
Endoglucanase is a generic term for cellulase that is characterized by starting hydrolysis from the middle part of the cellulose molecular chain, and a group of enzymes belonging to endoglucanase is listed under EC NOs: EC3.2.1.4, EC3.2.1.6, EC3.2.1.39, and EC3.2.1.73.
Exoglucanase is a generic term for cellulase that is characterized by starting hydrolysis from the terminus of the cellulose molecular chain, and a group of enzymes belonging to exoglucanase is listed under EC NOs: EC3.2.1.74 and EC3.2.1.58.
β-glucosidase is a generic term for cellulase that is characterized by acting on cellooligosaccharides or cellobiose, and a group of enzymes belonging to β-glucosidase is listed under EC No: EC3.2.1.21.
Xylanase is a generic term for cellulase that is characterized by acting on hemicellulose or, particularly, xylan, and a group of enzymes belonging to xylanase is listed under EC No: EC3.2.1.8.
Xylosidase is a generic term for cellulase that is characterized by acting on xylooligosaccharides, and a group of enzymes belonging to xylosidase is listed under EC No: EC3.2.1.37.
In the present invention, cellulose-containing biomass is used as a substrate of the enzymatic reaction. Cellulose-containing biomass is cellulose that is widely derived from plant biomass. More specifically, cellulose-containing biomass is bagasse, corn stover, corncob, switchgrass, rice straw, wheat straw, trees, wood materials, building material waste, newspaper, used paper, pulp, and the like. Although the above cellulose-containing biomass contains an impurity such as the macromolecular aromatic compound lignin and hemicellulose, cellulose-containing biomass in which lignin and hemicellulose are partially degraded by using an acid, an alkali, pressurized hot water, and the like as pre-treatment may also be used as cellulose. Here, pre-treated cellulose-containing biomass as described above is provided as “lignocellulose”, which can be used as a substrate of the enzymatic reaction.
As the cellulose-containing biomass used in the present invention, one that has been subjected to pre-treatment such as ammonia treatment, diluted sulfuric acid treatment, and hydrothermal treatment by a publicly known technique can be used.
For ammonia treatment, the methods described in JP Patent Publication (Kokai) No. 2008-161125 A and JP Patent Publication (Kokai) No. 2008-535664 A can be applied. Specifically, to biomass, ammonia is added at a concentration of 0.1 to 15 wt. %, and treatment is carried out at 4 to 200° C., preferably at 90 to 150° C. Ammonia to be added may be either in the liquid state or in the gaseous state. When ammonia is in the liquid state, either liquid ammonia or an aqueous solution of ammonia may be used. The number of treatment is not particularly limited, and it may be performed at least once. When the treatment is performed twice or more, the first treatment and the second treatment may be performed under different conditions. The product obtained by the ammonia treatment needs to be subjected to neutralization of ammonia or removal of ammonia before performing the hydrolysis reaction. Neutralization may be performed on a liquid that still contains a solid content or a liquid fraction from which the solid content has been separated. An acid reagent used for neutralization is not particularly limited. Ammonia can also be removed by volatilization in the gaseous state by keeping the ammonia-treated product under the reduced pressure condition. In that case, ammonia that has been removed may be recovered and recycled.
The hydrothermal treatment may be performed by, for example, adding water so that the cellulose-containing biomass is 0.1 to 50 wt. %, and treating the resulting solution at a temperature of 100 to 400° C. for one second to 60 minutes. The number of treatment is not particularly limited, and it may be performed at least once. When the treatment is performed twice or more, the first treatment and the second treatment may be performed under different conditions.
For diluted sulfuric acid treatment, for example, the concentration of sulfuric acid is preferably 0.1 to 15 wt. %, more preferably 0.5 to 5 wt. %. The reaction temperature can be set in a range of 100 to 300° C., preferably at 120 to 250° C. The reaction time can be set in a range of one second to 60 minutes. The number of treatment is not particularly limited, and it may be performed at least once. When the treatment is performed twice or more, the first treatment and the second treatment may be performed under different conditions. Because the hydrolysate obtained by the diluted sulfuric acid treatment contains an acid, it needs to be neutralized before using it in the hydrolysis reaction.
Regarding the condition of the enzymatic treatment of cellulose-containing biomass in the present invention, when an enzyme composition for saccharification of biomass containing a cellulase derived from filamentous fungi and the mutant glucosidase of the present invention is used, the treatment is preferably carried out at a temperature of 40° C. to 60° C., pH of 3 to 7, and a cellulose-containing biomass solid content concentration of 0.1 to 30%. By setting the condition of the enzymatic treatment in the above range, the cellulose degradation efficiency of a cellulase derived from filamentous fungi and a glucosidase derived from a thermophile can be maximized. Some of the glucosidase derived from a thermophile naturally have an optimum reaction temperature of near 100° C.; however, the glucosidase derived from a thermophile used in the present invention exhibits a sufficiently high specific activity even at 40° C. to 60° C. and can efficiently degrade cellulose-containing biomass in the co-presence of a cellulase derived from filamentous fungi. This enzymatic treatment may be carried out batch-wise or in a continuous manner.
Owing to a high β-glucosidase activity of the enzyme composition for saccharification of biomass containing the mutant glucosidase of the present invention, a sugar liquid obtained by hydrolysis of cellulosic biomass using this enzyme composition has characteristics of having small content of cellobiose but a large amount of glucose. Accordingly, a sugar liquid obtained by using the enzyme composition for saccharification of biomass according to the present invention can be favorably utilized as a carbon source for the growth of microorganisms or cultured cells or for fermentative production using these microorganisms or cultured cells. Examples of the microorganism or the cultured cell used here include yeast such as baker's yeast used in the fermentation industry, bacteria such as E. coli and the coryneform group of bacteria, filamentous fungi, actinomycetes, animal cells, and insect cells. The microorganisms and the cells to be used may be those isolated from natural environments or those having partially modified properties by mutation and genetic recombination. Also, because a sugar liquid derived from cellulose-containing biomass contains pentose such as xylose, a microorganism having an enhanced pentose metabolism pathway is preferably used. Also, using such a sugar liquid as the fermentation raw material, a chemical product can be produced. Specific examples of the chemical product include a substance that is mass-produced in the fermentation industry such as an alcohol, an organic acid, an amino acid, and a nucleic acid, for example, an alcohol such as ethanol, 1,3-propanediol, 1,4-butanediol, and glycerol, an organic acid such as acetic acid, lactic acid, pyruvic acid, succinic acid, malic acid, itaconic acid, and citric acid, a nucleoside such as inosine and guanosine, a nucleotide such as inosinic acid and guanylic acid, and an amine compound such as cadaverine. Further, the sugar liquid can also be applied to the production of an enzyme, an antibiotic, a recombinant protein, and so on.
