CN104450651A - Beta-glucosidase mutant and application thereof - Google Patents
Beta-glucosidase mutant and application thereof Download PDFInfo
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- CN104450651A CN104450651A CN201410753327.6A CN201410753327A CN104450651A CN 104450651 A CN104450651 A CN 104450651A CN 201410753327 A CN201410753327 A CN 201410753327A CN 104450651 A CN104450651 A CN 104450651A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2445—Beta-glucosidase (3.2.1.21)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01021—Beta-glucosidase (3.2.1.21)
Abstract
The invention aims at providing a beta-glucosidase mutant and application thereof. The amino acid sequence of the beta-glucosidase mutant is as shown in SEQ ID NO: 3, a nucleotide sequence of a coding gene is as shown in SEQ ID NO: 4, the optimum operative temperature is 50 DEG C and enzyme activity can be kept above 80% within 30-60 DEG C, and the mutant is stronger than a wild type in heat resistance; the optimum operative pH is 5.0 and the enzyme activity can be kept above 70% within 3.0-6.0, and the mutant is wider than the wild type in application scope under an acid condition; therefore, the beta-glucosidase mutant is conducive to the application in a cellulose raw material degrading process.
Description
Technical field
The invention belongs to genescreen renovation technique field, be specifically related to a kind of beta-glucoside enzyme mutant and application thereof.
Background technology
Beta-glucosidase, also known as β-D-Glucose glycosides glucose hydrolysis enzyme, another name gentiobiase, cellobiase (cellobias, CB or β-G) and amygdalase.It belongs to Mierocrystalline cellulose enzyme, is the important composition composition in cellulolytic enzyme system, can be hydrolyzed the β-D-Glucose key being incorporated into end irreducibility, discharge β-D-Glucose and corresponding aglucon simultaneously.
Mierocrystalline cellulose to be formed by connecting D-glucopyranose polymkeric substance by β-Isosorbide-5-Nitrae-glycosidic link, is the abundantest on the earth and reproducible resource.According to estimates, on the earth, annual photosynthesis produces 1.5 × 1,011 hundred million tons of Mierocrystalline celluloses (Ryu, D.D etc., 1980, Enzyme and Microbial Technology).In most cases, higher value application Mierocrystalline cellulose first effectively must to be degraded to it by cellulase.The biology of occurring in nature cellulase-producing is a lot, and mainly microorganism, as filamentous fungus, bacterium and actinomycetes etc.Therefore, general concern is just received to the screening of cellulase-producing microorganism or genetic modification Study and appliance.
It is generally acknowledged, cellulose substances generates glucose through enzymatic action at least needs three kinds of enzymes: endoglucanase, exocellobiohydrolase and beta-glucosidase.Endoglucanase and exocellobiohydrolase are first degraded into cellobiose Mierocrystalline cellulose, and it is resolved into glucose by beta-glucosidase again.Cellulose conversion, mainly by this three kinds of enzymes synergy, is finally glucose by cellulase.Beta-glucosidase plays a key effect in this process.And beta-glucosidase content is few in cellulose components, vigor is low, become the bottleneck of cellulase hydrolysis.Therefore, build engineering bacteria by gene recombination technology, secreting, expressing high reactivity beta-glucosidase is effectively degraded significant to Mierocrystalline cellulose.For this reason, a variety of beta-glucosidase gene has been had to be built in different engineering bacterias.And develop the more excellent beta-glucosidase of performance, and to improve its expression amount be still one of study hotspot of this area.
Summary of the invention
The object of this invention is to provide a kind of beta-glucoside enzyme mutant and application thereof.The present invention, by the screening of mass mutation, finally obtaining the mutant that a kind of thermotolerance is stronger, the scope of application is more wide in range under sour environment, and builds the Trichodermareesei engineering strain obtaining this mutant recombinant expressed, laying a good foundation for realizing its application.
One aspect of the present invention relates to a kind of beta-glucosidase, 93rd amino acids of to be aminoacid sequence the be beta-glucosidase of SEQ ID NO:1 becomes Ile from Leu, 133rd amino acids becomes Phe from Tyr, 222nd amino acids becomes Gly from Asn, 579th amino acids becomes Leu from Ile, and the 676th amino acids becomes Phe from Gln.
