JP5605331B2 - Protein having β-glucosidase activity and use thereof - Google Patents

Description

The present invention relates to a novel protein having β-glucosidase activity and use thereof.

In recent years, from the viewpoint of effective use of plant-based biomass, fermentative production of alcohol using fermented biomass of plant-based biomass resources and cellulose contained in unused resources, or fermentation of lactic acid, which is a raw material for biodegradable plastics Attempts have been made to produce them. For example, many reports have been made on attempts to produce lactic acid from glucose obtained by saccharification of cellulose by introducing L-lactic acid or D-lactic acid dehydrogenase genes into the genus Saccharomyces cerevisiae.

Here, the saccharification of cellulose requires the presence of a cellulase that hydrolyzes the β-D-glucopyranoside bond. Cellulases include cellobiohydrase (also called exoglucanase) that hydrolyzes high molecular weight cellulose in an exo form, endoglucanase that hydrolyzes in an endo form, cellobiohydrase and endoglucanase to some extent. Β-glucosidase that can be decomposed into D-glucose. Among these, as β-glucosidase, for example, β-glucosidase having excellent heat resistance derived from Thermoanaerobacter cellulolyticus has been reported (Patent Document 1). This β-glucosidase has an optimum pH of pH 5.0 to pH 6.0 and is stable in the range of pH 4.0 to 8.0. The temperature stability is stable up to 80 ° C. In addition, by recombining part of the β-glucosidase gene with low heat resistance to part of the gene with different heat resistance, β-glucosidase, which has a different origin, the heat resistance of the former β-glucosidase has been improved. It has been reported (Patent Document 2). This β-glucosidase has an optimum pH of 6.0 and is stable in the range of pH 4-9. Further, the optimum temperature is 40 to 45 ° C, and it is stable in the range of 25 to 35 ° C.

JP 10-52274 A JP-A-8-131168

When producing organic acids and alcohols such as lactic acid from cellulose, in order to reduce the complexity of the production equipment and production process and efficiently produce them, cellulose is decomposed and organic acids and alcohols are produced at the same time by fermentation. It is preferable. However, when an organic acid is produced by simultaneous fermentation, the organic acid increases in the medium as the fermentation progresses, and cellulase such as β-glucosidase is exposed to acidic conditions by this organic acid. For this reason, a cellulase having high activity under acidic conditions is required. However, no β-glucosidase having acid resistance has been found yet. In addition, considering the case of using yeast in producing an organic acid from cellulose, cellulase having high enzyme activity at the yeast growth temperature (for example, 30 ° C.) is required. However, for β-glucosidase, although the improvement in heat resistance has been studied, the activity in the vicinity of the growth temperature of yeast has not been studied. Therefore, an object of the present invention is to provide such a protein.

According to the present invention, at any pH of 30 ° C. and pH 5.0 or lower, β-glucosidase having the amino acid sequence of SEQ ID NO: 1 or 2 has an activity equal to or higher than β-glucosidase activity under the same conditions. A protein having β-glucosidase activity is provided. In this protein, it is preferable that the protein has an activity equal to or higher than β-glucosidase activity at the optimum pH of β-glucosidase consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2 at pH 4.0. Furthermore, at pH 3.0, it preferably has an activity equal to or higher than β-glucosidase activity at the optimum pH of β-glucosidase consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2.

Moreover, in the protein of this invention, it is a preferable aspect to have (beta) -glucosidase activity whose optimal pH in 30 degreeC is 5.0 or less.

Furthermore, the protein of the present invention has an activity equal to or higher than the β-glucosidase activity under the same conditions of β-glucosidase consisting of the amino acid sequence of SEQ ID NO: 1 at 30 ° C and any pH of 5.0 or lower. And one or several amino acid sequences of the amino acid sequence shown in SEQ ID NO: 1 have a substitution, deletion, addition and insertion sequence, and one or several amino acids in the vicinity of the active center of the β-glucosidase In a preferred embodiment, the amino acid residue is substituted with a more acidic amino acid residue. In the protein, the valine corresponding to position 169 in the amino acid sequence of SEQ ID NO: 1 is substituted with an acidic amino acid. More preferably, the acidic amino acid is aspartic acid.

Furthermore, in any one of the above proteins, one or several amino acids are preferably substituted with more polar amino acids in the vicinity of the surface of the β-glucosidase. In the described amino acid sequence, the amino acid residue corresponding to position 130 and / or position 382 or position 385 is preferably substituted with a polar amino acid, more preferably the polar amino acid is selected from neutral amino acids More preferably, the polar amino acid is glutamine or threonine.

Alternatively, the protein of the present invention has an activity equal to or higher than the β-glucosidase activity under the same conditions of β-glucosidase consisting of the amino acid sequence of SEQ ID NO: 2 at 30 ° C. and any pH of 5.0 or lower. The amino acid sequence of SEQ ID NO: 2 has one or more amino acid sequences substituted, deleted, added and inserted. In the protein, the amino acid sequence of SEQ ID NO: 2 In a preferred embodiment, the cysteine residue corresponding to the position is substituted with a polar and neutral amino acid residue other than cysteine, and the polar and neutral amino acid is preferably serine.

In any of the above proteins, the protein described in SEQ ID NO: 1 or 2 is preferably a thermostable β-glucosidase.

According to another aspect of the present invention, there is provided a cell that retains a polynucleotide encoding the protein described above in an expressible manner. In this cell, it is preferable that the protein can be expressed as a cell surface-presenting protein or a secreted protein. Preferably, the polynucleotide encoding one or more enzymes related to organic acid production is preferably expressed so that the organic acid is lactic acid, and the enzyme is lactate dehydrogenase. is there. Or it is a preferable aspect to hold | maintain the polynucleotide which codes 1 type, or 2 or more types of enzymes relevant to ethanol production so that expression is possible. In a preferred embodiment, the cell is yeast.

According to another aspect of the present invention, there is provided an organic acid production method comprising a step of producing an organic acid using any of the cells described above. According to still another embodiment, there is provided an ethanol production method comprising the step of producing ethanol using any of the cells described above.