The sugar liquid obtained by the hydrolysis of cellulosic biomass using the enzyme composition for saccharification of biomass according to the present invention can be used as a sugar liquid after removing the undegraded solid residues as needed, or be directly used as a sugar liquid with the solid residues still contained therein.
In the method for hydrolyzing biomass using the enzyme composition for saccharification of biomass according to the present invention, the used enzyme composition can be separated and recovered from the sugar liquid obtained by the enzymatic treatment of cellulose-containing biomass. Although the method of separation and recovery is not particularly limited, compared to a conventional unglycosylated glucosidase, the mutant glucosidase of the present invention has a characteristic of having a greatly reduced adsorptivity for cellulose-containing biomass, particularly for lignocellulose, and also for an ultrafiltration membrane. Therefore, for separation and recovery of the used enzyme composition, a method of subjecting the hydrolysate to solid-liquid separation as needed, filtering the sugar liquid thus obtained through an ultrafiltration membrane, and separating and recovering the enzyme composition as a non-permeable liquid is preferably used.
As the solid-liquid separation technique in the method for hydrolyzing biomass according to the present invention, either the filtration method or the centrifugation method can be used. Examples of the device for carrying out the solid-liquid separation include, but are not limited to, a belt filter, a screw decanter, a continuous centrifuge, a filter press, and a drum filter.
In the method for hydrolyzing biomass according to the present invention, as the ultrafiltration membrane used for separation and recovery of the enzyme composition, ones made of polyethersulfone (PES), polysulfone (PS), polyacrylonitrile (PAN), poly vinylidene difluoride (PVDF), regenerated cellulose, cellulose, a cellulose ester, sulfonated polysulfone, sulfonated polyethersulfone, polyolefin, polyvinyl alcohol, polymethyl methacrylate, polytetrafluoroethylene and the like can be used. Among them, from the viewpoint of long-term use, an ultrafiltration membrane made of a synthetic polymer other than cellulose is preferred. Generally, an ultrafiltration membrane made of a synthetic polymer has a problem that an enzyme (protein) has high adsorptivity for such a membrane. However, the enzyme composition separated and recovered in the present invention has reduced adsorptivity owing to the effect of glycosylation, and thus is preferably used. With regard to the molecular weight cutoff of the ultrafiltration membrane used in the present invention, an ultrafiltration membrane having a molecular weight cutoff of 500 Da to 100000 Da can be used. Among such ultrafiltration membranes, particularly, one with a molecular weight cutoff ranging from 10000 Da to 30000 Da, which can separate and recover both of the mutant glucosidase of the present invention and the cellulase component derived from filamentous fungi with good yield can be most preferably used.
The filtration method using an ultrafiltration membrane includes dead-end filtration and cross-flow filtration, and from the viewpoint of inhibition of membrane fouling, cross-flow filtration is preferred. Also, as the form of the membrane of ultrafiltration membrane to be used, an appropriately formed membrane such as a flat type membrane, a spiral type membrane, a tubular type membrane, and a hollow yarn type membrane can be used. Specific examples include G-5 type, G-10 type, G-20type, G-50 type, PW type, and HWS UF type, all of which are supplied by DESAL, HFM-180, HFM-183, HFM-251, HFM-300, HFM-116, HFM-183, HFM-300, HFK-131, HFK-328, MPT-U20, MPS-U20P, and MPS-U20S, all of which are supplied by Koch Membrane Systems Inc., SPE1, SPE3, SPE5, SPE10, SPE30, SPV5, SPV50, and SOW30, all of which are supplied by Synder Filtration, the product of microza(R) UF series manufactured by Asahi Kasei Corporation corresponding to a molecular weight cutoff of 3000 to 100000, and NTR7410 and NTR7450 manufactured by Nitto Denko Corporation.
EXAMPLES
Hereinbelow, the present invention will be specifically described with reference to Examples. However, the present invention is not limited to these Examples.
Comparative Example 1
Preparation of β-Glucosidase Derived from Pyrococcus furiosus (1)
A β-glucosidase derived from the hyperthermophilic archaea Pyrococcus furiosus (hereinbelow, referred to as “PfuBGL”) is so heat resistant that it is still active at 100° C. or higher, and it hydrolyzes various kinds of cellooligosaccharides to produce glucose; therefore, PfuBGL is anticipated for effective utilization of cellulosic biomass.
As to the PfuBGL gene, the gene shown in SEQ ID NO: 3 was entirely synthetized and linked to NcoI and BamHI of pET-11d using Ligation High (Toyobo Co., Ltd.), and the resulting vector was used for transformation of JM109 (Takara Bio Inc.). Screening was performed using an LB agar medium containing ampicillin as an antibiotic. From the transformed JM109 strain, the vector pET-PfuBGL thus prepared was isolated by the miniprep kit (QIAGEN) and subjected to a nucleotide sequence analysis. The pET-PfuBGL was used for transformation of the expression E. coli BL21(DE3)pLysS strain, whereby a BL21-PfuBGL strain was prepared. The BL21-PfuBGL strain was inoculated into 10 mL of an ampicillin-containing LB agar medium, followed by shaking culture (preculture) at 37° C. overnight. As the main culture, the bacteria obtained by the preculture were inoculated into 1 L of an ampicillin-containing LB agar medium, and shaking culture was performed until OD 600, the absorbance at a wavelength of 600 nm, reached 0.8. Subsequently, isopropyl-1-thio-β-D-galactoside (IPTG) was added so that the final concentration was 0.4 mM, and shaking culture was further continued at 25° C. overnight. After culturing, the bacteria were collected by centrifugation and resuspended in a 50 mM tris-HCl buffer (pH 8.0). The resulting solution was subjected to ultrasonic disintegration while ice-cooling, and the supernatant was collected by centrifugation as a cell-free extract. The cell-free extract thus obtained was kept warm at 85° C. for 15 minutes, and coagulation sedimentation of E. coli-derived proteins other than the glucosidase took place. The sediment was removed by centrifugation and the supernatant was dialyzed against a 50 mM acetate buffer (pH 5.0) through a dialysis membrane made of regenerated cellulose with a molecular weight cutoff of 10000 (manufactured by Spectrum Laboratories, Inc.). The protein solution thus obtained was used as the wild-type PfuBGL.
