A recombinant expression plasmid, genetically engineered bacteria and preparation method of (4s, 5r)-half ester

Abstract

This invention discloses a highly efficient and green method for preparing biotin intermediates in an aqueous system. Specifically, it utilizes recombinant microbial esterase to catalyze the asymmetric hydrolysis of racemic dimethyl biotin in a pure water system to generate (4S,5R)-hemimethyl ester. With a substrate concentration of 200 mM and a reaction time of 4 h, the (4S,5R)-hemimethyl ester exhibits an ee value greater than 99%, a yield of 100%, and a space-time yield exceeding 400 g / L / d. This preparation method demonstrates high catalytic efficiency, high substrate conversion, and high product optical purity. Furthermore, it uses pure water free of organic solvents as the reaction medium, making it potentially valuable for industrial applications.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology and relates to a method for preparing (4S,5R)-hemimethyl ester in a pure aqueous system using whole cells of recombinant Escherichia coli BL21(DE3) / pET21a-estsit01 with esterase as a biocatalyst. Background Technology

[0002] D-Biotin, also known as Vitamin B7 or Vitamin H, is a B vitamin and one of the essential water-soluble vitamins for maintaining normal physiological functions in animals. Biotin deficiency in humans can lead to nutritional disorders such as slow growth, dermatitis, loss of appetite, nausea, vomiting, hair loss, weight loss, anemia, elevated blood cholesterol, and depression. Therefore, D-Biotin is an important pharmaceutical product and feed additive with a huge market demand.

[0003] (4S,5R)-Biotin chiral hemimethyl ester is one of the key chiral intermediates in the synthesis of D-Biotin (a B vitamin), and its structural formula (1) is shown below:

[0004]

[0005] Currently, there are two methods for synthesizing (4S,5R)-biotin chiral hemimethyl ester.

[0006] One method is chemical resolution. Deng's group used acid anhydrides as substrates and chiral Lewis bases (cinchona alkaloid dimer DHQD-PHN) as catalysts to perform asymmetric alcoholysis of racemic acid anhydrides to obtain (4S,5R)-monoesters with an ee value of 93%. (Choi C, Tian SK, Deng L. Synthesis, 2001, (11): 1737-1741.). Subsequently, Chen Fen'er's group developed another catalyst, using a derivative of chloramphenicol byproduct [(1S,2S)-1-(4-nitrophenyl)-2-N,N-dimethylamino-3-triphenylmethoxy-1-propanol] as a catalyst, and acid anhydrides as substrates to perform asymmetric alcoholysis in organic solvents to obtain chiral half-esters, followed by reduction and cyclization to obtain chiral lactones, with a yield of 88% and an ee value as high as 98.5%. (Zhong Zheng, Wu Xuefen, Chen Fen'er. Organic Chemistry, 2012, 32: 1792-1802). However, these methods have drawbacks such as expensive catalysts and harsh reaction conditions.

[0007] In 1982, Tsuchihashi first employed a biocatalytic method. Using porcine liver esterase as a catalyst, he catalyzed the production of chiral hemimethyl esters in an organic solvent-water two-phase system, followed by reduction and cyclization to obtain a lactone with an ee value of 75% (Iriuchijima S, Hasegawa K, Tsuchihashi G. Agricultural and Biological Chemistry. 1982, 46(7): 1907-1910). Later, Chen et al. used polymer-immobilized porcine liver esterase (PLE) to catalyze the selective hydrolysis of meso-biotin dimethyl esters to obtain biotin hemimethyl esters in a methanol-water mixed solvent system, with a yield of 90% and an ee value of 91%. However, the low selectivity and high cost of the PLE catalyst limited its further industrial application (Chen FE, Chen XX, Dai HF, et al. Advanced Synthesis & Catalysis, 2005, 347(4): 549-554.).