It is a graph which shows the pH dependence of the beta-glucosidase activity of the modification enzyme A in Example 1, and is a figure which shows the beta-glucosidase activity of the protein of this invention. The figure which shows the amino acid sequence of the modification enzyme A in Example 1. FIG. It is a graph which shows the pH dependence of (beta) -glucosidase activity of the variant V169D and L130Q in Example 1, and is a figure which shows (beta) -glucosidase activity of the protein of this invention. It is a graph which shows the measurement result of the beta-glucosidase activity of the modification enzyme B in Example 2, and is a figure which shows the beta-glucosidase activity of the protein of this invention. It is a graph which shows the pH dependence of the beta-glucosidase activity of the modification enzyme B in Example 2, and is a figure which shows the beta-glucosidase activity of the protein of this invention. The figure which shows the amino acid sequence of the modification enzyme B in Example 2.

The protein having β-glucosidase activity of the present invention is a β-glucosidase activity under the same conditions of β-glucosidase comprising the amino acid sequence of SEQ ID NO: 1 or 2 at 30 ° C. and any pH of 5.0 or lower. It has the above activity. By using the β-glucosidase of the present invention, cellulose is more saccharified than the known β-glucosidase having the amino acid sequence of SEQ ID NO: 1 or 2 at 30 ° C. and any pH of 5.0 or lower. Can be done efficiently.

In addition, when the protein of the present invention can be expressed in a microorganism, even if the medium is grown at a low pH, contamination by other bacteria can be effectively prevented.

The protein of the present invention is any one selected from substitution, deletion, addition and insertion of one or more amino acids of thermostable β-glucosidase derived from Thermotoga maritima which is a thermophilic eubacteria It has an amino acid sequence called mutation. For this reason, it is estimated that the tolerance with respect to various physicochemical conditions is strong, and it is suitable also when using for industrial production of glucose from the cellulose as plant biomass.

Furthermore, in a cell capable of expressing the protein of the present invention and an enzyme related to organic acid production, when the organic acid is produced using a microorganism constituted by the cell, the pH of the medium is increased by the organic acid as a product. Even when it becomes low, saccharification of cellulose can be performed efficiently. Moreover, the process using an acid can be more actively performed in the production process of an organic acid.

Hereinafter, the protein of the present invention will be described, and a cell that holds the polynucleotide encoding the protein in an expressible manner will be described.

(Β-glucosidase consisting of the amino acid sequence described in SEQ ID NO: 1)
Β-glucosidase consisting of the amino acid sequence shown in SEQ ID NO: 1 is β-glucosidase derived from Thermotoga maritima, which is a thermophilic eubacteria. According to glycosyl hydrolase family (GHF), which is a protein structural classification classified by analyzing amino acid sequences of carbohydrate-related enzymes, this β-glucosidase is classified into GH1 family among the three families to which β-glucosidase belongs. Is done. As the physicochemical properties of this β-glucosidase, the optimum pH at 30 ° C. is pH 6.2, and the optimum temperature is 85 ° C.

(Β-glucosidase consisting of the amino acid sequence shown in SEQ ID NO: 2)
Β-glucosidase consisting of the amino acid sequence shown in SEQ ID NO: 2 is β-glucosidase derived from Thermotoga maritima which is a thermophilic eubacteria. According to glycosyl hydrolase family (GHF), this β-glucosidase is classified into GH3 family among the three families to which β-glucosidase belongs. Moreover, as a physicochemical property of this β-glucosidase, the optimum pH at 30 ° C. is pH 5, and the optimum temperature is 65 to 85 ° C.

The substrate on which β-glucosidase consisting of the amino acid sequences described in SEQ ID NOs: 1 and 2 acts is one or more sugars selected from short-chain cellooligosaccharides, cellobiose and β-glucoside, and derivatives thereof. Yes, it may be derived from animals or plants. When these saccharides are derived from plants, they can be obtained by hydrolysis of cellulose and hemicellulose constituting the carbohydrate material of plant biomass resources. Derivatives such as cellooligosaccharides include uronic acids such as galacturonic acid and glucuronic acid, ester derivatives in which hydroxyl groups are esterified, and alkyl derivatives in which hydroxyl groups are methylated as constituent monosaccharides.

In this specification, hemicellulose is a polysaccharide that constitutes a plant cell wall together with cellulose or a polysaccharide other than cellulose that constitutes a plant cell wall, and is a generic term for a homopolysaccharide or a heteropolysaccharide. Examples of hemicellulose include mannan, glucomannan, glucoxylan, galactan, xylan, arabinan, arabinoxylan, arabinogalactan, pectin, chitin, galactoglucomannan, clonoxylan, and xyloglucan.

(PH and β-glucosidase activity of the protein of the present invention)
The protein of the present invention has a β-glucosidase activity higher than that of a known β-glucosidase consisting of the amino acid sequence of SEQ ID NO: 1 or 2 under the same conditions at 30 ° C. and any pH of 5.0 or lower. -It has glucosidase activity. According to this protein, for example, when lactic acid is produced from cellulose using this protein, saccharification of cellulose can be efficiently carried out even if the pH in the culture solution is lowered by the produced lactic acid. The pH at which the β-glucosidase activity is higher than the β-glucosidase of SEQ ID NO: 1 or 2 is preferable in that contamination by other germs can be effectively prevented within the range of pH 5.0 or lower. The pH is any one of pH 4.0 or less. Moreover, since the process for neutralizing the lactic acid produced | generated when producing lactic acid from a cellulose becomes unnecessary, More preferably, it is any pH of 3.0 or less. Further, the ratio of the activity of SEQ ID NO: 1 or 2 to β-glucosidase is preferably 2.0 times or more, more preferably 2.5 times or more, and further preferably 3.0 times or more.

In addition, the β-glucosidase activity is preferably higher than the β-glucosidase activity at the optimum pH of the known β-glucosidase consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2 at 30 ° C. and pH 4.0. Preferably it is 1.2 times or more. Alternatively, at pH 3.0, it is preferably higher than the β-glucosidase activity at the optimum pH of the known β-glucosidase consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2, more preferably 1.3 times or more .

In addition, the β-glucosidase activity of the obtained protein can also be confirmed by a sugar hydrolase activity measurement using a chromogenic substrate known to those skilled in the art. As the chromogenic substrate, a known compound in which a chromogenic substance and a short-chain glucoside are bonded by a β-D-glucopyranoside bond can be used, for example, p-nitrophenyl group, o-nitrophenyl group, phenyl group, β-glucoside having a coloring group such as p-hydroxyphenyl group, p-chlorophenyl group, 6-bromo-2-naphthyl group, and a coloring substance such as phenolphthalein, 8-hydroxyquinoline, 8-hydroxyquinoline and β- The thing etc. which the glucoside couple | bonded can be mentioned. Among these, when p-nitrophenyl-β-D-glucopyranoside is used as a chromogenic substrate, the enzyme activity can be measured by measuring the absorbance of p-nitrophenol, for example, the absorbance at 405 nm. In addition, a method for measuring enzyme activity using a fluorescent substrate such as 4-methylumbelliferyl-β-D-glucoside, and glucose produced from the substrate using a Glucostat reagent capable of quantifying glucose For example, a method for measuring enzyme activity according to the amount.