Example 1
Determination of the N-Linked Glycosylation Sequence-Introduction Site in PfuBGL
First of all, the determination of the primary sequence and the tertiary structure was attempted to search for the glycosylation sequence-introduction site in PfuBGL.
To perform alignment with respect to a PfuBGL homologue, the homologue search server FUGUE was used. As a result, in terms of ZSCORE, which indicates the homology of the sequence, PfuBGL exhibited a maximum score of 71.65 with respect to Glycosyl hydrolase family 1 (ZSCORE≧6.0 indicates 99% confidence). In order to form the alignment of the Glycosyl hydrolase family 1 thus obtained, the JOY server was used. As a result, the sequence of PfuBGL corresponding to the position of the N-linked glycosylation sequence that is present at three sites in the β-glucosidase derived from Aspergillus niger (AspNgBGL) and in the β-glucosidase derived from Trichoderma reesei (TriReBGL) was found not to be the N-linked glycosylation sequence (three sites: H60, L61, and Y62, N148, L149, and Y150, and N374, G375, and M376).
An incomplete x-ray crystal structure of PfuBGL is reported (Thijis K. et al., Biochem. vol. 39, No. 17 (2000)), which has degradation ability of as low as 3.3 Å. A complete structural model has not yet been constructed, and such a model has not yet been registered at Protein Data Bank (PDB) either. In light of the above, in order to determine the tertiary structure of PfuBGL, detailed X-ray crystallography was attempted using new crystal conditions. New crystallization conditions were searched and crystallization was successfully achieved using phosphoric acid as a precipitating agent. An X-ray diffraction experiment was performed in the large synchrotron radiation facility SPring-8, and the structure of PfuBGL was determined with degradation ability of 2.5 Å, whereby a complete model of PfuBGL was successfully constructed. For structural determination, the molecular replacement method was used, and as a model molecule, the β-glucosidase derived from Themosphaera aggregans shown in SEQ ID NO: 8 (ThAggBGY, PDB ID: 1QVB) was used.
From the tertiary structure of PfuBGL thus obtained, in the aforementioned three sites corresponding to the position of the N-linked glycosylation sequence, a site that is exposed to the enzyme surface and not located near the active site, thereby presumably having a little impact on the enzyme activity, was only found in H60, L61, and Y62. Accordingly, this site was determined as the N-linked glycosylation sequence-introduction site, and a mutation of H60N, L61R, and Y62T was introduced by substitution, whereby the amino acid sequence shown in SEQ ID NO: 6 was obtained as the glycosylation mutant PfuBGL.
Example 2
Determination of the N-Linked Glycosylation Sequence-Introduction Site in a Glucosidase Having a Homologous Amino Acid Sequence to PfuBGL
To perform alignment between SEQ ID NO: 6 obtained in Example 1 and SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20, an alignment was formed using ClustalW and BOXSHADE, both of which are software that is well-known to those skilled in the art (FIG. 2-1 and FIG. 2-2). In SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 20, a site corresponding to the glycosylation sequence Asn-Arg-Thr (N-R-T) in SEQ ID NO: 6 was determined as the glycosylation sequence-introduction site in each sequence.
Example 3
Preparation of the Glycosylated Mutant PfuBGL (1)
The glycosylated mutant PfuBGL shown in SEQ ID NO: 6 was prepared.
First of all, using the mutation-introducing primers 5′-CCACATATTGGCACCTCTATAAGCAAGATCATG-3′ (SEQ ID NO: 21) and 5′-CATGATCTTGCTTAGTGCGGTTCCAATATGCTGG-3′ (SEQ ID NO: 22) for introducing the glycosylation sequence determined in Example 1 into PfuBGL shown in SEQ ID NO: 4, a N-linked glycosylation sequence mutation was introduced by site-directed mutagenesis. After confirming the sequence of the gene thus obtained, it was inserted between the EcoRI and NotI sites in the yeast expression vector pPIC9, which originally possesses the α factor secretion signal sequence. The mutation-introduced gene was used for transformation of E. coli, and the colony having the gene having the desired mutation was confirmed by a sequencer.
Yeast competent cells produced by a general technique were mixed with the mutant plasmids, whereby transformation was performed using GENE PULSERII (Bio-Rad Laboratories, Inc.) under the conditions of 1.7 kV, 25 μF, and 200Ω. The transformed yeast was streaked onto a RDB plate. Three days later, from the colonies that appeared on the plate, 10 colonies were selected and checked for the expression. A clone in which the expression of the desired protein was confirmed by polyacrylamide gel electrophoresis was selected.
As the seed culture, the yeast colony on the plate was inoculated into 2 mL of a BMGY medium and cultured for two days. Subsequently, as the main culture, 2 mL of the yeast seed culture liquid was added to 1 L of a BMGY medium and cultured for two days so as to allow yeast to sufficiently proliferate. Then, 1 L of the culture liquid was subjected to centrifugation once to precipitate yeast, and the BMGY medium was exchanged for a 2% methanol-containing BMMY medium. The yeast was then resuspended in the medium, followed by culturing for 48 hours. The medium containing the expressed protein was collected by centrifugation and filtered through a filter, followed by ammonium sulfate precipitation using 70% (w/v) ammonium sulfate. The precipitate was collected by centrifugation and dissolved in a buffer, and dialysis was performed and the desired protein was obtained.
The enzyme thus obtained was treated with EndoH and subjected to polyacrylamide gel electrophoresis (FIG. 3). The preparation of the N-linked glycosylated mutant PfuBGL was confirmed by a band shift before and after the EndoH treatment.
Example 4
Enzyme Activity of the Glycosylated Mutant PfuBGL (2)
The glycosylated mutant PfuBGL and PfuBGL were compared for the β-glucosidase activity. Using a 10 mM cellobiose/50 mM acetate buffer solution as a substrate, the enzymes prepared in Example 2 and Comparative Example 1 were each added at a final concentration of 0.23 mg/mL and the enzymatic reaction was carried out at 50° C. For quantitation of the product, Glucose Test Wako II (Wako Pure Chemical Industries, Inc.) was used.
One unit (U) of the β-glucosidase activity was calculated in accordance with the following formula.

