CN110577947B - Beta-glucosidase mutant and application thereof - Google Patents
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Abstract
The invention relates to a beta-glucosidase mutant and application thereof, wherein a single mutant library is prepared by carrying out site-directed mutagenesis on beta-glucosidase of original wild type Bacillus cereus W23Q through full-plasmid PCR; carrying out whole plasmid PCR by adopting a PCR system and a PCR program, eliminating a template by adopting Dpn I enzyme after the completion, then transforming the plasmid into escherichia coli Top10, culturing, extracting the plasmid, storing and sequencing; and after the success of mutation is determined, the plasmid is transformed into an expression host escherichia coli BL21, and the beta-glucosidase mutant is obtained after culture and induced expression. Compared with wild enzyme, the mutant can greatly improve the efficiency of catalyzing Rg3 to convert Rh 2. The Rh2 produced by the strain can reduce the cost and is convenient for industrial application.
Description
Technical Field
The invention belongs to the technical field of enzymology, and particularly relates to a beta-glucosidase mutant and application thereof.
Background
The ginseng is a famous and precious medicinal material, is also a traditional Chinese medicine, and has good medicinal value. The main active component of ginseng is ginsenoside, the main components of the ginseng comprise Rb1, rb2, rc, rd, rg3 and the like, wherein the content of Rh2 is very rare, so the ginseng is also called as rare saponin, rh2 has various effects, has synergistic attenuation with chemotherapeutic drugs, and in the process of treating tumors, the failure of clinical chemotherapy is often related to the drug resistance of tumor cells to the chemotherapeutic drugs, and the ginsenoside Rh2 can be used as a tumor drug resistance reversal agent and can improve the anti-tumor activity of the chemotherapeutic drugs. Thus, the natural product Rh2 has a wide market.
Although ginsenoside Rh2 has excellent anticancer performance, the ginsenoside Rh2 is a natural product, and the molecule is very complex, so that the ginsenoside Rh2 has no small difficulty in direct synthesis. The content of the natural product is very low, so the natural product is expensive, the water solubility is poor, the absorption is not easy, and the large-scale use of the natural product is limited. At present, ginsenoside Rh2 can be extracted and synthesized by whole-cell catalysis, acid hydrolysis and the like, but the efficiency is low, and the byproducts are more, so that the method is not suitable for industrial large-scale production.
From the application of beta-glucosidase in recent years, it has been found that its hydrolysis activity has been widely studied so far, and that it can hydrolyze natural products such as ginsenoside and oleuropein and has the ability to synthesize alkyl glycoside.
Disclosure of Invention
The invention aims to provide a beta-glucosidase mutant and a method for synthesizing ginsenoside Rh2 by using the mutant.
The technical scheme for realizing the purpose of the invention is as follows:
a beta-glucosidase mutant is derived from wild type Bacillus cereus W23Q, the nucleotide sequence of the beta-glucosidase mutant is shown as SEQ ID NO. 1, the amino acid sequence is shown as SEQ ID NO. 2, or the amino acid sequence shown as SEQ ID NO. 2 is obtained by at least one mutation condition:
mutating the 197 th alanine A to aspartic acid D, A197D for short;
or/and mutation of valine V at position 246 to aspartic acid D, referred to as V246D;
or/and mutation of aspartic acid D at the 295 th position into asparagine N, referred to as D295N for short;
or/and mutating tyrosine Y at position 304 into phenylalanine F, Y304F for short;
or/and mutation of asparagine N at position 360 into aspartic acid D, N360D for short;
or/and mutation of isoleucine I at the 489 th position into valine V, I489V for short.
The preparation method of the beta-glucosidase mutant comprises the following steps:
(1) Carrying out site-directed mutagenesis on beta-glucosidase of original wild type Bacillus cereus W23Q by reverse PCR to prepare a single mutant library;
(2) Carrying out whole plasmid PCR by adopting a PCR system and a PCR program, eliminating a template by adopting Dpn I enzyme after the completion, then transforming the plasmid into escherichia coli Top10, culturing, extracting the plasmid, storing and sequencing;
(3) And after the success of mutation is determined, the plasmid is transformed into an expression host escherichia coli BL21, and the beta-glucosidase mutant is obtained after culture and induced expression.