[0008] In 2010, Xu Yi et al. screened a strain of *Microbacterium chocolatum* (SIT101). The esterase produced by this strain can selectively catalyze the asymmetric hydrolysis of racemic biotin dimethyl ester to (4S,5R)-hemimethyl ester in a water-organic mixed solvent system, with a yield of 95% and an ee value greater than 99% (Xu Yi et al. A *Microbacterium chocolatum* and a method for preparing (4S,5R)-hemimethyl ester [P]. Chinese Patent. CN102120977A, 2010). In further work, the esterase from this strain was heterologously expressed in *E. coli*, constructing a high-yield recombinant esterase-producing *E. coli* BL21(DE3)-pET21a-estsit01.

[0009] Currently, all publicly available chemical or biosynthetic methods for preparing biotin intermediate (4S,5R)-half esters employ organic solvents or organic solvent-water mixed solvent systems. With increasing emphasis on environmental protection, it is necessary to develop a greener biocatalytic reaction system without the addition of organic solvents to achieve efficient and green preparation of (4S,5R)-half esters. Summary of the Invention

[0010] The technical problem this invention aims to solve is the recombinant esterase engineered strain derived from *Microbacterium chocolateii*, including its preparation method, the esterase, and its application in the selective hydrolysis of biotin-2-methyl ester. Because the constructed genetically engineered strains that efficiently express recombinant esterase exhibit poor stability in reaction systems containing organic solvents, the reaction process suffers from low substrate concentration, low catalytic efficiency, and easy catalyst deactivation. Furthermore, organic solvents pollute the environment, leading to increased downstream environmental treatment costs. By establishing a process for the hydrolysis of biotin-2-methyl ester in a pure aqueous system, the problem of catalyst deactivation can be alleviated, achieving high-yield, high-optical-purity (4S,5R)-hemimethyl ester. Moreover, this preparation process features short reaction time, mild conditions, and the use of pure water as the reaction medium, making it environmentally friendly, economical, and with low production costs. This aligns with the future development requirements of green pharmaceutical chemistry.

[0011] To achieve the above objectives, one of the technical solutions adopted by the present invention is as follows:

[0012] A recombinant expression plasmid, wherein the plasmid is pET-21a plasmid, characterized in that an esterase gene is linked to the restriction enzyme site of the pET-21a plasmid, the esterase gene sequence is SEQ ID NO:1, and the recombinant expression plasmid is named pET-21a-estsit01.

[0013] The construction method of the above recombinant expression plasmid includes: designing a pair of specific primers (SEQ ID NO:2, F5'-GGAATTCATGACCCTGTTCGACGGCATCACGTCT-3'; SEQ ID NO:3, R5'-CCCAAGCTTGTCGGCGGAGCGGATGATGATCGCCTC-3') based on the target gene sequence, performing PCR amplification using the extracted genomic DNA solution as a template, and then double-digesting the PCR product and the vector pET-21a with restriction endonucleases EcoRI and HindIII, respectively, to form complementary sticky ends, and then ligating them with T4 DNA ligase to form the recombinant expression plasmid pET-21a-estsit01 containing the esterase gene.

[0014] The second technical solution adopted by this invention is:

[0015] A recombinant expression transformant, characterized in that it comprises a host microorganism, wherein the host microorganism carries the aforementioned recombinant expression vector.

[0016] Preferably, the host microorganism is any conventional host microorganism in the art, as long as it can stably replicate the recombinant expression vector and effectively express the esterase gene it carries. The preparation method of the recombinant expression transformation includes: transforming the recombinant expression vector as described above into a host microorganism.

[0017] More preferably, the host microorganism is Escherichia coli (E. coli).

[0018] Furthermore, the host microorganism is Escherichia coli BL21(DE3).

[0019] A genetically engineered bacterium, characterized in that it is Escherichia coli BL21(DE3) carrying the above-mentioned recombinant expression plasmid pET-21a-estsit01, named E. coli BL21(DE3) / pET-21a-estsit01.

[0020] The method for preparing the genetically engineered bacteria includes: transforming the aforementioned recombinant expression plasmid pET-21a-estsit01 into E. coli BL21(DE3) to obtain the preferred genetically engineered strain of the present invention, namely E. coli BL21(DE3) / pET-21a-estsit01. The transformation method is a conventional method in the art, including chemical transformation, heat shock, or electroporation.