The aminoacid sequence of above-mentioned beta-glucoside enzyme mutant is SEQ ID NO:3, and the nucleotide sequence of its encoding gene is SEQ ID NO:4.
The present invention relates on the other hand and carries the recombinant plasmid that encoding sequence is the beta-glucoside enzyme mutant gene of SEQ ID NO:4.
The invention still further relates to a kind of engineering strain, it carries above-mentioned recombinant plasmid.
Described engineering strain is Trichodermareesei (Trichoderma reesei).
The invention still further relates to a kind of cellulase, being fermented by above-mentioned Trichodermareesei prepares.
The present invention obtains a kind of novel beta-glucoside enzyme mutant through a large amount of screening, and the optimum temperature of this mutant is 50 DEG C, and the enzyme of more than 80% can be kept within the scope of 30 DEG C-60 DEG C to live, and thermotolerance is stronger than wild-type; The suitableeest action pH is 5.0, and the enzyme of more than 70% can be kept within the scope of pH3.0-6.0 to live, and the scope of application is in acid condition more wide in range than wild-type, therefore advantageously in its application in cellulosic material degradation process.
Accompanying drawing explanation
Fig. 1: SDS-PAGE electrophoresis detection figure,
Wherein swimming lane 1 is T. reesei host fermented supernatant fluid, and swimming lane 2 is Trichodermareesei TR-BG fermented supernatant fluid, and swimming lane 3 is Trichodermareesei TR-BG5 fermented supernatant fluid, and arrow indication place is 95KDa;
Fig. 2: beta-glucosidase wild-type enzyme work-operative temperature relative to mutant graphic representation;
Fig. 3: beta-glucosidase wild-type enzyme work-action pH relative to mutant graphic representation.
Embodiment
Below in conjunction with example, method of the present invention is described further.The experimental technique of unreceipted actual conditions in an embodiment, usually can condition routinely, the condition as described in " Molecular Cloning: A Laboratory guide " that J. Pehanorm Brooker (Sambrook) etc. is write, or runs according to the condition that manufacturer advises.Those skill in the art related can understand better by embodiment and grasp the present invention.
The clone of embodiment 1 beta-glucosidase gene and the structure of mutant thereof
1.1 extract Aspergillus fumigatus total genomic dna
By Aspergillus fumigatus (Aspergillus fumigatus) incubated overnight, get appropriate thalline and be placed in centrifuge tube, the centrifugal 5min of 13000rpm, abandons supernatant; Add 400 μ l extraction buffers (100mM Tris-HCl, 100mMEDTA, 250mM NaCl, 1%SDS); Then add 100mg quartz sand or granulated glass sphere, beat instrument thermal agitation about 2min on pearl; After 65 DEG C of water-bath 20min, add 200 μ l10M NH4AC, ice bath 10min; The centrifugal 10min of 13000rpm, gets supernatant; Add the dehydrated alcohol of 2 times of volumes, place 30min for-20 DEG C; The centrifugal 10min of 13000rpm, abandons supernatant; By 70% washing with alcohol 2 times; Dry, add water dissolution, in-20 DEG C of preservations.
1.2 gene clone
With the genome DNA extracted in 1.1 for template, primer BG-F and BG-R is utilized to carry out pcr amplification respectively:
BG-F：AAAGGTACCATGAGATTCGGTTGGCTCGAGG；
BG-R：AAATCTAGACTAGTAGACACGGGGCAGAGG；
Pcr amplification condition is 95 DEG C of 4min; 94 DEG C of 30S; 58 DEG C of 30S, 72 DEG C 3min30 circulation; 72 DEG C, 7min; Gel reclaims test kit and reclaims pcr amplification product.
