CN114426961B - Beta-glucosidase mutant, encoding gene, expression strain and application thereof - Google Patents
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- 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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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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- 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)
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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 plasmid are induced to express, the beta-glucosidase with improved specific enzyme activity and stability is obtained. When pNPG and cellobiose are used as substrates, the specific enzyme activities of the mutants are respectively improved by 1.3 times and 1.5 times. The dynamic parameter measurement result shows that the mutant has k to the substrate pNPG and cellobiose cat The values are about 3.8 times and 2.7 times, respectively, that 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 the class of fibrohydrolase, mainly hydrolyzes beta-1, 4-glycosidic bonds in glycoside or oligosaccharide, simultaneously releases glucose and aglycone, has important application value in biomass conversion, food, medicine and other industries, and particularly in the field of new energy development mainly comprising cellulose degradation. The enzyme is cooperated with Endoglucanase (EG) and exoglucanase (CBH), and finally the produced glucose can be utilized by microorganisms such as yeast and the like to generate biofuel ethanol. The beta-glucosidase is a speed limiting enzyme in the cellulose degradation process, can relieve the inhibition of cellobiose on CBH and EG, and the activity of the beta-glucosidase directly determines the degradation efficiency of cellulose. Therefore, the beta-glucosidase with high specific activity obtained by adopting the protein engineering technology has potential application value for the efficient degradation of cellulose.
Disclosure of Invention
The invention provides a beta-glucosidase mutant, a coding gene, an expression strain and application thereof. The invention is based on beta-glucosidase from marine uncultured microorganism source, and the mutant gene is obtained by site-directed mutagenesis. After the engineering bacteria containing the mutant plasmid are induced to express, the beta-glucosidase with improved specific enzyme activity and stability is obtained. When p-nitrophenyl beta-glucopyranoside (pNPG) and cellobiose are used as substrates, the specific enzyme activities of the mutants are respectively improved by 1.3 times and 1.5 times. The dynamic parameter measurement result shows that the mutant has k to the substrate pNPG and cellobiose cat The values are about 3.8 and 2.7 times that of Bgl2A, respectively. The stability of the mutant is improved while the specific activity is improved. The stability of the mutant is improved by 1 time compared with Bgl2A under the conditions of 30-35 ℃ and pH 7.0-7.5. In cellobiose hydrolysis experiments, after 3 hours of reaction, the mutant enzyme produced about 1.35 times as much glucose as Bgl2A per mg of proteolytic cellobiose, 3 times as much as commercial enzyme28 times. 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, and the alanine at position 22 and the valine at position 224 in the amino acid sequence of the beta-glucosidase are mutated to serine, respectively.
The amino acid sequence of the beta-glucosidase mutant of the invention may also comprise nonsense mutations or a combination of synonymous mutations in the sequence.
The coding gene of the beta-glucosidase mutant has a nucleotide sequence shown in SEQ ID No: 2.
The mutant plasmid of the invention contains the sequence shown in SEQ ID No:2, and the coding gene of the beta-glucosidase mutant.
The strain expressing the beta-glucosidase mutant contains the mutant plasmid.
The invention discloses an engineering strain expressing a beta-glucosidase mutant, which is classified and named as Escherichia coli BL (DE 3)/pET 22b (+) -bgl2A (A22S/V224S) and is sent to China Center for Type Culture Collection (CCTCC) for preservation, wherein the preservation number is CCTCC NO: m20211098, storage time 2021, 8-30 days, address: university of chinese, martial arts.
The construction method of the engineering strain for expressing the beta-glucosidase mutant comprises the following steps:
firstly, using beta-glucosidase structure from bacillus polymyxa (paenibacillus polymyxa) as a template, the structure of beta-glucosidase Bgl2A was subjected to homology modeling by using Swiss-Model. By adopting a semi-rational design strategy, amino acid residues which are not completely conserved near the catalytic active center are selected as candidate sites, and the mutated target amino acid is determined through sequence conservation analysis.
According to the gene sequence of the beta-glucosidase Bgl2A, a mutation primer is designed and synthesized, a recombinant plasmid containing the beta-glucosidase Bgl2A gene is used as a template, the synthetic mutation primer is used as a primer, and site-directed mutagenesis is carried out based on an overlap extension PCR method, so that the mutant gene of the beta-glucosidase with improved specific activity and stability is obtained.