The invention also aims to provide application of the beta-glucosidase mutant in synthesis of ginsenoside Rh 2.
The method for synthesizing ginsenoside Rh2 by applying the beta-glucosidase mutant takes ginsenoside Rg3 as a substrate, and the ginsenoside Rh2 is generated by reaction under the catalysis of the beta-glucosidase mutant; the catalytic reaction temperature is 20-50 ℃; the concentration of the ginsenoside Rg3 in the catalytic reaction system is 1-10 percent w/v, the dosage of the beta-glucosidase mutant is 0.01-0.06 time of the weight of the ginsenoside Rg3 serving as a substrate, and the balance is water or phosphate buffer solution and a cosolvent for dissolving the ginsenoside Rg 3;
the pH value of the catalytic reaction system is 5.0-9.0.
The synthesis reaction is mild, the operation is simple, the byproducts are few, the enzymatic production belongs to green synthesis, and the industrial application of the enzymatic production of the ginsenoside Rh2 is facilitated.
The beta-glucosidase is derived from Bacillus cereus W23Q, 6 site-directed mutations are generated on the beta-glucosidase mutant gene compared with the original gene, and single-point or multi-point combined mutation can be carried out to artificially modify and evolve the wild enzyme. After mutation, the efficiency of enzyme catalysis Rg3 is higher, good activity is shown at 20-50 ℃, more simplicity and convenience are provided for industrial production operation, the temperature required by the reaction is not greatly different from the room temperature, the energy consumption of industrial production is reduced, and the production cost is reduced.
According to the beta-glucosidase mutant, through site-directed mutagenesis, the catalytic activity of the mutant on a substrate Rg3 is high, the conversion rate of the substrate Rg3 into Rh2 is over 91%, the conversion rate is high, byproducts are few, and the content of the prepared Rh2 is high.
Drawings
FIG. 1 shows the recombinant plasmid pET28a-bgl.
Detailed Description
The present invention will be further described with reference to the following examples, but the present invention is not limited thereto.
Example 1:
source of beta-glucosidase and obtaining of mutant thereof
Constructing a recombinant plasmid, and constructing a target gene into a pET-28a plasmid, wherein the restriction enzyme sites are BamH1 and Hind III.
The beta-glucosidase is derived from wild type Bacillus cereus W23Q, the nucleotide sequence of the beta-glucosidase mutant is shown as SEQ ID NO. 1, the amino acid sequence is shown as SEQ ID NO. 2, or the beta-glucosidase mutant is obtained by mutating at least one of the following amino acid sequences shown as SEQ ID NO. 2:
alanine A at position 197 is mutated into aspartic acid D and glutamic acid E, referred to as A197D for short;
or/and mutation of valine V at position 246 to aspartic acid D, referred to as V246D;
or/and mutating aspartic acid D at the 295 th position into asparagine N, referred to as D295N for short;
or/and mutating tyrosine Y at position 304 into phenylalanine F, Y304F for short;
or/and mutation of asparagine N at position 360 into aspartic acid D, N360D for short;
or/and mutating isoleucine I at the 489 th position into valine V, namely I489V for short.
Example 2:
the preparation method of the beta-glucosidase mutant comprises the following steps:
(1) Carrying out site-directed mutagenesis on beta-glucosidase by reverse PCR to prepare a single mutant library;
(2) Carrying out whole plasmid PCR by adopting a PCR system and a PCR program, eliminating a template by adopting Dpn I enzyme after the completion, transforming the plasmid into Escherichia coli DH5 alpha, culturing, extracting the plasmid, storing and sequencing;
(3) And after the success of mutation is determined, the plasmid is transformed into an expression host escherichia coli BL21, and the beta-glucosidase mutant is obtained after culture and induced expression.