1.3 expression vector establishment
Pcr amplification product restriction enzyme XbaI and KpnI in 1.2 is carried out enzyme cut; Meanwhile, carry out enzyme with restriction enzyme XbaI and KpnI to Trichoderma expression vector pTG to cut; Use gel purification kit by digestion products purifying, and with T4DNA ligase enzyme, above-mentioned two digestion products are connected; Connection product conversion is entered DH5a intestinal bacteria, selects with penbritin.Known by carrying out order-checking to obtained clone, the gene order of above-mentioned pcr amplification product is SEQ ID NO:2 (called after TR-BG), and the aminoacid sequence of its coding is SEQ ID NO:1.Find through NCBI BLAST sequence alignment, SEQ ID NO:1 is 100% with the amino acid sequence similarity of the beta-glucosidase deriving from Aspergillus fumigatus.
Amount in plasmid is used to prepare test kit purification of Recombinant plasmid from the correct escherichia coli cloning of sequencing result, this plasmid called after pTG-TR-BG.
The acquisition of 1.4 mutant
Applicant is in order to improve the thermotolerance of above-mentioned beta-glucosidase (aminoacid sequence is SEQ ID NO:1) further, to build the recombinant plasmid pTG-TR-BG that obtains in 1.3 for template, design primer respectively, carry out multiple spot rite-directed mutagenesis to goal gene, found that some sudden change does not affect its thermotolerance, some sudden change even makes thermotolerance poorer, also some sudden change in addition, although can improve thermotolerance, after sudden change, its pH scope of application narrows, all undesirable.Finally, applicant obtains and can significantly improve thermotolerance, can expand again the combination in the mutational site of the scope of application under acidic conditions: L93I, Y133F, N222G, I579L, Q676F five point mutation.
By above-mentioned five Point mutont called after TR-BG5, its aminoacid sequence is SEQ ID NO:3, with reference to this sequent synthesis coding nucleotide sequence SEQ ID NO:4.This sequence is optimum synthesis according to the codon bias of Trichodermareesei, and adds KpnI and XbaI two restriction enzyme sites respectively at composition sequence 5 ' and 3 ' two ends, and by Shanghai, Sheng Gong biotechnology limited-liability company completes.
Operation described in 1.3 is adopted to build the recombinant plasmid containing above-mentioned five Point mutont genes, called after pTG-TR-BG5.
Embodiment 2 transforms and screening
Draw Li's Trichoderma strains spore suspension in PDA plate center (9cm culture dish), treat that bacterium colony covers with whole culture dish, respectively cut the substratum of 1cm × 1cm size, be placed in 120mL YEG+U liquid nutrient medium, 30 DEG C, 200rpm, cultivate 14 ~ 16h.
Collect mycelium with aseptic Miracloth filter cloth, and clean once by solution A, aseptically cleaned mycelium is transferred to 40mL Protoplasting solution, 30 DEG C, 90rpm, cultivate 1-2h, detect protoplast transformation progress with microscopic examination.
With aseptic Miracloth filter-cloth filtering above-mentioned temperature body lotion body, gained filtrate is protoplast solution.Protoplast solution is sub-packed in the aseptic disposable centrifuge tube of two 50mL, and the volume solution B of every pipe is settled to 45mL, 3000rpm, centrifugal 10min, abandoning supernatant, obtain precipitation; By 5mL solution B washing and precipitating twice again; By pellet resuspended in 10mL solution B, and with blood counting chamber, protoplastis is counted.By protoplastis recentrifuge and abandoning supernatant, according to blood counting chamber count results, add the resuspended precipitation of appropriate solution B, make protoplastis number 1 × 10
8individual/about mL.
On ice, add in the aseptic 7mL centrifuge tube of precooling by the above-mentioned protoplast solution of 200 μ L, each conversion reaction 1 pipe, adds 10 μ g recombinant plasmid pTG-TR-BG5, adds 50 μ L solution C, places 20min on ice again after gentle mixing.
Conversion upper strata substratum is melted and remains on 55 DEG C; From ice, shift out above-mentioned 7mL centrifuge tube, and Xiang Guanzhong adds 2mL solution C and 4mL solution B, each pipe of gentle mixing, gained mixture is protoplast mixture; In each in 3 top-agar test tubes, add the above-mentioned protoplast mixture of 1mL, and topple over immediately and transform on lower floor flat board, and flat board is cultivated at 30 DEG C 5-7d and grow to there being transformant.
