CN114703165A

CN114703165A - Beta-glucosidase mutant and application thereof

Info

Publication number: CN114703165A
Application number: CN202210376425.7A
Authority: CN
Inventors: 李玉; 李庆刚; 王茂军; 史超硕; 刘逸寒; 路福平
Original assignee: Tianjin University of Science and Technology
Current assignee: Tianjin University of Science and Technology
Priority date: 2022-04-02
Filing date: 2022-04-02
Publication date: 2022-07-05
Anticipated expiration: 2042-04-02
Also published as: CN114703165B

Abstract

The invention mainly utilizes the site-directed mutagenesis technology to construct a beta-glucosidase mutant strain derived from the humicola insolens to obtain a beta-glucosidase mutant T331Q with improved glucose tolerance, and the amino acid sequence of the mutant is shown as SEQ ID NO: 4, respectively. Under the same condition, the mutant can reach the relative activity of 239.0 percent at the maximum under the activation of 400mmol/L glucose, and the relative activity can still reach 112.61 percent at the glucose concentration of 1.5mol/L, so that the mutant has good glucose tolerance. The mutant has higher application value in the fields of biofuel, food health care, biosynthesis and the like.

Description

Beta-glucosidase mutant and application thereof

Technical Field

The invention belongs to the technical field of microorganisms and genetic engineering, and particularly relates to a beta-glucosidase mutant with improved glucose tolerance and application thereof.

Background

beta-Glucosidases (beta-Glucosidases EC 3.2.1.21), collectively known as beta-D-glucoside hydrolases, belong to the class of hydrolases and are also known as cellobiase, gentiobiosidase or amygdalinase. The enzyme can hydrolyze the non-reducing beta-D-glycosidic bond bound to the terminal end, and release beta-D-glucose and the corresponding ligand. The beta-glucosidase can act on various bioactive substances because of having hydrolytic activity on various glucoside compounds, and most of ligands generated by hydrolysis are functional aglycones or aromatic components; some beta-glucosidase also has transglycosidation activity and can be converted into functional oligosaccharide, so the beta-glucosidase has wide application prospect in the fields of food health care, biological energy and the like.

In the actual production process, the enzyme is often in an adverse environment, and the beta-glucosidase is influenced by the product glucose, so that the production efficiency is low, the substrate is not sufficiently reflected and the like. Measures such as increasing enzyme amount and Synchronous Saccharification and Fermentation (SSF) are usually adopted to relieve the inhibition effect of the product, but the methods have complex process, large pollution and higher cost. In order to make the catalytic reaction proceed efficiently, it is now highly desirable to obtain β -glucosidase with high glucose tolerance. Meanwhile, glucose tolerance is increasingly regarded as a key property of beta-glucosidase in production processes of biofuels, food health care, biosynthesis and the like. The beta-glucosidase with high tolerance shows unique application potential because of reducing the inhibition effect of glucose on enzyme activity. CN102220302A is based on beta-glucosidase from marine uncultured microorganism, mutant gene is obtained by PCR fixed point mutation, and compared with wild beta-glucosidase protein, the glucose tolerance concentration of the obtained mutant protein is improved by 13 times. CN103266096A mutant Pbgl-W386C is obtained by mutating tryptophan at position 386 of beta-glucosidase gene Pbgl to cysteine, and the tolerance of glucose is 11.88 times higher than that before mutation and is increased by 10.88 times. CN105754973A the beta-glucosidase mutant was obtained by mutating tryptophan at position 233 of Streptomyces beta-glucosidase S-bgl6 molecule to aspartic acid, and the tolerance to glucose was improved 208.9 times compared with that before mutation. CN107142254A mutates amino acid 315 of beta-glucosidase from Alicyclobacillus acidocaldarius sp.A4 from histidine to arginine to obtain beta-glucosidase mutant with higher glucose tolerance, wherein under the same conditions, the promoting effects of glucose on the enzyme activities of the mutant and wild type are 212% and 163% respectively.

Disclosure of Invention

Aiming at the current industrial demand and the defects of the prior art, the invention mainly aims to construct a beta-glucosidase mutant with good glucose tolerance by a site-directed mutagenesis technology.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the invention provides a beta-glucosidase mutant, wherein the amino acid sequence of the beta-glucosidase mutant is shown as SEQ ID NO: 4, respectively.

In a second aspect, the present invention provides a gene encoding the β -glucosidase mutant.

In a third aspect, the present invention provides a vector comprising the gene of the β -glucosidase mutant.

In a fourth aspect, the present invention provides a recombinant strain comprising a gene or vector as described above.

In a fifth aspect, the present invention provides the use of a mutant β -glucosidase as described above, for hydrolysing the β -D-glucosidic bond at the non-reducing terminus of a glycoside or oligosaccharide compound, to generate β -D-glucose and the corresponding ligand; and, the mutant has improved glucose tolerance compared to the wild type.

In a sixth aspect, the present invention provides a method for preparing the β -glucosidase mutant as described above, by gene recombination and expression using the gene encoding the β -glucosidase mutant of the present invention or the vector of the present invention.