In the preparation method, the PCR system in the step (2) is as follows:
Takara PrimeSTAR ® HS DNA Polymerase 0.25μL
plasmid pET-28a-bgl 1. Mu.L
10 X Buffer 5μL
dNTPs 4μL
Upstream primer 1. Mu.L
Downstream primer 1. Mu.L
Sterile water to 50 μ L;
the PCR reaction program is as follows:
(1) denaturation: 4min at 98 ℃;
(2) 1, 10s at 98 ℃, 30s at 50-65 ℃, 7min at 72 ℃ and 25 cycles;
(3) fully extending for 10min at 72 ℃;
④4℃ 30min。
the recombinant plasmid pET28a-bgl is shown in FIG. 1.
The names and sequences of the upstream primer and the downstream primer are as follows:
example 3:
ginsenoside Rh2 is synthesized by using the beta-glucosidase mutant prepared in the example 2, 5g of ginsenoside Rg3 serving as a substrate is added into a 100mL three-necked flask, then 15mL of DMSO is used for dissolving, 85mL of phosphoric acid buffer solution with pH =7.0 is added continuously, 0.05g of enzyme is added continuously, stirring is carried out, water bath at 40 ℃ is carried out, pH is detected, and HCl/NaOH is used for adjusting the pH to be about 7.0. After 4h of reaction, the Rh2 yield was determined, and the results are shown in the following table.
As can be seen from the table, the conversion rate of wild-type Bacillus cereus W23Q enzyme for converting the substrate Rg3 into Rh2 is 85.51%, and the conversion rate of the enzyme mutant for converting the substrate Rg3 into Rh2 is more than 91% and as high as 99.63%, which proves that the prepared beta-glucosidase mutant can greatly improve the catalytic efficiency.
Sequence listing
<110> university of Guangxi Master
<120> beta-glucosidase mutant and application thereof
<141> 2019-11-01
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1560
<212> DNA
<213> Bacillus cereus (Bacillus cereus)
<400> 1
atgattgtta aacgtttatt aatgatatgt tgtatcgtca tcttttttat tcctttcact 60
ttcatttccc ctcactctac ttatgctgaa gctaatacta cgtataaaaa acatgaatta 120
cgtgctgtat ggatcgcatc tgttcttaat attgattggc cctcaaaaac cggcttaccc 180
atcgaaaatc aaaaacaaga gtttattaga ttattagacg atgtaaaaaa caccggtatg 240
aatgcagtcg ttgtacaaat caaaccaacc gctgatgctt tctatccttc aaattacggt 300
ccttggtctg aatacattac gggcacacaa ggaaaagatc ctggttatga cccactcgca 360
tttatggtcg atgaagcaca taaaagaaat atagaattcc acgcatggat taacccatac 420
cgaataacga tgaatcacac tgatataaat cgattatcaa ataatcaccc tgcaaaacaa 480
catcccgatt ggattgtacc ttatggcggg aagctatatt acaatcccgg tattccagaa 540
gtgaaaaaat ttataactga aggtgcttta gaaattgtgc aaaattatgc cattgatgcg 600
ctgcatatgg atgattattt ttatccatat aaattagcag gtgaacaatt ccccgatcaa 660
aaaacgtacg aaacgtataa taacggtaga tttacaaata tagaggattg gcgacgtaac 720
aatgtaaatg aacttgtaaa agatttaaat actgctataa aacaagaaaa atcatacgta 780
