FIELD OF THE INVENTION

The present invention relates to a mutant β-glucosidase.

BACKGROUND OF THE INVENTION

A technology is known for producing a saccharide from cellulose in a biomass material containing cellulose (hereinafter, sometimes refers to as “biomass”) and converting the saccharide to energies such as ethanol and chemical products using a fermentation method. Efforts made on environmental issues in recent years have helped advancement in various technological development for the industrial use of biomasses, and mass production of fuels and chemical products using biomasses has also been realized.

Biomass is made up of cellulose fibers, hemicelluloses surrounding the cellulose fibers and containing mainly xylan, and lignin. In the production of a saccharide using biomass as a raw material, it is important to increase saccharification efficiency of celluloses and hemicelluloses and the achievement thereof requires a biomass saccharification enzyme such as a cellulase and a hemicellulase which hydrolyzes cellulose and hemicellulose. For example, for efficiently degrading cellulose into glucose, at least three types of cellulases need to work in concert with each other, such as (1) a cellobiohydrolase (CBH) which cleaves off a saccharide in a cellobiose unit from the end of crystalline and non-crystalline cellulose fibers, (2) an endoglucanase (EG) which makes a cleavage into the cellulose chain by working on the non-crystalline cellulose, and (3) a β-glucosidase (BGL) which produces glucose by hydrolyzing the cellobiose produced by these enzymes.

Fungi well known for producing a cellulase used for the saccharification of biomass are microorganisms belonging to the genusTrichodermasuch asTrichoderma reeseiandTrichoderma viride. However, in the biomass saccharification using a cellulose derived from a microorganism belonging to the genusTrichoderma, relatively low β-glucosidase activity is noted (Non Patent Literature 1). A technology has been developed for increasing biomass saccharification activity of an enzyme derived from a microorganism belonging to the genusTrichodermaby expressing a β-glucosidase ofAspergillus aculeatusin the microorganism belonging to the genusTrichoderma(for example, Non Patent Literature 1, Patent Literature 1).(Patent Literature 1) International WO 2013/115305 A1(Non Patent Literature 1) MORIKAWA Yasushi, Research Frontier of Biomass Degrading Enzymes, 2012, CMC Publishing Co., Ltd., p 10-19

SUMMARY OF THE INVENTION

The present invention provides a mutant β-glucosidase selected from the group consisting of the following (i), (ii), and (iii):

(i) a polypeptide that consists of an amino acid sequence obtained by substituting, with Gln and Thr, an amino acid residue at a position corresponding to position 649 of SEQ ID NO: 1 and an amino acid residue at a position corresponding to the position 655 of SEQ ID NO: 1, respectively, in an amino acid sequence as set forth in SEQ ID NO: 1, or an amino acid sequence having at least 90% identity thereto, and has β-glucosidase activity;

(ii) a polypeptide that consists of an amino acid sequence obtained by substituting, with Tyr-Glu-Pro-Ala-Ser-Gly, an amino acid sequence of a region corresponding to the region from position 653 to position 658 of SEQ ID NO:1 in an amino acid sequence as set forth in SEQ ID NO: 1, or an amino acid sequence having at least 90% identity thereto; and

(iii) a polypeptide that comprises the polypeptide of (i) or (ii) and has β-glucosidase activity.

Further, the present invention provides a polynucleotide encoding the mutant β-glucosidase.

Further, the present invention provides a vector comprising the polynucleotide.

Further, the present invention provides a transformant comprising the polynucleotide or the vector.

Further, the present invention provides a biomass saccharification agent comprising the mutant β-glucosidase.

Further, the present invention provides a method for producing a saccharide comprising saccharifying a biomass using the mutant β-glucosidase.

The present invention further provides a method for producing a mutant β-glucosidase, comprising

(a) substituting, with Gln and Thr, an amino acid residue at a position corresponding to position 649 of SEQ ID NO: 1 and an amino acid residue at a position corresponding to position 655 of SEQ ID NO: 1, respectively, in a polypeptide that comprises the amino acid sequence as set forth in SEQ ID NO: 1, or an amino acid sequence having at least 90% identity thereto, and that has β-glucosidase activity or

(b) substituting, with Tyr-Glu-Pro-Ala-Ser-Gly, an amino acid sequence of a region corresponding to the region from position 653 to position 658 of SEQ ID NO:1 in a polypeptide that comprises the amino acid sequence as set forth in SEQ ID NO: 1, or an amino acid sequence having at least 90% identity thereto, and has β-glucosidase activity.