The optimum pH at 30 ° C. of the protein of the present invention is preferably pH 5.0 or less because it is shifted to the acidic side from the optimum pH of β-glucosidase of SEQ ID NO: 1. Moreover, since it has shifted to the acidic side from the optimum pH of β-glucosidase of SEQ ID NO: 2, pH 4.0 or less is preferable. In addition, for example, when the lactic acid is produced from cellulose using the protein of the present invention, the pH stability is such that cellulose can be efficiently saccharified even if the pH in the culture solution is lowered by the produced lactic acid. It is preferably stable at pH 2.5 or more, more preferably pH 2.0 or more.

Specific examples of the protein having β-glucosidase activity of the present invention include the following proteins.

One protein has an amino acid sequence having a mutation selected from substitution, deletion, addition and insertion of one or more amino acids of the amino acid sequence shown in SEQ ID NO: 1 or a combination thereof. Can be mentioned. Such a protein can be obtained, for example, by screening based on molecular evolution techniques such as mutant PCR and DNA shuffling. Further, from a DNA obtained by a general hybridization technique (Southern, EM., J Mol Biol, 1975, 98, 503) using a DNA encoding the amino acid sequence shown in SEQ ID NO: 1 or a part thereof as a probe. Can be synthesized. In addition, such a protein is obtained by PCR technology (Saiki, RK. Science, 1985, 230, 1350., et al., Etc.) using an oligonucleotide that specifically hybridizes to DNA encoding the amino acid sequence shown in SEQ ID NO: 1 as a primer. Saiki, RK. Et al., Science, 1988, 239, 487). In addition, if a condition with low stringency is selected as the hybridization condition, a base mutation can be easily introduced.

Furthermore, a mutation is artificially introduced into DNA encoding the amino acid sequence described in SEQ ID NO: 1 by the site-directed mutagenesis method (Kramer, W. & Fritz, HJ., Method Enzymol., 1987, 154, 350). Can also be obtained.

The homology with the amino acid described in SEQ ID NO: 1 is preferably 80% or more, more preferably 90% or more, and still more preferably 98% or more in the isolated protein.

It has an amino acid sequence having a mutation selected from substitution, deletion, addition and insertion of one or more amino acids of the amino acid sequence shown in SEQ ID NO: 1 or a combination thereof, and has β-glucosidase activity The protein is preferably one in which one or several amino acids are substituted with more acidic amino acids in the vicinity of the active center of β-glucosidase of SEQ ID NO: 1. Here, the “active center of β-glucosidase” specifically refers to positions 166 and 351 in SEQ ID NO: 1. The term “near the active center” refers to an amino acid residue located adjacent to or close to the amino acid residue of the active center estimated based on the three-dimensional structure of β-glucosidase. In contrast, domains involved in covalent bonds, ionic interactions, ion-dipole interactions, dipole-dipole interactions, hydrogen bonds, van der Waals forces, electrostatic interactions, hydrophobic interactions, etc. It is good. Specifically, such an amino acid residue is preferably the amino acid at position 169 (valine). Note that the three-dimensional structure and active center of β-glucosidase can be determined by Insight II (Accelrys), which is a known computer program for analyzing the three-dimensional structure of a protein. The charge of the protein can be obtained by Delphi software (Accelrys).

The “more acidic amino acid” refers to an amino acid having a lower isoelectric point than the amino acid to be substituted. When substituting the 169th amino acid (valine) in SEQ ID NO: 1 with a “more acidic amino acid”, as such amino acids, specifically, aspartic acid, glutamic acid, cysteine, glutamine, serine, methionine, phenylalanine, Examples include tyrosine and tryptophan, preferably acidic amino acids such as aspartic acid and glutamic acid, and more preferably aspartic acid.

Furthermore, it has an amino acid sequence having a mutation selected from substitution, deletion, addition and insertion of one or more amino acids of the amino acid sequence shown in SEQ ID NO: 1 or a combination thereof, and β-glucosidase activity As a protein having the above, it is preferable that one or several amino acids are substituted with more polar amino acids in the vicinity of the surface of β-glucosidase of SEQ ID NO: 1. Here, “near the surface of β-glucosidase” refers to an amino acid that is estimated to be located on or near the surface based on the three-dimensional structure of β-glucosidase. Specifically, the amino acid sequence described in SEQ ID NO: 1 The amino acid residue at position 130 (leucine) and / or position 382 (alanine) or position 385 (alanine) is preferred.

The term “more polar amino acid” refers to a more polar amino acid when the amino acid to be substituted is a polar amino acid, and polarity when the amino acid to be substituted is a nonpolar amino acid. An amino acid having a low hydrophobic group due to the amino acid or side chain. As such amino acids, specifically for leucine, in addition to polar amino acids such as glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, lysine, histidine, arginine, aspartic acid, glutamic acid, methionine, etc. The above-mentioned polar amino acids can be mentioned with respect to alanine. Of these, preferred are polar amino acids, more preferred are neutral amino acids selected from glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, and histidine, and more preferred is glutamine or threonine. In particular, those in which position 130 (leucine) is substituted with glutamine and position 382 (alanine) or position 385 (alanine) is substituted with threonine are preferred.

Examples of such a protein include a protein consisting of the amino acid sequence described in any one of SEQ ID NOs: 3 to 9. The protein of the present invention is obtained by using the above-described hybridization technique or PCR technique based on the base sequence of other microorganisms or a part thereof, in addition to these proteins, and has an equivalent β-glucosidase activity. It contains homologues of these proteins.

