A novel beta-glucosidase, an enzyme composition containing the same substance, and a method for producing a sugar solution using the same substance.

Abstract

The purpose of the present invention is to extract and provide a β-glucosidase gene having the effect of efficiently promoting saccharification in hydrolysis of cellulose-containing biomass from a hardly culturable symbiotic protozoan community in Coptotermes formosanus, and the present invention specifically relates to a β-glucosidase derived from protozoans of genus Pseudotrichonympha, comprising the amino acid sequence represented by SEQ ID NO: 1.

Description

Novel β-glucosidase, enzyme composition containing the same, and method for producing sugar solution using the same

[0001] The present invention relates to a novel β-glucosidase, an enzyme composition containing the β-glucosidase, and a method for producing a sugar solution from cellulose-containing biomass using the same.

[0002] As an alternative resource for crude oil, attempts to decompose renewable cellulose-containing biomass and use the obtained sugar to fermentatively produce biofuel or biopolymer raw materials have been actively studied both at home and abroad.

[0003] There are various methods for saccharifying cellulose, but the enzymatic saccharification method with low energy consumption and high sugar yield has become the mainstream of development. Multiple enzyme species are involved in the enzymatic decomposition of cellulose, and they can be roughly classified into three major types: cellobiohydrolase, endoglucanase, and β-glucosidase. Cellobiohydrolase is characterized by hydrolyzing from the terminal part of cellulose and is an enzyme capable of decomposing the crystalline region of cellulose. On the other hand, endoglucanase is characterized by hydrolyzing from the internal region of the cellulose molecular chain and is an enzyme that promotes the decrease in molecular weight due to cellulose decomposition.

[0004] β-glucosidase is an enzyme that mainly decomposes cellobiose, a disaccharide in which glucose is β-1,4-linked, and catalyzes the production of glucose, the final degradation product, and is an essential enzyme for sufficiently obtaining glucose useful as a fermentation raw material. Also, it is known that cellobiohydrolase or endoglucanase causes reaction inhibition due to the accumulation of cellobiose generated by cellulose decomposition. That is, β-glucosidase can significantly reduce the accumulation of cellobiose generated by cellulose decomposition, and thus has the effect of significantly improving the cellulose decomposition efficiency.

[0005] On the other hand, filamentous fungi are known as microorganisms that produce cellulase. Among filamentous fungi, the genus Trichoderma is known to produce large amounts of endo-type and exo-type cellulase extracellularly into the culture medium. Trichoderma-derived cellulase is most widely used for the enzymatic degradation of cellulose-containing biomass. However, some of the β-glucosidase produced by Trichoderma is localized in the cell wall of the fungus (Non-Patent Literature 1), and there have been problems with the amount and activity of β-glucosidase contained in cellulase prepared from Trichoderma culture medium being insufficient. In addition, it is known that many filamentous fungal-derived β-glucosidases are inhibited by glucose, and in the saccharification of cellulose-containing biomass, glucose generated in the saccharification reaction solution by cellulose degradation leads to inhibition of β-glucosidase activity, hindering the increase in glucose accumulation in the saccharification reaction solution (Non-Patent Literature 2).

[0006] Therefore, there is a need for high-quality β-glucosidase that efficiently promotes saccharification in the hydrolysis of cellulose-containing biomass, and the isolation of microbial-derived β-glucosidase has been carried out for some time.

[0007] Symbiotic protists of termites, which use wood as their sole source of nutrition, were known to have extremely high efficiency in decomposing cellulose. However, due to the difficulty in culturing these symbiotic protists, research has not progressed, and even in recent years, only a small amount of research has been conducted on the symbiotic protists and their cellulase (Patent Document 1). To date, there have been no examples of obtaining β-glucosidase genes derived from termite symbiotic protists.

[0008] Japanese Patent Publication No. 2003-70475

[0009] Messner, R et al. “Evidence for a single, specific β-glucosidase in cell wall from Tricoderma QM9414” Enzyme Microb. Technol. 1990, Vol. 21, 685-690 Andric, P. et al. “Reactor design for minimizing product inhibition during enzymatic lignocellulose Hydrolysis: I. Significance and mechanism of cellobiose and glucose inhibition on cellulolytic enzymes"Biotechnol. Adv. 2010 Volume 28 308-324

[0010] The object of the present invention is to provide a β-glucosidase gene that efficiently promotes saccharification in the hydrolysis of cellulose-containing biomass, by extracting it from a group of symbiotic protists of the difficult-to-culture termite species *Coptotermes formosanus*.

[0011] To solve the above problems, the inventors diligently conducted research and observed expressed genes from trace amounts of RNA of the difficult-to-culture symbiotic protist of the Formosan subterranean termite using single-cell transcriptome analysis. From the sequence information of the obtained cDNA library, candidate β-glucosidase sequences were obtained. The β-glucosidase activity of transformants containing the candidate β-glucosidase sequences and their effects on the saccharification of cellulose-containing biomass were investigated. By selecting sequences possessing β-glucosidase activity, the inventors discovered that a novel β-glucosidase derived from a protist of the genus Pseudotrichonymphha can be applied to the degradation of cellulose-containing biomass, thus completing the present invention.

[0012] In other words, the present invention consists of the following components: [1] Any one polypeptide from (A) to (C) below: (A) A polypeptide consisting of the amino acid sequence described in Sequence ID No. 1 (B) A polypeptide consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added in the amino acid sequence described in Sequence ID No. 1, and which has β-glucosidase activity (C) A polypeptide consisting of an amino acid sequence having at least 70% sequence identity with the amino acid sequence described in Sequence ID No. 1, and which has β-glucosidase activity [2] Any one polynucleotide from (a) to (d) below. (a) A polynucleotide comprising the base sequence described in Sequence ID No. 2. (b) A polynucleotide comprising a base sequence in which one or more bases are substituted, deleted, inserted and / or added in the base sequence described in Sequence ID No. 2, and which encodes a polypeptide having β-glucosidase activity. (c) A polynucleotide comprising a base sequence having at least 60% sequence identity with the base sequence described in Sequence ID No. 2, and which encodes a polypeptide having β-glucosidase activity. (d) A polynucleotide encoding the polypeptide described in [1]. [3] Any one of the following polynucleotides (a) to (d). (a) A polynucleotide comprising the nucleotide sequence described in Sequence ID No. 2. (b) A polynucleotide comprising a nucleotide sequence in which one or more nucleotides are substituted, deleted, inserted and / or added in the nucleotide sequence described in Sequence ID No. 2, and which encodes a polypeptide having β-glucosidase activity. (c) A polynucleotide comprising a nucleotide sequence having at least 50% sequence identity with the nucleotide sequence described in Sequence ID No. 2, and which encodes a polypeptide having β-glucosidase activity. (d) A polynucleotide encoding the polypeptide described in [1]. An expression vector comprising the polynucleotide described in [4], [2] or [3]. [5] A transformant having the polynucleotide described in [2] or [3] or the expression vector described in [4]. [6] A transformed Trichoderma filamentous fungus having the polynucleotide described in [2] or [3] or the expression vector described in [4].A method for producing an enzyme composition, comprising the step of culturing the transformant described in [7] [5] or the transformed Trichoderma filamentous fungus described in [6]. A method for producing a sugar solution from cellulose-containing biomass, comprising the step of producing the enzyme composition described in [7], wherein the enzyme composition obtained by the step is used. [9] A β-glucosidase derived from a protist of the genus Pseudotrichonymphha, characterized in that when the activity of β-glucosidase in the absence of glucose is set to 1, the β-glucosidase activity under conditions of a glucose concentration of 8 g / L is 0.5 or more.

[10] An enzyme composition comprising a β-glucosidase derived from a protist of the genus Pseudotrichonymphha and a cellulase derived from a filamentous fungus.

[11] The enzyme composition according to

[10] , characterized in that the filamentous fungus is a Trichoderma filamentous fungus. A method for producing a sugar solution from cellulose-containing biomass using the enzyme composition described in

[12] ,

[10] , or

[11] .

[13] A method for producing the sugar solution described in

[12] , comprising the step of recovering the enzyme composition described in

[10] or

[11] from the sugar solution.

[0013] This specification includes the disclosures of Japanese Patent Application No. 2017-165787, which forms the basis of the priority claim of this application.

[0014] The present invention provides a β-glucosidase that efficiently promotes saccharification in the hydrolysis of cellulose-containing biomass. The β-glucosidase of the present invention can be suitably used in the production of sugar solutions by hydrolysis of cellulose-containing biomass.

[0015] This is a photograph of the SDS-PAGE of the β-glucosidase according to the present invention (β-glucosidase containing the amino acid sequence described in SEQ ID NO: 1) expressed in Escherichia coli and purified in Example 7. This is a photograph of the SDS-PAGE of the β-glucosidase according to the present invention (β-glucosidase containing the amino acid sequence described in SEQ ID NO: 1) expressed in Trichoderma filamentous fungi in Example 10. This is a photograph of the SDS-PAGE of the saccharified supernatant in Example 14. This is a photograph of the SDS-PAGE of the β-glucosidase derived from Aspergillus filamentous fungi (β-glucosidase containing the amino acid sequence described in SEQ ID NO: 10) expressed in Trichoderma filamentous fungi in Comparative Example 1. This is a graph of the results of measuring the glucose-induced β-glucosidase activity inhibitory effect of the β-glucosidase and β-glucosidase mutants of the present invention in Example 18.