One unit (U) of the β-glucosidase activity=the concentration of glucose produced upon completion of the reaction (g/L)×1000/180/30

Further, a specific β-glucosidase activity per the amount of β-glucosidase (mg) was calculated by the following formula.

Specific β-glucosidase activity (U/mg protein)=β-glucosidase activity (U)/the amount of β-glucosidase added for the activity measurement

PfuBGL produced 1.58 g/L, while the glycosylated mutant PfuBGL produced 1.34 g/L glucose upon completion of the reaction. Also, the specific activity of the glycosylated mutant PfuBGL was 85% relative to the specific activity of PfuBGL, clearing indicating that the introduction of the glycosylation sequence mutation into the site determined in Examine 1 did not cause a loss of the enzyme activity. From this, it was confirmed that the glycosylated mutant PfuBGL is utilizable in place of PfuBGL.
Example 5
Heat Stability of the Glycosylated Mutant PfuBGL
The changes in the amount of glucose produced up to 24 hours when the product was kept warm at 50° C., 60° C., 70° C., 80° C., and 90° C. were measured.
The enzymes prepared in Example 2 and Comparative Example 1 (protein concentration of 1.0 mg/mL) were each kept warm at 50° C., 60° C., 70° C., 80° C., or 90° C. At each heat retention time, a substrate, which was a 10 mM cellobiose/50 mM acetate buffer solution, was added, and the enzymatic reaction was carried out for 30 minutes at the heat retention temperature. The solution after the reaction was collected and the product was quantitated using Glucose Test Wako II (Wako Pure Chemical Industries, Inc.) (FIG. 4 and FIG. 5).
By heating up to 24 hours, compared to PfuBGL, the glycosylated mutant PfuBGL exhibited more reduced enzyme inactivation at a high temperature. This finding confirmed that the glycosylated mutant PfuBGL achieved improved enzyme stability by acquiring a higher heat resistance than PfuBGL.
Reference Example 1
Preparation of Lignocellulose
Lignocelluloses 1 to 3 to be used for the hydrolysis using an enzyme composition containing the glycosylated mutant glucosidase were prepared as follows.
1. Preparation of Lignocellulose 1 (Ammonia Treatment)
As cellulose, rice straw was used. The above cellulose was placed in a small reactor (manufactured by Taiatsu Techno, TVS-N2 30 ml) and cooled with liquid nitrogen. To this reactor, ammonia gas was infused and the sample was completely immersed in liquid ammonia. The reactor was capped and left at room temperature for about 15 minutes. Further, it was treated in an oil bath of 150° C. for one hour. After the treatment, the reactor was taken out of the oil bath and ammonia gas was immediately leaked in a draft chamber. Subsequently, the inside of the reactor was dried by vacuuming down to 10 Pa by a vacuum pump. The resulting product was used in the following Examples as lignocellulose 1.
2. Preparation of Lignocellulose 2 (Diluted Sulfuric Acid Treatment)
As cellulose, rice straw was used. The cellulose was immersed in a 1% aqueous solution of sulfuric acid, followed by autoclave treatment (manufactured by Nitto koatsu K.K.) at 150° C. for 30 minutes. After the treatment, solid-liquid separation was performed to separate an aqueous solution of sulfuric acid (hereinbelow, diluted sulfuric acid treatment liquid) from sulfuric acid-treated cellulose. Subsequently, sulfuric acid-treated cellulose and the diluted sulfuric acid treatment liquid were mixed by stirring so that the solid content concentration was 10 wt. %. Subsequently, pH was adjusted to around 5 with sodium hydroxide. The resulting product was used in the following Examples as lignocellulose 2.
3. Preparation of Lignocellulose 3 (Hydrothermal Treatment)
As cellulose, rice straw was used. The above cellulose was immersed in water and subjected to autoclave treatment (manufactured by Nitto koatsu K.K.) at 180° C. for 20 minutes while stirring. In this treatment, the pressure was 10 MPa. After the treatment, the resulting product was subjected to solid-liquid separation by centrifugation (3000 G) to separate the solution component (hereinbelow, the hydrothermal treatment solution) from the treated biomass component. The resulting treated biomass component was used in the following Examples as lignocellulose 3.
Example 6
Hydrolysis of Lignocellulose Using the Enzyme Composition Composed of a Mixture of Cellulases Derived from Filamentous Fungi and the Glycosylated Mutant PfuBGL (1)
The changes in the amount of glucose produced by allowing the enzyme composition to act on the lignocellulose substrate were compared. A 50 mM acetate buffer (pH 5.0) in which 5 wt. % lignocellulose 1 (prepared in Reference Example 1) was suspended was used as a substrate. The reaction was allowed to proceed up to 28 hours at 50° C., and the reaction product was sampled as appropriate and measured for the glucose concentration (FIG. 6). As the mixture of cellulases derived from filamentous fungi, commercially available cellulase derived from Trichoderma reesei (Celluclast, Sigma) was used. As glucosidase, the glycosylated mutant PfuBGL prepared in Example 2 and PfuBGL prepared in Comparative Example 1 were each used. Cellulase was added at 0.5 mg/mL and glucosidase was added at 0.005 mg/mL ( 1/100 of the amount of cellulase).
Comparing the use of PfuBGL and the use of the glycosylated mutant PfuBGL, the amount of glucose produced by the glycosylated mutant PfuBGL after 28 hours of reaction was greatly increased, and it was 1.6 times as much as the amount produced by PfuBGL. It was revealed that the addition of the glycosylated mutant PfuBGL only in an amount of 1/100 the mount of cellulase was tremendously effective for increasing the production amount of glucose.
Reference Example 2
Preparation of a Cellulase Derived from Trichoderma 
A cellulase derived from Trichoderma was prepared by the following method.
1. Preculture
The following substances were added to distilled water in the amounts indicated below; corn steep liquor 5% (w/vol), glucose 2% (w/vol), ammonium tartrate 0.37% (w/vol), ammonium sulfate 0.14% (w/vol), potassium dihydrogen phosphate 0.2% (w/vol), calcium chloride dihydrate 0.03% (w/vol), magnesium sulfate heptahydrate 0.03% (w/vol), zinc chloride 0.02% (w/vol), iron (III) chloride hexahydrate 0.01% (w/vol), copper(II) sulfate pentahydrate 0.004% (w/vol), manganese chloride tetrahydrate 0.0008% (w/vol), boric acid 0.0006% (w/vol), and hexaammonium heptamolybdate tetrahydrate 0.0026% (w/vol). Then, 100 mL of the resulting solution was poured into a 500 mL triangle flask with a baffle and sterilized by autoclaving at 121° C. for 15 minutes. After naturally cooling, PE-M and Tween 80, which were each separately sterilized by autoclaving at 121° C. for 15 minutes, were each added at 0.1%. To the resulting preculture medium, Trichoderma reesei ATCC66589 was inoculated at 1×105/mL, and preculture was performed by shaking at 28° C. for 72 hours at 180 rpm (shaker: BIO-SHAKER BR-40LF manufactured by Taitec Corporation).
2. Main Culture
The following substances were added to distilled water in the amounts indicated below; corn steep liquor 5% (w/vol), glucose 2% (w/vol), cellulose (Avicel) 10% (w/vol), ammonium tartrate 0.37% (w/vol), ammonium sulfate 0.14% (w/vol), potassium dihydrogen phosphate 0.2% (w/vol), calcium chloride dihydrate 0.03% (w/vol), magnesium sulfate heptahydrate 0.03% (w/vol), zinc chloride 0.02% (w/vol), iron (III) chloride hexahydrate 0.01% (w/vol), copper(II) sulfate pentahydrate 0.004% (w/vol), manganese chloride tetrahydrate 0.0008% (w/vol), boric acid 0.0006% (w/vol), and hexaammonium heptamolybdate tetrahydrate 0.0026% (w/vol). Then, 2.5 L of the resulting solution was poured into a 5 L stirring jar (DPC-2A manufactured by ABLE & Biott Co., Ltd.) and sterilized by autoclaving at 121° C. for 15 minutes. After naturally cooling, PE-M and Tween 80, which were each separately sterilized by autoclaving at 121° C. for 15 minutes, were each added at 0.1%. In this, 250 mL of Trichoderma reesei ATCC66589 that had been precultured in the liquid culture medium by the aforementioned method was inoculated. Thereafter, the fungi were cultured at 28° C. for 87 hours at 300 rpm with an aeration rate of 1 vvm, followed by centrifugation, and the supernatant was filtered through a membrane (STERICUP-GV, material: PVDF, manufactured by Millipore Corporation).
Example 7
Hydrolysis of Lignocellulose Using the Enzyme Composition Composed of a Mixture of Cellulases Derived from Filamentous Fungi and the Glycosylated Mutant PfuBGL (2)
Hydrolysis of lignocelluloses 1 to 3 was performed in the same manner as Example 6 except for using lignocelluloses 1 to 3 prepared in Reference Example 1 as the substrate, the culture liquid of Trichoderma reesei prepared in Reference Example 2 as the mixture of cellulases derived from filamentous fungi, and adding cellulase at 1.0 mg/mL and glucosidase at 0.005 mg/mL ( 1/100 of the amount of cellulase).
In Table 2, the concentrations of glucose (g/L) produced after 28 hours of reaction were compared.










TABLE 2









Concentration of glucose




produced (g/L)












Wild-type
Glycosylated



Substrate
PfuBGL
mutant PfuBGL







Lignocellulose 1
5 g/L
15 g/L



(Ammonia treatment)



Lignocellulose 2
3 g/L
16 g/L



(Diluted sulfuric



acid treatment)



Lignocellulose 3
4 g/L
18 g/L



(Hydrothermal



treatment)










Comparing the use of PfuBGL and the use of the glycosylated mutant PfuBGL, the amount of glucose produced by the glycosylated mutant PfuBGL after 28 hours of reaction was greatly increased, and it was 1.8 times as much as the amount produced by PfuBGL. It was revealed that the addition of the glycosylated mutant PfuBGL only in an amount of 1/200 the mount of cellulase produced an effect of greatly increasing the production amount of glucose. It was revealed that not only commercially available cellulase such as the one used in Example 6 but also the culture liquid of Trichoderma reesei exerted an effect in use with a glycosylated mutant.
Example 8
Hydrolysis of Lignocellulose Using the Enzyme Composition Composed of a Mixture of Cellulases Derived from Filamentous Fungi and the Glycosylated Mutant PfuBGL (3)
Using the enzyme composition composed of a cellulase derived from filamentous fungi and the glycosylated mutant PfuBGL, the amounts of glucose produced were comparatively studied by varying the reaction temperature conditions of hydrolysis. By setting the reaction temperature at 30° C., 40° C., 50° C. (Example 7), or 60° C., the hydrolysis was performed by a similar procedure to Example 7, and the amount of glucose produced after 28 hours was measured. As the substrate, lignocellulose 1 was used.










