Preparation method and application of R-cyanohydrin lyase

文档序号：1841915 发布日期：2021-11-16 浏览：16次中文

阅读说明：本技术 一种r-氰醇裂解酶的制备方法及其应用 (Preparation method and application of R-cyanohydrin lyase ) 是由曹金辉宗匡喻海亮曾鹏刘建明陈文欢于 2021-08-26 设计创作，主要内容包括：本发明提供了一种新型的、来源于芒果的R-氰醇裂解酶的制备方法及其应用。具体地,通过通过构建随机突变和点饱和突变文库,以及高通量筛选获得多种突变的氰醇裂解酶,其方法安全、简单且易于操作。(The invention provides a preparation method and application of a novel R-cyanohydrin lyase derived from mango. Specifically, the method is safe, simple and easy to operate by constructing random mutation and point saturation mutation libraries and screening in high throughput to obtain various mutated cyanohydrin lyases.)

1. A preparation method of R-cyanohydrin lyase is characterized by comprising the following steps: comprises the steps of (a) preparing a mixture of a plurality of raw materials,

carrying out mutation treatment on the wild type gene of the R-cyanohydrin lyase: carrying out mutation treatment on an R-cyanohydrin lyase gene and a wild type gene sequence of the cyanohydrin lyase as shown in Seq ID No.1 to obtain a cyanohydrin lyase mutant gene, wherein the sequence of the cyanohydrin lyase mutant gene is shown in Seq ID No. 2;

adding enzyme cutting sites: inserting double enzyme cutting sites into a mutation gene of the cyanohydrin lyase;

preparing a recombinant plasmid: inserting the cyanohydrin lyase mutator gene into an expression vector to obtain a recombinant plasmid;

introducing a strain: introducing the recombinant plasmid with the cyanohydrin lyase gene into a strain to obtain a recombinant expression strain;

secretion and expression of the strains: adding signal peptide, inducing the recombinant expression strain in culture solution and collecting enzyme solution.

2. The process for producing R-cyanohydrin lyase as claimed in claim 1, wherein: the mode of mutation treatment is error-prone PCR.

3. The process for producing R-cyanohydrin lyase as claimed in claim 1, wherein: in the added enzyme cutting sites, the double enzyme cutting sites are NdeI/HindIII.

4. The process for producing R-cyanohydrin lyase as claimed in claim 1, wherein: in the recombinant plasmid preparation, the expression vector is pET26b (+), the N end of the expression vector is a signal peptide pelB leader, and the gene sequence of the signal peptide is shown as Seq ID No. 5.

5. The process for producing R-cyanohydrin lyase as claimed in claim 1, wherein: the introduced strain is E.coli BL21(DE 3).

6. The method for producing R-cyanohydrin lyase according to claim 1, wherein: in the secretion and expression of the strain, the culture solution is an LB culture medium.

7. The process for producing R-cyanohydrin lyase as claimed in claim 1, wherein: in the secretion and expression of the strain, induction culture is also included, when OD is in the culture medium₆₀₀After 1.0, 0.2mM IPTG was added and the temperature was maintained at 30 ℃ to induce expression for 4-5 h.

8. The use of the product of the preparation process of R-cyanohydrin lyase as claimed in claim 1 to 7, wherein: the use of said composition, comprising,

dissolving a substrate in tert-butyl methyl ether (MTBE) to obtain an MTBE solution of the substrate; diluting the obtained product in a phosphate-citric acid buffer solution, and adjusting the pH to 3.4 to obtain an enzyme solution; and fully mixing the MTBE solution of the substrate and the enzyme solution, cooling the system to 10 ℃, stirring into emulsion, and adding liquid hydrocyanic acid to react to obtain R-phenethyl cyanohydrin and R-o-methyl phenethyl cyanohydrin.

9. The method of using R-cyanohydrin lyase as claimed in claim 8, wherein: the substrate is one or two of phenethyl cyanohydrin and/or o-methyl phenethyl cyanohydrin; the MTBE solution concentration of the substrate was 8 microliters per milliliter of buffer; the volume ratio of the MTBE solution of the substrate to the enzyme solution is 2: 1.2 to 1.7; the ratio of the addition amount of the liquid hydrocyanic acid to the total volume of the MTBE solution of the substrate and the enzyme solution is 4-3.5: 1.2.

10. the R-phenylacetylcyanohydrin and R-o-tolylcyanohydrin prepared by the method of using R-cyanohydrin lyase as claimed in claim 8, wherein: the yield of the R-phenethyl cyanohydrin and the yield of the R-o-methyl phenethyl cyanohydrin are both more than or equal to 99.5 percent, and the chiral value (e.e value) is both more than or equal to 99.5 percent.

Technical Field

The invention relates to the technical field of biology, in particular to a preparation method and application of R-cyanohydrin lyase.