[0016] The embodiments of the present invention will be described in detail below.

[0017] In this invention, "β-glucosidase" refers to an enzyme that catalyzes the hydrolysis of β-glucosidic bonds in sugars. In this invention, β-glucosidase activity is measured by a reaction using p-nitrophenyl-β-D-glucopyranoside (pNP-Glc) as a substrate. Specifically, an enzyme solution is added to a substrate solution of pNP-Glc dissolved in 50 mM acetate-sodium acetate buffer (pH 5.0), and the reaction is allowed to proceed at 30°C for 10 minutes. Then, 1 / 10th of the reaction system volume of 2 M sodium carbonate is added and thoroughly mixed to stop the reaction, and the increase in absorbance at 405 nm is measured. If p-nitrophenol is released after the above reaction and the absorbance at 405 nm increases, it is determined that β-glucosidase activity is present.

[0018] The β-glucosidase of the present invention is characterized by being derived from a protist of the genus Pseudotrichonymphha, and specific examples include a polypeptide consisting of the amino acid sequence described in Sequence ID No. 1 or its homolog.

[0019] More specifically, the Pseudotrichonymphha-derived β-glucosidase of the present invention is one of the following polypeptides (A) to (C): (A) A polypeptide consisting of the amino acid sequence described in Sequence ID No. 1; (B) A polypeptide consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added in the amino acid sequence described in Sequence ID No. 1, and which has β-glucosidase activity; (C) A polypeptide consisting of an amino acid sequence having at least 70% sequence identity with the amino acid sequence described in Sequence ID No. 1, and which has β-glucosidase activity.

[0020] A polypeptide comprising the amino acid sequence described in Sequence ID No. 1 or its homolog can be prepared by known methods, and the method of preparation is not particularly limited as long as the polypeptide has β-glucosidase activity. A polypeptide comprising the amino acid sequence described in Sequence ID No. 1 or its homolog can be extracted from nature by known methods, or prepared using known methods known as peptide synthesis methods, or it can be prepared by genetic engineering using polynucleotides encoding the amino acid sequence of the polypeptide.

[0021] A homolog of a polypeptide comprising the amino acid sequence described in Sequence ID No. 1 may be a polypeptide having β-glucosidase activity, for example, a polypeptide comprising an amino acid sequence in which one or several, preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 or 2 amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence described in Sequence ID No. 1.

[0022] Furthermore, the homolog of the polypeptide described in SEQ ID NO: 1 may be a polypeptide having β-glucosidase activity, and the amino acid sequence may have at least 70%, preferably at least 80%, more preferably at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with the amino acid sequence described in SEQ ID NO: 1. For example, the polypeptide described in SEQ ID NO: 6 has an amino acid sequence with 88% sequence identity with SEQ ID NO: 1. Also, the polypeptide described in SEQ ID NO: 8 has an amino acid sequence with 80% sequence identity with SEQ ID NO: 1. As for the sequence identity between the amino acid sequence described in SEQ ID NO: 1 and the amino acid sequences of known β-glucosidases, for example, Trichoderma reesei-derived β-glucosidase I (BGLI) consists of 744 amino acids, but its sequence identity with SEQ ID NO: 1 is 29%.

[0023] The homolog of the polypeptide consisting of the amino acid sequence described in Sequence ID No. 1 may be a polypeptide derived from a protist of the genus Pseudotrichonymphha, preferably Pseudotrichonymphha hertwigi, Pseudotrichonymphha paulistana, or Pseudotrichonymphha grassi, as long as it has β-glucosidase activity.

[0024] The β-glucosidase derived from the Pseudotrichonymphha protist of the present invention preferably belongs to the GH3 family. In the present invention, "GH3 family" means polypeptides containing the Glycosyl hydroxyl family 3 active site. The Glycosyl hydroxyl family 3 active site is defined by the following amino acid sequence consisting of 18 amino acids. That is, Glycosyl hydroxyl family 3 active site is one amino acid selected from L (leucine), I (isoleucine), V (valine), and M (methionine) (a), one amino acid selected from either K (lysine) or R (arginine) (b), one amino acid selected from either E (glutamic acid), Q (glutamine), K (lysine), R (arginine), or D (aspartic acid) (c), one amino acid selected from either L (leucine), I (isoleucine), V (valine), M (methionine), F (phenylalanine), T (threonine), or C (cysteine) (d), and one amino acid selected from either L (leucine), I (isoleucine), V (valine), or T (threonine) ( Let e ​​represent one amino acid, f represent one amino acid selected from among amino acids L (leucine), I (isoleucine), V (valine), M (methionine), and F (phenylalanine), g represent one amino acid selected from among amino acids S (serine) and T (threonine), h represent one amino acid selected from among amino acids S (serine), G (glycine), A (alanine), D (aspartic acid), N (asparagine), I (isoleucine), and T (threonine), and x represent any amino acid. The 18 amino acid sequence aabxcxxxxGdefgDxxh is defined as a single amino acid a, b, c, d, e, f, g, h, x and amino acids G (glycine) and D (aspartic acid).The Glycosyl hydroxyl family 3 active site contained in the polypeptide can be easily looked up by anyone on the PROSITE (Database of protein domains, families and functional sites) website (http: / / prosite.expasy.org / ) (Christian, et al. 2002, Briefings in Bioinfomatics. VOL3. NO3. 265-274). Polypeptides consisting of the amino acid sequences described in SEQ ID NOs. 1, 6, or 8 belong to the GH3 family.

[0025] The β-glucosidase derived from the Pseudotrichonymphha genus of the present invention is preferably less susceptible to inhibition of β-glucosidase activity by glucose. Specifically, when the β-glucosidase activity in the absence of glucose is set to 1, the β-glucosidase activity under conditions of a glucose concentration of 8 g / L (or in the presence of 8 g / L glucose) is preferably 0.5 or higher, more preferably 0.6 or higher, more preferably 0.7 or higher, more preferably 0.8 or higher, more preferably 0.9 or higher, more preferably 1.0 or higher, more preferably 1.1 or higher, more preferably 1.2 or higher, more preferably 1.3 or higher, and particularly preferably 1.4 or higher. Furthermore, in addition to the β-glucosidase activity under the glucose concentration of 8 g / L, when the β-glucosidase activity in the absence of glucose is set to 1, the β-glucosidase activity under the glucose concentration of 20 g / L (or in the presence of 20 g / L of glucose) is preferably 0.5 or higher, more preferably 0.6 or higher, and particularly preferably 0.7 or higher. Here, the method for measuring β-glucosidase activity is the same as described above, except that 8 g / L or 20 g / L of glucose is added at the time of measurement, and the results obtained are used.

[0026] β-glucosidases whose activity is not easily inhibited by glucose are preferable in the saccharification of cellulose-containing biomass because they can maintain high β-glucosidase activity even when glucose is generated in the saccharification reaction solution due to cellulose decomposition.

[0027] The polynucleotide or its homolog consisting of the base sequence described in Sequence ID No. 2 is not particularly limited in its origin, as long as it encodes a polypeptide or its homolog consisting of the amino acid sequence described in Sequence ID No. 1. Here, "polynucleotide" refers to cDNA, genomic DNA, synthetic DNA, mRNA, synthetic RNA, replicon RNA, etc., regardless of its origin, but DNA is preferred. It may also be single-stranded or double-stranded with a complementary strand. It may also contain natural or artificial nucleotide derivatives.

[0028] The homolog of the polynucleotide described in Sequence ID No. 2 may be a polynucleotide comprising a base sequence in which one or several, preferably 1 to 40, more preferably 1 to 30, even more preferably 1 to 20, particularly preferably 1 to 10, and optimally preferably 1 to 5 bases are substituted, deleted, inserted and / or added in the base sequence described in Sequence ID No. 2.

[0029] Furthermore, the homolog of the polynucleotide described in Sequence ID No. 2 may be any polynucleotide encoding a polypeptide having β-glucosidase activity, including polynucleotides that hybridize under stringent conditions with the polynucleotide consisting of the base sequence described in Sequence ID No. 2, or the entire or a part of its complementary chain. Here, "polynucleotides that hybridize under stringent conditions" refers to polynucleotides that hybridize using known hybridization techniques (e.g., Current Protocols I Molecular Biology edit. Ausubel et al., (1987) Publish. John Wily & Sons Sectoin 6.3-6.4) as a probe, for example, by selecting one or more consecutive sequences of at least 20, preferably at least 25, more preferably at least 30, any polynucleotides from the original base sequence. Here, stringent conditions can be achieved, for example, by washing with a 0.1 to 2 times concentration SSC (saline-sodium citrate) solution (composition of 1x concentration SSC solution: 150 mM sodium chloride, 15 mM sodium citrate) at a hybridization temperature of 37°C in the presence of 50% formamide, 42°C for more stringent conditions, and 65°C for even more stringent conditions.