In a seventh aspect, the present invention provides a method of hydrolyzing a glycoside compound, in particular cellobiose or cellooligosaccharide, comprising the step of contacting a glycoside compound with a β -glucosidase mutant of the invention or with a recombinant strain of the invention, under conditions capable of hydrolyzing the β -D-glucosidic bond of the compound by enzymatic catalysis by the β -glucosidase mutant or the recombinant strain.

Has the advantages that:

the beta-glucosidase mutant obtained by the site-directed mutagenesis technology is recombined and expressed in a pichia pastoris system through a gene engineering technology, under the same condition, the mutant can reach the relative activity of 239.0 percent at the maximum under the activation of 400mmol/L glucose, and can still reach 112.61 percent at the relative activity when the glucose concentration is 1.5mol/L, so that the mutant has the obviously improved glucose tolerance compared with a wild type. The mutant of the invention has higher application value in the fields of biofuel, food health care, biosynthesis and the like.

Drawings

FIG. 1: PCR amplification electropherograms of the wild-type β -glucosidase gene in the examples; wherein: 1 is negative control, 2 is DNA Marker, and 3 is wild type bglii gene.

FIG. 2: in the examples, the relative enzyme activities of the wild type and the mutant under the activation of glucose at different concentrations are shown.

FIG. 3: electrophoresis verification of purified proteins of the mutants in the examples; wherein: 1 is protein Marker, 2 and 3 are recombinant strain GS115/pPIC 9K-bgliIM, and 4 is control strain GS115/pPIC 9K.

FIG. 4: the amino acid sequence of the beta-glucosidase mutant of the invention.

Detailed Description

The invention is further described below by means of specific embodiments. Unless otherwise specified, technical means, materials, and the like, which are referred to in the following embodiments, may be known to those skilled in the art, and may be selected as appropriate from known means and materials capable of solving the corresponding technical problems. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention.

According to a preferred embodiment of the invention, the nucleotide sequence of said gene is as shown in SEQ ID NO: 3, respectively.

In a third aspect, the present invention provides a vector comprising the gene of the β -glucosidase mutant. The vector may be one of vectors for producing a protein by gene recombination, such as an expression vector, known to those skilled in the art.

According to a preferred embodiment of the invention, the vector is the pPIC9K plasmid.

In a fourth aspect, the present invention provides a recombinant strain comprising a gene or vector as described above. The recombinant strain may be any host suitable for producing the β -glucosidase mutant of the present invention from the gene or vector of the present invention, such as pichia pastoris.

According to a preferred embodiment of the invention, the host is pichia pastoris GS 115.

According to another embodiment of the present invention, the host is Escherichia coli JM 109.

In a sixth aspect, the present invention provides a method for preparing the beta-glucosidase mutant as described above, by gene recombination and expression using the gene encoding the beta-glucosidase mutant of the present invention or the vector of the present invention. Genetic recombination methods and expression hosts known to those skilled in the art can be used, and media and culture conditions suitable for expression by the host are selected. The method may further comprise the step of recovering the β -glucosidase mutant, which may involve the step of isolating or purifying the β -glucosidase mutant from the culture or expression product of the host, and may be performed using any method known to those skilled in the art.

According to a preferred embodiment of the present invention, the mutant is prepared as follows: the mutant gene and a plasmid pPIC9K are subjected to double enzyme digestion through EcoR I and Avr II and are connected to obtain a recombinant vector, the recombinant vector is transformed into an escherichia coli host JM109, the plasmid is linearized by Sac I and is finally electrically transferred into pichia pastoris GS115 for expression, protein is purified through an anion exchange column and the like, and the enzyme activity of the protein is verified, so that the beta-glucosidase mutant with the glucose tolerance improved compared with a wild type is obtained.

The following definitions are used in the present invention:

1. nomenclature for amino acid and DNA nucleic acid sequences

Amino acid residues are identified using the IUPAC nomenclature, either in the form of three letter abbreviations or single letter symbols. DNA nucleic acid sequences employ the accepted IUPAC nomenclature.

2. Identification of beta-glucosidase mutants

"amino acid substituted at original amino acid position" is used to indicate a mutated amino acid in the β -glucosidase mutant. The mutant of the invention is shown as SEQ ID NO: 2 by substituting threonine (T) at position 331 with glutamine (Q), as represented by T331Q or Thr331 Gln. The gene mutation sites of the mutant are as follows:

the present invention will be described in more detail below by way of specific examples. Unless otherwise specified, in the following examples:

the culture medium and the enzyme activity determination method used by the invention are as follows:

LB culture medium: 10g/L of peptone, 5g/L of yeast powder, 10g/L of NaCl, 18g/L of agar added into a solid culture medium, and the other components are consistent.

MD solid medium: 20g/L glucose and 18g/L agar.

YPD medium: peptone 20g/L, yeast powder 10g/L, glucose 20g/L, solid medium added agar 18g/L, other components consistent.