Among the proteins consisting of the amino acid sequence set forth in any of SEQ ID NOs: 3 to 9, the protein consisting of the amino acid sequence set forth in any of SEQ ID NOs: 3, 6 and 9 is the 169th amino acid sequence set forth in SEQ ID NO: 1. This is preferable in that the amino acid at the position (valine) is substituted with aspartic acid, which is a more acidic amino acid. This is because the optimal pH is considered to shift to the acidic side of β-glucosidase consisting of the amino acid sequence described in SEQ ID NO: 1 due to this mutation. In the protein consisting of the amino acid sequence described in any of SEQ ID NOs: 4 to 9, the amino acid at position 130 (leucine) in the amino acid sequence described in SEQ ID NO: 1 is substituted with glutamine, which is a more polar amino acid. It is preferable in terms. This is because the optimum temperature is considered to have decreased due to this mutation. In the protein consisting of the amino acid sequence shown in any one of SEQ ID NOs: 4, 5, 7, 8, and 9, the amino acid at position 382 (alanine) of the amino acid sequence shown in SEQ ID NO: 1 is a more polar amino acid. In the protein substituted with threonine and having the amino acid sequence set forth in SEQ ID NO: 3 or 6, the amino acid at position 385 (alanine) in the amino acid sequence set forth in SEQ ID NO: 1 is replaced with threonine, which is a more polar amino acid. It is preferable in terms. Also in this case, it is considered that the optimum temperature is lowered by converting the amino acid on the protein surface from hydrophobic to hydrophilic.

Among the proteins consisting of the amino acid sequence set forth in any of SEQ ID NOs: 3 to 9, particularly in the protein consisting of the amino acid sequence set forth in SEQ ID NO: 6 or 9, the protein in the protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 The amino acid at position 169 is replaced with a more acidic amino acid, and the amino acid at position 130 is replaced with a more polar amino acid, and both effects of lowering the optimum pH and lowering the optimum temperature are obtained. It is preferable in terms. Further, in this protein, the amino acid at position 382 or the amino acid at position 385 is further substituted with a more polar amino acid, and β-glucosidase activity under acidic conditions is high and preferable.

As other proteins, those having an amino acid sequence having a mutation selected from substitution, deletion, addition and insertion of one or more amino acids of the amino acid sequence shown in SEQ ID NO: 2 or a combination thereof Can be mentioned. Such a protein can be synthesized from DNA obtained by using a hybridization technique or a PCR technique in addition to the screening based on the molecular evolution method of the protein described above. In addition, if a condition with low stringency is selected as the hybridization condition, a base mutation can be easily introduced. Furthermore, it can also be obtained by artificially introducing a mutation into the DNA encoding the amino acid sequence shown in SEQ ID NO: 2 by the above-mentioned Site-directed mutagenesis method.

The homology with the amino acid described in SEQ ID NO: 2 is preferably 80% or more in the isolated protein, more preferably 90% or more, and still more preferably 98% or more.

It has an amino acid sequence having a mutation selected from substitution, deletion, addition and insertion of one or more amino acids of the amino acid sequence shown in SEQ ID NO: 2 or a combination thereof, and has β-glucosidase activity As the protein, a protein in which the cysteine residue corresponding to position 161 in the amino acid sequence shown in SEQ ID NO: 2 is substituted with a polar and neutral amino acid residue other than cysteine is preferable. Here, specific examples of “polar and neutral amino acids other than cysteine” include glycine, serine, threonine, tyrosine, asparagine, glutamine, histidine and the like, preferably serine.

Examples of such a protein include a protein having the amino acid sequence set forth in any one of SEQ ID NOs: 10 to 15. The protein of the present invention is obtained by using the above-described hybridization technique or PCR technique based on the base sequence of other microorganisms or a part thereof, in addition to these proteins, and has an equivalent β-glucosidase activity. It contains homologues of these proteins.

Among the proteins consisting of the amino acid sequence set forth in any of SEQ ID NOs: 10 to 15, the protein consisting of the amino acid sequence set forth in any of SEQ ID NO: 10 to 14 is positioned at position 161 of the amino acid sequence set forth in SEQ ID NO: 2. The amino acid (cysteine) is preferable in that it is substituted with serine, which is a polar and neutral amino acid other than cysteine. Further, in the protein consisting of the amino acid sequence shown in any of SEQ ID NOs: 10 to 13, the amino acid at position 345 (leucine) in the amino acid sequence shown in SEQ ID NO: 2 is substituted with histidine that is more polar, and position 420 The amino acid (tryptophan) is substituted with a more polar arginine, and the amino acid at position 492 (lysine) is preferably substituted with a more acidic amino acid, glutamic acid. Furthermore, the protein consisting of the amino acid sequence described in any of SEQ ID NOs: 10 to 12 is preferable in that no mutation is introduced into the 89th amino acid (serine) of the amino acid sequence described in SEQ ID NO: 2.

The above protein having β-glucosidase activity can be expressed in cells by gene recombination. “By genetic recombination” means that various methods for introducing an exogenous polynucleotide encoding the protein into a host cell are used. The polynucleotide can be obtained, for example, by PCR so that a part of the polypeptide encoding the protein of SEQ ID NO: 1 or 2 is mutated, and can be obtained by various methods known to those skilled in the art. Can be obtained. The cell modified by genetic recombination so as to promote saccharification of cellulose or the like is preferably a transformant having the above-described polynucleotide introduced on a chromosome.

Specifically, various methods for introducing an exogenous polynucleotide include, for example, transformation methods, transfection methods, conjugation methods, protoplast methods, electroporation methods, lipofection methods, suitable host cells, A polynucleotide encoding the protein having β-glucosidase activity of the present invention by any of various appropriate means such as lithium acetate method, particle gun method, calcium phosphate precipitation method, Agrobacterium method, PEG method, and direct microinjection method Can be mentioned. Thereby, the cell of the present invention can be obtained. After introducing the polynucleotide encoding the protein having β-glucosidase activity of the present invention, the host cell is selected in a selective medium.

Cells for hosting the gene transfer are not particularly limited, but bacteria such as Eshrichia coli and Bacillus subtilis, Saccharomyces pombe yeasts such as Saccharomyces pombe, Pichia pastoris, etc. Yeast, insect cells such as sf9 and sf21, animal cells such as COS cells and Chinese hamster ovary cells (CHO cells), plant cells such as sweet potatoes and tobacco, and the like are preferable. Examples of the yeast include yeasts such as Saccharomyces cerevisiae, such as Saccharomyces cerevisiae, and specific examples include Saccharomyces cerevisiae IFO2260 strain and YPH strain.

Whether or not the target polynucleotide has been introduced into the host cell, or whether or not the target polynucleotide has been introduced into a desired site on the chromosome can be confirmed by PCR or Southern hybridization. . For example, it can be confirmed by preparing DNA from a transformant, performing PCR with an introduction site-specific primer, and detecting the expected band in electrophoresis for the PCR product. Alternatively, it can be confirmed by performing PCR with a primer labeled with a fluorescent dye or the like. These methods are well known to those skilled in the art.

