CN114426961A - Beta-glucosidase mutant, encoding gene thereof, expression strain and application thereof - Google Patents
Beta-glucosidase mutant, encoding gene thereof, expression strain and application thereof Download PDFInfo
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- 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
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- 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)
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Abstract
The invention discloses a beta-glucosidase mutant, a coding gene, an expression strain and application thereof. After the engineering bacteria containing the mutant plasmids are induced and expressed, the beta-glucosidase with improved specific activity and stability is obtained. When pNPG and cellobiose are taken as substrates, the specific enzyme activity of the mutant is respectively improved by 1.31.5 times. The kinetic parameter measurement result shows that the mutant has k to substrates pNPG and cellobiosecatThe values were about 3.8-fold and 2.7-fold, respectively, of the starting enzyme Bgl 2A. The stability of the mutant is improved while the specific activity is improved, and the mutant has potential application value in the field of biofuel based on cellulose degradation.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a beta-glucosidase mutant, a coding gene, an expression strain and application thereof.
Background
Beta-glucosidase (EC 3.1.2.21, beta-glucosidase) belongs to a fiber hydrolase, mainly hydrolyzes beta-1, 4-glycosidic bond in glucoside or oligosaccharide, releases glucose and glucoside ligand, has important application value in industries such as biomass conversion, food, medicine and the like, and especially in the field of new energy development mainly based on cellulose degradation. The enzyme is synergistically catalyzed by Endoglucanase (EG) and exoglucanase (CBH), and finally produced glucose can be utilized by microorganisms such as yeast to produce biofuel ethanol. Wherein the beta-glucosidase is a rate-limiting enzyme in the cellulose degradation process, can relieve the inhibition effect of cellobiose on CBH and EG, and the degradation efficiency of cellulose is directly determined by the activity of the beta-glucosidase. Therefore, the beta-glucosidase with high specific activity obtained by adopting a protein engineering technology has potential application value for efficiently degrading cellulose.
Disclosure of Invention
The invention provides a beta-glucosidase mutant, an encoding gene, an expression strain and application thereof. The invention obtains mutant genes by site-directed mutagenesis on the basis of beta-glucosidase from marine uncultured microorganisms. After the engineering bacteria containing the mutant plasmids are induced and expressed, the beta-glucosidase with improved specific activity and stability is obtained. When p-nitrophenyl beta-glucopyranoside (pNPG) and cellobiose are taken as substrates, the specific enzyme activity of the mutant is respectively improved by 1.3 and 1.5 times. The kinetic parameter measurement result shows that the mutant has k to substrates pNPG and cellobiosecatThe values were about 3.8 and 2.7 times larger than Bgl2A, respectively. The stability of the mutant is improved while the specific activity is improved. Under the conditions of 30-35 ℃ and pH7.0-7.5, the stability of the mutant is improved by 1 time compared with Bgl 2A. In the cellobiose hydrolysis experiment, after 3 hours of reaction, the amount of glucose produced by proteolysis of cellobiose per mg of mutant enzyme was about 1.35 times that of Bgl2A and 3.28 times that of the commercial enzyme. The mutant has potential application value in the field of biofuel based on cellulose degradation.
The amino acid sequence of the beta-glucosidase mutant is shown as SEQ ID No:1, wherein the alanine at position 22 is mutated to serine, and the valine at position 224 is mutated to serine.
The amino acid sequence of the beta-glucosidase mutant of the invention can also comprise nonsense mutation or combination of synonymous mutations in the sequence.
The nucleotide sequence of the coding gene of the beta-glucosidase mutant is shown as SEQ ID No:2, respectively.
The mutant plasmid of the invention is a plasmid containing the nucleotide sequence shown as SEQ ID No:2, and the coding gene of the beta-glucosidase mutant.
The strain for expressing the beta-glucosidase mutant contains the mutant plasmid.
The engineering strain expressing the beta-glucosidase mutant is classified and named as Escherichia coli BL21(DE3)/pET22b (+) -bgl2A (A22S/V224S), is delivered to China Center for Type Culture Collection (CCTCC) for preservation, and has the preservation number of CCTCC NO: m20211098, preservation time 2021, 8 months and 30 days, preservation address: china, Wuhan university.