One of them transformant incubated overnight of picking, gets appropriate thalline and is placed in centrifuge tube, and the centrifugal 5min of 13000rpm, abandons supernatant; Add 400 μ l extraction buffers (100mM Tris-HC, 100mM EDTA, 250mMNaCl, 1%SDS); Then add 100mg quartz sand or granulated glass sphere, beat instrument thermal agitation about 2min on pearl; After 65 DEG C of water-bath 20min, add 200 μ l10M NH
4aC, ice bath 10min; The centrifugal 10min of 13000rpm, gets supernatant; Add the dehydrated alcohol of 2 times of volumes, place 30min for-20 DEG C; The centrifugal 10min of 13000rpm, abandons supernatant; By 70% washing with alcohol 2 times; Dry, add water dissolution, in-20 DEG C of preservations.
Extract the genomic dna of above-mentioned transformant, and as template, utilize upstream and downstream primer to carry out pcr amplification, goal gene is verified.
Upstream primer sequence is: GGGGTACCATGGCTCTCTCCAAGCT;
Downstream primer sequence is: GCTCTAGATTACAAGCACTGCGAATAC;
Pcr amplification condition is 95 DEG C of 4min; 94 DEG C of 40S; 58 DEG C of 40S, 72 DEG C of 3min, 30 circulations; 72 DEG C of 7min.Utilize gel to reclaim test kit and reclaim pcr amplification product, and carry out sequencing analysis, the gene order of result display pcr amplification product is SEQ ID NO:4, thus illustrate that the present invention builds the Trichodermareesei engineering bacteria obtained containing beta-glucoside enzyme mutant TR-BG5 gene, by its called after Trichodermareesei TR-BG5 (Trichoderma reesei TR-BG5).
Above-mentioned same method is adopted also to be transformed in Trichodermareesei by the gene of wild-type beta-glucosidase TR-BG, build the Trichodermareesei engineering bacteria obtaining recombinant expressed wild-type beta-glucosidase TR-BG, by its called after Trichodermareesei TR-BG (Trichoderma reesei TR-BG).
Solution A: 2.5mL1M K
2hPO
4, 2.5mL1M KH
2pO
4, 48.156g MgSO
4, add dlH
2o is to final volume 500mL, degerming with the filtering with microporous membrane of 0.22 μm.
Solution B: 5mL1M Tris (pH7.5), 2.77g CaCl
2, 109.32g sorbyl alcohol, adds dlH
2o is to final volume 500mL, degerming with the filtering with microporous membrane of 0.22 μm.
Solution C: 250g PEG4000,2.77g CaCl
2, 5mL1M Tris (pH7.5), adds dlH
2o is to final volume 500mL, degerming with the filtering with microporous membrane of 0.22 μm.
Protoplasting solution: 0.4mg lyase (Lysing Enzyme from Trichodermaharzianum, Sigma) is dissolved in 40mL solution A, degerming with the filtering with microporous membrane of 0.22 μm.
Transform lower floor dull and stereotyped: 0.1%MgSO
4, 1%KH
2pO
4, 0.6% (NH
4)
2sO
4, 1% glucose, 0.35% agar powder.
Transform upper strata substratum: 0.1%MgSO
4, 1%KH
2pO
4, 0.6% (NH
4)
2sO
4, 1% glucose, 18.3% sorbyl alcohol, 0.35% agarose.
Trace element: at 250mL dlH
21g FeSO is added in O
47H
2o, 8.8g ZnSO
47H
2o, 0.4gCuSO
45H
2o, 0.15g MnSO
44H
2o, 0.1g Na
2b
4o
710H
2o, 50mg (NH
4)
6mo
7o
244H
2the dense HCl of O, 0.2mL, uses dlH after dissolving completely
2o is settled to 1L, degerming with the filtering with microporous membrane of 0.22 μm.
PDA is dull and stereotyped: 20g glucose, 200g potato cooking liquor, 15g agar, adds dlH
2o to final volume 1000mL, autoclaving.