Background

Chiral cyanohydrin is a chiral raw material with important position in industrial asymmetric synthesis production, and can be easily converted into medical and pesticide intermediates with important commercial values such as chiral alpha-hydroxy acid (ester), alpha-amino acid, beta-amino alcohol and the like, so the synthesis of chiral cyanohydrin is one of the focus of attention in academia and industry. The synthesis of chiral cyanohydrin has certain difficulty and challenge, and the asymmetric synthesis of chiral cyanohydrin needs to use chiral chemical or biological catalysts, wherein the biological catalyst (cyanohydrin) is far superior to the chemical catalyst due to the characteristics of rich sources, low cost, high catalytic efficiency, mild reaction conditions, high yield and optical purity, wide substrate application range and the like, and is widely used in the organic synthesis industry.

Cyanohydrin (lyase) enzyme catalyzes the addition of HCN and aldone to produce an alpha-chiral cyanohydrin product. The natural R-cyanohydrin enzyme exists in Rosaceae Prunus plants and some fruits, and at present, only plants derived from Rosaceae plants are cases of successful industrial application, such as Prunus amygdalus (Pa HNL), Prunus domestica (Pd HNL) or Prunus mume (Pm HNL), and the like, wherein the cyanohydrin lyase of Prunus amygdalus (Pa HNL) is taken as the main enzyme, so that the cyanohydrin enzyme from other sources is searched and applied to the industrial production of chiral cyanohydrin, and the practical significance is very important.

Disclosure of Invention

This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.

The present invention has been made in view of the above and/or problems occurring in the conventional preparation methods of R-cyanohydrin lyase.

Therefore, one of the objects of the present invention is to provide a method for preparing R-cyanohydrin lyase and the use thereof.

To solve the above technical problem, according to an aspect of the present invention, the present invention provides the following technical solutions: a preparation method of R-cyanohydrin lyase comprises the following steps,

adding enzyme cutting sites: inserting double enzyme cutting sites into a mutation gene of the cyanohydrin lyase;

preparing a recombinant plasmid: inserting the cyanohydrin lyase mutator gene into an expression vector to obtain a recombinant plasmid;

introducing a strain: introducing the recombinant plasmid with the cyanohydrin lyase gene into a strain to obtain a recombinant expression strain;

secretion and expression of the strains: inducing the recombinant expression strain in culture solution to express and collecting enzyme solution.

As a preferable embodiment of the process for producing R-cyanohydrin lyase of the present invention, wherein: the mode of mutation treatment is error-prone PCR.

As a preferable embodiment of the process for producing R-cyanohydrin lyase of the present invention, wherein: in the added enzyme cutting sites, the double enzyme cutting sites are NdeI/HindIII.

As a preferable embodiment of the process for producing R-cyanohydrin lyase of the present invention, wherein: in the recombinant plasmid preparation, the expression vector is pET26b (+), and the N end of the expression vector contains a signal peptide pelB leader.

As a preferable embodiment of the process for producing R-cyanohydrin lyase of the present invention, wherein: the introduced strain is E.coli BL21(DE 3).

As a preferable embodiment of the process for producing R-cyanohydrin lyase of the present invention, wherein: in the secretion and expression of the strain, the culture solution is an LB culture medium.

As a preferable embodiment of the process for producing R-cyanohydrin lyase of the present invention, wherein: in the secretion and expression of the strain, induction culture is also included, when OD is in the culture medium₆₀₀After 1.0, 0.2mM IPTG was added and the temperature was maintained at 30 ℃ to induce expression for 4-5 h.

The application of the product obtained by the preparation method of the R-cyanohydrin lyase comprises the following steps: the use of said composition, comprising,

The application of the product obtained by the preparation method of the R-cyanohydrin lyase comprises the following steps: the substrate is one or two of phenethyl cyanohydrin and/or o-methyl phenethyl cyanohydrin; the MTBE solution concentration of the substrate was 8 microliters per milliliter of buffer; the volume ratio of the MTBE solution of the substrate to the enzyme solution is 2: 1.2 to 1.7; the ratio of the addition amount of the liquid hydrocyanic acid to the total volume of the MTBE solution of the substrate and the enzyme solution is 4-3.5: 1.2.

the product prepared by the preparation method of the R-cyanohydrin lyase comprises the following steps: the yield of R-phenethyl cyanohydrin and R-o-methyl phenethyl cyanohydrin of the product is more than or equal to 95 percent, and the chiral value (e.e value) is more than or equal to 99.5 percent.

The invention has the beneficial effects that:

the invention provides a preparation method and application of a novel R-cyanohydrin lyase derived from mango. Specifically, various mutated cyanohydrin lyases are obtained by constructing random mutation libraries and point saturation mutation libraries and high-throughput screening, and the yield of the synthesized R-phenethyl cyanohydrin and R-o-methyl phenethyl cyanohydrin is more than or equal to 99.5 percent, and the chiral value (e.e value) is more than or equal to 99.5 percent. The method is safe, simple and easy to operate.

Detailed Description

In order to make the aforementioned objects, features and advantages of the present invention more comprehensible, specific embodiments thereof are described in detail below with reference to examples of the specification.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.

Furthermore, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.

In the examples of the present invention, R-selective cyanohydrin lyase derived from mango (Mangifera indica) was used.

The tert-butyl methyl ether and the liquid hydrocyanic acid used in the invention are analytically pure, and other raw materials and reagents are all commercially available if no special description is provided.