TABLE 3








Concentration of



Reaction temperature
glucose produced



of hydrolysis (° C.)
(g/L)









30° C.
 5 g/L



40° C.
13 g/L



50° C. (Example 7)
15 g/L



60° C.
 7 g/L










As shown in Table 3, it was revealed that it was preferable to set the reaction temperature in a range of 40° C. to 50° C. when a cellulase derived from filamentous fungi, particularly a cellulase derived from Trichoderma was used. This result reflects the fact that the optimum reaction temperature for a cellulase derived from Trichoderma is 40° C. to 50° C. That is, it was revealed that although the glucosidase derived from a thermophile according to the present invention was still highly active at 50° C. or higher, when it is used as an enzyme composition for saccharification of biomass containing a cellulase derived from filamentous fungi, the reaction was preferably carried out within a range of the optimum reaction temperature for the cellulase derived from filamentous fungi.
Example 9
Hydrolysis of Lignocellulose Using the Enzyme Composition Composed of a Mixture of Cellulases Derived from Filamentous Fungi and the Glycosylated Mutant PfuBGL (4)
Using the enzyme composition composed of a cellulase derived from filamentous fungi and the glycosylated mutant PfuBGL, the amounts of glucose produced were comparatively studied by varying the pH conditions of the hydrolysis reaction. By setting the pH of the hydrolysis reaction to 1.2, 3.5, 5.0 (Example 7), 7.0, or 8.2 by the addition of diluted sulfuric acid, the hydrolysis was performed by a similar procedure to Example 7, and the amount of glucose produced after 28 hours was measured. As the substrate, lignocellulose 1 was used.










TABLE 4








Concentration of



Reaction pH
glucose produced



of hydrolysis
(g/L)









1.2
 2 g/L



3.5
12 g/L



5 (Example 7)
15 g/L



7  
10 g/L



8.2
 5 g/L










As shown in Table 4, it was revealed that it was preferable to carry out the hydrolysis in a range of pH 3.5 to pH 7 when a cellulase derived from filamentous fungi, particularly a cellulase derived from Trichoderma is used.
Comparative Example 2
Preparation of a Glucosidase Homologous to PfuBGL (2)
Seven kinds of glucosidases having homologous amino acid sequences to PfuBGL prepared in Comparative Example 1 were prepared as follows.
DNA sequences of SEQ ID NOs: 23, 25, 27, 29, 31, 33, and 35 were each entirely synthesized and incorporated into the cloning site of pET-11d (between NcoI and BamHI), whereby the expression vector for each of the above DNA was constructed. Subsequently, in a similar manner to Comparative Example 1, the wild-type glucosidases represented by SEQ ID NOs: 24, 26, 28, 30, 32, 34, and 36 (ThAggBGY, CmGHFP, SaBGAL, SsoBGAL, PtBGAL, TvBGAL, and FnGHFP) were obtained.
Example 10
Preparation of a Glycosylated Mutant Glucosidase Homologous to PfuBGL
Based on the information of glycosylation sequence-introduction site in PfuBGL determined in Example 2, the sugar chain-introduction site in the wild-type glucosidases obtained in Comparative Example 2 (ThAggBGY, CmGHFP, SaBGAL, SsoBGAL, PtBGAL, TvBGAL, and FnGHFP) were determined, whereby the glycosylated mutant glucosidases were each prepared.
DNA sequences of SEQ ID NOs: 37, 39, 41, 43, 45, 47, and 49 encoding glycosylated mutant glucosidase were each entirely synthesized and incorporated between EcoRI and NotI of the pCU9 vector, whereby the expression vector for each of the above DNA was constructed. Subsequently, in a similar manner to Example 3, glycosylated mutant glucosidases represented by the amino acid sequences SEQ ID NOs: 38, 40, 42, 44, 46, 48, and 50 were obtained.
Example 11
Enzyme Activity of the Glycosylated Mutant Glucosidase
The enzyme activity of the glycosylated mutant glucosidases obtained in Example 10 was measured in a similar manner to Example 4 and compared with the enzyme activity of each wild-type glucosidase obtained in Comparative Example 2. Setting the enzyme activity of each wild-type glucosidase at 100, the enzyme activity of the glycosylated mutant was shown as relative activity (%) in Table 5.










TABLE 5








Relative activity (%)





of glycosylated





mutant glucosidase


Entry No.
SEQ ID NO
Glucosidase
to wild-type


















1 (Example 4)
6
PfuBGL
85%


2
38
ThAggBGY
90%


3
40
CmGHFP
83%


4
42
SaBGAL
87%


5
44
SsoBGAL
81%


6
46
PtBGAL
85%


7
48
TvBGAL
93%


8
50
FnGHFP
78%









In Table 5, the glycosylated mutant glucosidases were found to retain the enzyme activity in comparison with the wild-type glucosidase before glycosylation.
Example 12
Heat Stability of the Glycosylated Mutant Glucosidase
In a similar manner to Example 5, a heat stability test was performed on each wild-type glucosidase obtained in Comparative Example 2 and on each glycosylated mutant glucosidase obtained in Example 10. Regarding the heat stability, by setting the enzyme activity before applying heat at 100, the residual activity at each a heat retention time was shown as relative activity in FIGS. 8 to 20. It was revealed that in all glucosidases, the glycosylated mutants (FIG. 9, FIG. 11, FIG. 13, FIG. 15, FIG. 17, FIG. 19, and FIG. 21) exhibited improved heat stability compared to the wild-type (FIG. 8, FIG. 10, FIG. 12, FIG. 14, FIG. 16, FIG. 18, and FIG. 20).
Example 13
Hydrolysis of Lignocellulose Using the Enzyme Composition Composed of a Mixture of Cellulases Derived from Filamentous Fungi and the Glycosylated Mutant Glucosidase (5)
In a similar manner to Example 6, using each wild-type glucosidase obtained in Comparative Example 2 and each glycosylated mutant glucosidase obtained in Example 10, hydrolysis of lignocellulose 1 (ammonia treatment) prepared in Reference Example 1 was carried out. The amount of glucose produced by each glucosidase after 28 hours of reaction (g/L) was shown in Table 6.









TABLE 6









Concentration of glucose produced (g/L)













Glycosylated mutant


Entry

Wild-type glucosidase
glucosidase


No.
Glucosidase
(Comparative Example 2)
(Example 10)





2
ThAggBGY
5 g/L
10 g/L


3
CmGHFP
4 g/L
 9 g/L


4
SaBGAL
5 g/L
11 g/L


5
SsoBGAL
6 g/L
12 g/L


6
PtBGAL
5 g/L
 9 g/L


7
TvBGAL
4 g/L
10 g/L


8
FnGHFP
6 g/L
13 g/L









As shown in Table 6, in the hydrolysis of ammonia-treated lignocellulose 1, the sugar production by the glycosylated mutant glucosidases per unit time was greatly increased compared to the wild-type, confirming that the glycosylated mutant glucosidases had excellent cellulose degradation efficiency.
Example 14
Hydrolysis of Lignocellulose Using the Enzyme Composition Composed of a Mixture of Cellulases Derived from Filamentous Fungi and the Glycosylated Mutant Glucosidase (6)
In a similar manner to Example 6, using each wild-type glucosidase obtained in Comparative Example 2 and each glycosylated mutant glucosidase obtained in Example 10, hydrolysis of lignocellulose 2 (diluted sulfuric acid treatment) prepared in Reference Example 1 was carried out. The amount of glucose produced by each glucosidase after 28 hours of reaction (g/L) was shown in Table 7.