[0030] Furthermore, the homolog of the polynucleotide described in SEQ ID NO: 2 may be a polynucleotide that encodes a polypeptide having β-glucosidase activity, and may consist of a base sequence having at least 50%, preferably at least 60%, more preferably at least 80%, even more preferably at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with the base sequence described in SEQ ID NO: 2. For example, the polynucleotide described in SEQ ID NO: 3 is a polynucleotide consisting of a base sequence with 66% sequence identity with SEQ ID NO: 2. Also, the polynucleotide described in SEQ ID NO: 7 or 9 is a polynucleotide consisting of a base sequence with 53% sequence identity with SEQ ID NO: 2.

[0031] As used herein, the term "identity" refers to the degree of similarity between two different amino acid sequences or nucleotide sequences when they are aligned and compared using a sequence alignment program. Specifically, it refers to the percentage of identical amino acids relative to the total number of amino acids in Sequence ID No. 1, or the percentage of identical nucleotides relative to the total number of nucleotides in Sequence ID No. 2. The sequence alignment program used to align and compare the two sequences is BLAST (blastn, blastp), a widely used software in this field. BLAST is available to anyone on the NCBI (National Center for Biotechnology Information) website, and identity can be easily checked using default parameters.

[0032] The homolog of the polynucleotide consisting of the base sequence described in Sequence ID No. 2 may be a polynucleotide that codes for a polypeptide having β-glucosidase activity, and may be derived from protists of the genus Pseudotrichonymphha, preferably Pseudotrichonymphha hertwigi, Pseudotrichonymphha paulistana, or Pseudotrichonymphha grassi.

[0033] The polynucleotide consisting of the base sequence described in Sequence ID No. 2 can be prepared by cloning from protists of the genus Pseudotrichonymphha, but it can also be chemically synthesized. Cloning from protists of the genus Pseudotrichonymphha can be performed using commonly known methods. For example, the entire ORF sequence can be determined from cDNA reverse-transcribed from RNA isolated from cells of the genus Pseudotrichonymphha, and then amplified by PCR. It is also possible to chemically synthesize this directly using a DNA synthesizer.

[0034] An expression vector containing the polynucleotide can be produced by ligating the polynucleotide downstream of a promoter expressible in a host cell using restriction enzymes and DNA ligase. In the present invention, the expression vector may be any vector that introduces a target gene into a host cell so that it can be expressed. It may be an autonomously replicating plasmid in which the target gene is introduced outside the host genome, or it may be a DNA fragment in which the target gene is introduced into the host genome. Examples of expression vectors include bacterial plasmids, yeast plasmids, phage DNA such as lambda phage, viral DNA such as retroviruses, baculoviruses, vaccinia viruses, and adenoviruses, and Agrobacterium as a vector. For example, when the host cell is E. coli, pUC, pET, pBAD, etc., can be used as examples. The promoter may be any promoter appropriate to the host cell used for gene expression. For example, when the host cell is E. coli, examples include the lac promoter, Trp promoter, PL promoter, PR promoter, etc. When the host cell is a cellulase-producing filamentous fungus, preferred promoters include cellulose-inducible promoters, more preferably cbh promoters, egl promoters, bgl promoters, xyn promoters, and bxl promoters.

[0035] Preferred host cells in transformants having the polynucleotide or expression vector of the present invention include Escherichia coli, bacterial cells, yeast cells, fungal cells, insect cells, plant cells, and animal cells. Examples of yeast cells include those of the genera Pichia, Saccharomyces, and Schizosaccharomyces. Examples of insect cells include Sf9, examples of plant cells include dicotyledonous plants, and examples of animal cells include CHO, HeLa, and HEK293. More preferably, filamentous fungal cells are used, more preferably filamentous fungi of the genus Aspergillus, and more preferably filamentous fungi of the genus Trichoderma. By using filamentous fungi of the genus Trichoderma as host cells, it becomes possible to produce more of the β-glucosidase of the present invention.

[0036] Transformation or transfection can be carried out by known methods such as the calcium phosphate method, electroporation method, or Agrobacterium method.

[0037] The β-glucosidase of the present invention can be obtained by expressing it under the control of a promoter in host cells transformed or transfected as described above, and then recovering the product. For expression, the host cells are proliferated or grown to an appropriate cell density, the promoter is induced by a temperature shift or chemical induction method using culture medium components, and the cells are cultured for a certain period of time.

[0038] In the present invention, the enzyme composition refers to a mixture of β-glucosidase derived from a protist of the genus Pseudotrichonymphha and one or more other enzymes. The enzyme composition may be obtained by separately producing the β-glucosidase and one or more other enzymes and then mixing them, or it may be a culture of a transformant having a polynucleotide or expression vector encoding a polypeptide having β-glucosidase activity, and containing the β-glucosidase and one or more enzymes derived from host cells. The culture includes the culture supernatant, as well as the transformant or lysates of the transformant.

[0039] Cellulase is preferred as the enzyme to be mixed with the β-glucosidase. The cellulase referred to here is not particularly limited as long as it is an enzyme that has the activity to break down cellulose, and may be a mixture of two or more enzymes. Examples of such enzymes include cellulase, hemicellulase, cellobiohydrolase, endoglucanase, exoglucanase, xylanase, and mannanase. The activity of cellobiohydrolase and endoglucanase contained in the cellulase is measured using p-nitrophenyl-β-D-lactopyranoside (pNP-Lac) as a substrate, and the activity of β-xylosidase is measured using p-nitrophenyl-β-D-xylopyranoside (pNP-Xyl) as a substrate. Specifically, an enzyme solution is added to a substrate solution of pNP-Lac dissolved in 50 mM acetate-sodium acetate buffer (pH 5.0), and the reaction is allowed to proceed at 30°C for 10 minutes. Then, 1 / 10th of the reaction system volume of 2 M sodium carbonate is added and mixed well to stop the reaction, and the increase in absorbance at 405 nm is measured. If, after the above reaction, p-nitrophenol is released and the absorbance at 405 nm increases, it is determined that cellobiohydrolase and endoglucanase activity is present. For β-xylosidase activity, the reaction is carried out in the same manner as above using pNP-Xyl as the substrate, and if, after the reaction, p-nitrophenol is released and the absorbance at 405 nm increases, it is determined that β-xylosidase activity is present.

[0040] The cellulase is preferably a cellulase derived from filamentous fungi. The cellulase derived from filamentous fungi is a mixture comprising at least endoglucanase and cellobiohydrolase. Examples of microorganisms that produce the cellulase derived from filamentous fungi include those of the genera Trichoderma, Aspergillus, Cellulomonas, Clostridium, Streptomyces, Humicola, Acremonium, Irpex, Mucor, and Talaromyces. These microorganisms produce cellulase in the culture medium, and the culture medium may be used as is as unpurified cellulase derived from filamentous fungi, or the culture medium may be purified and formulated and used as a mixture of cellulase derived from filamentous fungi.

[0041] The filamentous fungus-derived cellulase described above is preferably a cellulase derived from the genus Trichoderma. The genus Trichoderma produces a cellulase in culture medium comprising at least two types of endoglucanases and at least two types of cellobiohydrolases, and the cellulase prepared from such a culture medium can be preferably used in the present invention. That is, when the β-glucosidase of the present invention is used as an enzyme composition together with a cellulase derived from the genus Trichoderma, it can increase the sugar yield in the saccharification of cellulose-containing biomass.

[0042] When cellulose-containing biomass is enzymatically treated with the β-glucosidase of the present invention, the β-glucosidase of the present invention with high residual activity can be recovered from the saccharified solution obtained by the enzymatic treatment. Furthermore, the β-glucosidase of the present invention with high residual activity can also be recovered from the saccharified solution obtained by enzymatically treating cellulose-containing biomass using an enzyme composition containing the β-glucosidase of the present invention and the filamentous fungus-derived cellulase described above. Moreover, with regard to the filamentous fungus-derived enzyme composition, the filamentous fungus-derived cellulase obtained by enzymatically treating cellulose-containing biomass using an enzyme composition containing the β-glucosidase of the present invention and the filamentous fungus-derived cellulase described above can recover a filamentous fungus-derived cellulase with higher residual activity compared to a saccharified solution obtained by enzymatically treating cellulose-containing biomass using only the filamentous fungus-derived cellulase. The filamentous fungus-derived cellulase is preferably Trichoderma-derived endoglucanase, cellobiohydrase, or β-xylosidase, with β-xylosidase being particularly preferred.

