阅读说明：本技术 一种重组表达质粒与基因工程菌及(4s,5r)-半酯的制备方法 (Recombinant expression plasmid, genetic engineering bacterium and preparation method of (4S,5R) -half-ester ) 是由 徐毅 沈海云 徐长利 马宝娣 吴小梅 于 2019-09-23 设计创作，主要内容包括：本发明公开一种水相体系中高效绿色制备生物素中间体的方法,即采用重组微杆菌酯酶整细胞在纯水体系中催化内消旋生物素二甲酯不对称水解生成(4S,5R)-半甲酯。当底物浓度为200mM,反应时间为4h,(4S,5R)-半甲酯e.e.值大于99％,产率达100％,时空产率超过400g/L/d。该制备方法催化效率高、底物转化率高、产品光学纯度高,并且以不含有机溶剂的纯水作为反应介质,具有潜在的工业应用价值。(The invention discloses a method for efficiently and environmentally preparing a biotin intermediate in a water phase system, namely, a recombinant microbacterium esterase whole cell is adopted to catalyze meso-biotin dimethyl ester to be asymmetrically hydrolyzed in a pure water system to generate (4S,5R) -half methyl ester. When the substrate concentration is 200mM and the reaction time is 4h, the e.e. value of (4S,5R) -half methyl ester is more than 99%, the yield reaches 100%, and the space-time yield exceeds 400 g/L/d. The preparation method has high catalytic efficiency, high substrate conversion rate and high optical purity of the product, and has potential industrial application value by taking pure water without organic solvent as a reaction medium.)

1. A recombinant expression plasmid is a pET-21a plasmid and is characterized in that an esterase gene is connected to the restriction enzyme site of the pET-21a plasmid, the sequence of the esterase gene is SEQ ID NO. 1, and the recombinant expression plasmid is named as pET-21 a-estsite 01.

2. A genetically engineered bacterium, which is Escherichia coli BL21(DE3) carrying the recombinant expression plasmid pET-21a-estsit01 described in claim 1, and which is named E.coli BL21(DE3)/pET-21a-estsit 01.

3. A method for preparing a (4S,5R) -half-ester, comprising the steps of:

step 1: adding E.coli BL21(DE3)/pET21a-estsit01 thallus of claim 2 as a biocatalyst into pure water to obtain a thallus suspension; then adding a substrate biotin dimethyl ester, controlling the reaction temperature to be 30 ℃, carrying out asymmetric hydrolysis reaction, and dropwise adding 20% (w/v) ammonia water to control the pH of the reaction solution to be 7.5-8.0, wherein the reaction time is 2-24 h, so as to obtain a reaction solution;

step 2: and (2) centrifuging the reaction solution obtained in the step (1), separating cells, taking supernatant, adding 2mol/L HCl to adjust the pH to 2.0, extracting the acidizing fluid for 3 times by using equal volume of ethyl acetate, combining organic phases, drying by using anhydrous sodium sulfate, then decompressing and evaporating the organic solvent, and drying the sample in vacuum at 70 ℃ to obtain a target product, namely the (4S,5R) -half methyl ester solid.

4. The process for producing (4S,5R) -half-ester according to claim 3, wherein the amounts of wet cell weight, biotin dimethyl ester and pure water used in step 1 are as follows: biotin dimethyl ester: 10-30 g of pure water: 1-2L of 100-300 mmol.

5. The process for the preparation of (4S,5R) -half-ester according to claim 3, wherein the centrifugation speed in step 2 is 1000rpm and the centrifugation time is 20 min.

Technical Field

The invention belongs to the technical field of biological engineering, and relates to a method for preparing (4S,5R) -half methyl ester in a pure water phase system by using esterase-producing recombinant escherichia coli BL21(DE3)/pET21 a-estist 01 whole cells as a biocatalyst.

Background

D-biotin belongs to the B vitamins, also known as Vitamin B7 or Vitamin H, and is one of water-soluble vitamins necessary for maintaining normal physiological functions in animal bodies. If the human body lacks biotin, the nutrient diseases such as slow growth and the like, dermatitis, inappetence, nausea, vomiting, alopecia, weight reduction, anemia, blood cholesterol increase, mental depression and the like can be caused, so the D-biotin is an important medical product and feed additive, and the market demand is huge.