The construction method of the engineering strain for expressing the beta-glucosidase mutant comprises the following steps:
firstly, the structure of beta-glucosidase Bgl2A was modeled by using a beta-glucosidase structure from Bacillus polymyxa (Paenibacillus polymyxa) as a template and using a Swiss-Model. And (3) adopting a semi-rational design strategy, selecting non-completely conserved amino acid residues near a catalytic active center as candidate sites, and determining mutated target amino acids through sequence conservation analysis.
Designing and synthesizing a mutation primer according to a gene sequence of beta-glucosidase Bgl2A, and carrying out site-directed mutagenesis by taking a recombinant plasmid containing the beta-glucosidase Bgl2A gene as a template and the synthesized mutation primer as a primer based on an overlap extension PCR method to obtain the mutant gene of the beta-glucosidase with improved specific activity and stability.
The mutant gene is connected with pEASY-T3 plasmid and transformed into Escherichia coli Trans1-T1 competent cell, positive clone is picked, and DNA sequence determination is carried out on the mutant gene. Selecting a clone with a correct sequence to extract a plasmid, obtaining a pEASY-T3 recombinant plasmid containing a mutant beta-glucosidase gene, carrying out double enzyme digestion on the pEASY-T3 recombinant plasmid and an expression plasmid vector by Nde I and Xho I, and then connecting the mutated gene after enzyme digestion and the expression plasmid vector by using T4 DNA ligase to obtain a ligation product; the ligation product is transformed into host bacteria, and positive clones are screened to obtain the engineering strain containing the mutant gene.
The expression plasmid vector in the above construction method includes pCold, pET15, pET22 or pET28, etc.
The host bacteria in the construction method comprise E.coli BL21(DE3), E.coli DH5 alpha, E.coli JM109 or E.coli Rosetta and the like.
The beta-glucosidase mutant can be obtained by fermenting the engineering strain.
The application of the beta-glucosidase mutant is to apply the beta-glucosidase mutant to the cellulose degradation process, and when pNPG and cellobiose are taken as substrates, the specific enzyme activity of the mutant is respectively improved by 1.3 and 1.5 times under the conditions of 30-35 ℃ and pH7.0-7.5. The kinetic parameter measurement result shows that the mutant has k to substrates pNPG and cellobiosecatThe value is greatly increased compared to Bgl 2A. The stability of the mutant is improved while the specific activity is improved. Under the conditions of 30-35 ℃ and pH7.0-7.5, the stability of the mutant is improved by 1 time compared with that of the starting enzyme Bgl 2A. In the experiment of cellobiose hydrolysis, after 3 hours of reaction, the amount of glucose produced by proteolysis of cellobiose per mg of mutant enzyme was about 1.35 times that of the starting enzyme Bgl2A, and the commercial enzyme3.28 times of the total weight of the powder. The mutant has potential application value in the field of biofuel based on cellulose degradation.
The invention determines and compares the specific enzyme activity, the optimum pH, the optimum temperature, the kinetic parameters, the stability and the like of the mutant protein and the original wild type protein. The determination result shows that when pNPG and cellobiose are used as substrates, the specific enzyme activity of the mutant obtained by the invention is respectively improved by 1.3 times and 1.5 times compared with that of the starting enzyme Bgl 2A. The kinetic parameter measurement result shows that the mutant has k to substrates pNPG and cellobiosecatThe values were 3.8-fold and 2.7-fold respectively for the starting enzyme Bgl 2A. The stability of the mutant is improved while the specific activity is improved. Under the conditions of 30-35 ℃ and pH7.0-7.5, the half-life period of the mutant is prolonged by 1 time compared with that of the starting enzyme Bgl2A, and the mutation does not cause the change of the optimum temperature and the optimum pH.
Drawings
FIGS. 1 and 2 are electropherograms of PCR amplification products of the present invention: in FIG. 1, lanes are DNAmarker, PCR-amplified fragments V224S-S, V224S-X, A22S-S and A22S-X, respectively; in FIG. 2, lanes are DNAmarker, PCR amplification products using V224S-S and V224S-X as templates, and PCR amplification products using A22S-S and A22S-X as templates, respectively.
FIG. 3 is an SDS-PAGE pattern of purified mutein and the starting enzyme Bgl2A: 1 is Bgl2A broken supernatant, 2 is Bgl2A pure enzyme, 3 is Bgl2A: A22S/V224S broken supernatant, 4 is Bgl2A: A22S/V224S pure enzyme, and M is protein marker.
In FIG. 4, a is the optimum temperature measurement result, and b is the optimum pH measurement result.