[0043] Among these Trichoderma species, cellulases derived from Trichoderma reesei are more preferred. Examples of cellulase mixtures derived from Trichoderma reesei include cellulase mixtures derived from Trichoderma reesei QM9414, Trichoderma reesei QM9123, Trichoderma reesei Rut-30, Trichoderma reesei PC3-7, Trichoderma reesei CL-847, Trichoderma reesei MCG77, Trichoderma reesei MCG80, and Trichoderma viride QM9123. Further, it may be a cellulase mixture derived from a mutant strain that is derived from the above-mentioned Trichoderma genus and has been subjected to mutation treatment with a mutagen or ultraviolet irradiation, etc., and has improved cellulase productivity.

[0044] In the present invention, the method for producing an enzyme composition including a step of culturing a transformant into which a polynucleotide or an expression vector encoding a polypeptide having the β-glucosidase activity is introduced may be any method as long as it includes a culturing step capable of expressing the β-glucosidase. When the transformant is cultured, the expression of a polypeptide having β-glucosidase activity in the transformant is newly imparted or enhanced, and as a result, an enzyme composition containing the β-glucosidase and one or more enzymes derived from the host cell can be obtained from the culture. The culture may be any of a culture supernatant, a transformant, or a disrupted product of the transformant.

[0045] There are no particular limitations on the method for culturing the transformant, and known methods can be employed. Various culture methods can be used for cultivation, such as shaking culture, stirring culture, stirring and shaking culture, static culture, and continuous culture. As the culture medium for culturing the transformant, any medium that contains a carbon source, nitrogen source, inorganic salts, etc. that the transformant can assimilate, and that can efficiently cultivate the transformant, can be used, whether a natural medium or a synthetic medium. If the transformant is a filamentous fungus of the genus Trichoderma, it is more preferable to cultivate it in a medium containing cellulose-containing biomass. By culturing a transformed filamentous fungus of the genus Trichoderma, into which a polynucleotide or expression vector encoding the β-glucosidase of the present invention has been introduced, in a cellulose-containing biomass medium, Trichoderma-derived cellulase can be expressed, and an enzyme composition containing Trichoderma-derived cellulase and the β-glucosidase can be produced.

[0046] In this invention, cellulose-containing biomass is not limited to materials that contain at least cellulose. Specifically, it includes bagasse, corn stover, corn cob, switchgrass, rice straw, wheat straw, trees, timber, waste building materials, newspaper, recycled paper, pulp, etc. Although these cellulose-containing biomass materials contain impurities such as high-molecular aromatic compounds lignin and hemicellulose, materials that have been pretreated with acids, alkalis, pressurized hot water, etc., to decompose or remove all or part of the lignin and hemicellulose can also be used as cellulose-containing biomass.

[0047] The method for producing a sugar solution from cellulose-containing biomass using the enzyme composition described above is not particularly limited. The production of the sugar solution using this enzyme composition may be carried out in a batch or continuous manner. Furthermore, the enzyme composition used can be separated and recovered from the saccharified solution obtained by enzymatic treatment of cellulose-containing biomass. The method for separating and recovering the enzyme is not particularly limited, but it can be recovered on the impermeable side by filtering the saccharified solution with an ultrafiltration membrane or the like. If necessary, solid matter may be removed from the saccharified solution as a pre-filtration step. The recovered enzyme composition can be used again in the saccharification reaction.

[0048] (Reference Example 1) Protein Concentration Measurement Method A commercially available protein concentration measurement reagent (Quick Start Bradford Protein Assay, manufactured by Bio-Rad) was used. 5 μL of a cellulase solution derived from filamentous fungi diluted in 250 μL of the protein concentration measurement reagent returned to room temperature was added, and the absorbance at 595 nm after standing at room temperature for 5 minutes was measured using a microplate reader. BSA was used as a standard, and the protein concentration was calculated by referring to the calibration curve.

[0049] (Reference Example 2) β-Glucosidase Activity Measurement Method 10 μL of an enzyme dilution solution was added to 90 μL of a 50 mM acetate buffer containing 1 mM p-nitrophenyl-β-glucopyranoside (manufactured by Sigma-Aldrich Japan), and the reaction was carried out at 30 °C for 10 minutes. Then, 10 μL of 2 M sodium carbonate was added and mixed well to stop the reaction, and the increase in absorbance at 405 nm was measured. The activity to release 1 μmol of p-nitrophenol per minute was defined as 1 U. For the blank, 10 μL of 2 M sodium carbonate was added to 90 μL of a 50 mM sodium acetate buffer containing 1 mM p-nitrophenyl-β-glucopyranoside and mixed well, and then 10 μL of the enzyme dilution solution was added and the reaction was carried out at 30 °C for 30 minutes. Then, the increase in absorbance at 405 nm was measured. At this time, the enzyme solution was diluted so that the absorbance at 405 nm did not exceed 1. Also, for the calibration curve, p-nitrophenol solutions were prepared to have concentrations of 0.1 mM, 0.2 mM, 1 mM, and 2 mM. Instead of the enzyme dilution solution, 10 μL was added, 10 μL of 2 M sodium carbonate was added and mixed well to develop color, and it was created from the measured absorbance.

[0050] (Reference Example 3) Method for Measuring β-Xylosidase Activity 90 μL of 50 mM acetate buffer containing 1 mM p-nitrophenyl-β-xylopyranoside (Sigma-Aldrich Japan) was mixed with 10 μL of enzyme diluent and reacted at 30°C for 30 minutes. Then, 10 μL of 2 M sodium carbonate was added and mixed well to stop the reaction, and the increase in absorbance at 405 nm was measured. The activity that releases 1 μmol of p-nitrophenol per minute was defined as 1 U. For the blank, 90 μL of 50 mM acetate buffer containing 1 mM p-nitrophenyl-β-xylopyranoside was mixed with 10 μL of 2 M sodium carbonate and mixed well, then 10 μL of enzyme diluent was added and reacted at 30°C for 30 minutes. Then, the increase in absorbance at 405 nm was measured. At this time, the enzyme solution was diluted so that the absorbance at 405 nm did not exceed 1. Furthermore, the calibration curve was created by preparing p-nitrophenol solutions to concentrations of 0.1 mM, 0.2 mM, 1 mM, and 2 mM, adding 10 μL of each solution instead of the enzyme diluent, adding 10 μL of 2 M sodium carbonate, mixing well to develop a color, and then measuring the absorbance.

[0051] (Reference Example 4) Method for Measuring Cellobiohydrolase Endoglucanase Activity 90 μL of 50 mM acetate buffer containing 1 mM p-nitrophenyl-β-lactopyranoside (Sigma-Aldrich Japan) was mixed with 10 μL of enzyme diluent and reacted at 30°C for 60 minutes. Then, 10 μL of 2 M sodium carbonate was added and mixed well to stop the reaction, and the increase in absorbance at 405 nm was measured. The activity that releases 1 μmol of p-nitrophenol per minute was defined as 1 U. For the blank, 90 μL of 50 mM acetate buffer containing 1 mM p-nitrophenyl-β-lactopyranoside was mixed with 10 μL of 2 M sodium carbonate and mixed well, then 10 μL of enzyme diluent was added and reacted at 30°C for 30 minutes. Then, the increase in absorbance at 405 nm was measured. At this time, the enzyme solution was diluted so that the absorbance at 405 nm did not exceed 1. Furthermore, the calibration curve was created by preparing p-nitrophenol solutions to concentrations of 0.1 mM, 0.2 mM, 1 mM, and 2 mM, adding 10 μL of each solution instead of the enzyme diluent, adding 10 μL of 2 M sodium carbonate, mixing well to develop a color, and then measuring the absorbance.

[0052] (Reference Example 5) Measurement of sugar concentration: Glucose and cellobiose were quantitatively analyzed using the ACQUITY UPLC system (Waters) under the following conditions. Quantitative analysis was performed based on calibration curves created using glucose and cellobiose standards. If the cellobiose value was lower than 1 g / L, it was considered below the detection limit. Column: AQUITY UPLC BEH Amide 1.7 μm 2.1 × 100 mm Column Separation method: HILIC Mobile phase: Mobile phase A: 80% acetonitrile, 0.2% TEA (triethylamine) aqueous solution, Mobile phase B: 30% acetonitrile, 0.2% TEA (triethylamine) aqueous solution, following the gradient described below. The gradient was a linear gradient reaching the mixing ratio corresponding to the following times. Starting conditions: (A 99.90%, B 0.10%), 2 minutes after start: (A 96.70%, B 3.30%), 3.5 minutes after start: (A 95.00%, B 5.00%), 3.55 minutes after start: (A 99.90%, B 0.10%), 6 minutes after start: (A 99.90%, B 0.10%). Detection method: ELSD (Evaporative Light Scattering Detector) Flow rate: 0.3 mL / min Temperature: 55°C

[0053] (Reference Example 6) SDS-PAGE For SDS-PAGE, 15% polyacrylamide gel e-PAGE (ATTO) was used. 5 μg of the β-glucosidase-transformed Trichoderma strain enzyme was mixed with an equal volume of sample buffer Ez-apply (ATTO), and heated at 95°C for 10 minutes to prepare the electrophoresis sample. Precision Plus Protein Dual Color Standards (Bio-Rad) was used as the molecular weight marker. The electrophoresis buffer consisted of 25 mM Tris, 192 mM glycine, and 0.1% SDS aqueous solution, and electrophoresis was performed at a constant current of 20 mA for 90 minutes. The gel after electrophoresis was stained with Bio-Safe Commassie G-250 Stain (Bio-Rad) and destained with pure water.