The (4S,5R) -Biotin chiral semi-methyl ester is one of key chiral intermediates for synthesizing D-Biotin (D-Biotin, B vitamins), and the structural formula (1) is shown as follows:

the current synthesis methods of (4S,5R) -biotin chiral half methyl ester can be divided into two types.

One is a chemical resolution method. The Deng group takes anhydride as a substrate, chiral Lewis base (cinchona alkaloid dimer DHQD-PHN) as a catalyst to carry out asymmetric alcoholysis reaction on inner racemic anhydride to obtain (4S,5R) -monoester, and the e.e value is 93%. (Choi C, Tian S K, Deng L. Synthesis,2001, (11): 1737-1741.). Then, another catalyst was developed by chen fennel topic group, using the derivative of chloramphenicol byproduct [ (1S,2S) -1- (4-nitrophenyl) -2-N, N-dimethylamino-3-triphenylmethoxy-1-propanol ] as a catalyst, using acid anhydride as a substrate, performing asymmetric alcoholysis in an organic solvent to obtain chiral half ester, and then performing reduction and ring closure to obtain chiral lactone, wherein the yield is 88%, and the e.e value is as high as 98.5%. (Bell, Wuxuefen, Chenxiang. organic chemistry, 2012, 32: 1792-. However, these methods have the disadvantages of expensive catalyst and harsh reaction conditions.

In 1982, Tsuchihashi used a biocatalytic approach for the first time. Takes pig liver esterase as a catalyst, obtains chiral half methyl ester in an organic solvent-water two-phase system through catalysis, and obtains lactone with the e.e value of 75 percent through reduction and ring closure (Iriuchijima S, Hasegawa K, Tsuchihashi G.Agricultural and biological chemistry 1982,46(7): 1907-. Later, Chen et al utilized polymer immobilized pig liver esterase PLE to catalyze the selective hydrolysis of meso-biotin dimethyl ester in a methanol-water mixed solvent system to give biotin hemi-methyl ester in 90% yield with an e.e value of 91%. However, the use of PLE as a catalyst is limited in its further industrial application because of its low selectivity and high cost (Chen F E, Chen X X, Dai H F, et al, advanced Synthesis & Catalysis,2005,347(4): 549-.

In 2010, Xuyi et al screened a Microbacterium chocolate (Microbacterium chocolateum SIT101), the esterase produced by the strain can catalyze meso-biotin dimethyl ester to generate (4S,5R) -half methyl ester in a water-organic mixed solvent system in a highly selective manner, the yield is 95%, and the e.e value is more than 99% (Xuyi et al, a Microbacterium chocolate and a method for preparing (4S,5R) -half ester [ P ]. Chinese patent No. CN102120977A, 2010). In the further work, the esterase from the strain is expressed heterologously in escherichia coli, and escherichia coli E.coli BL21(DE3) -pET21a-estsit01 capable of highly producing recombinant esterase is constructed.

The chemical or biological synthesis method for preparing the biotin intermediate (4S,5R) -half-ester which is disclosed at present adopts an organic solvent or an organic solvent-water mixed solvent system, and with the attention on environmental protection, a more green biocatalytic reaction system without organic solvent addition needs to be developed to realize the efficient green preparation of the (4S,5R) -half-ester.

Disclosure of Invention

The technical problem to be solved by the invention is a recombinant esterase engineering bacterium from Microbacterium chocolates, a preparation method of the recombinant engineering bacterium, the esterase and application of the engineering bacterium in selective hydrolysis of dimethyl biotin. Because the constructed genetic engineering strain for efficiently expressing the recombinant esterase has poor stability in a reaction system containing an organic solvent, the problems of low substrate concentration, low catalytic efficiency, easy inactivation of a catalyst, environment pollution caused by the organic solvent, increased downstream environmental-friendly treatment cost and the like exist in the reaction process. By establishing the process for hydrolyzing the biotin dimethyl ester in a pure water phase system, the problem of catalyst deactivation can be relieved, the (4S,5R) -half methyl ester with high optical purity can be obtained with high yield, the preparation process has short reaction time and mild conditions, pure water is selected as a reaction medium, and the preparation process is environment-friendly, economic and low in production cost. Meets the development requirements of green pharmaceutical chemicals in the future.