aagtttggca taagtccgtt cggcgtatgg cgaaatatag ctgacgatcc aactggctct 840
aacacaactg caggtcaaag gaactatgat gacctttacg ctgatacacg tgagtggata 900
caaaaaggat atattgacta cattacaccg caaatctatt ggaatattgg tttcacacca 960
gctgcatatg acatcttagt agattggtgg gtaaaagaaa caaataataa accgattcac 1020
ctatacatcg gtcaggcggc ctataaaatt aataataatt ctgtcccagc ttggtctaat 1080
ccagaagaat acccaagaca aattgaatta aatcggttat atcctgaaat aaaaggtagt 1140
atgcatttta gcttaaaaga tattaataac aatccactag gaataaaaga tagactctca 1200
aaagacatat ataaatatcc tccattaatc ccctctatgc cttggcttga tcatgatcca 1260
ccaaaacaac cgactttaaa aggtgctatt ccaagagatg aaggtattgc tataggcatt 1320
attgacgata gagagaatga ttctgcttat tacaccattt accgcgcgaa tggaaaaaat 1380
gaagtggata tacaaaaccc aaaaaattta cttactactg taaggaaaac aaaacttgga 1440
gaaatttatg tagataaaac agctatctct ggagaaacgt atacgtatgt agtaactgca 1500
gttgatcgat tgcataatga aagcgttgca tctagtcatg ctaccgttaa agcaaaataa 1560
<210> 2
<211> 519
<212> PRT
<213> Bacillus cereus (Bacillus cereus)
<400> 2
Met Ile Val Lys Arg Leu Leu Met Ile Cys Cys Ile Val Ile Phe Phe
1 5 10 15
Ile Pro Phe Thr Phe Ile Ser Pro His Ser Thr Tyr Ala Glu Ala Asn
20 25 30
Thr Thr Tyr Lys Lys His Glu Leu Arg Ala Val Trp Ile Ala Ser Val
35 40 45
Leu Asn Ile Asp Trp Pro Ser Lys Thr Gly Leu Pro Ile Glu Asn Gln
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Lys Gln Glu Phe Ile Arg Leu Leu Asp Asp Val Lys Asn Thr Gly Met
65 70 75 80
Asn Ala Val Val Val Gln Ile Lys Pro Thr Ala Asp Ala Phe Tyr Pro
85 90 95
Ser Asn Tyr Gly Pro Trp Ser Glu Tyr Ile Thr Gly Thr Gln Gly Lys
100 105 110
Asp Pro Gly Tyr Asp Pro Leu Ala Phe Met Val Asp Glu Ala His Lys
115 120 125
Arg Asn Ile Glu Phe His Ala Trp Ile Asn Pro Tyr Arg Ile Thr Met
130 135 140
Asn His Thr Asp Ile Asn Arg Leu Ser Asn Asn His Pro Ala Lys Gln
145 150 155 160
His Pro Asp Trp Ile Val Pro Tyr Gly Gly Lys Leu Tyr Tyr Asn Pro
165 170 175
Gly Ile Pro Glu Val Lys Lys Phe Ile Thr Glu Gly Ala Leu Glu Ile
180 185 190
Val Gln Asn Tyr Ala Ile Asp Ala Leu His Met Asp Asp Tyr Phe Tyr
195 200 205
Pro Tyr Lys Leu Ala Gly Glu Gln Phe Pro Asp Gln Lys Thr Tyr Glu
210 215 220
Thr Tyr Asn Asn Gly Arg Phe Thr Asn Ile Glu Asp Trp Arg Arg Asn
225 230 235 240
Asn Val Asn Glu Leu Val Lys Asp Leu Asn Thr Ala Ile Lys Gln Glu
245 250 255
Lys Ser Tyr Val Lys Phe Gly Ile Ser Pro Phe Gly Val Trp Arg Asn
260 265 270
Ile Ala Asp Asp Pro Thr Gly Ser Asn Thr Thr Ala Gly Gln Arg Asn
275 280 285
Tyr Asp Asp Leu Tyr Ala Asp Thr Arg Glu Trp Ile Gln Lys Gly Tyr
290 295 300
Ile Asp Tyr Ile Thr Pro Gln Ile Tyr Trp Asn Ile Gly Phe Thr Pro
305 310 315 320
Ala Ala Tyr Asp Ile Leu Val Asp Trp Trp Val Lys Glu Thr Asn Asn
325 330 335