[0021] The third technical solution adopted in this invention is: using the above-mentioned recombinant expression transformant or genetically engineered bacteria as a biocatalyst to selectively hydrolyze biotin dimethyl ester.

[0022] This invention provides a method for preparing (4S,5R)-half esters, characterized by comprising the following steps:

[0023] Step 1: The above E. coli BL21(DE3) / pET21a-estsit01 bacterial cells were used as a biocatalyst and added to pure water to obtain a bacterial cell suspension; then, the substrate dimethyl biotin was added, and the reaction temperature was controlled at 30℃ to carry out an asymmetric hydrolysis reaction. The pH of the reaction solution was controlled between 7.5 and 8.0 by adding 20% ​​(w / v) ammonia water dropwise, and the reaction time was 2 to 24 hours to obtain the reaction solution;

[0024] Step 2: Centrifuge the reaction solution obtained in Step 1, separate the cells and take the supernatant, add 2 mol / L HCl to adjust the pH to 2.0, extract the acidified solution three times with an equal volume of ethyl acetate, combine the organic phases and dry with anhydrous sodium sulfate, then evaporate the organic solvent under reduced pressure, and dry the sample under vacuum at 70℃ to finally obtain the target product, namely (4S,5R)-hemimethyl ester solid.

[0025] Preferably, in step 1, the amounts of bacterial cell wet weight, biotin dimethyl ester, and purified water are calculated according to the following ratio: bacterial cell wet weight: biotin dimethyl ester: purified water = 10-30g: 100-300mmol: 1-2L.

[0026] Preferably, the centrifugation speed in step 2 is 1000 rpm and the centrifugation time is 20 min.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] (1) This invention provides a process for preparing biotin intermediates in an aqueous system. The process involves the asymmetric hydrolysis of dimethyl biotin to generate (4S,5R)-hemimethyl ester using a self-constructed recombinant *E. coli* BL21(DE3) / pET21a-estsit01 cells. Pure water is used as the reaction system. This process solves the problems of poor enzyme stability, low catalytic efficiency, and environmental pollution caused by the addition of organic solvents. At a substrate concentration of 200 mM, the space-time yield is greater than 400 g / L / d. The reaction process exhibits high conversion rate and high optical purity.

[0029] (2) The preparation process of biotin intermediate in aqueous system provided by the present invention is convenient to cultivate, the raw materials are inexpensive, and pure water is used as the reaction medium in the reaction process to avoid the use of organic solvents. It is green, environmentally friendly and economical, has good enzyme stability and high catalytic efficiency, and has potential industrial application value. Attached Figure Description

[0030] Figure 1 It is an enzyme-catalyzed asymmetric synthesis of biotin intermediate (4S,5R)-hemimethyl ester in a pure water reaction system. Detailed Implementation

[0031] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0032] The sources of the materials in the following examples are:

[0033] The recombinant genetically engineered bacterium *E. coli* BL21(DE3) / pET21a-estsit01 was constructed in the laboratory previously. The target gene was amplified using conventional PCR techniques, and then ligated to the vector pET-21a using enzyme digestion and ligation techniques. The ligation solution was transformed into *E. coli* DH5α, and after amplification, positive clones were selected for culture, and the plasmid was extracted to obtain the recombinant plasmid pET-21a-estsit01. This recombinant plasmid was then transformed into *E. coli* BL21(DE3) competent cells, and positive clones were selected to obtain the recombinant expression transformant *E. coli* BL21(DE3) / pET21a-estsit01.

[0034] All reagents used in the embodiments of this invention are of analytical grade or HPLC grade.

[0035] In the various embodiments of the present invention, the conversion rate of biotin dimethyl ester asymmetric hydrolysis reaction was measured using a Shimadzu SPD-20A high-performance liquid chromatograph (Shimadzu Instruments Co., Ltd.), with a reverse-phase C18 column (Diamonsil plus, 4.6 mm × 250 mm × 5 μm, Beijing Dicoma Technology Co., Ltd.); the specific analytical conditions are as follows:

[0036] The mobile phase ratio was methanol:water = 65:35 (v / v), the flow rate was 1.0 mL / min, the detection wavelength was 210 nm, and the column temperature was 20 °C.