YEG+U liquid nutrient medium: 20g glucose, 5g yeast extract, 1g uridine, adds dlH
2o to final volume 1000mL, autoclaving.
Embodiment 3 is fermented and is verified
The T. reesei host, the Trichodermareesei TR-BG5 and Trichodermareesei TR-BG that do not import foreign gene are inoculated in MM fermention medium (1.5% glucose, 1.7% lactose, 2.5% corn steep liquor, 0.44% (NH respectively
4)
2sO
4, 0.09%MgSO
4, 2%KH
2pO
4, 0.04%CaCl
2, 0.018% tween-80,0.018% trace element) cultivate, cultivate 48 hours for 30 DEG C, then cultivate 48 hours for 25 DEG C.Respectively fermented liquid is carried out centrifugal, get supernatant liquor and carry out SDS-PAGE electrophoresis detection.Result as shown in Figure 1, the theoretical molecular size deriving from the beta-glucosidase of Aspergillus fumigatus of the present invention is about 95KDa, the i.e. position of arrow indication in Fig. 1, swimming lane 1 is Host Strains fermented supernatant fluid, it does not almost have protein band at arrow pointed location, and the Trichodermareesei TR-BG5 fermented supernatant fluid of the Trichodermareesei TR-BG of swimming lane 2 and swimming lane 3 respectively has a protein band clearly at arrow indication place, thus the Trichodermareesei engineering bacteria TR-BG energy effective expression wild-type beta-glucosidase TR-BG that the present invention builds is described, Trichodermareesei engineering bacteria TR-BG5 can effective expression beta-glucoside enzyme mutant TR-BG5.
Embodiment 4 beta-glucosidase enzyme activity determination
1, enzyme activity determination method
1) unit of enzyme activity's definition
50 DEG C, under pH value is the condition of 4.8 (neutrality is 6.0), the per minute enzyme amount required for reducing sugar that release is equivalent to 1 micromoles glucose of degrading from the saligenin solution that concentration is 0.5% is an enzyme activity unit U.
2) measuring principle
Beta-glucosidase can generate glucose by the glucoside bond in hydrolysis fiber disaccharides.Glucose under boiling water bath condition can with 3,5-dinitrosalicylic acid (DNS) reagent generation color reaction.The glucose amount that the degree of depth and the enzymolysis of reaction solution color produce is directly proportional, and the growing amount of glucose is directly proportional to the vigor of beta-glucosidase in reaction solution.Therefore, by the intensity of spectral colorimetric assaying reaction liquid color, the vigor of beta-glucosidase in reaction solution can be calculated.
3) determination step
Get four 25mL colorimetric cylinders (blank tube, three sample hoses).Respectively in four arms, accurately add saligenin solution (5.3) 2.00mL by corresponding pH buffer preparation.Accurately add the enzyme liquid 0.50mL to be measured (blank tube does not add) in three sample hoses diluted respectively, with eddy blending machine mixing, capping plug.Four test tubes are placed in 50 ± 0.1 DEG C of water-baths simultaneously, accurate timing, reaction 15min.In each pipe, add DNS reagent 3.OmL rapidly, exactly, in blank tube, accurately add the enzyme liquid 0.50mL to be measured diluted, shake up.Four arms are put into boiling water bath simultaneously, accurate timing, heating 10min, takes out, is cooled to room temperature rapidly, is settled to 25mL with water.With blank tube zeroing, under spectrophotometer wavelength 540nm, use 10mm cuvette, measure respectively.In three sample hoses, the absorbancy of sample liquid, averages.By looking into typical curve or obtaining the content of reducing sugar with equation of linear regression.
4) enzyme work is calculated
Enzyme activity calculation formula: X1=A × (10
6/ 180.2/10
3) × 1/0.5 × n/15=0.74 × A × n
In formula:
X l-β glucuroide enzyme activity, u/g (or u/mL);
The reducing sugar amount that A-absorbancy checks on typical curve (or calculating), mg;
10
6/ 180.2/10
3-glucose content is converted into micromole;
1/0.5-be converted into enzyme liquid 1ml;
The extension rate of n-enzyme sample;
15-time scale factor.