Example 1

The specific principle of the design of the invention is to obtain various mutant cyanohydrin lyase by constructing random mutation libraries and point saturation mutation libraries and high-throughput screening.

The mutant source of the R-cyanohydrin lyase is wild-type cyanohydrin lyase, and the specific synthetic method comprises the following steps: adding enzyme cutting sites NdeI and HindIII into a cyanohydrin lyase wild gene shown as Seq ID No.1 or a cyanohydrin lyase mutant gene shown as Seq ID No.2, carrying out DNA enzyme cutting purification, inserting the purified cyanohydrin lyase mutant gene into an expression vector pET26b (+) to obtain a recombinant plasmid, and transforming the recombinant plasmid into E.coli BL21(DE3) to construct a recombinant expression strain. Culturing the recombinant strain in LB culture medium to OD₆₀₀And (3) after 1.0, adding 0.2mM IPTG, carrying out induction culture at 30 ℃ for 4-5 hours, centrifuging and collecting wild type or mutant cells, and carrying out ultrasonic cell lysis to obtain a crude enzyme solution.

Example 2

The preparation method of the wild-type cyanhydrin lyase mutant comprises the following steps:

the method comprises the steps of introducing random mutation and (or) introduced point saturation mutation based on enzyme protein structure/simulated substrate docking into a wild gene sequence of cyanohydrin lyase of mango (Mangifera indica) as shown in Seq ID No.1 in an error-prone PCR mode, and then carrying out high-throughput screening to obtain a wild-type mutant of the cyanohydrin lyase in the invention as shown in Seq ID No. 2.

The protein sequence after transcription and translation by a wild gene cyanohydrin lyase of mango (Mangifera indica) is shown as Seq ID No.3, the protein sequence translated by a mutant of wild-type cyanohydrin lyase is shown as Seq ID No.4, the mutant of wild-type cyanohydrin lyase has the difference that isoleucine at position 109 is mutated into methionine, asparagine at position 110 is mutated into alanine or serine, isoleucine at position 321 is mutated into threonine or alanine, proline at position 354 is mutated into isoleucine, serine at position 355 is mutated into leucine compared with the wild cyanohydrin lyase, Seq ID No.4 shows the possibility of one of the protein sequences, but the possibility of mutation of other various protein sequences has the same performance.

Except for Seq ID No.2, the R-cyanohydrin lyase mutant gene is changed into ATG at position 325-327, GCT or AGT at position 328-330, ACT or GCT at position 961-963, ATA at position 1060-1062, TTA or TTG at position 1063-1065, and any condition that any position is changed.

Example 3

The high-throughput screening of cyanohydrin enzyme comprises the following steps: at 25 ℃, 130 mu L of 100mM potassium phosphate-citric acid buffer solution with pH value of 5.0 and 20 mu L of diluted cyanohydrin lyase solution are sequentially added into a 96-well plate, and finally 50 mu L of substrate solution of phenethyl cyanohydrin or o-methyl phenethyl cyanohydrin is added, and the change of the absorbance value is read for 5 minutes at 280nm by using an enzyme-linked immunosorbent assay, wherein the change of the absorbance value represents the enzyme activity.

The substrate solution: the buffer was prepared at a concentration of 8. mu.l/ml using 100mM pH 3.5 potassium phosphate-citric acid buffer.

Example 4

Application of R-cyanohydrin lyase: the process for synthesizing R-phenethyl cyanohydrin and R-o-methyl phenethyl cyanohydrin is as follows.

15mmol of the substrate was dissolved in 2.1mL of tert-butyl methyl ether (MTBE) to give an MTBE solution of the substrate. The resultant cyanohydrin enzyme was diluted in 10mM phosphate-citric acid buffer solution to make the total volume 3.7mL, and the pH was adjusted to 3.4. And fully mixing the MTBE solution of the substrate and the enzyme solution, cooling the system to 10 ℃, stirring into emulsion, and adding 1.2ml of liquid hydrocyanic acid to start reaction.

Samples were taken at different time points and derivatized with acetic anhydride in the presence of pyridine and dichloromethane and analyzed by GC on a cyclodextrin column (CP-Chirasil-Dex CB) to obtain substrate conversion and enantiomeric values.

After the substrate conversion is finished, adding 1 time of MTBE in the volume of the water phase, extracting for three times, combining the organic phases, dehydrating by anhydrous sodium sulfate, and concentrating under reduced pressure to obtain the product. The relationship between the product yield and the product chiral value at 15mmol of substrate addition and the cyanohydrin addition is shown in Table 1.

TABLE 1 relationship between product yield, product chiral value and cyanohydrin enzyme addition

As can be seen from Table 1, the yields of the synthesized R-phenethyl cyanohydrin and R-o-methyl phenethyl cyanohydrin are both more than or equal to 99.5%, and the chiral values (e.e values) are both more than or equal to 99.5%.

The invention provides a preparation method and application of a novel R-cyanohydrin lyase derived from mango. Specifically, various mutant cyanohydrin lyase are obtained by constructing random mutation and point saturation mutation libraries and high-throughput screening, and the method is safe, simple and easy to operate.

It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.

Sequence listing

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