[0037] The conversion rate of the product (4S,5R)-hemimethyl ester was calculated as follows:

[0038] Conversion rate W = 0.60 × (X - X0) × B / C × 100%

[0039] Where X represents the peak area of ​​the product in the reaction solution, X0 represents the peak area of ​​the substrate dimethyl ester spontaneously hydrolyzed 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 a constant coefficient between the product peak area and concentration.

[0040] In each embodiment of the present invention, the yield of (4S,5R)-hemimethyl ester is calculated by dividing the actual mass of (4S,5R)-hemimethyl ester produced by the theoretical mass of (4S,5R)-hemimethyl ester produced.

[0041] In this embodiment of the invention, the cell enzyme activity unit is defined as: the amount of enzyme required to catalyze the production of 1.0 μmol of (4S,5R)-hemimethyl ester per minute at 30°C and pH 7.5 is 1 activity unit, or 1 U.

[0042] The optical purity of the product (4S,5R)-hemimethyl ester was determined using a Shimadzu SPD-20A high-performance liquid chromatograph (HPLC), manufactured by Shimadzu Instruments Co., Ltd. A chiral column (4.6 mm × 250 mm × 550 mm, CHIRALCEL OJ-H, purchased from Daicel Chiral Technology (Shanghai) Co., Ltd.) was used. Specific analytical conditions are as follows:

[0043] The mobile phase was hexane:isopropanol:trifluoroacetic acid = 97:3:0.2 (v / v), the flow rate was 0.5 mL / min, the detection wavelength was 220 nm, and the column temperature was 20 °C.

[0044] Example 1

[0045] Construction of recombinant plasmids and recombinant engineered bacteria: A pair of specific primers (F5'-GGAATTCATGACCCTGTTCGACGGCATCACGTCT-3'; R5'-CCCAAGCTT GTCGGCGGAGCGGATGATGATCGCCTC-3') were designed based on the target gene for PCR amplification. The PCR system consisted of: 25 μl of 2×pfu PCR Master Mix, 0.5 μl each of the upstream and downstream primers (0.25 μmol / L), 1 μl of DNA template (approximately 0.05 μg of plasmid pET21a-estsit01), 20 μl of ddH2O, and 3 μl of dimethyl sulfoxide (DMSO). PCR reaction procedure: template denaturation at 95℃ for 8 min; template denaturation at 95℃ for 30 s, annealing at 55℃ for 45 s, primer extension at 72℃ for 150 s, this process was repeated 30 times; primer extension along the template continued at 72℃ for 7 min. The sequence of the esterase gene is shown in SEQ ID NO:1.

[0046] After purification and recovery of the PCR products by agarose gel electrophoresis, they were double-digested with EcoRI and HindIII for 30 min. The digested products were ligated using T4 DNA ligase, and the ligation solution was transformed into E. coli DH5α competent cells. Positive clones were selected, and a large number of recombinant expression vector plasmids pET21a-estsit01 were obtained by plasmid extraction.

[0047] The recombinant plasmid pET21a-estsit01 was transformed into E. coli BL21(DE 3) competent cells, and positive clones were selected to obtain the recombinant expression transformant E. coli BL21(DE3) / pET21a-estsit01, which was used for subsequent induction of expression. The recombinant expression transformant E. coli BL21(DE3) / pET21a-estsit01 is E. coli BL21(DE3) carrying the aforementioned recombinant expression vector pET21a-estsit01.

[0048] Example 2

[0049] Culture of E. coli BL21(DE3) / pET21a-estsit01 cells:

[0050] Seed culture: Take one loopful of Escherichia coli BL21(DE3) / pET21a-estsit01 slant culture stored at 4℃ and inoculate it into a 250mL shake flask containing 50mL seed culture medium. Control the temperature of the shaker to 30℃ and the rotation speed to 180rpm for 12h to obtain the seed culture.