YPG medium: peptone 20g/L, yeast powder 10g/L, and glycerol 20 g/L.

BMGY medium: 10g of yeast powder and 20g of peptone are dissolved in 700mL of water and sterilized at 121 ℃ for 20min, and 100mL of pH 6.0, 1mol/L potassium phosphate buffer, 100mL of 10 XYNB, 2mL of 500 Xbiotin and 100mL of 10 Xglycerol are added at 60 ℃ for use.

BMMY medium: 10g of yeast powder and 20g of peptone are dissolved in 700mL of water and sterilized at 121 ℃ for 20min, and 100mL of 1mol/L potassium phosphate buffer pH 6.0, 100mL of 10 XYNB, 2mL of 500 Xbiotin and 2.5mL of methanol are added at 60 ℃ in use.

MES buffer: 3.91g MES was weighed and dissolved in water, the pH was adjusted to 6.5 with Tris alkali solution, and water was added to a constant volume of 1L.

buffer A: the final concentration was 20mmol/L, pH 6.5.5 MES buffer, and the membrane was sterilized for further use.

buffer B: 20mmol/L, pH 6.5.5 MES buffer and 1mol/L NaCl solution, and the solution is passed through a 0.22 μm sterile membrane for further use.

G418 screening medium: melting YPD culture medium according to concentration (0.5mg/mL, 1mg/mL, 2mg/mL), adding at about 55 deg.C, and mixing; other resistance selection media added resistance at the corresponding concentrations.

Escherichia coli competence preparation culture medium

And E.coli reviving and LB culture medium.

Washing culture medium: adding CaCl into each liter₂ 11.1g；

Heavy suspension culture medium: 1.5mL of glycerol, 8.5mL of 11.1g/L CaCl₂A solution;

a pichia pastoris competence preparation culture medium:

YPD medium: peptone 20g/L, yeast powder 10g/L, glucose 20 g/L;

sterile water: sterilizing with membrane water at 121 deg.C for 20 min;

1mol/L sorbitol: dissolving 18.21g sorbitol in 100mL deionized water, sterilizing at 121 deg.C for 20min, and storing at 4 deg.C for use.

The enzyme activity detection of the beta-glucosidase is mainly based on the ligand and glucose released by enzymolysis reaction, a synthetic substrate p-nitrophenyl-beta-D-glucopyranoside (pNPG) is used as a substrate, hydrolysis products of the p-nitrophenyl-beta-D-glucopyranoside (pNPG) and glucose are obtained, and the p-nitrophenyl has a specific light absorption value under 400 nm.

The specific detection method comprises the following steps: mixing 100 μ L of diluted enzyme solution with 2.9mL of substrate solution (pNPG dissolved in disodium hydrogen phosphate-citric acid buffer solution with final concentration of 3mmol/L and pH of 5.0), reacting at 50 deg.C for 4min, adding 1mL of 1mol/L Na₂CO₃The solution stops the reaction. The supernatant of inactivated fermentation broth boiled at 100 ℃ for 10min was used as a control. Measurement of OD of reaction solution₄₀₀The pNP content was calculated with reference to the standard curve as a value of nm. The amount of enzyme required to produce 1. mu. mol of p-nitrobenzene (pNP) per minute in the reaction system was defined as 1 enzyme activity unit (U).

The p-nitrobenzene (pNP) standard curve is as follows; preparing 0.1mg/mL p-nitrophenol mother liquor, and respectively diluting to the concentration of 0.002 mg/mLmL, 0.004mg/mL, 0.006mg/mL, 0.008mg/mL, 0.010mg/mL, and absorbance at 400nm was measured using sterile water as a blank. Taking OD value as ordinate and pNP concentration (mg/mL) as abscissa, making pNP standard curve, and obtaining linear regression equation Y of 40.8X-0.026; r is²＝0.9988。

Wherein, before the enzyme solution and the substrate solution are mixed, the substrate solution needs to be preheated in a water bath at 50 ℃ for more than 2 min.

The beta-glucosidase enzyme activity calculation formula is as follows: enzyme activity S ═ X times V₁×1000×n/139×t×V₂

Wherein X is the amount of p-NP (mg/mL); v₁Volume of reaction solution (mL); v₂Volume of enzyme solution (mL); n is the dilution multiple of the enzyme solution; t is the reaction time (min).

Definition of enzyme activity: the amount of enzyme required to produce 1. mu. mol of p-nitrobenzene (pNP) per minute in the reaction system at 50 ℃ was defined as 1 unit of enzyme activity (U).