TABLE 7









Concentration of glucose produced (g/L)













Glycosylated mutant


Entry

Wild-type glucosidase
glucosidase


No.
Glucosidase
(Comparative Example 2)
(Example 10)





2
ThAggBGY
5 g/L
10 g/L


3
CmGHFP
4 g/L
 9 g/L


4
SaBGAL
5 g/L
11 g/L


5
SsoBGAL
6 g/L
12 g/L


6
PtBGAL
5 g/L
 9 g/L


7
TvBGAL
4 g/L
10 g/L


8
FnGHFP
6 g/L
13 g/L









As shown in Table 7, in the hydrolysis of diluted sulfuric acid-treated lignocellulose 2, the sugar production by the glycosylated mutant glucosidases per unit time was greatly increased compared to the wild-type, confirming that the glycosylated mutant glucosidases had excellent cellulose degradation efficiency.
Example 15
Hydrolysis of Lignocellulose Using the Enzyme Composition Composed of a Mixture of Cellulases Derived from Filamentous Fungi and the Glycosylated Mutant Glucosidase (7)
In a similar manner to Example 6, using each wild-type glucosidase obtained in Comparative Example 1 and Comparative Example 2 and each glycosylated mutant glucosidase obtained in Example 1 and Example 10, hydrolysis of lignocellulose 3 (hydrothermal treatment) prepared in Reference Example 1 was carried out. The amount of glucose produced by each glucosidase after 28 hours of reaction (g/L) was shown in Table 8.









TABLE 8









Concentration of glucose produced (g/L)













Glycosylated mutant


Entry

Wild-type glucosidase
glucosidase


No.
Glucosidase
(Comparative Example 1, 2)
(Example 1, 10)





1
PfuBGL
4 g/L
11 g/L


2
ThAggBGY
4 g/L
12 g/L


3
CmGHFP
5 g/L
14 g/L


4
SaBGAL
5 g/L
14 g/L


5
SsoBGAL
4 g/L
12 g/L


6
PtBGAL
5 g/L
15 g/L


7
TvBGAL
4 g/L
13 g/L


8
FnGHFP
6 g/L
15 g/L









As shown in Table 8, in the hydrolysis of hydrothermally treated lignocellulose 3, the sugar production by the glycosylated mutant glucosidases per unit time was greatly increased compared to the wild-type, confirming that the glycosylated mutant glucosidases had excellent cellulose degradation efficiency.
Example 16
Hydrolysis of Industrial Pulp Using the Enzyme Composition Composed of a Mixture of Cellulases Derived from Filamentous Fungi and the Glycosylated Mutant Glucosidase
In the hydrolysis of industrial pulp, the amount of glucose produced by the action of each wild-type glucosidase obtained in Comparative Example 1 and Comparative Example 2 and that produced by the action of each glycosylated mutant glucosidase obtained in Example 1 and Example 10 were comparatively studied. Hydrolysis was performed in the same manner as Example 6 except for using a 50 mM acetate buffer (pH 5.0) in which 5 wt. % industrial pulp (manufactured by To a Kasei Co., Ltd.) was suspended as a substrate. The amount of glucose produced by each glucosidase after 28 hours of reaction (g/L) was shown in Table 9.









TABLE 9









Concentration of glucose produced (g/L)













Glycosylated mutant


Entry

Wild-type glucosidase
glucosidase


No.
Glucosidase
(Comparative Example 1, 2)
(Example 1, 10)





1
PfuBGL
9 g/L
12 g/L


2
ThAggBGY
8 g/L
11 g/L


3
CmGHFP
8 g/L
11 g/L


4
SaBGAL
10 g/L 
13 g/L


5
SsoBGAL
10 g/L 
12 g/L


6
PtBGAL
9 g/L
11 g/L


7
TvBGAL
8 g/L
10 g/L


8
FnGHFP
11 g/L 
13 g/L









As shown in Table 9, also in the hydrolysis of industrial pulp, the sugar production by the glycosylated mutant glucosidases per unit time was greatly increased compared to the wild-type, confirming that the glycosylated mutant glucosidases had excellent cellulose degradation efficiency.
Example 17
Selection of the Glycosylation Mutation-Induction Site
Based on the information of the tertiary structure of PfuBGL obtained in Example 1, a further search for glycosylation site was conducted. Protection of the catalytic site by glycosylation was attempted by introducing the glycosylation sequence around the cleft where the enzymatic reaction takes place. First of all, the potential glycosylation mutation-introduction site was limited to, in the part other than the sugar chain mutation-introduction site in PfuBGL described in Example 1, the part that is exposed to the surface of the enzyme. Subsequently, such a position that would not cause the steric hindrance and/or structural deformation by introduction of mutation of the glycosylation sequence Asn-Xaa-Thr (N—X-T) was searched. Also, in the part where the active site of glucosidase was avoided, three mutation-introduction sites were selected.
As a result, in addition to the glycosylation site selected in Example 1, three mutation-introduction sites were newly selected, which were H37-D38-K39 (mutation A), S230-F231-E232 (mutation C), and A364-Y365-E366 (mutation E).
Example 18
Preparation of the Glycosylated Mutant PfuBGL (2)
In order to introduce the glycosylation sequence into the mutation-introduction site determined in Example 17 in the glycosylated mutant PfuBGL shown in SEQ ID NO: 6, using primers for introducing mutation A shown in SEQ ID NO: 57 and SEQ ID NO: 58, N-linked glycosylation sequence mutation was introduced by site-directed mutagenesis. As a result, the glycosylated mutant PfuBGL2A gene of SEQ ID NO: 51 was obtained. Also, as to mutation C and mutation E, using primers for introducing mutation C shown in SEQ ID NO: 59 and SEQ ID NO: 60 and primers for introducing mutation E shown in SEQ ID NO: 61 and SEQ ID NO: 62, respectively, N-linked glycosylation sequence mutation was introduced by site-directed mutagenesis in a similar manner, whereby the glycosylated mutant PfuBGL2C gene shown in SEQ ID NO: 53 and the glycosylated mutant PfuBGL2E gene shown in SEQ ID NO: 55 were each obtained. Using the glycosylated mutant genes prepared as above, the glycosylated mutant PfuBGL2A represented by SEQ ID NO: 52, the glycosylated mutant PfuBGL2C represented SEQ ID NO: 54, and the glycosylated mutant PfuBGL2E represented by SEQ ID NO:56 were each obtained by following the same steps as in Example 3.
Example 19
Enzyme Activity of the Glycosylated Mutant PfuBGL (2)
In a similar manner to Example 4, the enzyme activity of the glycosylated mutant glucosidase PfuBGL2A (SEQ ID NO: 52), the glycosylated mutant PfuBGL2C (SEQ ID NO: 54), and the glycosylated mutant PfuBGL2E (SEQ ID NO: 56) obtained in Example 18 was measured in comparison with the wild-type PfuBGL. Setting the enzyme activity of the wild-type PfuBGL at 100, the enzyme activity of the glycosylated mutant was shown as relative activity (%) in Table 10.










TABLE 10








Relative activity (%)





of glycosylated mutant


Entry No.
SEQ ID NO
Glucosidase
glucosidase to wild-type


















9
52
PfuBGL2A
84


10
54
PfuBGL2C
81


11
56
PfuBGL2E
85









As shown in Table 10, it was found that the mutant glucosidase having two or more sugar chains attached thereto retained the enzyme activity in comparison with the wild-type glucosidase before glycosylation.
Example 20
Hydrolysis of Lignocellulose Using the Enzyme Composition Composed of a Mixture of Cellulases Derived from Filamentous Fungi and Various Kinds of Glycosylated Mutant Glucosidases (8)
In a similar manner to Example 6, using the glycosylated mutant glucosidases (PfuBGL2A, PfuBGL2C, and PfuBGL2E) obtained in Example 18, hydrolysis of lignocellulose 2 (diluted sulfuric acid treatment) prepared in Reference Example 1 was carried out. The amount of glucose produced after 28 hours of reaction (g/L) was shown in Table 11.