FIG. 5 shows the stability at 30-35 ℃ and pH 7.0-7.5.
Detailed Description
The following examples are carried out in the conventional manner unless otherwise specified.
(one) construction of expression Strain containing mutant Gene of beta-glucosidase of the present invention
1. Selection of mutation sites of beta-glucosidase gene
Based on the sequence alignment, Bgl2A was most similar to beta-glucosidase BglB (PDB code:2O9R) from Bacillus polymyxa Paenibacillus polymyxa, with 43% amino acid sequence identity. The structure of beta-glucosidase Bgl2A was homologously modeled using Swiss-Model (http:// swissmodel. expasy. org.; Kiefer F, Arnold K, Kunzli M, Bordoli L, Schwede T. The SWISS-MODEL reproducibility and associated resources. nucleic Acids research.2009,37, D387-392.) using The BglB structure as a template.
According to the simulated structure and the multi-sequence alignment analysis, the alanine A at the site 22 and the valine V at the site 224 of site-specific mutation are determined, the alanine A at the site 22 is replaced by the serine S in the mutation direction, and the valine V at the site 224 is replaced by the serine S.
2. Design of mutant primers and PCR amplification of mutant genes
According to the gene sequence of beta-glucosidase Bgl2A: SEQ ID No. 2 (the amino acid sequence is shown as SEQ ID No. 1), and selected mutation sites 22A and 224V, the following 6 site-directed mutagenesis primers were designed (Table 1).
The recombinant plasmid containing beta-glucosidase Bgl2A gene is used as template plasmid, and the synthetic mutation primers are paired, and respectively comprise: the amplification products of the bgl2A-F, A22S-R, A22S-F, bgl2A-R, bgl2A-F, V224S-R, V224S-F and bgl2A-R serving as PCR primer pairs are named A24S-S, A24S-X, V224S-S and V224S-X in sequence; and recovering 4 amplified fragments, adding A22S-S and A22S-X into the same reaction system, adding V224S-S and V224S-X into another reaction system to serve as PCR reaction templates in the respective reaction systems, and amplifying the full-length sequence of the bgl2A mutant gene by using a bgl2A-F, bgl2A-R primer pair as an amplification primer and recovering the full-length sequence for later use.
TABLE 1 primer sequences
3. Construction of expression vectors
Connecting the PCR amplification product obtained in the step 2 with pEASY-T3 plasmid (TaKaRa) to establish the following enzyme digestion system: 10ng of pEASY-T3 vector, 70ng of PCR amplification product, and ligation at 25 ℃ for 15 min. The ligation product is thermally shocked to transform escherichia coli Trans1-T1 competent cells, and sequencing of the obtained transformant verifies whether mutation exists; selecting a clone with a correct sequence to extract a plasmid, and obtaining a pEASY-T3 recombinant plasmid containing the beta-glucosidase mutant gene; the recombinant plasmid pEASY-T3 and the vector pET-22b (+) which are obtained by double enzyme digestion of NdeI and XhoI are used, and then the mutated gene after enzyme digestion is connected with an expression plasmid vector by using T4 DNA ligase, so as to establish an enzyme digestion system as follows: 25ng of pET-22b (+) vector, 50ng of mutant gene enzyme digestion fragment, 2 mu L of 10 × ligation buffer and 1 mu LT4 DNA ligase (TaKaRa), adding water to 20 mu L, and connecting for 1h at 22 ℃ to obtain a connecting product; the obtained transformant is sequenced to verify whether the transformant is mutated or not, and the transformant with the correct sequence is selected to obtain the engineering strain Escherichia coli BL21(DE3)/pET-22b (+) -bgl2A (A22S/V224S) containing the mutant gene.
The strain Escherichia coli BL21(DE3)/pET-22b (+) -bgl2A (A22S/V224S) is sent to the China Center for Type Culture Collection (CCTCC) for preservation. The preservation number is NO: CCTCC M20211098, preservation time is 2021, 8, month and 30 days, preservation unit address: china center for type culture Collection, Wuhan university, China.