Example 1: construction of wild type beta-glucosidase bglii recombinant strain

1.1 Synthesis and amplification of the wild-type beta-glucosidase Gene bglii

The wild type beta-glucosidase bglihi gene sequence from Humicola insolens (Humicola insolens) is obtained according to the GenBank KF588650.1, in order to be suitable for pichia pastoris expression, the codon is optimized, and the optimized nucleotide sequence is shown as SEQ ID NO: 1, the organism company was entrusted with the synthesis of the sequence and the amplification by PCR, and the primer sequences were as follows:

primer P1: F5'-ATGTCTTTGCCTCCAGACTTC-3';

primer P2: R5'-CGACTCTTTGATTAGAAAGGAGTAA-3';

amplifying by taking P1 and P2 as upstream and downstream primers and a wild-type gene of bglii as a template;

the reaction system for amplification is as follows:

upstream primer P1	1.5μL
		Downstream primer P2	1.5μL
DNA template	2μL
		PrimeStar enzyme	25μL
ddH₂O	20μL

The setting of the amplification program is as follows: pre-denaturation: 5min at 95 ℃; denaturation: 10s at 98 ℃; annealing: 20s at 50 ℃; extension: 10s at 72 ℃; reacting for 32 cycles; extension: 10min at 72 ℃.

The PCR product is subjected to agarose gel electrophoresis, a band of wild beta-glucosidase gene can be seen, the band is 1431bp (shown in figure 1), and then the PCR product is recovered by a small amount of DNA recovery kit, so that the wild beta-glucosidase gene, namely bglii, is obtained.

1.2 linearization of expression vectors

The pPIC9K plasmid was extracted according to the kit manual. Carrying out agarose gel electrophoresis after double enzyme digestion by EcoR I and Avr II, and then recovering the product by a DNA gel recovery kit to obtain a linearized vector sequence.

1.3 construction of recombinant strains

The target fragment (bglii) which is subjected to double enzyme digestion by EcoR I and Avr II and the vector fragment are connected to form a recombinant plasmid pPIC 9K-bglii, the recombinant plasmid is transformed into Escherichia coli JM109, and the sequence is verified to be shown in SEQ ID NO: 1.

example 2: beta-glucosidase mutant obtained by site-directed mutagenesis method

2.1 carrying out directional mutation based on site-directed mutagenesis technology to construct a novel beta-glucosidase mutant, designing primers as follows:

primer P3: 5'-CAGTTGTTCTACAACAAGTACGGTGATTGTATCGGTCCA G-3', respectively;

primer P4: R5'-CTCCAAGTTTCCCAAAAAGTCGTCCTC-3';

in a site-directed mutagenesis PCR reaction system, the directional mutagenesis PCR is carried out by taking P3 and P4 as upstream and downstream primers and taking the recombinant plasmid pPIC 9K-bglii as a template.

The reaction system for amplification is as follows:

upstream primer P3	1.5μL
		Downstream primer P4	1.5μL
DNA template	2μL
		Primerstar enzyme	25μL
ddH₂O	20μL

The amplification conditions were: pre-denaturation: 5min at 95 ℃; denaturation: 10s at 98 ℃; annealing: 20s at 62 ℃; extension: 1min at 72 ℃; reacting for 32 cycles; extension: 10min at 72 ℃.

2.2 the obtained linear plasmid with beta-glucosidase mutant gene is self-interlinked to obtain recombinant plasmid pPIC 9K-bgliim, which is transformed into Escherichia coli JM109, and the recombinant plasmids pPIC 9K-bglii and pPIC 9K-bgliim are transformed into Pichia pastoris to obtain recombinant strain GS115/pPIC 9K-bgliim capable of expressing beta-glucosidase mutant.

Example 3: validation of beta-glucosidase mutants

3.1 Shake flask validation

The recombinant strain obtained in example 2 is referred to a Pichia expression manual, a single colony is streaked and separated from the screened high-copy recombinant yeast, and the single colony and a single colony of a reference bacterium P.pastoris GS115/pPIC9K are respectively inoculated into 30mL of YPG medium (containing 50 mu g/mL of kanamycin at the final concentration) and cultured for 16-18h at 30 ℃ and 180 r/min; inoculating the seed liquid into 50mL BMGY liquid culture medium according to the inoculation amount of 2%, culturing at 30 deg.C and 180r/min to OD₆₀₀To 5-6 (about 16-20 h); collecting the bacterial liquid in a sterilized 50mL centrifuge tube, centrifuging for 5min at 8000r/min, pouring off the supernatant, re-suspending the thallus with 20mL BMMY culture medium for cell washing, repeating the washing for 3 times, and finally re-suspending the thallus with BMMY culture medium (final concentration OD of the thallus)₆₀₀1); after starvation for 1h, adding an inducer methanol, adding 0.5% methanol every 24h for induction expression, and sampling and analyzing at regular time to prepare the beta-glucosidase enzyme solution.

Respectively measuring the glucose tolerance of the beta-glucosidase in the enzyme solution, and comparing the tolerance of the mutant and the wild beta-glucosidase under different glucose concentrations to obtain 1 strain of the strain of which the glucose tolerance is obviously stronger than that of the wild beta-glucosidase.