The mutant gene is connected with pEASY-T3 plasmid and transformed into colibacillus Trans1-T1 competent cell, positive clone is selected, and DNA sequence determination is carried out on the mutant gene. Selecting a clone extraction plasmid with a correct sequence to obtain a pEASY-T3 recombinant plasmid containing a mutant beta-glucosidase gene, carrying out double 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 with the expression plasmid vector by T4 DNA ligase to obtain a connection product; the connection product is transformed into host bacteria, and positive clones are screened to obtain engineering strains containing the mutant genes of the invention.
The expression plasmid vector in the construction method comprises pCold, pET15, pET22 or pET28 and the like.
The host bacteria in the construction method comprise E.coli BL21 (DE 3), 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 that when the beta-glucosidase mutant is applied to the cellulose degradation process and pNPG and cellobiose are used 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 pH 7.0-7.5. The dynamic parameter measurement result shows that the mutant has k to the substrate pNPG and cellobiose cat The value is greatly increased compared to Bgl 2A. The stability of the mutant is improved while the specific activity is improved. The stability of the mutant is improved by 1 time compared with that of the starting enzyme Bgl2A under the conditions of 30-35 ℃ and pH of 7.0-7.5. In cellobiose hydrolysis experiments, after 3 hours of reaction, the amount of glucose produced per mg of mutant enzyme proteolytic cellobiose was about 1.35 times that of the starting enzyme 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 invention measures and compares the specific enzyme activity, the optimal pH, the optimal temperature, the kinetic parameters, the stability and the like of the mutant protein and the original wild type protein. The measurement result shows that pNPG and cellobiose are used as substratesWhen the specific enzyme activity of the mutant obtained by the invention is improved by 1.3 times and 1.5 times compared with that of the starting enzyme Bgl 2A. The dynamic parameter measurement result shows that the mutant has k to the substrate pNPG and cellobiose cat The values were 3.8 times and 2.7 times, respectively, that of the starting enzyme Bgl 2A. The stability of the mutant is improved while the specific activity is improved. The half-life of the mutant is prolonged by 1 time relative to the starting enzyme Bgl2A at 30-35 ℃ and pH7.0-7.5, and the mutation does not cause the change of the optimal temperature and the optimal pH.
Drawings
FIGS. 1 and 2 show electropherograms of PCR amplification products of the present invention: lanes of FIG. 1 are DNAmarker, PCR amplified fragments V224S-S, V S-X, A22S-S and A22S-X, respectively; each lane in FIG. 2 shows the PCR amplification products using DNAmaror, V224S-S and V224S-X combinations as templates, and the PCR amplification products using A22S-S and A22S-X combinations as templates.
FIG. 3 is an SDS-PAGE profile of purified muteins and of the starting enzyme Bgl2A: 1 is Bgl2A crushed supernatant, 2 is Bgl2A pure enzyme, 3 is Bgl2A: A22S/V224S crushed supernatant, 4 is Bgl2A: A22S/V224S pure enzyme, and M is protein marker.
In FIG. 4, a is the measurement result of the optimum temperature, and b is the measurement result of the optimum pH.
FIG. 5 shows stability at 30-35deg.C and pH 7.0-7.5.
Detailed Description
The methods of implementation in the following examples are conventional, unless otherwise specified.
Construction of an expression Strain containing the beta-glucosidase mutant Gene of the invention
1. Selection of mutation sites of beta-glucosidase gene
Based on the sequence alignment, bgl2A was most similar to the beta-glucosidase BglB (PDB code:2O 9R) from Paenibacillus polymyxa, with an amino acid sequence identity of 43%. The structure of The β -glucosidase Bgl2A was homologously modeled using The structure of BglB as a template using Swiss-Model (http:// swissmodel. Expasy. Org/; kiefer F, arnold K, kunzli M, bordoli L, schwede T. The SWISS-MODEL Repository and associated resources.nucleic Acids research.2009,37, D387-392.).
According to the simulated structure and the multi-sequence alignment analysis, the site-directed mutation sites are 22-site alanine A and 224-site valine V, the mutation direction is that the 22-site alanine A is replaced by serine S, and the 224-site valine V is replaced by 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 (its 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 the beta-glucosidase Bgl2A gene is used as a template plasmid, and the synthetic mutation primers are paired, wherein the synthetic mutation primers are respectively as follows: bgl2A-F and A22S-R, A S-F and bgl2A-R, bgl2A-F and V224S-R, V224S-F and bgl2A-R, as PCR primer pairs, the amplified products were named A24S-S and A24S-X, V224S-S and V224S-X in sequence; 4 amplified fragments are recovered, A22S-S and A22S-X are added into the same reaction system, V224S-S and V224S-X are added into another reaction system to serve as PCR reaction templates in the respective reaction systems, and bgl2A-F, bgl A-R primer pairs are used as amplification primers to amplify the bgl2A mutant gene full-length sequences and are recovered for standby.