(II) expression and protein purification of the gene engineering bacteria containing the beta-glucosidase mutation
The engineered strain Escherichia coli BL21(DE3)/pET22b (+) -bgl2A (A22S/V224S) obtained in the step (A) was inoculated into 400mL of LB liquid medium containing ampicillin, and the medium was cultured at 37 ℃ and 200rpm to OD6000.6(UNICO UV2102 UV visible spectrophotometer with culture LB medium as blank); adding IPTG with final concentration of 0.2mM for induction, and culturing at 16 deg.C and 120rpm for 16 hr; collecting thallus at 4 deg.C and 8000g, adding Bindingbuffer with 3 times of bacterial liquid volume, performing 350W ultrasonic treatment under ice bath condition for 30min to break cells, and collecting supernatant at 12000g to obtain crude enzyme solution. The crude enzyme solution is purified by Ni-NTA column chromatography. The imidazole concentration in the eluent was 60mM, eluting 3 column volumes. The obtained protein is detected to reach the purity of SDS-PAGE.
The pNPG is used as a substrate, the optimum pH value of the mutant is 6.5, and the enzyme can show more than 70% of enzyme activity within the pH value range of 6.0-7.5. The mutant has catalytic activity within the range of 30-55 ℃, and the optimal temperature is 45 ℃.
(III) detection of specific activity of the beta-glucosidase mutant of the invention
1. Enzyme activity determination by taking pNPG as substrate
The reaction system was 500. mu.L of citrate-Na at pH6.52HPO4Adding pNPG with final concentration of 5mM into buffer solution, preheating at 45 deg.C for 5min, adding enzyme solution, reacting for 5min, adding 500 μ L1 MNa2CO3The reaction was terminated. Experiment groups were subjected to 3 parallel experiments, and the control group was prepared by replacing enzyme solution with buffer solution. The absorbance at 405nm was determined by zeroing the control. Enzyme activity (U) is defined as: the amount of enzyme used to produce 1. mu. mol pNP per minute.
2. Enzyme activity determination using cellobiose as substrate
The reaction system was 500. mu.L of citrate-Na at pH6.52HPO4Adding cellobiose with the final concentration of 100mM into the buffer solution, preheating for 5min at 45 ℃, adding the enzyme solution, reacting for 5min, placing the reaction system in a boiling water bath, boiling for 5min to completely inactivate the enzyme, operating according to the operation instruction of the glucose determination kit, paralleling 3 experimental groups, and replacing the glucose solution with deionized water in a control group. The absorbance at 505nm was determined by zeroing the control. Enzyme activity (U) is defined as: the amount of enzyme used to produce 1. mu. mol glucose per minute.
The determination result shows that when pNPG and cellobiose are used as substrates, the specific enzyme activity of the mutant obtained by the invention is respectively improved by 1.3 times and 1.5 times compared with that of the original enzyme Bgl 2A.
(IV) determination of kinetic parameters of the beta-glucosidase of the invention
The kinetic parameters of β -glucosidase for the artificial substrate pNPG and the natural substrate cellobiose are shown in table 2. The assay buffer was 50mM, pH6.5 citrate-Na2HPO4A buffer solution in which the pNPG concentration is in the range of 0 to 20mM and the cellobiose concentration is in the range of 0 to 200mM, as a specific activation method, in accordance with embodiment (III) of the present invention. The Michaelis-Menten equation isOn the basis, the abscissa is the substrate concentration and the ordinate is the reaction speed, and the software Origin 8.5 is used for carrying out nonlinear fitting to obtain Km、Vmax、kcat。
The results showed that the affinity of the mutant for the substrates pNPG and cellobiose was significantly reduced, but k was lower than that of the mutantcatThe values show different increases, where k is the substrate pNPGcatA value of about 3.8 times that of the starting enzyme Bgl2A, k for cellobiosecatThe value is 2.7 times of that of the starting enzyme Bgl2A
TABLE 2 kinetic parameters of beta-glucosidase
(V) detection of the stability of the beta-glucosidase of the invention
Carrying out heat treatment on Bgl2A and the mutant at the temperature of 30-35 ℃ and the pH value of 7.0-7.5, sampling every half hour or one hour, calculating the enzyme activity residual rate after heat treatment for a certain time by taking the initial enzyme activity as 100%, wherein the formula is as follows: the residual enzyme activity is equal to (enzyme activity before heat treatment-lost enzyme activity)/enzyme activity after heat treatment is multiplied by 100%.
The determination result shows that the half-life period of the mutant under the condition is 3h, and is improved by 1 time compared with the wild type.