3.2 Shake flask review, purification and enzyme Activity Studies

Inoculating the recombinant strain and single colony of control bacterium P.pastoris GS115/pPIC9K into 30mL YPG medium (containing kanamycin with final concentration of 50 μ g/mL) respectively, and culturing at 30 deg.C and 180r/min for 16-18 h; inoculating the seed liquid into 50mL BMGY liquid culture medium according to the inoculation amount of 2%, culturing at 30 deg.C and 180r/min to OD₆₀₀To 5-6 (about 16-20 h); collecting bacterial liquid in a sterilized 50mL centrifuge tube, 8000r/min, centrifuging for 5min, pouring off supernatant, and resuspending with 20mL BMMY culture mediumThe cells were washed repeatedly 3 times, and the cells were resuspended in BMMY medium (final cell concentration OD)₆₀₀1); after starvation for 1h, adding an inducer methanol, adding 0.5% methanol every 24h to induce expression, and culturing for 96 h. Placing the fermentation liquor in a 50mL sterile centrifuge tube, centrifuging at 4 ℃ and 12000r/min for 20min, and removing thalli; filtering the supernatant of the fermentation liquid with a 0.22 μm sterilizing membrane, placing the supernatant in a 30kD ultrafiltration tube for ultrafiltration concentration, and centrifuging at 4 ℃ and 5000r/min for 60min to obtain about 1mL of concentrated solution. Transferring the concentrated fermentation liquor into a dialysis bag for dialysis, putting the dialysis bag into a beaker filled with deionized water, and dialyzing under magnetic stirring at 4 ℃ to remove salt ions in the fermentation liquor. Placing the dialyzed enzyme solution in a 10mL centrifuge tube, centrifuging at 8000r/min and 4 ℃ for 10min, removing insoluble bottom impurities, and removing insoluble impurities through a 0.22-micron water system film; equilibrium anion exchange column (SourseS): using 20mmol/L MES buffer solution as buffer A, washing the column by using the buffer A, and washing 3-5 column volumes to balance the anion exchange column; loading: all samples were loaded onto the equilibrated column, the loading flow rate was set at 0.5mL/min, 3-5 column volumes were washed with buffer A, and the protein peak was collected from the completion of loading. After the sample equilibrated, elution was performed with buffer B and different gradients were set until 100% NaCl concentration was reached. The elution flow rate is set as 1mL/min, every time an elution peak appears, the collecting pipe is replaced, and all the elution peaks are collected and numbered respectively. The purified enzyme solution was subjected to polyacrylamide gel electrophoresis (SDS-PAGE) analysis using 5% concentrated gel and 12% separation gel, and the results are shown in FIG. 3. Lane 4 of figure 3 is the fermentation supernatant of control strain GS115/pPIC9K, the control group has substantially no protein secretion because pichia pastoris GS115 has been specifically engineered to have little or no extracellular protein secretion, and

lane

2 or 3 is the purified enzyme solution of recombinant strain GS115/pPIC 9K-bgliim, with a visible protein band with an apparent molecular weight of about 60 kDa. Measuring the enzyme activity and glucose tolerance of the purified enzyme solution to obtain a beta-glucosidase mutant T331Q, wherein the enzyme activity of the beta-glucosidase can reach 239.0% of relative activity under the activation of 400mmol/L glucose, and the phase activity of the beta-glucosidase mutant is 1.5mol/L glucose112.61% of the activity can still be achieved (the enzyme activity is shown in figure 2).

Example 4: sequencing of beta-glucosidase mutants

The recombinant strain is used for extracting a beta-glucosidase gene sequence and sequencing (Jinzhi biotechnology limited), and the result shows that the nucleotide sequence of the beta-glucosidase mutant gene obtained by amplification is shown as SEQ ID NO: 3, the coding gene was designated as bglHiM.

And (2) respectively comparing the amino acid sequence of the obtained beta-glucosidase bglHiM with the amino acid sequence of wild-type beta-glucosidase bglHi, namely SEQ ID NO: 1, comparative analysis is carried out, and the results show that: the 331 st amino acid of β -glucosidase bglHiM was mutated from Thr to Gln compared to wild-type β -glucosidase bglHi (as shown in fig. 4).

Although the present invention has been disclosed in the form of preferred embodiments, it is not intended to limit the present invention, and those skilled in the art may make various changes, modifications, substitutions and alterations in form and detail without departing from the spirit and principle of the present invention, the scope of which is defined by the appended claims and their equivalents.