In order to achieve the purpose, one of the technical schemes adopted by the invention is as follows:

a recombinant expression plasmid is a pET-21a plasmid and is characterized in that an esterase gene is connected to the restriction enzyme site of the pET-21a plasmid, the sequence of the esterase gene is SEQ ID NO. 1, and the recombinant expression plasmid is named as pET-21 a-estsite 01.

The construction method of the recombinant expression plasmid comprises the following steps: a pair of specific primers (SEQ ID NO:2, F5 '-GGAATTCATGACCCTGTTCGACGGCATCACGTCT-3'; SEQ ID NO:3, R5 '-CCCAAGCTTGTCGGCGGAGCGGATGATGATCGCCTC-3') are designed according to a target gene sequence, PCR amplification is carried out by taking an extracted genome DNA solution as a template, a PCR product and a vector pET-21a are subjected to double digestion by restriction enzymes EcoRI and HindIII respectively to form complementary cohesive ends, and then the complementary cohesive ends are connected by T4 DNA ligase to form a recombinant expression plasmid pET-21 a-estsite 01 containing an esterase gene.

The second technical scheme adopted by the invention is as follows:

a recombinant expression transformant comprising a host microorganism carrying the recombinant expression vector.

Preferably, the host microorganism is any host microorganism that is conventional in the art, provided that the recombinant expression vector is stably self-replicating and the carried esterase gene can be efficiently expressed. The preparation method of the recombinant expression transformation comprises the following steps: the recombinant expression vector is transformed into a host microorganism.

More preferably, the host microorganism is Escherichia coli (E.coli),

further, the host microorganism is Escherichia coli BL21(DE 3).

A genetically engineered bacterium is characterized in that the Escherichia coli BL21(DE3) carrying the recombinant expression plasmid pET-21a-estsit01 is named E.coli BL21(DE3)/pET-21a-estsit 01.

The preparation method of the genetic engineering bacteria comprises the following steps: the recombinant expression plasmid pET-21a-estsit01 is transferred into E.coli BL21(DE3), and the preferred genetic engineering strain of the invention, namely E.coli BL21(DE3)/pET-21a-estsit01, can be obtained. The transformation method is conventional in the art and includes chemical transformation, thermal shock or electrical transformation.

The third technical scheme adopted by the invention is as follows: the recombinant expression transformant or the genetically engineered bacterium is used as a biocatalyst to selectively hydrolyze the biotin dimethyl ester.

The invention provides a preparation method of (4S,5R) -half ester, which is characterized by comprising the following steps:

step 1: adding the E.coli BL21(DE3)/pET21a-estsit01 thallus serving as a biocatalyst into pure water to obtain a thallus suspension; then adding a substrate biotin dimethyl ester, controlling the reaction temperature to be 30 ℃, carrying out asymmetric hydrolysis reaction, and dropwise adding 20% (w/v) ammonia water to control the pH of the reaction solution to be 7.5-8.0, wherein the reaction time is 2-24 h, so as to obtain a reaction solution;

step 2: and (2) centrifuging the reaction solution obtained in the step (1), separating cells, taking supernatant, adding 2mol/L HCl to adjust the pH to 2.0, extracting the acidizing fluid for 3 times by using equal volume of ethyl acetate, combining organic phases, drying by using anhydrous sodium sulfate, then decompressing and evaporating the organic solvent, and drying the sample in vacuum at 70 ℃ to obtain a target product, namely the (4S,5R) -half methyl ester solid.

Preferably, in the step 1, the wet weight of the bacterial cells, the amounts of the biotin dimethyl ester and the pure water are as follows: biotin dimethyl ester: the amount of pure water is 10-30 g, 100-300 mmol, 1-2L.

Preferably, the centrifugation speed in the step 2 is 1000rpm, and the centrifugation time is 20 min.