Lys Pro Ile His Leu Tyr Ile Gly Gln Ala Ala Tyr Lys Ile Asn Asn
340 345 350
Asn Ser Val Pro Ala Trp Ser Asn Pro Glu Glu Tyr Pro Arg Gln Ile
355 360 365
Glu Leu Asn Arg Leu Tyr Pro Glu Ile Lys Gly Ser Met His Phe Ser
370 375 380
Leu Lys Asp Ile Asn Asn Asn Pro Leu Gly Ile Lys Asp Arg Leu Ser
385 390 395 400
Lys Asp Ile Tyr Lys Tyr Pro Pro Leu Ile Pro Ser Met Pro Trp Leu
405 410 415
Asp His Asp Pro Pro Lys Gln Pro Thr Leu Lys Gly Ala Ile Pro Arg
420 425 430
Asp Glu Gly Ile Ala Ile Gly Ile Ile Asp Asp Arg Glu Asn Asp Ser
435 440 445
Ala Tyr Tyr Thr Ile Tyr Arg Ala Asn Gly Lys Asn Glu Val Asp Ile
450 455 460
Gln Asn Pro Lys Asn Leu Leu Thr Thr Val Arg Lys Thr Lys Leu Gly
465 470 475 480
Glu Ile Tyr Val Asp Lys Thr Ala Ile Ser Gly Glu Thr Tyr Thr Tyr
485 490 495
Val Val Thr Ala Val Asp Arg Leu His Asn Glu Ser Val Ala Ser Ser
500 505 510
His Ala Thr Val Lys Ala Lys
515
<210> 3
<211> 40
<212> PRT
<213> Bacillus cereus (Bacillus cereus)
<400> 3
Thr Gly Thr Gly Cys Ala Ala Ala Ala Thr Thr Ala Thr Gly Ala Cys
1 5 10 15
Ala Thr Thr Gly Gly Cys Ala Gly Cys Gly Cys Ala Thr Cys Ala Ala
20 25 30
Thr Gly Thr Cys Ala Thr Ala Ala
35 40
<210> 4
<211> 45
<212> PRT
<213> Bacillus cereus (Bacillus cereus)
<400> 4
Gly Thr Ala Ala Cys Ala Ala Thr Gly Ala Ala Cys Thr Thr Gly Ala
1 5 10 15
Cys Ala Ala Ala Gly Gly Cys Ala Gly Thr Ala Thr Thr Thr Ala Ala
20 25 30
Ala Thr Cys Gly Thr Thr Thr Gly Thr Cys Ala Ala Gly
35 40 45
<210> 5
<211> 41
<212> PRT
<213> Bacillus cereus (Bacillus cereus)
<400> 5
Gly Ala Cys Cys Thr Thr Thr Ala Cys Gly Cys Thr Ala Ala Thr Ala
1 5 10 15
Cys Ala Cys Thr Cys Cys Ala Cys Thr Cys Ala Cys Gly Thr Gly Thr
20 25 30
Ala Thr Thr Cys Ala Gly Cys Gly Thr
35 40
<210> 6
<211> 41
<212> PRT
<213> Bacillus cereus (Bacillus cereus)
<400> 6
Gly Gly Ala Thr Ala Cys Ala Ala Ala Ala Ala Gly Gly Ala Thr Thr
1 5 10 15
Thr Ala Thr Thr Gly Gly Thr Ala Ala Thr Gly Thr Ala Gly Thr Cys
20 25 30
Ala Ala Thr Ala Ala Ala Thr Cys Cys
35 40
<210> 7
<211> 40
<212> PRT
<213> Bacillus cereus (Bacillus cereus)
<400> 7
Gly Thr Cys Cys Cys Ala Gly Cys Thr Thr Gly Gly Thr Cys Thr Gly
1 5 10 15
Ala Thr Cys Cys Ala Gly Gly Gly Gly Thr Ala Thr Thr Cys Thr Gly
20 25 30
Gly Ala Thr Cys Ala Gly Ala Ala
35 40
<210> 8
<211> 41
<212> PRT
<213> Bacillus cereus (Bacillus cereus)
<400> 8
Gly Thr Ala Gly Ala Thr Ala Ala Ala Ala Cys Ala Gly Cys Thr Gly
1 5 10 15
Thr Cys Thr Cys Thr Cys Cys Gly Thr Thr Thr Cys Thr Cys Cys Ala
20 25 30
Gly Ala Gly Ala Cys Ala Gly Cys Thr
35 40
Claims (1)
1. An application of a beta-glucosidase mutant in synthesizing ginsenoside Rh2, wherein the amino acid sequence of the beta-glucosidase mutant is that alanine A at the 197 th position of a sequence shown as SEQ ID NO. 2 is mutated into aspartic acid D, A197D for short;
