阅读说明：本技术 一种β-葡萄糖苷酶突变体及其应用 (Beta-glucosidase mutant and application thereof ) 是由 杨峰 张振雷 王晓军 李文娟 庞金蕙 于 2019-11-01 设计创作，主要内容包括：本发明涉及一种β-葡萄糖苷酶突变体及其应用,通过全质粒PCR对原有野生型Bacillus cereus W23Q 的β-葡萄糖苷酶进行定点突变,制备单突变体库；采用PCR体系和程序进行全质粒PCR,结束后采用Dpn I酶消除模板,再将质粒转化到大肠杆菌Top10中,培养提取质粒保存并送测序；确定突变成功后,将质粒转化到表达宿主大肠杆菌BL21中,培养、诱导表达,即得到β-葡萄糖苷酶突变体。该突变体较野生酶能大幅度提高催化Rg3转化Rh2的效率。应用该菌种生产Rh2,能降低成本,便于工业化应用。(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 whole 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.)

1. a β -glucosidase mutant, characterized in that: 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 the amino acid sequence shown as SEQ ID NO. 2 by at least one of the following conditions:

Alanine A at position 197 is mutated into aspartic acid D, A197D for short;

Or/and mutation of valine V at position 246 to aspartic acid D, which is V246D for short;

Or/and mutation of aspartic acid D at position 295 to asparagine N, abbreviated as D295N;

or/and mutation of 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 position 489 into valine V, I489V for short.

2. the method for producing the β -glucosidase mutant of claim 1, comprising the steps of:

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.

3. the method for preparing a β -glucosidase mutant as claimed in claim 2, wherein the PCR system in step (2) is:

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:

Pre-denaturation: 4min at 98 ℃;

② at 98 ℃ for 10s, at 50-65 ℃ for 30s, at 72 ℃ for 7min, 25 cycles;

③ fully extending the temperature to 72 ℃ for 10 min;

④4℃ 30min。

4. The method for preparing a mutant β -glucosidase as claimed in claim 3, wherein the names and sequences of the upstream and downstream primers are as follows:

。

5. the use of the mutant β -glucosidase of claim 1 for the synthesis of ginsenoside Rh 2.

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 and 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 small, so the ginseng is also called as rare saponin, Rh2 has various effects, has synergistic attenuation with chemotherapeutic drugs, and has the advantages that 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 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, the molecule is very complex, and direct synthesis has no small difficulty. The content of the natural product is extremely low, so the natural product is expensive, the water solubility is poor, the natural product is not easy to absorb, and the use of the natural product in large quantity is limited. At present, the extraction and synthesis of ginsenoside Rh2 have the modes of whole-cell catalysis, acid hydrolysis and the like, but the efficiency is very low, and the byproducts are more, so 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:

Alanine A at position 197 is mutated into aspartic acid D, A197D for short;

Or/and mutation of valine V at position 246 to aspartic acid D, which is V246D for short;

Or/and mutation of aspartic acid D at position 295 to asparagine N, abbreviated as D295N;

Or/and mutation of 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 position 489 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% 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 a phosphoric acid 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, the beta-glucosidase mutant gene has 6 site-directed mutations compared with the original gene, and can be subjected to single-point or multi-point combined mutation to artificially modify and evolve wild enzyme, after mutation, the enzyme catalysis Rg3 has higher efficiency, shows good activity at 20 ~ 50 ℃, provides more simplicity for industrial production operation, and simultaneously has little difference between the temperature required by the reaction and room temperature, reduces the energy consumption of industrial production, and reduces the production cost.

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 converting the substrate Rg3 into Rh2 reaches 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 pET28 a-bgl.

Detailed Description

The present invention will be further described with reference to the following examples, but the present invention is not limited thereto.