Sequence listing

<120> beta-glucosidase mutant and application thereof

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1431

<212> DNA

<213> Humicola insolens

<400> 1

atgtctttgc ctccagactt caagtgggga tttgctactg ccgcctacca gattgagggt 60

tccgttaacg aggatggtcg tggtccatct atctgggaca ccttctgcgc catcccagga 120

aagattgctg acggttcttc tggtgctgtc gcttgcgact cttacaagcg tactaaggaa 180

gatattgcct tgttgaagga attgggtgct aactcctacc gtttctccat ctcctggtcc 240

agaattatcc ctttgggtgg acgtaacgac ccaatcaatc aaaagggaat cgatcattac 300

gttaagttcg ttgatgactt gatcgaagcc ggtattaccc ctttcattac cttgttccac 360

tgggacttgc cagacgcttt ggacaaaaga tacggaggtt ttttgaacaa ggaagagttt 420

gccgctgact tcgagaacta cgctagaatt atgttcaagg ctatccctaa atgtaagcat 480

tggattacct ttaacgagcc atggtgctcc gccatcttgg gatacaacac cggttacttt 540

gccccaggtc acacttccga cagatccaag tctccagttg gtgattctgc cagagagcct 600

tggatcgttg gtcataacat cttgatcgcc cacgccagag ctgttaaggc ctaccgtgag 660

gatttcaagc caacccaggg tggtgagatc ggtatcaccc ttaacggaga cgctactttg 720

ccttgggatc cagaagaccc agctgacatt gaggcttgcg atagaaagat cgagttcgct 780

atctcctggt tcgccgaccc aatctacttc ggaaagtacc cagactccat gagaaagcag 840

ttgggagaca gattgccaga gttcactcca gaggaagtcg ccttggttaa gggttctaat 900

gatttttacg gtatgaacca ctacaccgcc aactacatca agcacaagac tggtgtccct 960

ccagaggacg actttttggg aaacttggag actttgttct acaacaagta cggtgattgt 1020

atcggtccag agacccaatc cttttggttg cgtccacacg ctcaaggatt cagagacttg 1080

ttgaattggt tgtctaagag atacggttac ccaaaaattt acgttactga gaacggtacc 1140

tccttgaagg gtgagaacga catgcctttg gagcaggtct tggaggacga cttcagagtt 1200

aagtacttta acgactacgt tagagctatg gctgccgctg ttgctgagga cggatgcaac 1260

gttcgtggtt atttggcctg gtctttgctt gacaactttg agtgggccga gggatacgag 1320

accagattcg gtgtcaccta cgttgattac gccaacgacc agaagcgtta cccaaagaag 1380

tccgctaagt ccttgaaacc acttttcgac tctttgatta gaaaggagta a 1431

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<211> 476

<212> PRT

<213> Humicola insolens

<400> 2

Met Ser Leu Pro Pro Asp Phe Lys Trp Gly Phe Ala Thr Ala Ala Tyr

1 5 10 15

Gln Ile Glu Gly Ser Val Asn Glu Asp Gly Arg Gly Pro Ser Ile Trp

20 25 30

Asp Thr Phe Cys Ala Ile Pro Gly Lys Ile Ala Asp Gly Ser Ser Gly

35 40 45

Ala Val Ala Cys Asp Ser Tyr Lys Arg Thr Lys Glu Asp Ile Ala Leu

50 55 60

Leu Lys Glu Leu Gly Ala Asn Ser Tyr Arg Phe Ser Ile Ser Trp Ser

65 70 75 80

Arg Ile Ile Pro Leu Gly Gly Arg Asn Asp Pro Ile Asn Gln Lys Gly

85 90 95

Ile Asp His Tyr Val Lys Phe Val Asp Asp Leu Ile Glu Ala Gly Ile

100 105 110

Thr Pro Phe Ile Thr Leu Phe His Trp Asp Leu Pro Asp Ala Leu Asp

115 120 125

Lys Arg Tyr Gly Gly Phe Leu Asn Lys Glu Glu Phe Ala Ala Asp Phe

130 135 140

Glu Asn Tyr Ala Arg Ile Met Phe Lys Ala Ile Pro Lys Cys Lys His

145 150 155 160

Trp Ile Thr Phe Asn Glu Pro Trp Cys Ser Ala Ile Leu Gly Tyr Asn

165 170 175

Thr Gly Tyr Phe Ala Pro Gly His Thr Ser Asp Arg Ser Lys Ser Pro

180 185 190

Val Gly Asp Ser Ala Arg Glu Pro Trp Ile Val Gly His Asn Ile Leu

195 200 205

Ile Ala His Ala Arg Ala Val Lys Ala Tyr Arg Glu Asp Phe Lys Pro

210 215 220

Thr Gln Gly Gly Glu Ile Gly Ile Thr Leu Asn Gly Asp Ala Thr Leu

