CN104745557B - From the thermophilic tiny bacillus halide alcohol dehalogenase mutant of cleaning and its application - Google Patents

Abstract

The invention provides application of the mutant and mutant of the thermophilic tiny bacillus halide alcohol dehalogenase of cleaning in the synthesis of Atorvastatin intermediate, the present invention is by setting up high pass screening scheme, to halide alcohol dehalogenase directional transformation, mutant F176M/A187R is obtained, its vigor improves 3.11 times relative to wild type halide alcohol dehalogenase.The 3-hydroxyethyl butyrate of mutant F176M/A187R whole-cell catalytics 100g/L (S) 4 chlorine 3 synthesizes the 3-hydroxyethyl butyrate of (R) 4 cyano group 3, can obtain 86% molar yield.

Description

Halohydrin dehalogenase mutant derived from Leptophilus cleanophilus and its application

(1) Technical field

The invention relates to the fields of molecular biology and biotechnology, in particular to a method for preparing halohydrin dehalogenase mutants using gene mutation technology, a high-throughput screening method for a library of halohydrin dehalogenase mutants, and the obtained mutation Body and its application in the preparation of atorvastatin intermediate (R)-4-cyano-3-hydroxybutyric acid ethyl ester.

(2) Background technology

Cardiovascular disease (CVD) is the leading cause of death worldwide. Cardiovascular diseases such as coronary heart disease, stroke, and hypertension have become the number one killer of the Chinese population. In the primary and secondary prevention of cardiovascular and cerebrovascular diseases, statins can effectively inhibit cholesterol synthesis and significantly reduce the incidence of cardiovascular and cerebrovascular diseases, and have become the basic drugs for the treatment of atherosclerotic diseases. In addition, statins also dominate the global cholesterol-lowering drugs market. According to the global drug sales data released by IMS in 2009, atorvastatin ranked first in the world's best-selling drugs with global sales of US$12.3 billion, and became the only drug in the world with annual sales exceeding US$10 billion.

Atorvastatin (Figure 1) is a fully synthetic high-efficiency statin drug, and β, δ-dihydroxyvaleric acid is the key active structure. Atorvastatin efficiently inhibits hepatic cholesterol synthesis, promotes the increase of hepatic low-density lipoprotein (LDL) receptors, and changes the formation of very low-density lipoprotein particles. Atorvastatin reduces low-density lipoprotein cholesterol levels by 41-61% in the dose range of 10-80 mg/d, and reduces serum triglyceride levels in patients with hypertriglyceridemia by 43%. In addition, statins provide very good cardiovascular protection for people with high or low cholesterol and no cardiovascular and cerebrovascular diseases, and help patients with diabetes and hypertension to reasonably control cholesterol levels. Standard therapeutic doses of statins can achieve very Significant protective effect. Atorvastatin is an important category for the treatment of cardiovascular diseases in my country, and its importance and status as the first choice drug for the treatment of cardiovascular diseases will be further highlighted.

The domestic and foreign markets of atorvastatin have been monopolized by large foreign companies, and the domestic patent protection period of atorvastatin is about to expire. Foreign products monopolize the domestic market. This variety has been monopolized by foreign pharmaceutical giants for a long time, resulting in high prices. The price of a piece of "Lipitor" is as high as 10 yuan, and the burden of medication is heavy for patients. Therefore, the development of new technologies for the production of atorvastatin with independent intellectual property rights and cost advantages, the realization of the localization of major varieties of drugs for the treatment of cardiovascular diseases, and the reduction of the production and sales costs of drugs are the key to solving the problem of "difficult and expensive medical treatment" for the people. "The urgency of the question.

(R)-Ethyl 4-cyano-3-hydroxybutyrate (HN) is the key chiral synthetic precursor of atorvastatin (Figure 2). Mastering advanced HN synthesis technology can control the supply of raw materials for atorvastatin production, enhance the core competitiveness of products, and enhance the company's voice and pricing power in the international atorvastatin drug market. There are many synthetic pathways for HN in the literature, among which there are three mainstream industrialization routes.

1) The traditional HN chemical synthesis process starts from (S)-4-chloro-3-hydroxybutyrate ethyl ester ((S)-CHBE), after the protection of the hydroxyl group, it reacts with sodium cyanide, and the cyano group replaces the chlorine atom. Finally, HN was deprotected by HCl, and the total yield of the three steps was 68.6%.