Compared with the prior art, the invention has the beneficial effects that:

(1) the invention provides a preparation process of a biotin intermediate in an aqueous phase system, which is characterized in that (4S,5R) -half methyl ester is generated by carrying out asymmetric hydrolysis reaction on self-constructed recombinant Escherichia coli E.coli BL21(DE3)/pET21 a-estsite 01 cells catalyzing biotin dimethyl ester, and pure water is used as a reaction system. Solves the problems of poor enzyme stability, low catalytic efficiency, environmental pollution and the like caused by adding an organic solvent. The space-time yield is greater than 400g/L/d at a substrate concentration of 200 mM. The conversion rate and the optical purity in the reaction process are high.

(2) According to the preparation process of the biotin intermediate in the water phase system, the used biocatalyst Escherichia coli E.coli BL21(DE3)/pET21 a-estsite 01 is independently constructed in the laboratory, the culture is convenient, the raw materials are cheap, and pure water is used as a reaction medium in the reaction process, so that the use of an organic solvent is avoided, and the preparation process is green, environment-friendly, economical, good in enzyme stability, high in catalysis efficiency and has potential industrial application value.

Drawings

FIG. 1 shows the asymmetric synthesis of biotin intermediate (4S,5R) -hemi-methyl ester by enzyme catalysis in a pure water reaction system.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.

The material sources in the following examples are:

the recombinant genetically engineered bacterium E.coli BL21(DE3)/pET21a-estsit01 is constructed in the early stage of a laboratory. Amplifying a target gene by using a conventional PCR technology, connecting the target gene with a vector pET-21a by using enzyme digestion and connection technologies, transforming a connecting solution into Escherichia coli E.coli DH5 alpha, culturing positive clones after amplification, and extracting plasmids to obtain a recombinant plasmid pET-21 a-estsite 01. And transforming the recombinant plasmid into a competent cell of Escherichia coli E.coli BL21(DE3), and selecting positive clones to obtain a recombinant expression transformant E.coli BL21(DE3)/pET21 a-estsite 01.

The reagents used in the examples of the present invention were either analytically pure or HPLC pure.

In the embodiments of the present invention, the conversion rate of asymmetric hydrolysis reaction of biotin dimethyl ester and the like were measured by Shimadzu high performance liquid chromatograph SPD-20A from Shimadzu instruments, and the chromatographic column used was a reverse chromatographic column C18 column (Diamonsil plus,4.6 mm. times.250 mm. times.5 μm, Beijing Dike Mac technologies Co., Ltd.); the specific analysis conditions were as follows:

the mobile phase ratio is methanol: water 65: 35(v/v), a flow rate of 1.0mL/min, a detection wavelength of 210nm, and a column temperature of 20 ℃.

The conversion of the product (4S,5R) -hemi methyl ester was calculated as follows:

conversion W ═ 0.60 × (X-X)₀)×B/C×100％

Wherein X represents the peak area of the product in the reaction solution, and X represents₀Represents the area of the peak of spontaneous hydrolysis of the substrate dimethyl ester in the blank reaction solution, C represents the concentration of the substrate dimethyl ester, and B represents the dilution factor of the reaction solution. 0.60 is the coefficient constant of the peak area and concentration of the product.

The yield of (4S,5R) -hemi-methyl ester in each example of the invention was calculated from the mass of the actual product (4S,5R) -hemi-methyl ester divided by the mass of the theoretical product (4S,5R) -hemi-methyl ester;

the definition of the unit of cell enzyme activity referred to in the examples of the present invention is: the amount of enzyme required to catalyze the formation of 1.0. mu. mol of (4S,5R) -hemi methyl ester per minute at 30 ℃ and pH7.5 is 1 activity unit, i.e., 1U.

The detection of the optical purity value of the product (4S,5R) -half methyl ester adopts Shimadzu high performance liquid chromatograph SPD-20A, which is from Shimadzu instruments; the column used was a chiral column (4.6 mm. times.250 mm. times.550, CHIRALCEL OJ-H, available from Dailuo pharmaceutical chiral technology (Shanghai) Co., Ltd.). The specific analysis conditions were as follows:

the mobile phase is n-hexane: isopropyl alcohol: trifluoroacetic acid 97:3:0.2(v/v), flow rate 0.5mL/min, detection wavelength 220nm, column temperature 20 ℃.