(VI) application of beta-glucosidase mutant in cellobiose hydrolysis
The reaction system included cellobiose at a final concentration of 200g/L, 50mM, pH6.5 citrate-Na2HPO4The buffer and 100U beta-glucosidase mutant were subjected to shaking reaction in a water bath at 35 ℃ and 200rpm, and periodically sampled, and the resulting glucose was detected with a glucose assay kit, and Bgl2A and commercial beta-glucosidase (purchased from Sigma, G0395) were added in the same amount as the enzyme to prepare a control group.
After 3h of reaction, the amount of glucose produced per mg of mutant proteolytically hydrolyzed cellobiose was about 1.35 times that of Bgl2A and 3.28 times that of the commercial enzyme.
SEQ ID No:1
MTKISLPTCSPLLTKEFIYGVSTSSFQIEGGSAHRLPCIWDTFCDTPGKIADNSNGHVACDHY NNWKQDIDLIESLGVDAYRLSISWPRVITKSGELNPEGVKFYTDILDELKKRNIKAFVTLYH WDLPQHLEDEGGWLNRETAYAFAHYVDLITLAFGDRVHSYATLNEPFCSAFLGYEIGIHAPG KVGKQYGRKAAHHLLLAHGLAMTVLKQNSPTTLNGISLNFTPCYSISEDADDIAATAFADD YLNQWYMKPIMDGTYPAIIEQLPSAHLPDIHDGDMAIISQSIDYLGINYYTRQFYKAHPTEIY EPIEPTGPLTDMGWEIYPKSFTELLVTLNNTYTLPPIFITENGAAMPDSYNNGEINDVDRLDY YNSHLNAVHNATEQGVRIDGYFAWSLMDNFEWAEGYLKRFGIVYVDYSTQQRTIKNSGLA YKALISNR
SEQ ID No:2
ATGACTAAAATATCTTTACCAACTTGTTCACCTCTATTAACAAAAGAGTTTATTTATGGTG TAAGCACAtcaTCTTTCCAAATAGAAGGTGGCTCAGCTCACCGTCTGCCGTGTATCTGGGA TACCTTTTGTGATACTCCAGGTAAAATAGCTGATAACTCAAATGGGCATGTTGCATGCGAT CATTACAATAATTGGAAACAAGACATAGATTTAATCGAATCATTAGGAGTAGATGCTTAC AGACTTTCTATTTCTTGGCCTCGTGTTATTACAAAAAGTGGTGAGCTTAACCCTGAAGGC GTAAAGTTTTACACCGACATCTTAGATGAACTGAAAAAGCGCAATATTAAAGCGTTTGTC ACGTTATACCACTGGGATTTACCTCAACACCTAGAAGACGAAGGGGGTTGGTTAAATCG AGAAACAGCTTACGCGTTTGCTCACTATGTTGATTTAATTACCTTGGCATTCGGTGACCG GGTGCATTCATACGCTACCTTAAACGAACCCTTTTGCAGTGCTTTTTTAGGTTACGAAATT GGCATTCATGCGCCAGGGAAAGTCGGCAAACAATACGGGCGCAAAGCCGCCCACCATT TGTTATTAGCACATGGCCTTGCCATGACCGTATTAAAGCAAAACTCACCGACGACTTTAA ACGGTATCTCCCTTAACTTTACTCCCTGTTATAGCATCTCTGAAGACGCTGATGACATTGC TGCAACAGCGTTTGCAGATGACTACTTAAACCAGTGGTACATGAAACCCATCATGGATG GTACATACCCAGCAATTATTGAACAATTACCTTCAGCACATCTGCCAGATATTCACGATGG TGACATGGCCATTATTTCACAATCAATTGATTATTTAGGTATTAACTaTTATACCCGTCAATT TTATAAAGCGCACCCTACTGAAATATATGAGCCAATAGAGCCTACTGGCCCGCTAACCGA TATGGGCTGGGAAATTTACCCTAAGTCGTTTACAGAGTTATTAGTCACACTTAACAATAC CTATACCCTACCGCCTATTTTTATTACTGAAAATGGCGCAGCTATGCCCGACAGCTATAAT AATGGTGAAATCAATGATGTAGATCGACTAGACTACTACAACAGTCACCTAAATGCCGTT CACAATGCAACAGAGCAAGGCGTTAGAATAGACGGCTATTTTGCCTGGAGCCTAATGGA TAACTTTGAATGGGCAGAAGGTTACTTAAAAAGATTTGGTATAGTTTATGTAGATTACAG CACACAGCAACGTACTATAAAAAATAGTGGCCTAGCCTATAAAGCATTAATCTCAAATAG ATAA
Sequence listing
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<120> Title, beta-glucosidase mutant, coding gene, expression strain and application thereof
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gtaagcacat catctttcca aatagaaggt ggctcagctc accgtctgcc gtgtatctgg 180
gatacctttt gtgatactcc aggtaaaata gctgataact caaatgggca tgttgcatgc 240
gatcattaca ataattggaa acaagacata gatttaatcg aatcattagg agtagatgct 300
tacagacttt ctatttcttg gcctcgtgtt attacaaaaa gtggtgagct taaccctgaa 360
ggcgtaaagt tttacaccga catcttagat gaactgaaaa agcgcaatat taaagcgttt 420