TABLE 1 primer sequences
3. Construction of expression vectors
The PCR amplified product obtained in the step 2 is connected with pEASY-T3 plasmid (TaKaRa), and the following digestion system is established: 10ng of pEASY-T3 vector, 70ng of PCR amplification product, and 15min at 25 ℃. Performing heat shock transformation on escherichia coli Trans1-T1 competent cells by using a connection product, and sequencing the obtained transformant to verify whether mutation occurs; selecting a clone extraction plasmid with a correct sequence to obtain a pEASY-T3 recombinant plasmid containing the beta-glucosidase mutant gene; the resulting pEASY-T3 recombinant plasmid and pET-22b (+) vector were digested with NdeI and XhoI, and the digested mutant gene was ligated with the expression plasmid vector using T4 DNA ligase to construct the following digestion system: 25ng pET-22b (+) vector, 50ng mutant gene restriction enzyme fragment, 2. Mu.L 10 Xligation buffer, 1. Mu.L 4 DNA ligase (TaKaRa), and water supply to 20. Mu.L, 22℃for 1h to obtain ligation product; the ligation product is transformed into host bacteria, the obtained transformant is sequenced to verify whether mutation exists, and the transformant with the correct sequence is selected to obtain the engineering strain Escherichia coli BL (DE 3)/pET-22 b (+) -bgl2A (A22S/V224S) containing the mutant gene of the invention.
The strain Escherichia coli BL (DE 3)/pET-22 b (+) -bgl2A (A22S/V224S) of the invention is sent to China center for type culture collection (China Center for Type Culture Collection, CCTCC) for preservation. The preservation number is NO: CCTCC M20211098, with a preservation time of 2021, 8 months and 30 days, a preservation unit address: china center for type culture Collection of university of Wuhan, china.
(II) expression and protein purification of genetically engineered bacteria containing the beta-glucosidase mutation of the invention
The engineering strain Escherichia coli BL (DE 3)/pET 22b (+) -bgl2A (A22S/V224S) of the gene obtained in the step (I) is inoculated into 400mL LB liquid medium containing ampicillin, and is placed at 37 ℃ and cultured at 200rpm until the OD 600 0.6 (UNICO UV2102 UV visible spectrophotometer, culture LB medium as blank); adding IPTG with the final concentration of 0.2mM for induction, and continuously culturing at 16 ℃ and 120rpm for 16 hours; cells were disrupted by adding a 3-fold volume of Bindingbuffer at 4℃and 8000g of the cells were collected by centrifugation, followed by 350W of ultrasound for 30min under ice bath conditions, and the supernatant was collected by 12000g of centrifugation to obtain a crude enzyme solution. The crude enzyme solution was purified by Ni-NTA column chromatography. The imidazole concentration in the eluate was 60mM, and 3 column volumes were eluted. The obtained protein reaches SDS-PAGE purity through detection.
The pNPG is used as a substrate, the optimal pH of the mutant is 6.5, and the enzyme can show more than 70% of enzyme activity within the pH range of 6.0-7.5. The mutant has catalytic activity within the range of 30-55 ℃ and the optimal temperature of the mutant is 45 ℃.
(III) detection of specific Activity of mutant containing beta-glucosidase of the invention
1. Determination of enzyme Activity Using pNPG as substrate
The reaction system was 500. Mu.L, and the reaction was performed using citate-Na at pH6.5 2 HPO 4 Adding pNPG with final concentration of 5mM into buffer solution, preheating at 45deg.C for 5min, adding enzyme solution, reacting for 5min, adding 500 μl 1MNA 2 CO 3 The reaction was terminated. The experimental group was subjected to 3 parallel experiments, and the control group replaced the enzyme solution with the buffer solution. The absorbance at 405nm was measured by zeroing the control group. The enzyme activity (U) is defined as: the amount of enzyme used to produce 1. Mu. Mol of pNP per minute.
2. Determination of enzyme Activity Using cellobiose as substrate
The reaction system was 500. Mu.L, and the reaction was performed using citate-Na at pH6.5 2 HPO 4 The cellobiose with the final concentration of 100mM is added into the buffer solution, the enzyme solution is added after the buffer solution is preheated for 5min at 45 ℃, the reaction system is placed into a boiling water bath for boiling for 5min after the reaction is carried out for 5min, the enzyme is fully deactivated, the operation is carried out according to the operation instruction of the glucose determination kit, 3 experimental groups are parallel, and deionized water is used for replacing the glucose solution in the control group. The absorbance at 505nm was measured with the control set zeroed. The enzyme activity (U) is defined as: the amount of enzyme used to produce 1. Mu. Mol glucose per minute.