[0054] (Reference Example 7) Cellulase production by culturing Trichoderma filamentous fungi Trichoderma filamentous fungi spores 1.0 × 10 ７The spore solution was diluted with physiological saline to a concentration of 1 / mL, and 2.5 mL of this diluted spore solution was inoculated into 250 mL of culture medium with the composition shown in Table 1, placed in a 1 L baffled flask. The culture was then incubated for 3 days at 28°C and 160 rpm (pre-culture). For the main culture, 250 mL of the pre-culture medium was inoculated into 2.5 L of the main culture medium shown in Table 2, each placed in a 5 L mini-jar. The culture was incubated for 4 days at 28°C, 700 rpm, 1 vvm, and pH 5. Neutralization was performed using 10% ammonia and 1N sulfuric acid. After centrifugation of the culture medium 4 days after the start of cultivation, the supernatant was filtered through an ultrafiltration membrane to remove bacterial cells and obtain cellulase derived from Trichoderma filamentous fungi.

[0055]

[0056]

[0057] (Reference Example 8) Method for Measuring the Inhibitory Effect of Glucose on β-Glucosidase Activity A mixture containing 1 mM p-nitrophenyl-β-glucopyranoside (Sigma-Aldrich Japan), 50 mM acetate buffer, and glucose at concentrations of 0, 4, 8, or 20 g / L was prepared by adding the enzyme solution to a total reaction solution volume of 100 μL, and the mixture was reacted at 30°C for 100 minutes. Then, 10 μL of 2 M sodium carbonate was added and the mixture was thoroughly mixed to stop the reaction, and the increase in absorbance at 405 nm was measured. The activity that releases 1 μmol of p-nitrophenol per minute was defined as 1 U. The blank was prepared by adding 10 μL of 2 M sodium carbonate to a mixture containing 1 mM p-nitrophenyl-β-glucopyranoside, 50 mM sodium acetate buffer, and glucose in the same volume as the reaction mixture before enzyme solution addition as described above. After mixing thoroughly, the enzyme solution was added to bring the total volume of the blank to 110 μL, and the mixture was reacted at 30°C for 30 minutes. Subsequently, the increase in absorbance at 405 nm was measured.

[0058] (Example 1) RNA-Seq analysis and β-glucosidase candidate sequence analysis from one cell of a difficult-to-culture termite symbiotic organism. From the group of difficult-to-culture termite symbiotic protists contained in the intestinal tract extract of the Formosan subterranean termite, Pseudotrichonymphha grassi cells were selected, fractionated using a microcapillary, and RNA extraction, reverse transcription, cDNA amplification, conversion to a library, and sequencing were performed from one cell using the Quartz-seq method developed by Sasagawa et al. (Sasagawa, Y. et al.: Genome Biol., 14: R31, 2013). Based on the sequencing results, a base sequence encoding a candidate β-glucosidase was selected. The signal sequence portion of the amino acid sequence encoded by the aforementioned base sequence was predicted using the protein signal sequence prediction tool SignalP (http: / / www.cbs.dtu.dk / services / SignalP / ). As a result, the amino acid sequence described in SEQ ID NO: 1 was obtained as the β-glucosidase candidate sequence excluding the signal sequence, and the base sequence of SEQ ID NO: 2 was obtained as the base sequence encoding the amino acid sequence. When the amino acid sequence family described in SEQ ID NO: 1 was examined using PROSITE, it was found that the amino acid sequence of SEQ ID NO: 5, which corresponds to Glycosyl hydroxyl family 3 active site, is present from position 259 to 276 of SEQ ID NO: 1.

[0059] (Example 2) Preparation of β-glucosidase gene-transformed Escherichia coli A plasmid was synthesized by linking the polynucleotide described in Sequence ID No. 2 (the nucleotide sequence encoding a candidate β-glucosidase gene from a protist of the genus Pseudotrichonymphha), obtained in Example 1, to the NdeI and XhoI restriction enzyme sites of pET14b using an artificial gene synthesis service (Genscript). Here, the structure of the plasmid sequence was designed so that the transformant would express a β-glucosidase having the amino acid sequence described in Sequence ID No. 1, with a His-tag added to the N-terminus. The plasmid was used to transform Escherichia coli (Rossetta 2 (DE3)) strain.

[0060] (Example 3) Preparation of cell-free extract of β-glucosidase gene-transformed Escherichia coli The β-glucosidase gene-transformed Escherichia coli prepared in Example 2 was inoculated into 10 mL of ampicillin-containing LB medium and cultured overnight at 37°C with shaking (pre-culture). For the main culture, the cells obtained in the pre-culture were inoculated into ampicillin-containing LB medium and cultured at 37°C with shaking until the turbidity OD600 at a wavelength of 600 nm was 0.8. Then, isopropyl-1-thio-β-D-galactoside (IPTG) was added to a final concentration of 0.1 mM, and the culture was further incubated overnight at 16°C. After culturing, the cells were collected by centrifugation and resuspended in 50 mM Tris-HCl buffer (pH 7.6). This solution was sonicated while being cooled on ice, and the supernatant was collected as a cell-free extract by centrifugation. When the β-glucosidase activity of the cell-free extract of E. coli transformed with the β-glucosidase candidate gene was measured, it was found to be 0.14 U per 1 mg of protein contained in the cell-free extract. Furthermore, the total β-glucosidase activity obtained from 1 L of culture medium of E. coli transformed with the β-glucosidase gene was 21.6 U. As a control, cell-free extracts of transformants with plasmids that did not contain the β-glucosidase candidate gene were similarly prepared, and when the β-glucosidase activity was measured, no β-glucosidase activity was detected. In other words, since β-glucosidase activity was detected only from cell-free extracts expressing a polypeptide in which a His-tag was added to the amino acid sequence described in SEQ ID NO: 1, it was found that the polypeptide consisting of the amino acid sequence described in SEQ ID NO: 1 is a β-glucosidase sequence derived from a protist of the genus Pseudotrichonymphha.

[0061] (Example 4) Preparation of β-glucosidase mutant gene-transformed Escherichia coli Similar to Example 2, plasmids were synthesized by artificial gene synthesis service (Genscript) in which the polynucleotides described in SEQ ID NO: 7, which has 53% sequence identity with SEQ ID NO: 2, and SEQ ID NO: 9, which also has 53% sequence identity with SEQ ID NO: 2, were linked to the NdeI and XhoI restriction enzyme sites of pET14b, respectively. Here, the structure of the plasmid sequence was designed so that in the transformant, β-glucosidase having the amino acid sequences described in SEQ ID NO: 6 and SEQ ID NO: 8, with a His-tag added to the N terminus, would be expressed, respectively. The plasmid was transformed into Escherichia coli (Rossetta 2 (DE3)) strain.

[0062] (Example 5) Preparation of cell-free extract of β-glucosidase mutant gene-transformed Escherichia coli The β-glucosidase mutant gene-transformed Escherichia coli prepared in Example 4 was inoculated into 10 mL of ampicillin-containing LB medium and cultured overnight at 37°C with shaking (pre-culture). For the main culture, the cells obtained in the pre-culture were inoculated into ampicillin-containing LB medium and cultured at 37°C with shaking until the turbidity OD600 at a wavelength of 600 nm was 0.8. Then, isopropyl-1-thio-β-D-galactoside (IPTG) was added to a final concentration of 0.1 mM, and the culture was further cultured overnight at 16°C. After culturing, the cells were collected by centrifugation and resuspended in 50 mM Tris-HCl buffer (pH 7.6). While cooling this solution with ice, ultrasonic disruption was performed, and the supernatant was collected as a cell-free extract by centrifugation. After reducing the extract volume to 1 / 12 using an ultrafiltration membrane unit, the presence or absence of β-glucosidase activity was examined. As a control, cell-free extracts of plasmid transformants that did not contain the β-glucosidase candidate gene were prepared in the same way, and the presence or absence of β-glucosidase activity was examined, but no β-glucosidase activity was detected. In other words, β-glucosidase activity was detected only from cell-free extracts expressing polypeptides in which His-tags were added to the amino acid sequences described in SEQ ID NO: 6 (88% sequence identity with SEQ ID NO: 1) and SEQ ID NO: 8 (80% sequence identity with SEQ ID NO: 1). Therefore, it was found that polypeptides consisting of the amino acid sequences described in SEQ ID NO: 6 and SEQ ID NO: 8 possess β-glucosidase activity. The results of the β-glucosidase activity measurement are shown in Table 3.