[0051] The seed culture medium contained, per liter, 10g sodium chloride, 10g tryptone, 5g yeast extract, with an initial pH of 7.0, and the remainder being water.

[0052] Shake-flask fermentation: Inoculate the seed culture into the fermentation medium. The amount of seed culture used for inoculation should be based on the OD of the fermentation broth after inoculation. 600 =0.1 ratio calculation; after inoculation, control the shaker temperature at 30℃ and the rotation speed at 180rpm for fermentation. After culturing for 2 hours, add IPTG to a final concentration of 0.2mM and continue fermentation for 8 hours. Centrifuge the fermentation broth, wash the cell pellet twice with physiological saline to obtain wet cells.

[0053] The fermentation medium, calculated per liter, contains 25g of glycerol, 15g of yeast extract, 10g of sodium chloride, 2g of K2HPO4, 1.2g of MgSO4·7H2O, with an initial pH of 7.2, and the remainder is water.

[0054] Example 3

[0055] Take 0.1 g of wet E. coli BL21(DE3) / pET21a-estsit01 cells obtained in Example 2, suspend them in 9.0 mL of Tris-HCl buffer solution (0.2 M, pH 7.5), add 1 mmol of biotin dimethyl ester, and then add 1.0 mL of ethanol. The reaction mixture is shaken in a constant temperature shaker at 30 °C and 150 rpm, and samples are taken intermittently. Extract with ethyl acetate, and analyze the enantiomeric excess and yield using chiral liquid chromatography. After 24 hours of reaction, the yield of (4S,5R)-hemimethyl ester is 65%, and the enantiomeric excess (ee) is greater than 99%.

[0056] Example 4

[0057] 0.1 g of wet E. coli BL21(DE3) / pET21a-estsit01 cells obtained in Example 2 were suspended in 8.0 mL of Tris-HCl buffer solution (0.2 M, pH 7.5). 1 mmol of biotin dimethyl ester was added, followed by 2.0 mL of dimethyl sulfoxide. The reaction mixture was shaken intermittently in a constant temperature shaker at 30 °C and 150 rpm. Samples were taken and extracted with ethyl acetate. The enantiomeric excess and yield were analyzed by chiral liquid chromatography. After 24 hours of reaction, the yield of (4S,5R)-hemimethyl ester was 55%, and the enantiomeric excess (ee) was greater than 99%.

[0058] Example 5

[0059] 0.1 g of wet E. coli BL21(DE3) / pET21a-estsit01 cells obtained in Example 2 were suspended in 10.0 mL of Tris-HCl buffer solution (0.2 M, pH 7.5). 1 mmol of biotin dimethyl ester was added, and the mixture was shaken intermittently in a constant temperature shaker at 30 °C and 150 rpm. The mixture was extracted with ethyl acetate, and the enantiomeric excess and yield were analyzed by chiral liquid chromatography. After 24 hours of reaction, the yield of (4S,5R)-hemimethyl ester was 70%, and the enantiomeric excess (ee) was greater than 99%.

[0060] Example 6

[0061] 0.1 g of wet E. coli BL21(DE3) / pET21a-estsit01 cells obtained in Example 2 were suspended in 10.0 mL of pure water. 1 mmol of dimethyl ester substrate was added. The reaction mixture was shaken on a constant-temperature shaker at 30°C and 150 rpm. Samples were taken periodically during the reaction, and the conversion rate was analyzed by HPLC. The pH was adjusted with 20% (v / v) ammonia to maintain the pH of the reaction solution between 7.5 and 8.0. The reaction was considered complete when the pH no longer changed. The enantiomeric excess and yield of the product were analyzed by chiral liquid chromatography. After 12 hours of reaction, the conversion rate of (4S,5R)-hemimethyl ester reached 90%, the enantiomeric excess (ee) was greater than 99%, and the space-time yield was 66.24 g / L / d.