The measurement 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.
(IV) determination of kinetic parameters of beta-glucosidase containing the invention
Kinetic parameters of β -glucosidase on the artificial substrate pNPG and the natural substrate cellobiose are shown in table 2. The assay buffer was 50mM, pH6.5 citrate-Na 2 HPO 4 Buffers, wherein the pNPG concentration ranges from 0 to 20mM and the cellobiose concentration ranges from 0 to 200mM, are described in the third embodiment of the present invention. Based on Michaelis-Menten equation, the abscissa is substrate concentration, the ordinate is reaction speed, and nonlinear fitting is performed by using software Origin 8.5 to obtain K m 、V max 、k cat 。
The results show that the affinity of the mutant to the substrates pNPG and cellobiose is obviously reduced, but k is reduced cat The values exhibit different degrees of growth, among othersK when pNPG is used as a substrate cat The value is about 3.8 times that of the starting enzyme Bgl2A, and the k of cellobiose is calculated cat The value is 2.7 times of that of the starting enzyme Bgl2A
TABLE 2 kinetic parameters of beta-glucosidase
(V) detection of stability of beta-glucosidase containing the invention
Performing heat treatment on Bgl2A and the mutant at 30-35 ℃ and pH7.0-7.5, sampling every half hour or one hour, and 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: enzyme activity remaining ratio= (enzyme activity before heat treatment-loss enzyme activity)/heat treatment enzyme activity×100%.
The measurement result shows that the half life of the mutant under the condition is 3h, and is improved by 1 time compared with the wild type.
Use of mutant containing beta-glucosidase in cellobiose hydrolysis
The reaction system comprises cellobiose with a final concentration of 200g/L, 50mM, pH6.5 citate-Na 2 HPO 4 The buffer and 100U of the beta-glucosidase mutant were subjected to shaking reaction in a water bath at 35℃and 200rpm, periodically sampled, and the resultant glucose was detected by a glucose assay kit while adding Bgl2A and a commercial beta-glucosidase (purchased from Sigma Co., ltd., G0395) in the same amount as a control group.
After 3h of reaction, the amount of glucose produced per mg of mutant proteolytic cellobiose was about 1.35 times that of Bgl2A, 3.28 times that of 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
Organization Applicant
----------------------
Street :
City :
State :
Country :
PostalCode :
PhoneNumber :
FaxNumber :
EmailAddress :
<110> OrganizationName university Anhui
Application Project
-------------------
<120> Title. Beta. -glucosidase mutant, coding gene, expression strain and use thereof
<130> AppFileReference :
<140> CurrentAppNumber :
<141> CurrentFilingDate : ____-__-__
Sequence
--------
<213> OrganismName :
<400> PreSequenceString :
MTKISLPTCSPLLTKEFIYGVSTSSFQIEGGSAHRLPCIWDTFCDTPGKIADNSNGHVACDHYNNWKQDIDLIESL
GVDAYRLSISWPRVITKSGELNPEGVKFYTDILDELKKRNIKAFVTLYHWDLPQHLEDEGGWLNRETAYAFAHYVDLITL
AFGDRVHSYATLNEPFCSAFLGYEIGIHAPGKVGKQYGRKAAHHLLLAHGLAMTVLKQNSPTTLNGISLNFTPCYSISED
ADDIAATAFADDYLNQWYMKPIMDGTYPAIIEQLPSAHLPDIHDGDMAIISQSIDYLGINYYTRQFYKAHPTEIYEPIEP
TGPLTDMGWEIYPKSFTELLVTLNNTYTLPPIFITENGAAMPDSYNNGEINDVDRLDYYNSHLNAVHNATEQGVRIDGYF
AWSLMDNFEWAEGYLKRFGIVYVDYSTQQRTIKNSGLAYKALISNR
<212> Type : PRT
<211> Length : 442
SequenceName : SEQ ID No:1
SequenceDescription :
Sequence
--------
<213> OrganismName :
<400> PreSequenceString :
atgactaaaa tatctttacc aacttgttca cctctattaa caaaagagtt tatttatggt 60
gtaagcacat catctttcca aatagaaggt ggctcagctc accgtctgcc gtgtatctgg 120
gatacctttt gtgatactcc aggtaaaata gctgataact caaatgggca tgttgcatgc 180