[0063]

[0064] (Example 6) Preparation of an enzyme composition containing a cell-free extract of β-glucosidase gene-transformed Escherichia coli and cellulase derived from Trichoderma filamentous fungi and saccharification of wood raw material powder cellulose Trichoderma reesei was cultured according to Reference Example 7 to produce cellulase derived from Trichoderma filamentous fungi. An enzyme composition was prepared by mixing the cell-free extract of β-glucosidase gene-transformed Escherichia coli prepared in Example 3 with the cellulase derived from Trichoderma filamentous fungi and used in the saccharification reaction. The enzyme composition was mixed so that the protein concentration of Trichoderma filamentous fungi cellulase was 0.2 g / L and the protein concentration of the cell-free extract of β-glucosidase gene-transformed Escherichia coli was 2.1 g / L per 1 mL of saccharification reaction solution. As the biomass to be saccharified, wood-based powdered cellulose Arbocel® (J. Rettenmaier & Sohne) was used. The saccharification reaction was carried out as follows: 50 mg of biomass was placed in a 2 mL tube, sodium acetate buffer (pH 5.2) was added to a final concentration of 50 mM, and pure water was added so that the solid content concentration of the wood-based powdered cellulose was 5% by weight at the start of the reaction. Furthermore, the enzyme composition was added to the prepared solution, and the reaction was started at a reaction condition of 35°C using a heat block rotator. After 24 hours of saccharification, the sample was centrifuged for 5 minutes under conditions of 10,000 × g, the supernatant was separated, and 1 / 10th the volume of the supernatant was added to a 1N sodium hydroxide aqueous solution to stop the saccharification reaction. The supernatant was filtered through a 0.22 μm filter, and the filtrate was subjected to sugar analysis according to Reference Example 5. As a control, cell-free extracts of E. coli transformed with a vector lacking the β-glucosidase gene were used. These were mixed with cellulase derived from Trichoderma filamentous fungi to achieve a protein concentration of 2.1 g / L per 1 mL of saccharification reaction solution, and the glycation reaction and sugar analysis of the glycation supernatant were performed in the same manner as described above. The β-glucosidase activity of the enzyme composition containing the cell-free extract of β-glucosidase-transformed E. coli and cellulase derived from Trichoderma filamentous fungi used in the above saccharification reaction was 2.6 times that of cellulase derived from Trichoderma filamentous fungi without the cell-free extract of β-glucosidase-transformed E. coli.As a result of the glycation reaction, when an enzyme composition containing a cell-free extract of β-glucosidase gene-transformed E. coli and cellulase derived from Trichoderma filamentous fungi was added, the amount of glucose accumulated increased by approximately 2.7 times, and the amount of cellobiose accumulated decreased, compared to when cellulase derived from Trichoderma filamentous fungi without the β-glucosidase of the present invention was added. The results of glucose and cellobiose analysis are shown in Table 4.

[0065]

[0066] (Example 7) His-tag crude purification of β-glucosidase His-tag purification was performed on the cell-free extract of β-glucosidase gene-transformed Escherichia coli whose enzyme activity was confirmed in Example 3. His-tag purification was performed using the His-Bind Purification Kit (Merck Millipore) according to the batch method in the instructions. SDS-PAGE was performed on the purified fraction according to Reference Example 6, and the densest band was detected in the eluted fraction between 75 kDa and 100 kDa, confirming that His-tag-tagged β-glucosidase with a theoretical molecular weight of 83.9 kDa had been purified. A photograph of the SDS-PAGE gel is shown in Figure 1. Using a gel filtration column PD-10 (GE Healthcare), the buffer of the eluted fraction was replaced with 20 mM Tris-HCl (pH 7.6) according to the instructions, and this was obtained as crude purified β-glucosidase. The protein concentration and β-glucosidase activity of the crude purified β-glucosidase were measured, and it was found to be 3.50 U per 1 mg of protein contained in the crude purified β-glucosidase.

[0067] (Example 8) Preparation of an enzyme composition containing crude purified β-glucosidase and cellulase derived from Trichoderma filamentous fungi and saccharification of wood raw material powder cellulose Trichoderma reesei was cultured according to Reference Example 7 to produce cellulase derived from Trichoderma filamentous fungi. An enzyme composition was prepared by mixing the crude purified β-glucosidase produced in Example 7 with the cellulase derived from Trichoderma filamentous fungi and used in the saccharification reaction. The enzyme composition was mixed so that the protein concentration of Trichoderma filamentous fungi-derived cellulase was 0.2 g / L and the protein concentration of crude purified β-glucosidase was 0.017 g / L per 1 mL of saccharification reaction solution. The saccharification reaction and sugar analysis of the saccharified supernatant were performed in the same manner as in Example 6, except that crude purified β-glucosidase was used in the enzyme composition. As a control, only cellulase derived from Trichoderma fungi was used, and the saccharification reaction and sugar analysis of the saccharified supernatant were performed in the same manner as above, by adding Trichoderma fungus-derived cellulase so that the protein concentration per 1 mL of saccharification reaction solution was 2.1 g / L. The β-glucosidase activity of the enzyme composition containing the crude purified β-glucosidase and Trichoderma fungus-derived cellulase used in the above saccharification reaction was 1.6 times that of Trichoderma fungus-derived cellulase alone. As a result of the saccharification reaction, when the enzyme composition of Trichoderma cellulase mixed with crude purified β-glucosidase was added, the amount of glucose accumulated increased by approximately 1.8 times and the amount of cellobiose accumulated decreased compared to when only Trichoderma fungus-derived cellulase was added. Table 5 shows the results of glucose and cellobiose analysis.

[0068]

[0069] (Example 9) Production of a β-glucosidase gene-transformed Trichoderma filamentous fungus A plasmid was synthesized by artificial gene synthesis service (Genscript Inc.) in which the nucleotide sequence described in Sequence ID No. 2 (the nucleotide sequence encoding the β-glucosidase gene from a protist of the genus PseudoTrichonympha) and the polynucleotide described in Sequence ID No. 3, which has 66% sequence identity, were linked to the NdeI and XhoI restriction enzyme sites of pET14b. From the plasmid, the nucleotide sequence portion of Sequence ID No. 3 was amplified by PCR and linked downstream of the endoglucanase 1 promoter derived from Trichoderma reesei in a frame with the β-glucosidase secretion signal sequence of Aspergillus aculeatus. The sequence and the hygromycin resistance gene of the transformant were cloned between the T-DNA borders of the pBI101 plasmid. The sequence between the T-DNA borders of the constructed plasmid is shown in Sequence ID No. 4. Here, Sequence ID No. 4 is designed so that when the above sequence is introduced into the genome of a host cell, a β-glucosidase having the amino acid sequence described in Sequence ID No. 1, encoded by base pairs 978 to 3161 (Sequence ID No. 3), is expressed and secreted. The structure of Sequence ID No. 4 is shown below.

[0070] LB = Left T-DNA border: base numbers 1-26 Pegl1 = Endoglucanase 1 promoter from Trichoderma reesei: base numbers 27-920 Sbgl = β-glucosidase secretion signal from Aspergillus aculeatus: base numbers 921-977 bgl = β-glucosidase from Pseudotrichonymphha protists: base numbers 978-3161 (SEQ ID NO: 3) Tegl1 = Endoglucanase 1 terminator from Trichoderma reesei: base numbers 3162-4019 PamdS = Acetamidase promoter from Aspergillus nidulans: base numbers 4020-5027 hygR = Hygromycin B phosphotransferase derived from Streptomyces hygroscopicus: base numbers 5028-6065 TamdS = Acetamidase terminator derived from Aspergillus nidulans: base numbers 6066-6786 RB = Right T-DNA border: base numbers 6787-6810.

[0071] The prepared plasmid was introduced into Agrobacterium tumefaciens AGL1 strain, and Trichoderma reesei was infected with the transformed Agrobacterium to obtain a β-glucosidase-transformed Trichoderma strain. The transformation of Trichoderma with Agrobacterium was performed based on the method of Marcel et al. (Marcel, et al. 2006, Nat Biotechnol 16:839-842). The transformed Agrobacterium was cultured for 8 hours in an induction medium (IM) liquid medium containing glucose and acetosyringone. The transformed Agrobacterium was then mixed with a spore solution of Trichoderma reesei, and cultured for 3 days on cellophane placed on an induction medium (IM) solid medium. After that, the cellophane was transferred to a potato dextrose agar plate (selective medium) containing cefotaxime and hygromycin. Colonies that grew on the selective medium were picked and re-seeded onto the selective medium. This purification culture was repeated twice to obtain the transformed Trichoderma strain.