[0062] Example 7

[0063] 0.2 g of wet E. coli BL21(DE3) / pET21a-estsit01 cells obtained in Example 2 were suspended in 10.0 mL of pure water. 1 mmol of dimethyl ester substrate was added. The reaction mixture was shaken on a constant temperature shaker at 30 °C and 150 rpm. Samples were taken periodically during the reaction, and the conversion rate was analyzed by HPLC. The pH was adjusted with 20% (v / v) ammonia to maintain the pH of the reaction solution between 7.5 and 8.0. The reaction was considered complete when the pH no longer changed. The enantiomeric excess and yield of the product were analyzed by chiral liquid chromatography. After 2 hours of reaction, the conversion of (4S,5R)-hemimethyl ester reached 100%, the enantiomeric excess (ee) was greater than 99%, and the space-time yield was 443 g / L / d.

[0064] Example 8

[0065] 0.2 g of wet *E. coli* BL21(DE3) / pET21a-estsit01 cells obtained in Example 2 were suspended in 10.0 mL of pure water. 2 mmol of dimethyl ester substrate was added. The reaction mixture was reacted on a shaker at 30 °C and 150 rpm. Samples were taken periodically during the reaction, and the conversion rate was analyzed by HPLC. The pH was adjusted with 20% (w / v) ammonia to maintain the pH of the reaction solution between 7.5 and 8.0. The reaction was considered complete when the pH no longer changed. The enantiomeric excess and yield of the product were analyzed by chiral liquid chromatography. After 4 hours of reaction, the conversion of (4S,5R)-hemimethyl ester reached 100%, the enantiomeric excess (ee) was greater than 99%, and the space-time yield was 442 g / L / d.

[0066] Example 9

[0067] 0.2 g of wet *E. coli* BL21(DE3) / pET21a-estsit01 cells obtained in Example 2 were suspended in 10.0 mL of pure water. 3 mmol of dimethyl ester substrate was added. The reaction mixture was shaken on a constant-temperature shaker at 30°C and 150 rpm. Samples were taken periodically during the reaction, and the conversion rate was analyzed by HPLC. The pH was adjusted with 20% (w / v) ammonia to maintain the pH of the reaction solution between 7.5 and 8.0. The reaction was considered complete when the pH no longer changed. The enantiomeric excess and yield of the product were analyzed by chiral liquid chromatography. After 6 hours of reaction, the reaction ceased, the conversion of (4S,5R)-hemimethyl ester reached 83%, and the enantiomeric excess (ee) was greater than 99%.

[0068] Example 10

[0069] Take 0.7 g of wet E. coli BL21(DE3) / pET21a-estsit01 cells prepared in Example 2, suspend them in 40 mL of pure water, add 2 g (5 mmol) of dimethyl ester substrate, and place in a shaker at 30 °C and 150 rpm for whole-cell catalytic reaction. During the reaction, take samples periodically and adjust the pH with 20% (v / v) ammonia to maintain the pH between 7.5 and 8.0 until the reaction pH no longer decreases, at which point the reaction is terminated.