gatcattaca ataattggaa acaagacata gatttaatcg aatcattagg agtagatgct 240
tacagacttt ctatttcttg gcctcgtgtt attacaaaaa gtggtgagct taaccctgaa 300
ggcgtaaagt tttacaccga catcttagat gaactgaaaa agcgcaatat taaagcgttt 360
gtcacgttat accactggga tttacctcaa cacctagaag acgaaggggg ttggttaaat 420
cgagaaacag cttacgcgtt tgctcactat gttgatttaa ttaccttggc attcggtgac 480
cgggtgcatt catacgctac cttaaacgaa cccttttgca gtgctttttt aggttacgaa 540
attggcattc atgcgccagg gaaagtcggc aaacaatacg ggcgcaaagc cgcccaccat 600
ttgttattag cacatggcct tgccatgacc gtattaaagc aaaactcacc gacgacttta 660
aacggtatct cccttaactt tactccctgt tatagcatct ctgaagacgc tgatgacatt 720
gctgcaacag cgtttgcaga tgactactta aaccagtggt acatgaaacc catcatggat 780
ggtacatacc cagcaattat tgaacaatta ccttcagcac atctgccaga tattcacgat 840
ggtgacatgg ccattatttc acaatcaatt gattatttag gtattaacta ttatacccgt 900
caattttata aagcgcaccc tactgaaata tatgagccaa tagagcctac tggcccgcta 960
accgatatgg gctgggaaat ttaccctaag tcgtttacag agttattagt cacacttaac 1020
aatacctata ccctaccgcc tatttttatt actgaaaatg gcgcagctat gcccgacagc 1080
tataataatg gtgaaatcaa tgatgtagat cgactagact actacaacag tcacctaaat 1140
gccgttcaca atgcaacaga gcaaggcgtt agaatagacg gctattttgc ctggagccta 1200
atggataact ttgaatgggc agaaggttac ttaaaaagat ttggtatagt ttatgtagat 1260
tacagcacac agcaacgtac tataaaaaat agtggcctag cctataaagc attaatctca 1320
aatagataa 1329
<212> Type : DNA
<211> Length : 1329
SequenceName : SEQ ID No:2
SequenceDescription :
Claims (4)
1. A mutant β -glucosidase characterized by:
the amino acid sequence of the beta-glucosidase mutant is shown in SEQ ID No: 1.
2. A gene encoding the β -glucosidase mutant of claim 1, characterized in that:
the nucleotide sequence of the coding gene is shown as SEQ ID No: 2.
3. An engineered strain expressing the β -glucosidase mutant of claim 1, characterized in that:
the classification of the engineering strains is named asEscherichia coli BL21(DE3)/pET22b(+)-bgl2A(A22S/V224S) sent to China Center for Type Culture Collection (CCTCC) for preservation, wherein the preservation number is CCTCC NO: m20211098, storage time 2021, 8-30 days, address: university of chinese, martial arts.
4. Use of a β -glucosidase mutant according to claim 1, characterized in that:
the beta-glucosidase mutant is applied to a cellulose degradation process.
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| CN101955922A (en) * | 2010-08-02 | 2011-01-26 | 安徽大学 | Glucose-tolerant beta-glucosidase and expression thereof |
| CN102220302A (en) * | 2011-05-20 | 2011-10-19 | 安徽大学 | Beta-glucosidase mutant, recombined expression plasmid and converted engineering strain |
| CN102844430A (en) * | 2010-02-11 | 2012-12-26 | 南方化学知识产权有限公司 | Optimized cellulase enzymes |
| CN104726435A (en) * | 2015-04-13 | 2015-06-24 | 安徽大学 | Beta-glucosidase mutant, recombinant expression plasmid thereof and transformed engineering strain |
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| CN102844430A (en) * | 2010-02-11 | 2012-12-26 | 南方化学知识产权有限公司 | Optimized cellulase enzymes |
| CN101955922A (en) * | 2010-08-02 | 2011-01-26 | 安徽大学 | Glucose-tolerant beta-glucosidase and expression thereof |
| CN102220302A (en) * | 2011-05-20 | 2011-10-19 | 安徽大学 | Beta-glucosidase mutant, recombined expression plasmid and converted engineering strain |
| CN104726435A (en) * | 2015-04-13 | 2015-06-24 | 安徽大学 | Beta-glucosidase mutant, recombinant expression plasmid thereof and transformed engineering strain |
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| Improving the cellobiose hydrolysis activity of glucose-stimulating β-glucosidase Bgl2A;Shuaifeng Liu et al.;《Enzyme and Microbial Technology》;第169卷;第1-10页 * |
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