2) American Codexis uses the halohydrin dehalogenase HheC as the starting strain, and uses the directed evolution method of ProSAR to increase the dehalogenation and cyanation activity of the halohydrin dehalogenase by about 4000 times, and the volumetric yield of the reaction is increased by 2500 times. The substrate concentration can reach 140g/L, the amount of enzyme used is 1.2g/L, the reaction is 5h, and the yield is 92.4%.

3) Foreign scholar Dan E.Robertson screened a nitrilase gene that can asymmetrically degrade 3-hydroxyglutaronitrile to form (R)-4-cyano-3-hydroxybutyric acid from Acer Group, and then saturate it at a fixed point Mutation technique The BD9570 mutant was obtained after directed evolution of the nitrilase, the catalytic substrate concentration was increased from 100mM to 3M (330g/L), the enzyme usage was 6% (wt), and the reaction was 12h. The obtained product (R)-4-cyano-3-hydroxybutyric acid is extracted, and then reacted with ethanol to obtain HN through one-step esterification.

At present, the synthesis processes of the above-mentioned HN have all achieved industrial production scale in enterprises. Compared with the chemical method, the enzymatic method has short synthesis steps, high yield and less environmental pollution. Comparing the synthesis of HN by halohydrin dehalogenase and nitrile hydrolysis, although the substrate concentration of halohydrin dehalogenase is only 140g/L, the amount of enzyme used in this process is 1.2g/L, and the reaction time is 5h, which is far away. Less than nitrilase 60g/L, reaction time 12h. Therefore, compared with the above, the process of synthesizing HN by halohydrin dehalogenase has greater advantages. However, this key halohydrin dehalogenase has been protected by foreign patents. If the industrial application of this step is to be realized in China, a new halohydrin dehalogenase needs to be developed. Recently, our laboratory cloned a new type of halohydrin dehalogenase gene HHDH-PL from Leptophilus sclerosophila, and realized soluble expression in Escherichia coli. higher vitality.

(3) Contents of the invention

The object of the present invention is to provide a halohydrin dehalogenase mutant derived from the food cleaner Corynebacterium parvum and the application of the mutant to prepare the key chiral intermediate HN of atorvastatin using (S)-CHBE as a substrate , the present invention screens the mutant obtained by the high-throughput method, and shows higher activity in the process of (S)-CHBE synthesis of HN.

Technical scheme of the present invention is:

The present invention provides a halohydrin dehalogenase mutant derived from Coryne parvum, which is a food cleaner. The halohydrin dehalogenase mutant is the amino acid sequence shown in SEQ ID NO: 2 (the nucleotide sequence is SEQ ID NO: 1) amino acid at position 176 and/or position 187 is mutated.

Further, the mutant is to mutate the 176th phenylalanine in the amino acid sequence shown in SEQ ID NO: 2 into methionine.

Further, the mutant is to mutate alanine at position 187 of the amino acid sequence shown in SEQ ID NO: 2 into arginine or serine.

Further, the halohydrin dehalogenase mutant is one of the following: (1) the 176th phenylalanine in the amino acid sequence shown in SEQ ID NO: 2 is mutated into methionine (the amino acid sequence is SEQ ID NO: 3); (2) mutating the 187th alanine in the amino acid sequence shown in SEQ ID NO: 2 to arginine (the amino acid sequence is shown in SEQ ID NO: 4); (3) replacing The 187th alanine in the amino acid sequence shown in SEQ ID NO: 2 is mutated into serine (the amino acid sequence is shown in SEQ ID NO: 5); (4) the 176th in the amino acid sequence shown in SEQ ID NO: 2 Phenylalanine is mutated into methionine, and the 187th alanine in the amino acid sequence shown in SEQ ID NO: 2 is mutated into arginine at the same time (the amino acid sequence is shown in SEQ ID NO: 6); (5 ) mutate the 176th phenylalanine in the amino acid sequence shown in SEQ ID NO: 2 to methionine, and simultaneously mutate the 187th alanine in the amino acid sequence shown in SEQ ID NO: 2 to serine (amino acid The sequence is shown in SEQ ID NO: 7).