Thus, cells produced by genetic recombination can retain the protein having the β-glucosidase activity. Therefore, if this cell is used, saccharification of cellulose can be efficiently performed at 30 ° C. and any pH of 5.0 or less.

In addition, when β-D-glucose is produced using the above-described cells, treatment with an acid can be performed. For example, when producing β-D-glucose directly from cellulose or a plant biomass resource containing cellulose, sulfuric acid, hydrochloric acid, phosphoric acid, nitric acid are used for the purpose of making the cellulose amorphous in the presence of cellulase such as β-glucosidase. It becomes possible to perform partial hydrolysis of cellulose by adding an acid such as. Therefore, simplification of operation in the case of producing β-D-glucose from cellulose can be achieved.

The polynucleotide encoding the protein of the present invention is preferably provided so that the protein of the present invention can be expressed as a cell surface display protein or a secreted protein. In particular, the protein of the present invention is preferably retained on the cell surface. β-glucosidase is retained in the cell surface and decomposes the cellooligosaccharide and the like on the cell surface, so that the cell can quickly use the cellooligosaccharide and the like. For secretion of β-glucosidase or cell surface display, in addition to the coding region of β-glucosidase, the polynucleotide is a secreted protein according to the type of microorganism used, or a protein for retaining the secreted protein and the cell surface layer. It is preferable to provide a region that encodes. The technology for displaying the desired protein on the cell surface is described in WO 01/79483, Fujita et al. (Fujita et al., 2004. Appl Environ Microbiol 70: 1207-1212 and Fujita et al., 2002. Appl Environ Microbiol 68: 5136-5141.), Murai et al., 1998. Appl Environ Microbiol 64: 4857-4861. And can be carried out based on these descriptions.

Examples of the secreted protein include a secretion signal of a glucoamylase gene of Rhizopus oryzae. Examples of the protein that can be retained on the cell surface include an aggregating protein or a part thereof, for example, from 320 amino acid residues in the 5 ′ region of the SAG1 gene encoding α-agglutinin, which is a sex agglutinin protein. There is a peptide that

In a cell capable of expressing such a protein having β-glucosidase activity of the present invention, a polyenzyme encoding an enzyme related to organic acid production (organic acid-generating enzyme) or an enzyme related to ethanol production (ethanol-generating enzyme) is used. It is preferable to hold the nucleotide so that it can be expressed. When an organic acid producing enzyme is expressed in cells, β-D-glucose is produced from cellulose or the like by the protein having β-glucosidase activity of the present invention, and β-D-glucose is degraded by the organic acid producing enzyme. Thus, an organic acid can be produced directly from cellulose or the like. Thereby, the saccharification process by the enzyme of enzyme of polysaccharide, fermentation, etc. can be skipped, and the complexity of a manufacturing apparatus or a manufacturing process is reduced. In addition, the protein having β-glucosidase activity of the present invention exhibits good β-glucosidase activity even when the production amount of organic acid is increased and the medium is shifted to the acidic side during saccharification of cellulose or the like. be able to.

When ethanol-producing enzyme is expressed in cells, β-D-glucose is produced from cellulose or the like by the protein having β-glucosidase activity of the present invention, and D-glucose is degraded by the ethanol-producing enzyme. Thus, ethanol can be produced directly from cellulose or the like.

In the present specification, the “organic acid” is an organic compound that exhibits acidity, and is a free acid or a salt thereof. The acidic group included in the “organic acid” is preferably a carboxylic acid group. Examples of such “organic acids” include lactic acid, butyric acid, acetic acid, pyruvic acid, succinic acid, formic acid, malic acid, citric acid, malonic acid, propionic acid, ascorbic acid, and adipic acid. These “organic acids” may be DL form in addition to D form and L form. The “organic acid” is preferably lactic acid. In addition to being a raw material for biodegradable plastics, lactic acid is useful as a raw material for various drugs, foods, and feeds. The organic acid producing enzyme means an enzyme that acts in the process of producing the organic acid, such as lactate dehydrogenase that produces lactic acid from pyruvate, succinate dehydrogenase that produces fumaric acid from succinic acid, etc. And citrate synthase that produces citrate from oxaloacetate. The organic acid-generating enzyme is not particularly limited, and the organic acid-generating enzyme derived from various organisms can be used. These organic acid producing enzymes may be used alone or in combination of two or more depending on the type of organic acid to be obtained and the type of microorganism used.

The ethanol producing enzyme means an enzyme that acts in the process of producing ethanol, and examples thereof include pyruvate decarboxylase that produces acetaldehyde from pyruvate and alcohol dehydrogenase that produces ethanol from acetaldehyde. Ethanol-producing enzymes can be used from various organisms without any particular limitation such as origin. These ethanol-producing enzymes may be used alone or in combination of two or more depending on the type of microorganism used.

Various organic acid-producing enzymes or ethanol-producing enzymes are mutated by substitution, deletion, insertion and / or addition of one or more amino acid residues in the amino acid sequence of the organic acid-generating enzyme or ethanol-generating enzyme. Those modified can be used in the same manner.

The cells of the present invention as described above can be obtained by a well-known genetic engineering technique. For example, it can be performed according to Molecular Cloning: A Laboratory Manual (T. Maniatis, et al., Cold Spring Harbor Laboratory). The DNA construct for recombination for introducing the target polynucleotide into the host cell is not particularly limited, and is a linear DNA fragment, plasmid (DNA), virus (DNA), retrotransposon (DNA), artificial A chromosome (YAC) can be selected according to the introduction form of a foreign gene (extrachromosomal or intrachromosomal) or the like to take a form of a vector.

In addition to the polynucleotides described above, DNA constructs include promoters such as glyceraldehyde triphosphate dehydrogenase gene promoter (TDH promoter) and pyruvate decarboxylase gene promoter (PDC promoter), CYC1 terminator and TDH3 terminator. In addition to terminators such as cis elements, cis elements such as enhancers, splicing signals, poly A addition signals, selectable markers, and ribosome binding sequences (SD sequences) can be ligated as necessary. The selectable marker is not particularly limited, and various known selectable marker genes such as drug resistance genes and auxotrophic genes can be used.