225 230 235 240

Pro Trp Asp Pro Glu Asp Pro Ala Asp Ile Glu Ala Cys Asp Arg Lys

245 250 255

Ile Glu Phe Ala Ile Ser Trp Phe Ala Asp Pro Ile Tyr Phe Gly Lys

260 265 270

Tyr Pro Asp Ser Met Arg Lys Gln Leu Gly Asp Arg Leu Pro Glu Phe

275 280 285

Thr Pro Glu Glu Val Ala Leu Val Lys Gly Ser Asn Asp Phe Tyr Gly

290 295 300

Met Asn His Tyr Thr Ala Asn Tyr Ile Lys His Lys Thr Gly Val Pro

305 310 315 320

Pro Glu Asp Asp Phe Leu Gly Asn Leu Glu Thr Leu Phe Tyr Asn Lys

325 330 335

Tyr Gly Asp Cys Ile Gly Pro Glu Thr Gln Ser Phe Trp Leu Arg Pro

340 345 350

His Ala Gln Gly Phe Arg Asp Leu Leu Asn Trp Leu Ser Lys Arg Tyr

355 360 365

Gly Tyr Pro Lys Ile Tyr Val Thr Glu Asn Gly Thr Ser Leu Lys Gly

370 375 380

Glu Asn Asp Met Pro Leu Glu Gln Val Leu Glu Asp Asp Phe Arg Val

385 390 395 400

Lys Tyr Phe Asn Asp Tyr Val Arg Ala Met Ala Ala Ala Val Ala Glu

405 410 415

Asp Gly Cys Asn Val Arg Gly Tyr Leu Ala Trp Ser Leu Leu Asp Asn

420 425 430

Phe Glu Trp Ala Glu Gly Tyr Glu Thr Arg Phe Gly Val Thr Tyr Val

435 440 445

Asp Tyr Ala Asn Asp Gln Lys Arg Tyr Pro Lys Lys Ser Ala Lys Ser

450 455 460

Leu Lys Pro Leu Phe Asp Ser Leu Ile Arg Lys Glu

465 470 475

<210> 3

<211> 1431

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atgtctttgc ctccagactt caagtgggga tttgctactg ccgcctacca gattgagggt 60

tccgttaacg aggatggtcg tggtccatct atctgggaca ccttctgcgc catcccagga 120

aagattgctg acggttcttc tggtgctgtc gcttgcgact cttacaagcg tactaaggaa 180

gatattgcct tgttgaagga attgggtgct aactcctacc gtttctccat ctcctggtcc 240

agaattatcc ctttgggtgg acgtaacgac ccaatcaatc aaaagggaat cgatcattac 300

gttaagttcg ttgatgactt gatcgaagcc ggtattaccc ctttcattac cttgttccac 360

tgggacttgc cagacgcttt ggacaaaaga tacggaggtt ttttgaacaa ggaagagttt 420

gccgctgact tcgagaacta cgctagaatt atgttcaagg ctatccctaa atgtaagcat 480

tggattacct ttaacgagcc atggtgctcc gccatcttgg gatacaacac cggttacttt 540

gccccaggtc acacttccga cagatccaag tctccagttg gtgattctgc cagagagcct 600

tggatcgttg gtcataacat cttgatcgcc cacgccagag ctgttaaggc ctaccgtgag 660

gatttcaagc caacccaggg tggtgagatc ggtatcaccc ttaacggaga cgctactttg 720

ccttgggatc cagaagaccc agctgacatt gaggcttgcg atagaaagat cgagttcgct 780

atctcctggt tcgccgaccc aatctacttc ggaaagtacc cagactccat gagaaagcag 840

ttgggagaca gattgccaga gttcactcca gaggaagtcg ccttggttaa gggttctaat 900

gatttttacg gtatgaacca ctacaccgcc aactacatca agcacaagac tggtgtccct 960

ccagaggacg actttttggg aaacttggag cagttgttct acaacaagta cggtgattgt 1020

atcggtccag agacccaatc cttttggttg cgtccacacg ctcaaggatt cagagacttg 1080

ttgaattggt tgtctaagag atacggttac ccaaaaattt acgttactga gaacggtacc 1140

tccttgaagg gtgagaacga catgcctttg gagcaggtct tggaggacga cttcagagtt 1200

aagtacttta acgactacgt tagagctatg gctgccgctg ttgctgagga cggatgcaac 1260

gttcgtggtt atttggcctg gtctttgctt gacaactttg agtgggccga gggatacgag 1320

accagattcg gtgtcaccta cgttgattac gccaacgacc agaagcgtta cccaaagaag 1380

tccgctaagt ccttgaaacc acttttcgac tctttgatta gaaaggagta a 1431

<210> 4

<211> 476

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Met Ser Leu Pro Pro Asp Phe Lys Trp Gly Phe Ala Thr Ala Ala Tyr