the application is as follows: ginsenoside Rg3 is used as a substrate, and is reacted to generate ginsenoside Rh2 under the catalysis of a beta-glucosidase mutant; the catalytic reaction temperature is 40 ℃; the concentration of the ginsenoside Rg3 in the catalytic reaction system is 1-10% w/v, the dosage of the beta-glucosidase mutant is 0.01-0.06 times of the weight of the ginsenoside Rg3 serving as a substrate, and the balance is water or a phosphoric acid buffer solution and a cosolvent for dissolving the ginsenoside Rg 3;
the pH of the catalytic reaction system was 7.0.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20130055217A (en) * | 2011-11-18 | 2013-05-28 | 한방바이오 주식회사 | NOVEL β-GLUCOSIDASE PROTEIN AND USE THEREOF |
| CN104726432A (en) * | 2014-12-22 | 2015-06-24 | 江苏大学 | Beta-glucosidase D mutant as well as expression plasmid and recombinant bacteria thereof |
| KR20180040269A (en) * | 2016-10-12 | 2018-04-20 | 주식회사 케이바이오팜 | Method for producing rare ginsenosides from ginseng berry |
| CN109182439A (en) * | 2018-09-15 | 2019-01-11 | 东北林业大学 | The bioconversion method of the rare saponin(e Rg3 of ginseng |
| KR101968004B1 (en) * | 2017-10-13 | 2019-04-10 | 재단법인 지능형 바이오 시스템 설계 및 합성 연구단 | Mass producing method of ginsenoside Rh2 mix |
| CN110004129A (en) * | 2018-01-04 | 2019-07-12 | 安徽正方生物科技有限公司 | A kind of β-glucosidase mutants and its application |
-
2019
- 2019-11-01 CN CN201911058974.4A patent/CN110577947B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20130055217A (en) * | 2011-11-18 | 2013-05-28 | 한방바이오 주식회사 | NOVEL β-GLUCOSIDASE PROTEIN AND USE THEREOF |
| CN104726432A (en) * | 2014-12-22 | 2015-06-24 | 江苏大学 | Beta-glucosidase D mutant as well as expression plasmid and recombinant bacteria thereof |
| KR20180040269A (en) * | 2016-10-12 | 2018-04-20 | 주식회사 케이바이오팜 | Method for producing rare ginsenosides from ginseng berry |
| KR101968004B1 (en) * | 2017-10-13 | 2019-04-10 | 재단법인 지능형 바이오 시스템 설계 및 합성 연구단 | Mass producing method of ginsenoside Rh2 mix |
| CN110004129A (en) * | 2018-01-04 | 2019-07-12 | 安徽正方生物科技有限公司 | A kind of β-glucosidase mutants and its application |
| CN109182439A (en) * | 2018-09-15 | 2019-01-11 | 东北林业大学 | The bioconversion method of the rare saponin(e Rg3 of ginseng |
Non-Patent Citations (2)
| Title |
|---|
| Comparative analysis of the expression level of recombinant ginsenoside-transforming β-glucosidase in GRAS hosts and mass production of the ginsenoside Rh2-Mix;Muhammad Zubair Siddiqi 等;《PLOS ONE》;20170419;1-15 * |
| NCBI GENBANK:LC456422.1;FIKRY,A.E.等;《GENBANK》;20190117;1-2 * |
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