[0072] (Example 10) Production of an enzyme composition by culturing β-glucosidase gene-transformed Trichoderma filamentous fungi The β-glucosidase gene-transformed Trichoderma filamentous fungi prepared in Example 9 were cultured in the same manner as in Reference Example 7. After centrifugation of the culture medium four days after the start of culture, the supernatant was filtered through an ultrafiltration membrane to remove bacterial cells, thereby preparing an enzyme composition containing the β-glucosidase of the present invention expressed in Trichoderma filamentous fungi and cellulase derived from Trichoderma filamentous fungi. The protein concentration of the enzyme composition was measured according to Reference Example 1, and SDS-PAGE was performed according to Reference Example 6 to confirm the expressed protein. As a comparative control, only cellulase derived from Trichoderma filamentous fungi obtained by culturing non-transformed Trichoderma filamentous fungi was used, and culture, supernatant collection, culture supernatant filtration, and SDS-PAGE were performed in the same manner. SDS-PAGE results showed that in the enzyme composition containing the β-glucosidase and cellulase derived from the genus Trichoderma, the band for the β-glucosidase of the present invention was observed as being more than one-tenth the total number of bands of the enzyme composition that could be confirmed by SDS-PAGE. This confirmed that the β-glucosidase of the present invention is expressed in large quantities in the β-glucosidase gene-transformed Trichoderma filamentous fungi. A photograph of the SDS-PAGE gel is shown in Figure 2.

[0073] Furthermore, the β-glucosidase activity of the enzyme composition containing the β-glucosidase and cellulase derived from Trichoderma filamentous fungi was measured according to Reference Example 2. As a result, the β-glucosidase activity of the enzyme composition containing the β-glucosidase and cellulase derived from Trichoderma filamentous fungi was 2.0 times that of the β-glucosidase activity of Trichoderma filamentous fungi cellulase alone. In addition, the total β-glucosidase activity obtained from 1 L of culture medium of the β-glucosidase gene-transformed Trichoderma filamentous fungi was approximately 1.80 × 10⁻⁶. ３In the case of β-glucosidase expression in Trichoderma filamentous fungi, approximately 850 times more β-glucosidase was obtained from the same volume of culture medium compared to the case of β-glucosidase expression in Escherichia coli in Example 3, indicating that the productivity of the β-glucosidase of the present invention is high.

[0074] (Example 11) Saccharification of microcrystalline cellulose using an enzyme composition containing β-glucosidase and cellulase derived from Trichoderma filamentous fungi The enzyme composition containing β-glucosidase and cellulase derived from Trichoderma filamentous fungi prepared in Example 10 was used in the saccharification reaction. Cellulose microcrystalline cellulose (Merck) was used as the biomass to be saccharified. The saccharification reaction and sugar analysis of the saccharified supernatant were performed in the same manner as in Example 6, except for the biomass used. The amount of enzyme added was 8 mg / g-biomass. As a control, only cellulase derived from Trichoderma filamentous fungi was used, and the saccharification reaction and sugar analysis of the saccharified supernatant were performed in the same manner. As a result, saccharification using the enzyme composition containing the aforementioned β-glucosidase and cellulase derived from Trichoderma filamentous fungi resulted in approximately 1.6 times greater glucose accumulation and a decrease in cellobiose accumulation compared to saccharification using only Trichoderma filamentous fungi cellulase. The glucose and cellobiose results from the sugar analysis are shown in Table 6.

[0075]

[0076] (Example 12) Saccharification of wood raw material powder cellulose using an enzyme composition containing β-glucosidase and cellulase derived from Trichoderma filamentous fungi The enzyme composition containing β-glucosidase and cellulase derived from Trichoderma filamentous fungi prepared in Example 10 was used in the saccharification reaction. Arbocel® (J. Rettenmaier & Sohne), a wood raw material powder cellulose, was used as the biomass to be saccharified. The saccharification reaction and sugar analysis of the saccharified supernatant were carried out in the same manner as in Example 11, except for the biomass used. As a comparative control, only cellulase derived from Trichoderma filamentous fungi was used, and saccharification and sugar analysis of the saccharified supernatant were carried out in the same manner. As a result, saccharification using the enzyme composition containing the aforementioned β-glucosidase and cellulase derived from Trichoderma filamentous fungi resulted in approximately 1.8 times greater glucose accumulation compared to saccharification using only Trichoderma filamentous fungi cellulase, and no cellobiose accumulation was detected. The results of glucose and cellobiose analysis are shown in Table 7.

[0077]

[0078] (Example 13) Alkali-treated bagasse saccharification using an enzyme composition containing β-glucosidase and cellulase derived from Trichoderma filamentous fungi The enzyme composition containing β-glucosidase and cellulase derived from Trichoderma filamentous fungi prepared in Example 10 was used in the saccharification reaction. Bagasse that had undergone alkali treatment (pretreatment) was used as the biomass to be saccharified. The saccharification reaction and sugar analysis of the saccharified supernatant were carried out in the same manner as in Example 11, except for the biomass used. As a control, only cellulase derived from Trichoderma filamentous fungi was used, and saccharification and sugar analysis of the saccharified supernatant were carried out in the same manner. As a result, in saccharification using the enzyme composition containing β-glucosidase and cellulase derived from Trichoderma filamentous fungi, the amount of glucose accumulated increased by approximately 1.8 times compared to saccharification using only cellulase derived from Trichoderma filamentous fungi, and no accumulation of cellobiose was detected. The results of the glucose and cellobiose analysis are shown in Table 8.

[0079]

[0080] (Example 14) Confirmation of residual components in the saccharification supernatant of an enzyme composition containing β-glucosidase and cellulase derived from Trichoderma filamentous fungi The enzyme composition containing β-glucosidase and cellulase derived from Trichoderma filamentous fungi prepared in Example 10 was used in the saccharification reaction. As the biomass to be saccharified, wood raw material powder cellulose Arbocel® (J. Rettenmaier & Sohne), alkali-treated bagasse, and untreated bagasse were used. The saccharification reaction was carried out in the same manner as in Example 11 under reaction conditions of 35°C, and the sample after 24 hours of saccharification was centrifuged for 5 minutes under conditions of 10,000 × g to collect the saccharification supernatant. 3.5 μL of the saccharification supernatant was taken out, mixed with an equal volume of sample buffer, heated at 95°C for 10 minutes, and SDS-PAGE was performed according to Reference Example 6. To confirm the band of the enzyme subjected to saccharification, the enzyme composition diluted to the same concentration as in the saccharification reaction solution was also subjected to SDS-PAGE in the same manner as the saccharification supernatant. As a control, only cellulase derived from Trichoderma filamentous fungi obtained by culturing untransformed Trichoderma filamentous fungi was used, and saccharification, recovery of the saccharification supernatant, and SDS-PAGE were performed in the same manner. As a result, in all cases of saccharification of wood raw material powder cellulose, alkali-treated bagasse saccharification, and untreated bagasse saccharification, the band of the β-glucosidase of the present invention was clearly observed in the saccharification supernatant of the enzyme composition containing the β-glucosidase and Trichoderma filamentous fungi-derived cellulase, confirming that the β-glucosidase of the present invention is easier to recover as a saccharification supernatant than many other cellulase components derived from Trichoderma filamentous fungi. Furthermore, in the saccharification of wood raw material powder cellulose, in the saccharification supernatant of an enzyme composition containing β-glucosidase and cellulase derived from Trichoderma filamentous fungi, more bands of many cellulase components derived from Trichoderma filamentous fungi were observed compared to the saccharification supernatant of only Trichoderma filamentous fungi-derived cellulase. This confirmed that when the β-glucosidase of the present invention is used together with Trichoderma filamentous fungi-derived cellulase as an enzyme composition, the Trichoderma filamentous fungi-derived cellulase is also more easily recovered as part of the saccharification supernatant. A photograph of the SDS-PAGE gel is shown in Figure 3.

[0081] (Example 15) Measurement of residual activity of the cellulose saccharification supernatant of an enzyme composition containing β-glucosidase and cellulase derived from Trichoderma filamentous fungi The cellulose saccharification supernatant of the enzyme composition, recovered in Example 14, was measured for enzyme activity according to Reference Examples 2, 3, and 4. The enzyme composition diluted to the same concentration as in the saccharification reaction solution in Example 14 was also measured for enzyme activity. The percentage of the enzyme activity in the saccharification supernatant, with the enzyme activity of the enzyme composition diluted to the same concentration as in the saccharification reaction solution set to 100%, is shown in Table 9 as the residual activity in the saccharification supernatant. From the results of the residual activity of β-glucosidase, it was confirmed that the β-glucosidase of the present invention is easier to recover as saccharification supernatant than β-glucosidase derived from Trichoderma filamentous fungi. Furthermore, results from the residual activity of β-xylosidase and cellobiohydrolase / endoglucanase confirmed that when the β-glucosidase of the present invention is used as an enzyme composition together with cellulase derived from Trichoderma filamentous fungi, the Trichoderma filamentous fungi-derived β-xylosidase, cellobiohydrolase, and endoglucanase can be easily recovered as a saccharified supernatant.