1 5 10 15

Gln Ile Glu Gly Ser Val Asn Glu Asp Gly Arg Gly Pro Ser Ile Trp

20 25 30

Asp Thr Phe Cys Ala Ile Pro Gly Lys Ile Ala Asp Gly Ser Ser Gly

35 40 45

Ala Val Ala Cys Asp Ser Tyr Lys Arg Thr Lys Glu Asp Ile Ala Leu

50 55 60

Leu Lys Glu Leu Gly Ala Asn Ser Tyr Arg Phe Ser Ile Ser Trp Ser

65 70 75 80

Arg Ile Ile Pro Leu Gly Gly Arg Asn Asp Pro Ile Asn Gln Lys Gly

85 90 95

Ile Asp His Tyr Val Lys Phe Val Asp Asp Leu Ile Glu Ala Gly Ile

100 105 110

Thr Pro Phe Ile Thr Leu Phe His Trp Asp Leu Pro Asp Ala Leu Asp

115 120 125

Lys Arg Tyr Gly Gly Phe Leu Asn Lys Glu Glu Phe Ala Ala Asp Phe

130 135 140

Glu Asn Tyr Ala Arg Ile Met Phe Lys Ala Ile Pro Lys Cys Lys His

145 150 155 160

Trp Ile Thr Phe Asn Glu Pro Trp Cys Ser Ala Ile Leu Gly Tyr Asn

165 170 175

Thr Gly Tyr Phe Ala Pro Gly His Thr Ser Asp Arg Ser Lys Ser Pro

180 185 190

Val Gly Asp Ser Ala Arg Glu Pro Trp Ile Val Gly His Asn Ile Leu

195 200 205

Ile Ala His Ala Arg Ala Val Lys Ala Tyr Arg Glu Asp Phe Lys Pro

210 215 220

Thr Gln Gly Gly Glu Ile Gly Ile Thr Leu Asn Gly Asp Ala Thr Leu

225 230 235 240

Pro Trp Asp Pro Glu Asp Pro Ala Asp Ile Glu Ala Cys Asp Arg Lys

245 250 255

Ile Glu Phe Ala Ile Ser Trp Phe Ala Asp Pro Ile Tyr Phe Gly Lys

260 265 270

Tyr Pro Asp Ser Met Arg Lys Gln Leu Gly Asp Arg Leu Pro Glu Phe

275 280 285

Thr Pro Glu Glu Val Ala Leu Val Lys Gly Ser Asn Asp Phe Tyr Gly

290 295 300

Met Asn His Tyr Thr Ala Asn Tyr Ile Lys His Lys Thr Gly Val Pro

305 310 315 320

Pro Glu Asp Asp Phe Leu Gly Asn Leu Glu Gln Leu Phe Tyr Asn Lys

325 330 335

Tyr Gly Asp Cys Ile Gly Pro Glu Thr Gln Ser Phe Trp Leu Arg Pro

340 345 350

His Ala Gln Gly Phe Arg Asp Leu Leu Asn Trp Leu Ser Lys Arg Tyr

355 360 365

Gly Tyr Pro Lys Ile Tyr Val Thr Glu Asn Gly Thr Ser Leu Lys Gly

370 375 380

Glu Asn Asp Met Pro Leu Glu Gln Val Leu Glu Asp Asp Phe Arg Val

385 390 395 400

Lys Tyr Phe Asn Asp Tyr Val Arg Ala Met Ala Ala Ala Val Ala Glu

405 410 415

Asp Gly Cys Asn Val Arg Gly Tyr Leu Ala Trp Ser Leu Leu Asp Asn

420 425 430

Phe Glu Trp Ala Glu Gly Tyr Glu Thr Arg Phe Gly Val Thr Tyr Val

435 440 445

Asp Tyr Ala Asn Asp Gln Lys Arg Tyr Pro Lys Lys Ser Ala Lys Ser

450 455 460

Leu Lys Pro Leu Phe Asp Ser Leu Ile Arg Lys Glu

465 470 475

<210> 5

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atgtctttgc ctccagactt c 21

<210> 6

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

cgactctttg attagaaagg agtaa 25

<210> 7

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

cagttgttct acaacaagta cggtgattgt atcggtccag 40

<210> 8

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

ctccaagttt cccaaaaagt cgtcctc 27

Claims

1. The beta-glucosidase mutant is characterized in that the amino acid sequence of the beta-glucosidase mutant is shown as SEQ ID NO: 4, respectively.

2. A gene encoding the β -glucosidase mutant of claim 1.

3. A recombinant vector comprising the gene of claim 2.

4. A recombinant strain comprising the gene of claim 2 or the recombinant vector of claim 3.

5. The recombinant vector of claim 3, wherein the vector is the pPIC9K plasmid.

6. The recombinant strain of claim 4, wherein the strain is Pichia pastoris GS115 or Escherichia coli JM 109.

7. Use of the mutant β -glucosidase of claim 1 for hydrolyzing β -D-glucosidic bonds at the non-reducing end of a glycosidic or oligosaccharaide compound to form β -D-glucose and corresponding ligands.

8. A method for preparing the β -glucosidase mutant of claim 1, comprising the steps of:

(1) the nucleotide sequence is shown as SEQ ID NO: 3 and a linearization vector pPIC9K are subjected to double enzyme digestion by EcoR I and Avr II and then connected to obtain a recombinant vector containing the beta-glucosidase mutant gene;

(2) the recombinant vector is transformed into Pichia pastoris GS115 to obtain a recombinant strain containing a beta-glucosidase mutant gene;

(3) culturing the recombinant strain under proper conditions, inducing expression, collecting expression products, and purifying to obtain the beta-glucosidase mutant.

9. A method of hydrolyzing a glycoside compound, comprising the step of contacting a glycoside compound with the β -glucosidase mutant of claim 1, under conditions effective to hydrolyze a β -D-glucosidic bond of the compound by enzymatic catalysis by the β -glucosidase mutant.