(Method of producing organic acid and ethanol)
By culturing the cell of the present invention using a medium containing cellulose as at least a part of the carbon source, an organic acid or ethanol can be produced. At this time, if the cell of the present invention is a cell that can express a polynucleotide encoding one or more enzymes related to organic acid production, the organic acid can be produced and related to ethanol production. Ethanol can be produced by using cells that hold a polynucleotide encoding one or more enzymes that can be expressed. In culturing the cells of the present invention, culturing conditions can be selected according to the cell type. In the production of the organic acid, neutralization of the product organic acid or the like may be performed as necessary, or treatment such as continuous removal of the organic acid or ethanol may be performed. As a medium for culturing the cells, a natural medium, a synthetic medium may be used as long as it contains a nitrogen source, inorganic salts, etc. in addition to cellulose corresponding to the cell of the present invention and can culture the cell efficiently. Any of the media can be used.

As the nitrogen source, ammonium salt of inorganic acid or organic acid such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate or other nitrogen-containing compounds, peptone, meat extract, corn steep liquor, etc. may be used. it can. Examples of inorganic substances that can be used include potassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.

The carbon source preferably includes a fraction separated or extracted from plant biomass resources. Here, the plant biomass resource means an organic resource derived from a plant and excluding fossil resources. In addition, plant biomass resources include waste and unused resources. Among them, in the present invention, it is preferable to use a saccharide-based plant biomass resource. Examples of the saccharide-based plant biomass resource include woody plant materials such as conifers and broad-leaved trees, kenaf, hemp, cotton, Herbaceous plant materials including various crop plants such as sugarcane and other millet plants, algae including various seaweeds, marine plant materials such as seaweeds, etc., and waste and unused materials discharged when using these Is mentioned. Waste and unused materials include paper, paper products, sawdust, chips and other sawmill waste, construction waste, agricultural waste such as bagasse, rice straw, wheat straw, rice straw, tea waste, vegetable waste, etc. Examples include forest waste materials such as food waste, thinned wood, and damaged trees. When these saccharide plant biomass resources are used as the carbon source of the present invention, it is preferable that polysaccharides contained therein are easily decomposed by a degrading enzyme such as β-glucosidase, including saccharides. Thus, it is preferable that the fraction is separated or extracted.

Moreover, in the plant biomass resource containing a cellulose or a cellulose, since the cellulose often has a crystal structure, it is preferable to make the cellulose amorphous. Amorphization of cellulose often involves a reduction in molecular weight at the same time. For example, cellulose can be made amorphous or low molecular by partially hydrolyzing cellulose under acidic conditions with an inorganic acid such as sulfuric acid, hydrochloric acid, phosphoric acid, and nitric acid. In addition, the cellulose can be made amorphous or low molecular by treatment with supercritical water, alkali, pressurized hot water, or the like.

Moreover, in a culture process, the oligosaccharide or monosaccharide produced | generated by a decomposing enzyme from cellulose or a cellulose can be added as a part of carbon source. By doing so, it is possible to effectively supply monosaccharides to the microorganism, particularly from the beginning of the culture to the initial stage of the culture. The saccharide is added to such an extent that it does not inhibit the degrading enzyme. Preferably, the saccharide is added only during a certain period from the beginning of the culture to the beginning of the culture (within about 2 to 10 hours from the start of the culture).

In addition, stationary culture, shaking culture, aeration stirring culture, etc. can be used for culture | cultivation, and it can be made for 6 to 72 hours at 30 to 35 degreeC on anaerobic conditions or microaerobic conditions. The pH can be adjusted using an inorganic or organic acid, an alkaline solution, or the like. During the culture, antibiotics such as ampicillin and tetracycline can be added to the medium as necessary.

After completion of the culturing step, the organic acid or ethanol can be obtained by performing a step of separating the organic acid or ethanol from the culture. In the present invention, the culture includes cultured cells or microbial cells, or crushed cells or cells in addition to the culture supernatant.

In order to separate the organic acid or ethanol from the culture, a general purification means can be used after obtaining a crude extract fraction containing the organic acid or ethanol. For example, when organic acid or ethanol is produced in the microorganism, the cells are destroyed by ultrasonic destruction treatment, grinding treatment, pressure crushing, etc. by conventional methods, and the organic matter separated from the cell components is separated. An acid-containing crude extract fraction can be obtained. In this case, protease is added as necessary. In addition, when organic acid or ethanol is produced outside the cells, the culture solution or the like can be filtered, centrifuged, etc. to remove the solid content to obtain a crude extract containing organic acid or ethanol. it can. About these containing crude extraction fractions, an organic acid or ethanol can be refine | purified using conventionally well-known various refinement | purification separation methods. Moreover, various organic acid derivatives can be obtained by performing esterification etc. with respect to the said crude extraction fraction and its refined | purified material as needed. When the organic acid to be produced is lactic acid, a polylactic acid precursor can be obtained by esterification.

As described above, according to the method for producing an organic acid or ethanol of the present invention, the organic acid or ethanol can be obtained directly from cellulose using a predetermined microorganism. For this reason, it is possible to obtain organic acid or ethanol from cellulose more efficiently than in the prior art, and it is possible to suppress the conversion of carbon resources to CO ₂ and to make better recycling.

EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated concretely, this invention is not limited to these Examples. The gene recombination operation described below was performed according to Molecular Cloning: A Laboratory Manual (T. Maniatis, et al., Cold Spring Harbor Laboratory). Further, gene amplification by PCR was performed using KOD plus DNA polymerase (TOYOBO) according to the attached protocol unless otherwise stated. As a PCR amplification apparatus, Gene Amp PCR system 9700 (PE Applied Biosystems) was used. For the ligation reaction, LigaFast Rapid DNA Ligation System (Promega) was used. Yeast genomic DNA was prepared using Fast DNA Kit (Bio 101) according to the attached protocol. Yeast transformation was performed using Frozen-EZ Yeast Transformation II (ZYMO RESEARCH) according to the attached protocol.

(First screening for modified enzyme A)
The β-glucosidase gene bglA (GH1 family, Genebank: X74163) derived from Thermotoga maritima represented by SEQ ID NO: 1 was subjected to error-prone PCR (10 mM Tris-HCl pH 9.0, 50 mM KCl, 0.1% Triton X-100, 2-6 mM) _{MaCl 2, 0.2-0.6mM MnCl 2, 0.2mM} dATP, 0.2mM dGTP, amplifies 1mMdCTP, 1mM dTTP, 1-100ng / μl MnP, by 0.3μM primer, 25 mU / μl Promega Taq DNA polymerase), 100 bases per A library in which an average of one mutation (error rate 1%) was randomly introduced was prepared. In this amplification reaction by error-prone PCR, after heat treatment at 95 ° C for 3 minutes, 94 ° C for 30 seconds, 60 ° C for 30 seconds and 72 ° C for X minutes (X: size of amplified gene is 1 per kb) 1 cycle), this was repeated 20 cycles, and finally 4 ° C. 80 μl of enzyme activity assay solution (100 mM sodium lactate buffer containing 2 mM p-nitrophenyl β-D-glucopyranoside) prepared to pH 3.0, 4.0, 6.5 was added to the library and incubated at 30 ° C for 60 minutes Thereafter, the presence or absence of coloration of p-nitrophenol produced by decomposition was confirmed by measuring the absorbance at 405 nm, and those showing high activity at 30 ° C. and pH 3.0, 4.0, 6.5 were screened. As a result, two clones (Lib1-33 and Lib1-91, respectively) were obtained.