[0082]

[0083] (Example 16) Measurement of residual activity of untreated bagasse saccharification supernatant of enzyme composition containing β-glucosidase and cellulase derived from Trichoderma filamentous fungi The untreated bagasse saccharification supernatant of the enzyme composition recovered in Example 14 was measured for enzyme activity according to Reference Examples 2, 3, and 4. The enzyme composition diluted to the same concentration as in the saccharification reaction solution in Example 14 was also measured for enzyme activity in the same manner. The percentage of enzyme activity in the saccharification supernatant, with the enzyme activity of the enzyme composition diluted to the same concentration as in the saccharification reaction solution set to 100%, is shown in Table 10 as the residual activity in the saccharification supernatant. From the results of the residual activity of β-glucosidase, it was confirmed that the β-glucosidase of the present invention is easier to recover as saccharification supernatant than β-glucosidase derived from Trichoderma filamentous fungi. Furthermore, results from the residual activity of β-xylosidase and cellobiohydrolase / endoglucanase confirmed that when the β-glucosidase of the present invention is used as an enzyme composition together with cellulase derived from Trichoderma filamentous fungi, the Trichoderma filamentous fungi-derived β-xylosidase, cellobiohydrolase, and endoglucanase can be easily recovered as a saccharified supernatant.

[0084]

[0085] (Example 17) Measurement of residual activity of alkali-treated bagasse saccharification supernatant of enzyme composition containing β-glucosidase and cellulase derived from Trichoderma filamentous fungi The alkali-treated bagasse saccharification supernatant of the enzyme composition recovered in Example 14 was measured for enzyme activity according to Reference Examples 2, 3, and 4. The enzyme composition diluted to the same concentration as in the saccharification reaction solution in Example 14 was also measured for enzyme activity in the same manner. The percentage of enzyme activity in the saccharification supernatant, when the enzyme activity of the enzyme composition diluted to the same concentration as in the saccharification reaction solution is taken as 100%, is shown in Table 11 as the residual activity in the saccharification supernatant. From the results of the residual activity of β-glucosidase, it was confirmed that the β-glucosidase of the present invention is easier to recover as saccharification supernatant than β-glucosidase derived from Trichoderma filamentous fungi. Furthermore, results from the residual activity of β-xylosidase and cellobiohydrolase / endoglucanase confirmed that when the β-glucosidase of the present invention is used as an enzyme composition together with cellulase derived from Trichoderma filamentous fungi, the Trichoderma filamentous fungi-derived β-xylosidase, cellobiohydrolase, and endoglucanase can be easily recovered as a saccharified supernatant.

[0086]

[0087] (Comparative Example 1) Production of Trichoderma filamentous fungi transformed with β-glucosidase gene from Aspergillus filamentous fungi and production of enzyme composition by culturing the transformants Similar to Example 9, a plasmid was prepared designed so that the nucleotide sequence described in SEQ ID NO: 11 (the nucleotide sequence encoding the β-glucosidase gene from Aspergillus filamentous fungi) is introduced into the genome of a host cell, and β-glucosidase having the amino acid sequence described in SEQ ID NO: 10 is expressed and secreted. The plasmid was introduced into Agrobacterium tumefaciens AGL1 strain, and Trichoderma reesei was infected with the transformed Agrobacterium to obtain a β-glucosidase-transformed Trichoderma strain. Similar to Example 10, the Aspergillus-derived β-glucosidase gene-transformed Trichoderma filamentous fungi prepared above were cultured in the same manner as in Reference Example 7. After centrifugation of the culture medium four days after the start of culture, the supernatant was filtered through an ultrafiltration membrane to remove bacterial cells, thereby preparing an enzyme composition containing Aspergillus-derived β-glucosidase and Trichoderma-derived cellulase expressed in the Trichoderma filamentous fungi. SDS-PAGE was performed on the enzyme composition according to Reference Example 6 to confirm the expressed proteins. As a comparative control, only Trichoderma-derived cellulase obtained by culturing untransformed Trichoderma filamentous fungi was used, and culture, supernatant collection, culture supernatant filtration, and SDS-PAGE were performed in the same manner. SDS-PAGE results confirmed the expression of β-glucosidase derived from Aspergillus filamentous fungi. A photograph of the SDS-PAGE gel is shown in Figure 4. Following Reference Example 8, the glucose-mediated inhibition of β-glucosidase activity by an enzyme composition containing the Aspergillus filamentous fungus-derived β-glucosidase and Trichoderma filamentous fungus-derived cellulase was measured, and the results were used for comparison with Example 18.

[0088] (Example 18) Method for Measuring the Inhibitory Effect of Glucose on β-Glucosidase Activity Following Reference Example 8, the inhibitory effect of glucose on β-glucosidase activity was measured for an enzyme composition containing β-glucosidase prepared in Example 10, cellulase derived from Trichoderma filamentous fungi, and cell-free extract of β-glucosidase mutant gene-transformed Escherichia coli prepared in Example 5. As a control, only cellulase derived from Trichoderma filamentous fungi was used, and the inhibitory effect of glucose on β-glucosidase activity was measured in the same manner. Figure 5 shows the relative activity values ​​in the presence of glucose, when the β-glucosidase activity under the condition of a glucose concentration of 0 g / L in the reaction solution is standardized to 1. Furthermore, Table 12 shows the relative activity values ​​under the condition of 8 g / L glucose, and Table 13 shows the relative activity values ​​under the condition of 20 g / L glucose, when the β-glucosidase activity under the condition of 0 g / L glucose in the reaction solution is standardized to 1. As a result, the enzyme composition containing β-glucosidase and cellulase derived from Trichoderma filamentous fungi, and the cell-free extract of β-glucosidase mutant gene-transformed Escherichia coli, all showed less reduction in β-glucosidase activity due to glucose compared to the enzyme composition containing only Trichoderma filamentous fungus cellulase and the enzyme composition containing Aspergillus filamentous fungus β-glucosidase and Trichoderma filamentous fungus cellulase in Comparative Example 1. In other words, the enzyme composition containing β-glucosidase and cellulase derived from Trichoderma filamentous fungi of the present invention, and the cell-free extract of β-glucosidase mutant gene-transformed Escherichia coli, were both less susceptible to inhibition of β-glucosidase activity by glucose compared to cellulase derived from Trichoderma filamentous fungi alone and the enzyme composition containing β-glucosidase derived from Aspergillus filamentous fungi and cellulase derived from Trichoderma filamentous fungi of Comparative Example 1.

[0089]

[0090]

[0091] All publications, patents, and patent applications cited herein shall be incorporated herein by direct reference.

Claims

1. Polypeptides of either (A) to (C) are: (A) a polypeptide whose amino acid sequence is represented by SEQIDNO:1; (B) a polypeptide obtained by replacing, removing, inserting and / or adding one or more amino acids to the amino acid sequence represented by SEQIDNO:1 and containing beta-glucosidase activity; and (C) a polypeptide that has at least 70% sequence similarity to the amino acid sequence represented by SEQIDNO:1 and contains this beta-glucosidase activity. 2.Polynucleotides that are one of the following (a) to (d): (a) a polynucleotide containing a nucleotide sequence represented by SEQIDNO:2; (b) a polynucleotide containing a nucleotide sequence obtained by replacing, removing, inserting and / or adding one or more nucleotides to the nucleotide sequence represented by SEQIDNO:2 and encoding a polypeptide containing this beta-glucosidel activity; (c) a polynucleotide containing a nucleotide sequence that has at least 60% sequence similarity to the nucleotide sequence represented by SEQIDNO:2 and encoding a polypeptide containing this beta-glucosidel activity; and (d) a polynucleotide encoding a polypeptide according to claim 13.Polynucleotides that are one of the following (a) to (d): (a) a polynucleotide containing a nucleotide sequence represented by SEQIDNO:2; (b) a polynucleotide containing a nucleotide sequence obtained by replacing, removing, inserting and / or adding one or more nucleotides to the nucleotide sequence represented by SEQIDNO:2 and that encodes a polypeptide with beta-glucosidase activity; (c) a polynucleotide containing a nucleotide sequence that has at least 50% sequence similarity to the nucleotide sequence represented by SEQIDNO:

2. (d) polynucleotides encoding polypeptides according to claim 1; 4. Expression vectors composed of polynucleotides according to claim 2 or 3; 5. Transformants composed of polynucleotides according to claim 2 or 3, or expression vectors according to claim 4; 6. Transformed filamentous fungi of the genus Trichoderma composed of polynucleotides according to claim 2 or 3, or expression vectors according to claim 4; 7.Methods for the production of enzymatic components incorporating the steps of the transformation culture according to claim 5 or filamentous fungi of the genus Trichoderma according to claim 68. Methods for the production of sugar solutions from cellulose-containing biomass, incorporating the steps of the production of enzymatic components according to claim 7, where the enzymatic components obtained by the steps are used to produce sugar solutions.

9. Beta-glucosidase derived from protists of the genus Pseudotrichonympha, where the activity of beta-glucosidase...

10. An enzyme composition consisting of beta-glucosidase derived from protists of the genus Pseudorichonympha and cellulase derived from filamentous fungi.

11. An enzyme composition according to claim 10 where the filamentous fungus is a filamentous fungus of the genus Trichoderma.

12. A method for the production of sugar solutions from cellulose-containing biomass using enzyme compositions according to claim 10 or 11. 13.The method for producing the sugar solution according to claim 12, which includes the procedure for recovering the enzyme components according to claim 10 or 11 from the sugar solution;

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