[0070] The resulting reaction solution was centrifuged (1000 rpm, 20 min), cells were separated, and the supernatant was collected. The pH was adjusted to 2.0 with 2 mol / L HCl, and the acidified solution was extracted three times with an equal volume of ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The organic solvent was then recovered under reduced pressure. The residue was dried under vacuum at 70 °C to obtain the target product, (4S,5R)-hemimethyl ester, as a white powder (1.76 g), with a separation yield of 91.5%. The melting point of (4S,5R)-hemimethyl ester was determined to be 149-151 °C, and the ee value was 99.1%. SEQUENCE LISTING <110> Shanghai University of Applied Technology <120> A method for preparing a recombinant expression plasmid, genetically engineered bacteria, and (4S, 5R)-half ester. <130> BCN1192403 <160> 3 <170> PatentIn version 3.5 <210> 1 <211> 1110 <212> DNA <213> Artificial Sequence <220> <223> Esterase gene <400> 1 atgaccctgt ttgatggcat tacctctcgt attgtggata ccgatcgcct gaccgttaat 60 attctggaac gcgcagcaga tgatccgcag accccgccgg atcgtaccgt tgtgtttgtt 120 catggtaatg tgtctagcgc cctgttttgg caggaaatta tgcaggatct gccgagcgat 180 ttacgcgcca ttgccgtgga tttacgcggc tttggcggct ctgaacatgc cccggttgat 240 gcaacccgcg gcgttcgtga tttttcagat gatttacatg ccaccttaga agccttagat 300 attccggttg cccatttagt gggctggagt atgggtggcg gcgtggttat gcagtatgca 360 ctggatcatc cggttctgtc actgacctta cagtctccgg ttagtccgta tggctttggc 420 ggtacccgtc gcgatggtag tcgcttaacc gatgatgatg caggctgtgg tggcggcggt 480 gccaatccgg attttattca gcgcttaatt gatcatgata ccagcgatga tgcacagacc 540 tctccgcgta gcgtgtttcg cgcaggctat gttgcctcag attataccac cgatcatgaa 600 gatgtttggg ttgaatcaat gttaaccacc tcaaccgccg atggtaatta tccgggtgat 660 gccgttccga gcgataattg gccgggcttt gccgcaggtc gtcatggtgt gctgaatacg 720 atggccccgc agtattttga tgtgtcaggc attgtggatt tagccgaaaa accgccgatt 780 ctgtggattc atggtaccgc agatgcaatt gtgagcgatg cctcctttta tgatctgaat 840 tatctgggcc agttaggcat tgttccgggt tggccgggcg aagatgttgc cccggcacag 900 gaaatggtga gtcagacccg tgatgtgctg ggtcgctatg cagcaggcgg tggtaccgtg 960 accgaagttg ccgttgaagg tgcaggtcat agtgcacatc tggaacgtcc ggcagtgttt 1020 cgtcatgcac tgctggaaat tattggctat gtgggcgcag cagccgatcc ggccccgccg 1080 accgaagcca ttattattcg tagcgcagat 1110 <210> 2 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 2 ggaattcatg accctgttcg acggcatcac gtct 34 <210> 3 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Reversed primer <400> 3 cccaagcttg tcggcggagc ggatgatgat cgcctc 36

Claims

1. A process for the preparation of (4S, 5R)-half-ester, characterized in that, Includes the following steps: Step 1: An esterase gene is ligated to the restriction site of the pET-21a plasmid to obtain a recombinant expression plasmid; the esterase gene sequence is SEQ ID NO:1, and the recombinant expression plasmid is named pET-21a-estsit01; Step 2: Transform the recombinant expression plasmid pET-21a-estsit01 into Escherichia coli BL21(DE3) to obtain E. coli BL21(DE3) / pET-21a-estsit01 cells; Step 3: The E. coli BL21(DE3) / pET21a-estsit01 bacterial cells were used as a biocatalyst and added to pure water to obtain a bacterial cell suspension. Then, the substrate dimethyl biotin was added, and the reaction temperature was controlled at 30°C to carry out an asymmetric hydrolysis reaction. The pH of the reaction solution was controlled between 7.5 and 8.0 by adding 20% ​​w / v ammonia dropwise, and the reaction time was 2 to 24 hours to obtain the reaction solution. Step 4: Centrifuge the reaction solution obtained in Step 3, separate the cells and take the supernatant, add 2 mol / L HCl to adjust the pH to 2.0, extract the acidified solution three times with an equal volume of ethyl acetate, combine the organic phases and dry with anhydrous sodium sulfate, then evaporate the organic solvent under reduced pressure, and dry the sample under vacuum at 70℃ to finally obtain the target product, namely (4S,5R)-hemimethyl ester solid. The amounts of bacterial cell wet weight, biotin dimethyl ester, and purified water used are calculated as follows: bacterial cell wet weight: biotin dimethyl ester: purified water = 10-30g: 100-300mmol: 1-2L. In step 4, the centrifugation speed is 1000 rpm and the centrifugation time is 20 min.

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