(Second screening using the result of the first screening as the parent strain)
Next, a random mutation secondary library was prepared again using a mixture of equal amounts of the obtained two types of clone genes. As a result of screening, a clone having an activity at pH 4.0 higher than that at the optimum pH of the wild strain (Wild) was obtained. The five representative clones are called Lib2-3, Lib2-12, Lib2-26, Lib2-30, and Lib2-93, respectively.

(Confirmation of pH dependency)
The clones Lib1-33 and Lib1-91 obtained by the first screening and the clones Lib2-3, Lib2-12, Lib2-26, Lib2-30 and Lib2-93 obtained as a result of the second screening have a pH of 30 ° C. Dependency was examined. The result is shown in FIG. As shown in FIG. 1, in Lib1-91, the β-glucosidase activity was improved about 2-fold at 30 ° C. and at pH 3.0, 4.0, and 6.5, respectively, as compared with the wild type (Wild). In Lib1-33, the optimum pH was shifted from pH 5.5 to pH 5 to the acidic side compared to the wild type (Wild). Of the clones Lib2-3, Lib2-12, Lib2-26, Lib2-30, and Lib2-93 obtained by the second screening, Lib2-12, Lib2-26, Lib2-30, and Lib2-93 The optimum pH was shifted to the acidic side. Among these, in Lib2-12, Lib2-26, and Lib2-93, the specific activity at the optimum pH was increased about 2.5 times that of the wild type (Wild).

(Determining amino acid sequence)
The amino acid sequences of these mutants were determined. The results are shown in FIG. 2 and SEQ ID NOs: 3-9. In the second screening, since the mutations (Lib1-33, Lib1-91) obtained in the first screening are introduced as parent strains, which mutant is derived in FIG. Indicated. That is, the one having Lib1-33 as the parent strain is Lib2-12, and the one having Lib1-91 as the parent strain is Lib2-3, Lib2-26, Lib2-30, and Lib2-93. Moreover, the cell in which the alphabet which represents the kind of amino acid in FIG. 2 is not described represents that it is the same amino acid as a wild strain (wild). Looking at Lib2-12, Lib2-26, and Lib2-93, which showed good results in confirming the pH dependency in FIG. 1, first, the mutant Lib2-12 derived from Lib1-33 is the same as Lib1-91. A mutation was introduced, and the 130th amino acid residue was mutated from leucine (L) to glutamine (Q). In addition, in Lib1-91-derived mutant Lib2-93, a mutation was introduced at the same position as Lib1-33 (the 169th amino acid residue was mutated from valine (V) to aspartic acid (D)), and Lib2 Even at -26, the 169th amino acid residue was mutated from valine (V) to alanine (A). From these facts, it is speculated that the two amino acids (130th and 169th) in which mutations were commonly introduced contributed to acid resistance. In Lib2-93, the 382nd amino acid residue was mutated from alanine (A) to threonine (T). In Lib2-12, the 385th amino acid residue was mutated from alanine (A) to threonine (T). I found out. Any of these mutations was also observed in other clones (Lib1-33, Lib1-91, Lib2-3, Lib2-26, Lib2-30). From this, it is speculated that the 382nd or 385th amino acid also contributes to acid resistance.

(Production of mutant V169D and L130Q and measurement of activity)
A mutant obtained by converting the 160th valine (V) to aspartic acid (D) (hereinafter referred to as V169D) and a mutant obtained by converting the 130th leucine (L) to glutamine (Q) (hereinafter referred to as L130Q) ) And the activity at pH 3.0, pH 4.0, and pH 6.5 at 30 ° C. was measured. The result is shown in FIG. In V169D, the activity at pH 4.0 was improved over pH 6.5, indicating that it was directly involved in the shift of the optimum pH to the acidic side. In addition, L130Q overall improved the activity compared to the wild type, and it was thought that it contributed to the optimum temperature decrease.

(Modeling of protein structure)
To examine the relationship between the amino acid sequence and the three-dimensional structure, referring to the PDB (10DO) data of the wild-type enzyme, three-dimensional structure modeling of the mutant enzyme A using Lib2-93 was performed using Insight II software. It was. The charge near the active center was evaluated using Delphi software. From the results of the three-dimensional structure modeling, it was found that the 169th amino acid was located inside the pocket and the 130th amino acid was located on the molecular surface. Of these, the amino acid at the 169th is adjacent to the amino acid at the active center (166th glutamic acid (E) and 351st glutamic acid (E)), and the surface charge of the active center is mutated to acidic aspartic acid (D). Was estimated to have shifted to the acidic side. This suggests the possibility that the optimum pH has decreased. As for the 130th amino acid, the specific activity is improved in the mutants (Lib1-91, Lib2-3) that are located on the surface of the molecule and in which the 130th amino acid is mutated and the 169th amino acid is not mutated. Although an optimum pH shift was not observed, it was suggested that the surface amino acid was converted from hydrophobic to hydrophilic, which may have affected the optimum temperature decrease. In addition, since the 382nd and 385th amino acids were also located on the surface of the molecule, it was suggested that, similarly to the 130th amino acid, it may have affected the optimum temperature decrease.