2, enzyme activity determination result
Mensuration T. reesei host fermented supernatant fluid enzyme is lived and is only 5.5U/mL according to the method described above, the enzyme of the fermented supernatant fluid of Trichodermareesei TR-BG is lived as 242U/mL, and the enzyme of Trichodermareesei TR-BG5 fermented supernatant fluid is lived as 291U/mL, thus proving that further Host Strains itself produces the amount of beta-glucosidase seldom, recombinant bacterium Trichodermareesei TR-BG and TR-BG5 then can the beta-glucosidase of high expression external source.
Embodiment 5 characterization analysis
1, optimum temperature
Respectively at 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C, 70 DEG C, the fermented supernatant fluid enzyme measuring above-mentioned Trichodermareesei TR-BG and TR-BG5 under pH6.0 condition is lived, live as 100% with the highest enzyme, calculate relative enzyme to live, do temperature-enzyme curve alive relatively.Result as shown in Figure 2, the optimum temperature of wild-type beta-glucosidase TR-BG is 40 DEG C, and the optimum temperature of beta-glucoside enzyme mutant TR-BG5 is 50 DEG C, and the enzyme of more than 80% can be kept within the scope of 30 DEG C-60 DEG C to live, thus after sudden change is described, the thermotolerance of beta-glucosidase is significantly improved.
2, Optimun pH
The fermented supernatant fluid of above-mentioned Trichodermareesei TR-BG and TR-BG5 is diluted respectively with the damping fluid that pH value is 3.0,3.5,4.0,4.5,5.0,5.5,6.0,6.5,7.0,7.5,8.0, under 50 DEG C of conditions, measure its enzyme live, live as 100% with the highest enzyme, calculate relative enzyme to live, do the relative enzyme of pH-curve alive.Result as shown in Figure 3, the suitableeest action pH of wild-type beta-glucosidase TR-BG is 6.0, and the suitableeest action pH of beta-glucoside enzyme mutant TR-BG5 is 5.0, and the enzyme of more than 70% can be kept within the scope of pH3.0-6.0 to live, thus beta-glucosidase adaptive faculty in acid condition been significantly enhanced after sudden change is described, the scope of application is more wide in range.
To sum up, the expression amount of beta-glucoside enzyme mutant in Trichodermareesei that the present invention obtains is significantly higher than wild-type, and the thermotolerance of mutant is stronger, the scope of application is in acid condition more wide in range, advantageously in its application in cellulose materials enzymolysis process.
Claims (8)
1. a beta-glucosidase, it is characterized in that, 93rd amino acids of described beta-glucosidase to be aminoacid sequence the be beta-glucosidase of SEQ ID NO:1 becomes Ile from Leu, 133rd amino acids becomes Phe from Tyr, 222nd amino acids becomes Gly from Asn, 579th amino acids becomes Leu from Ile, and the 676th amino acids becomes Phe from Gln.
2. beta-glucosidase as claimed in claim 1, it is characterized in that, the aminoacid sequence of described beta-glucosidase is SEQ ID NO:3.
3. a gene, is characterized in that, described genes encoding beta-glucosidase according to claim 1.
4. gene as claimed in claim 3, it is characterized in that, the nucleotide sequence of described gene is SEQ IDNO:4.
5. a recombinant plasmid, is characterized in that, described recombinant plasmid carries gene according to claim 3.
6. an engineering strain, is characterized in that, described engineering strain carries recombinant plasmid according to claim 4.
7. engineering strain as claimed in claim 6, it is characterized in that, described engineering strain is Trichodermareesei (Trichoderma reesei).
8. prepare a method for beta-glucosidase according to claim 1, it is characterized in that, described method is for beta-glucosidase by engineering strain fermentation according to claim 6.
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| CN105567663A (en) * | 2016-01-28 | 2016-05-11 | 中国农业科学院饲料研究所 | Beta-glucosidase mutant F67Y with improved catalyst efficiency and encoding gene and application thereof |
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