TABLE 11









Concentration of glucose produced (g/L)













Glycosylated mutant


Entry

Wild-type glucosidase
glucosidase


No.
Glucosidase
(Comparative Example 1)
(Example 18)













1
PfuBGL
3 g/L
—


9
PfuBGL2A
—
18 g/L


10
PfuBGL2C
—
18 g/L


11
PfuBGL2E
—
19 g/L









As shown in Table 11, it was revealed that the mutant glucosidases having two or more sugar chains attached thereto (SEQ ID NO: 52, SEQ ID NO: 54, and SEQ ID NO: 56) also had greatly improved lignocellulose degradation efficiency compared to an unglycosylated wild-type glucosidase (Comparative Example 1; the amount of glucose produced: 3 g/L).
Example 21
Evaluation of the Adsorptivity of the Glycosylated Mutant Glucosidase to Lignocellulose
The adsorptivity of the glycosylated mutant glucosidase to crystalline cellulose (Avicel) and to lignocelluloses 1 to 3 prepared in Reference Example 1 was evaluated. As the glucosidase, the glycosylated mutant PfuBGL prepared in Example 1 and the wild-type PfuBGL prepared in Comparative Example 1 were used, and the enzyme solutions were adjusted so as to have equal specific activities, and further prepared at a final concentration of 100 mM by the addition of an acetate buffer (pH 5). The resulting enzyme solutions were each added to the crystalline cellulose and to lignocelluloses 1 to 3 at a final concentration (solid concentration) of 7.5 wt. %, and the resulting mixtures were kept warm and stirred at 50° C. for one hour. Subsequently, the mixtures after reaction were centrifuged at 15000 rpm for 10 minutes, and the supernatant thus obtained was measured for the cellobiose degradation activity. Setting the enzyme activity of an enzyme solution without crystalline cellulose and lignocelluloses 1 to 3 at 100, the cellobiose degradation activity was evaluated in terms of relative activity (%).










TABLE 12









Relative activity (%) of




cellobiose degradation



activity (%)












Wild-type
Glycosylated



Cellulose
PfuBGL
mutant PfuBGL







Crystalline cellulose
85%
95%



(Avicel)



Lignocellulose 1
23%
84%



(Diluted sulfuric



acid treatment)



Lignocellulose 2
62%
84%



(Ammonia treatment)



Lignocellulose 3
34%
81%



(Hydrothermal



treatment)










As shown in Table 12, it was revealed that the cellobiose degradation activity in the supernatant of the glycosylated mutant glucosidase was increased in the presence of any of the lignocelluloses 1 to 3, compared to the wild-type glucosidase. Also, the cellobiose degradation activity in the supernatant was slightly increased also for Avicel, which was crystalline cellulose. From the above results, it was revealed that the glycosylated mutant glucosidase had low adsorptivity particularly for lignocellulose (pre-treatment cellulose product), and thus would be recovered with good yield from the supernatant after solid-liquid separation.
Example 22
Evaluation of the Adsorptivity of the Glycosylated Mutant Glucosidase to an Ultrafiltration Membrane
The adsorptivity of the glycosylated mutant glucosidase to an ultrafiltration membrane was evaluated. As the ultrafiltration membrane, VIVASPIN20 (SARTORIUS K.K., made of polyethersulfone, a molecular weight cut off of 10000 Da) was used. As the glucosidase, the glycosylated mutant PfuBGL prepared in Example 1 and the PfuBGL prepared in Comparative Example 1 were used, and the enzyme solutions were adjusted so as to have equal specific activities, and further prepared at a final concentration of 50 mM by the addition of an acetate buffer (pH 5). From these enzyme solutions, 5 mL was transferred to the ultrafiltration membrane VIVASPIN20, followed by centrifugation at 4000 G for 10 minutes. After centrifugation, to the solution remained on the non-permeable side of the ultrafiltration membrane (about 100 μL or less), 3 mL of a 50 mM acetate buffer was added, and the residual products left on the surface of the ultrafiltration membrane and on the inner wall of the container were collected by pipetting. The residual product thus collected was made up to 5 mL, and from this, 10 μL was sampled for the cellobiose degradation activity. The above series of operations was repeated seven times, and setting the initial activity at 100(%), the cellobiose degradation activity was calculated as relative activity (%) at each operation. The results thus obtained are shown in Table 13.










TABLE 13









Relative activity (%) of




cellobiose degradation



activity (%)









Number of

Glycosylated


repetition of
Wild-type
mutant


operation
PfuBGL
PfuBGL












0 (initial)
100% 
100%


1
 21%
114%


2
5.4%
115%


3
1.8%
112%


4
ND
116%


5
ND
110%


6
ND
101%


7
ND
98%









As shown in Table 13, it was revealed that the activity of the unglycosylated wild-type PfuBGL was decreased as the number of repetition of the operation was increased (the activity disappeared at the fourth and subsequent operations). Meanwhile, the glycosylated mutant PfuBGL remained active even after seven repeated operations. That is, it was revealed that the glycosylated mutant glucosidase had low adsorptivity for the ultrafiltration membrane, and thus was recovered with good yield when it was recovered using an ultrafiltration membrane after the hydrolysis reaction.
Example 23
Recovery of the Enzyme Composition in the Hydrolysate Using an Ultrafiltration Membrane
From the product of hydrolysis of lignocelluloses 1 to 3 in Example 7, the enzyme composition was recovered as follows. First of all, 10 mL of the hydrolysate was centrifuged and 5 mL of the supernatant was obtained. Subsequently, the supernatant was filtered through a precise membrane filter having an average pore diameter of 0.2 μm (PVDF membrane, manufactured by Millipore Corporation), and the filtrate was collected. The total volume of the filtrate thus obtained was transferred to the ultrafiltration membrane VIVASPIN20 (made of polyethersulfone, a molecular weight cutoff of 10000 Da), followed by centrifugation. The residual product remained on the non-permeable side of the ultrafiltration membrane was collected and measured for the cellobiose degradation activity. By setting the activity of the introduced enzyme at the initial enzyme activity of 100%, the enzyme activity of the enzyme composition recovered as the residual product was calculated in terms of relative value (%).










TABLE 14









Relative activity (%) of




cellobiose degradation



activity of the recovered



enzyme













Glycosylated




Wild-type
mutant



Cellulose
PfuBGL
PfuBGL







Lignocellulose 1
21%
69%



(Diluted sulfuric



acid treatment)



Lignocellulose 2
11%
51%



(Ammonia treatment)



Lignocellulose 3
7.7% 
48%



(Hydrothermal



treatment)