(Screening of modified enzyme B)
The β-glucosidase gene bglB (GH3 family, Genebank: AE001690) derived from Thermotoga maritima represented by SEQ ID NO: 2 was subjected to error-prone PCR (10 mM Tris-HCl pH 9.0, 50 mM KCl, 0.1% TritonX-100, 2-6 mM) _{MaCl 2, 0.2-0.6mM MnCl 2, 0.2mM} dATP, 0.2mM dGTP, amplifies 1mMdCTP, 1mM dTTP, 1-100ng / μl MnP, by 0.3μM primer, 25 mU / μl Promega Taq DNA polymerase), 100 bases per A library in which an average of 2 mutations (error rate 2%) was randomly introduced was prepared. In this amplification reaction by error-prone PCR, after heat treatment at 95 ° C for 3 minutes, 94 ° C for 30 seconds, 60 ° C for 30 seconds and 72 ° C for X minutes (X: size of amplified gene is 1 per kb) 1 cycle), this was repeated 20 cycles, and finally 4 ° C. 80 μl of enzyme activity assay solution (100 mM sodium lactate buffer containing 2 mM p-nitrophenyl β-D-glucopyranoside) prepared to pH 2.5, 3.5, 6.5, respectively, was added to the library and incubated at 30 ° C. for 60 minutes Thereafter, the presence or absence of coloration of p-nitrophenol produced by decomposition was confirmed by measuring the absorbance at 405 nm, and those showing high activity at 30 ° C. and pH 2.5, 3.5, 6.5 were screened. As a result, 6 clones (referred to as Lib1-14, Lib1-16, Lib1-20, Lib1-24, Lib1-25, Lib1-32) with improved activity on the acidic side compared to the wild strain (Wild) Obtained.

(Confirmation of pH dependency)
The pH dependence of Lib1-14, Lib1-16, Lib1-20, Lib1-24, Lib1-25, and Lib1-32 obtained by screening was confirmed at 30 ° C. The results are shown in FIGS. FIG. 4 shows the BGL activity at pH 2.5, 3.5, 6.5 in each clone, and FIG. 5 shows the pH dependency in each clone. Among the obtained clones, Lib1-24 had an activity at pH 2.5 slightly less than that of the wild type, and an activity at pH 3.5 was improved by about 3.5 times. As for the optimum pH, the optimum pH of the wild type was pH 5.0, whereas in Lib1-24, the optimum pH was shifted to the pH side to pH 4.0. In Lib1-32, the activity at pH2.5 is almost the same, but the activity at pH5.0 is improved about 2 times, and the activity at pH5.0 is improved about 5 times in Lib1-16. .

(Determining amino acid sequence)
The amino acid sequences of these mutants were determined. The results are shown in FIG. 6 and SEQ ID NOs: 10-15. In addition, the cell in which the alphabet showing the kind of amino acid is not described in FIG. 6 represents that it is the same amino acid as a wild strain (wild). As for Lib1-16Lib1-24 and Lib1-25, which showed good results in the confirmation of pH dependency in FIG. 4, the 161st amino acid residue was mutated from cysteine (C) to serine (S). The first amino acid residue is mutated from leucine (L) to histidine (H), the 420th amino acid residue is mutated to tryptophan (W) to arginine (R), and the 492th amino acid residue is lysine (K) To glutamate (E). The same mutation was introduced in Lib1-14, but only in Lib1-14, the 89th serine (S) was mutated to proline (P), and this mutation decreased β-glucosidase activity. It is speculated that this led to

(Presentation of modified enzymes A and B to the surface of yeast)
The DNA encoding the mutant obtained in Examples 1 and 2 was introduced into the pB7-TDH3p vector for expression by the TDC3 promoter, and this was transformed to express L-lactic acid synthase 2002-362891 A strain that expresses β-glucosidase on the cell surface (hereinafter referred to as a TmBGLB mutant) was prepared by integrating into the yeast T165 strain described in the publication No. 1/2001. Transformants were selected using YPD chloramphenicol medium. Moreover, it confirmed that the transformant was expressing (beta) -glucosidase activity with the following method. Cells cultured overnight in YPD medium and washed with sterilized water were suspended in a phosphate buffer (50 mM sodium phosphate, pH 5.0) to obtain a cell solution. Suspend 2 mM p-nitrophenyl β-D-glucopyranoside and bacterial cell solution in phosphate buffer so that OD ₆₀₀ = 0.5, react at 30 ° C for 10 minutes, and then add 1M Na ₂ CO ₃ to stop the reaction. I let you. The absorbance at a wavelength of 405 nm of the supernatant after removing the cells by centrifugation at 15,000 rpm for 5 minutes was measured. As a result, it was confirmed that also in the TmBGLB mutant, the same pH dependency as that in which the β-glucosidase activity was improved in Examples 1 and 2 as compared with the wild type strain was observed.

SEQ ID NOs: 3-15: β-glucosidase variants

Claims (11)

A mutation that has β-glucosidase activity and is selected from amino acid substitution, deletion, addition, and insertion, or a combination thereof, in the amino acid sequence of SEQ ID NO: 2 with no more than 17 amino acids A protein comprising an amino acid sequence having all the mutations described in (1) to (12) below:
In the amino acid sequence set forth in SEQ ID NO: 2,
(1) The amino acid corresponding to position 161 is serine.
(2) The amino acid corresponding to position 345 is histidine.
(3) The amino acid corresponding to position 420 is arginine.
(4) The amino acid corresponding to position 492 is glutamic acid.
(5) The amino acid corresponding to position 20 is alanine.
(6) The amino acid corresponding to position 74 is serine.
(7) The amino acid corresponding to position 407 is isoleucine.
(8) The amino acid corresponding to position 525 is valine.
(9) The amino acid corresponding to position 605 is phenylalanine.
(10) The amino acid corresponding to position 650 is isoleucine.
(11) The amino acid corresponding to position 660 is glutamic acid.
(12) Arginine-phenylalanine-lysine-proline at positions 718 to 721 is serine-serine-asparagine. Furthermore, the protein of Claim 1 which consists of an amino acid sequence provided with substitution as described in the following (13) and (14) in the amino acid sequence of sequence number 2.
(13) The amino acid corresponding to position 4 is valine.
(14) The amino acid corresponding to position 59 is serine.   The protein of Claim 1 or 2 which has the amino acid sequence in any one of sequence number 10-13.   The cell which hold | maintains the polynucleotide which codes the protein in any one of Claims 1-3 so that expression is possible.   The cell according to claim 4, wherein the protein can be expressed as a cell surface display protein or a secreted protein.   The cell according to claim 4 or 5, which holds a polynucleotide encoding one or more organic acid-generating enzymes in an expressible manner.   The cell according to claim 6, wherein the organic acid is lactic acid and the enzyme is lactate dehydrogenase.   The cell according to claim 4 or 5, which holds a polynucleotide encoding one or more ethanol-producing enzymes in an expressible manner.   The cell according to any one of claims 4 to 8, wherein the cell is yeast.   A method for producing an organic acid, comprising a step of producing an organic acid using the cell according to claim 6. A method for producing ethanol, comprising the step of producing ethanol using the cell according to claim 8 .