gtcacgttat accactggga tttacctcaa cacctagaag acgaaggggg ttggttaaat 480
cgagaaacag cttacgcgtt tgctcactat gttgatttaa ttaccttggc attcggtgac 540
cgggtgcatt catacgctac cttaaacgaa cccttttgca gtgctttttt aggttacgaa 600
attggcattc atgcgccagg gaaagtcggc aaacaatacg ggcgcaaagc cgcccaccat 660
ttgttattag cacatggcct tgccatgacc gtattaaagc aaaactcacc gacgacttta 720
aacggtatct cccttaactt tactccctgt tatagcatct ctgaagacgc tgatgacatt 780
gctgcaacag cgtttgcaga tgactactta aaccagtggt acatgaaacc catcatggat 840
ggtacatacc cagcaattat tgaacaatta ccttcagcac atctgccaga tattcacgat 900
ggtgacatgg ccattatttc acaatcaatt gattatttag gtattaacta ttatacccgt 960
caattttata aagcgcaccc tactgaaata tatgagccaa tagagcctac tggcccgcta 1020
accgatatgg gctgggaaat ttaccctaag tcgtttacag agttattagt cacacttaac 1080
aatacctata ccctaccgcc tatttttatt actgaaaatg gcgcagctat gcccgacagc 1140
tataataatg gtgaaatcaa tgatgtagat cgactagact actacaacag tcacctaaat 1200
gccgttcaca atgcaacaga gcaaggcgtt agaatagacg gctattttgc ctggagccta 1260
atggataact ttgaatgggc agaaggttac ttaaaaagat ttggtatagt ttatgtagat 1320
tacagcacac agcaacgtac tataaaaaat agtggcctag cctataaagc attaatctca 1380
aatagataa 1344
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Claims (5)
1. A β -glucosidase mutant, characterized in that:
the amino acid sequence of the beta-glucosidase mutant is shown as SEQ ID No:1, wherein the alanine at position 22 is mutated to serine, and the valine at position 224 is mutated to serine.
2. A gene encoding the β -glucosidase mutant as claimed in claim 1, characterized in that:
the nucleotide sequence of the coding gene is shown as SEQ ID No:2, respectively.
3. An engineered strain expressing the β -glucosidase mutant of claim 1, characterized in that:
the engineering strain is classified and named as Escherichia coli BL21(DE3)/pET22b (+) -bgl2A (A22S/V224S), and is delivered to China Center for Type Culture Collection (CCTCC) for preservation, and the preservation number is CCTCC NO: m20211098, preservation time 2021, 8 months and 30 days, preservation address: china, Wuhan university.
4. Use of a β -glucosidase mutant as claimed in claim 1, wherein:
applying the beta-glucosidase mutant to a cellulose degradation process.
5. Use according to claim 4, characterized in that:
respectively taking pNPG and cellobiose as substrates, the specific enzyme activity of the mutant is improved by 1.3 and 1.5 times compared with that of the starting enzyme; the stability of the enzyme is improved by 1 time compared with the original enzyme under the conditions of 30-45 ℃ and pH 6.0-7.5.
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| CN115896071A (en) * | 2022-09-07 | 2023-04-04 | 中国科学院青岛生物能源与过程研究所 | Beta-glucosidase mutant with high enzyme activity and high glucose tolerance and application thereof |
| CN117402858A (en) * | 2023-12-13 | 2024-01-16 | 北京科为博生物科技有限公司 | Beta-glucosidase mutant with improved heat resistance |
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| CN117402858B (en) * | 2023-12-13 | 2024-03-15 | 北京科为博生物科技有限公司 | Beta-glucosidase mutant with improved heat resistance |
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