As shown in Table 14, it was revealed that the activity of the recovered glycosylated mutant glucosidase was greatly increased compared to the mutant glucosidase. This was assumed to be attributable to decreased adsorptivity of the glycosylated mutant glucosidase for lignocellulose as demonstrated in Example 21, and also to decreased adsorptivity of the glycosylated mutant glucosidase for the ultrafiltration membrane as demonstrated in Example 22.
Example 24
Analysis of the Sugar Chain Component of the Glycosylated Mutant Glucosidase
The sugar chain structure of the glycosylated PfuBGL described in Example 1 was analyzed. To 1.33 mg of a sample, which was freeze-dried (−80° C.) glycosylated PfuBGL, 1.33 mL of purified water was added to prepare a 1 mg/mL sample solution. The neutral sugar and amino sugar in this sample solution were quantitated by the following procedure.
1. Neutral Sugar
Into a test tube, 100 μL of the 1 mg/mL sample solution was transferred, which was dried under reduced pressure, to which 200 μL of 2 M trifluoroacetic acid was added. The resulting test tube was subjected to nitrogen substitution and then sealed under reduced pressure. Subsequently, the hydrolysis was performed at 100° C. for six hours, and the resulting product was dried again under reduced pressure. To the residue thus obtained, 200 μL of purified water was added, in which the residue was dissolved. The resulting solution was filtered through a filter of 0.22 μm. A sample obtained by diluting the filtrate 10-fold with purified water was analyzed under the following conditions.
As the analytical instrument, the HPLC system LC20A system (Shimadzu Corporation) and the spectrofluorometer RF-10AXL (Shimadzu Corporation) were used.
The analytical conditions were as follows; TSK-gel Sugar AXG 4.6 mm I.D.×15 cm (Tosoh Corporation), the column temperature, 70° C., the mobile phase, a 0.6 M potassium borate buffer (pH 8.7), and the mobile phase flow rate, 0.4 mL/min.
Using 1 wt. % arginine and 3 wt. % boric acid as the reaction reagent, post-column labeling was performed at a reaction reagent flow rate of 0.5 mL/min and a reaction temperature of 150° C. Also, as to the detection wavelength, the excitation and the detection were performed at 320 nm and 430 nm, respectively. The neutral sugar was quantitated in comparison with each standard product of neutral sugar.
2. Amino Sugar
Into a test tube, 100 μL of the 1 mg/mL sample solution was transferred, which was dried under reduced pressure, to which 200 μL of 4 M trihydrochloric acid was added. The resulting test tube was subjected to nitrogen substitution and then sealed under reduced pressure. Subsequently, the hydrolysis was performed at 100° C. for six hours, and then the resulting product was dried again under reduced pressure. To the residue thus obtained, 200 μL of purified water was added, in which the residue was dissolved. The resulting solution was filtered through a filter of 0.22 μm.
As the analytical instrument, the HPLC system LC20A system (Shimadzu Corporation) and the spectrofluorometer RF-10AXL (Shimadzu Corporation) were used.
The analytical conditions were as follows; TSK-gel SCX 6 mm I.D.×15 cm (Tosoh Corporation), the column temperature, 60° C., the mobile phase, a 0.16 M boric acid potassium borate buffer (pH 7.6), and the mobile phase flow rate, 0.3 mL/min.
Using 1 wt. % arginine and 3 wt. % boric acid as the reaction reagent, post-column labeling was performed at a reaction reagent flow rate of 0.5 mL/min and a reaction temperature of 150° C. Also, as to the detection wavelength, the excitation and the detection were performed at 320 nm and 430 nm, respectively. The neutral sugar was quantitated in comparison with each standard product of neutral sugar.









TABLE 15






Concentration
Composition


Sugar
(nmol/mg)
ratio (*1)


















Neutral sugar
Rhamnose
30
1



Ribose
ND
—



Mannose
576 
18 



Arabinose
24
1



Galactose
75
2



Xylose
ND
—



Glucose
54
2


Amino sugar
Glucosamine
62
2



Galactosamine
37
1





(*1) By setting the value of the concentration B of glucosamine at “2” as the standard, the concentrations B of other sugars were calculated as relative ratio. In calculating, the digits after the decimal point were rounded to an integer.






As shown in Table 15, it was revealed that the main component of the neutral sugar composing the sugar chain of the sugar chain mutant PfuBGL obtained in Example 1 was mannose. Also, from the composition ratio of mannose to N-glucosamine, the sugar chain mutant PfuBGL was found to have a high mannose type sugar chain.
Industrial Applicability
The glycosylated mutant glucosidase derived from a thermophile according to the present invention can be used for the production of a sugar liquid by cellulose degradation. Because the glycosylated mutant glucosidase derived from a thermophile has an effect of greatly increasing the cellulose degradation efficiency, it is capable of considerably reducing the cost of enzyme. In light of the above, the glycosylated mutant glucosidase derived from a thermophile according to the present invention is extremely industrially beneficial.
All of the publications, patents, and patent applications cited in the present specification are hereby incorporated by reference in their entirety.

## Claims

1. A method of producing a mutant glucosidase derived from a thermophile having a sugar chain bound to an amino acid side chain of a peptide sequence of the mutant glucosidase by covalent binding and having glucosidase activity, wherein the mutant glucosidase demonstrates enhanced ability to increase saccharification of a lignocellulose substrate which has been subjected to pre-treatement when added to cellulase as compared to the corresponding unglycosylated wild-type glucosidase of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20, which comprises:
(i) preparing DNA encoding the mutant glucosidase derived from a thermophile by introducing a DNA sequence encoding Asn-X-Ser or Asn-X-Thr (wherein, X is any amino acid except proline) into DNA encoding a glucosidase derived from a thermophile originally devoid of a glycosylation sequence and adding a DNA sequence encoding a secretion signal sequence to the DNA encoding the mutant glucosidase,
wherein the glucosidase derived from the thermophile is a protein comprising:
(a) any of the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20, or
(b) an amino acid sequence having 95% or more identity with any of the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20,
and β-glucosidase activity,
the DNA sequence encoding Asn-X-Ser or Asn-X-Thr is substituted for amino acids of the glucosidase derived from a thermophile corresponding to amino acids of H60-L61-Y62 in the amino acid sequence shown in SEQ ID NO: 4,
(ii) introducing the DNA encoding the mutant glucosidase to which the DNA sequence encoding the secretion signal sequence has been added into an eukaryotic microorganism so that a mutant glucosidase encoded by the DNA of the mutant glucosidase is expressed as a secretory protein, and
(iii) isolating and purifying the mutant glucosidase thus expressed as a secretory protein.

2. The method according to claim 1, wherein the sugar chain is a high mannose type sugar chain.

3. The method according to claim 2, wherein the eukaryotic microorganism is Pichia pastoris.

4. The method according to claim 2, wherein the secretion signal sequence is an α factor secretion signal sequence.

5. The method according to claim 1, wherein the eukaryotic microorganism is Pichia pastoris.

6. The method according to claim 1, wherein the secretion signal sequence is an α factor secretion signal sequence.

7. The method according to claim 1, wherein the mutant glucosidase derived from a thermophile comprises an amino acid sequence shown in any of SEQ ID NO: 6, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, and SEQ ID NO: 50.

8. The method according to claim 1, wherein the lignocellulose substrate is rice straw.

9. The method according to claim 1, wherein the pre-treatment is ammonia treatment, diluted sulfuric acid treatment or hydrothermal treatment.

10. The method according to claim 1, wherein the cellulase is derived from Trichoderma reesei.

11. The method according to claim 1, in step (i), when the glucosidase derived from the thermophile is a protein comprising:
(a) the amino acid sequence shown in SEQ ID NO: 4, or
(b) the amino acid sequence having 95% or more identity with the amino acid sequence shown in SEQ ID NO: 4 and a β glucosidase activity,
the DNA sequence encoding Asn-X-Ser or Asn-X-Thr is substituted for amino acids of the glucosidase derived from a thermophile corresponding to amino acids selected from H37-D38-K39, S230-F231-E232, and A364-Y365-E366 in addition to H60-L61-Y62 in the amino acid sequence shown in SEQ ID NO: 4.

12. The method according to claim 11, wherein the mutant glucosidase derived from a thermophile comprises an amino acid sequence shown in any of SEQ ID NO: 52, SEQ ID NO: 54, and SEQ ID NO: 56.