The present invention also provides an application of the halohydrin dehalogenase mutant in the preparation of ethyl (R)-4-cyano-3-hydroxybutyrate, specifically the application is: dehalogenating the halohydrin The wet thallus or the supernatant obtained after ultrasonic disruption of the recombinant genetically engineered bacterium with the gene encoding the enzyme mutant is used as a catalyst, and (S)-4-chloro-3-hydroxybutyric acid ethyl ester As the substrate, use PBS buffer solution with pH 7.5 as the reaction medium, control the pH value of the reaction solution to 7.5 by adding a mass concentration of 30% NaCN aqueous solution, and carry out the reaction at 500rpm and 40°C. After the reaction, the reaction solution is separated Purification affords ethyl (R)-4-cyano-3-hydroxybutyrate.

Further, the amount of the catalyst is 70-100g/L buffer solution, preferably 100g/L based on the weight of wet cells, and the initial concentration of the substrate is 100-120g/L buffer solution, preferably 120g/L.

Further, the wet bacteria were prepared as follows: inoculate the recombinant genetically engineered bacteria containing the gene encoding the halohydrin dehalogenase mutant into LB liquid medium containing ampicillin at a final concentration of 50 mg/L, and culture at 37°C for 12 hours, Obtain the seed liquid; then inoculate the seed liquid into fresh LB liquid medium containing a final concentration of 50 mg/L kanamycin with an inoculum volume concentration of 1%, and cultivate at 37°C until the cell concentration _OD600 is 0.4-0.8 , and then add IPTG with a final concentration of 0.2mM to the culture medium, induce culture at 24°C for 12h, centrifuge at 5000rpm for 10min at 4°C, and collect the wet cells.

Further, the conditions for the sonication of the wet cells are: resuspend the wet cells with 200 mM PBS, pH 7.5 (wt/vol=1:10), take 50 mL of the resuspended cell solution, and Crushed for 30 minutes.

Further, the method for separating and purifying the reaction liquid is as follows: after the reaction is completed, filter the reaction liquid, extract the filtrate with ethyl acetate, take the organic phase and dry it with anhydrous sodium sulfate, and distill the organic phase of ethyl acetate under reduced pressure , the residual dark yellow substance is the product HN.

The present invention uses the expression plasmid containing the parental halohydrin dehalogenase gene, then designs and synthesizes appropriate primers, uses the expression vector containing the halohydrin dehalogenase HHDH-PL gene as a template, and uses error-prone PCR and MEGAWHOP technology to carry out random Mutation, the amplified product is transformed into host cells, and a random mutation library is constructed. After the mutants were induced and expressed, they were screened by chromogenic method to obtain halohydrin dehalogenase mutants with higher catalytic activity. These mutants can use (S)-CHBE as a substrate, which is the key to efficient biocatalytic production of atorvastatin Chiral intermediate HN (Figure 2).

The present invention obtains a variety of halohydrin dehalogenase mutants. Taking the amino acid sequence shown in SEQ ID NO: 2 as a reference sequence, a single or double mutation is performed on the 176th and 187th positions, and these mutants are identified as (S )-CHBE as a substrate has an activity at least 3.1 times higher than that of the parent, while the selectivity and thermal stability remain unchanged. Preferably, the phenylalanine (Phe) at position 176 in the parent sequence is mutated into methionine (Met) and the alanine (Ala) at position 187 in the parent sequence is mutated into Arginine (Arg) or Serine (Ser).

The halohydrin dehalogenase mutants of the present invention can be used in the form of whole cells of engineering bacteria, or in the form of partially purified or completely purified enzymes. If necessary, the halohydrin dehalogenase mutants of the present invention can also be made into immobilized enzymes or immobilized enzymes in the form of immobilized cells by using immobilization techniques known in the art.

The plasmid containing the full-length mutation is transformed into an appropriate host cell, and the positive mutant with high halohydrin dehalogenase activity is selected through culturing, inducing expression, and screening. Finally, the plasmid DNA was extracted from the positive mutants and subjected to DNA sequencing analysis to determine the mutation information of the mutants. In the preparation method of the halohydrin dehalogenase mutant of the present invention, pET32a(+) vector and Escherichia coli BL21(DE3) host cells can be used. In the method for preparing the halohydrin dehalogenase mutant of the present invention, the obtained halohydrin dehalogenase mutant gene can be expressed in prokaryotic cells or eukaryotic cells, and any other suitable method known in the art can also be used. The method realizes extracellular expression in prokaryotic cells or eukaryotic cells.

In order to screen a large number of halohydrin dehalogenase mutant libraries, the present invention establishes a high-throughput screening method based on azide ion detection, which can be applied to screen halohydrin dehalogenases with good activity for the preparation of β- Alcohol replacement.

The term "parent" used in the text of this application refers to the halohydrin dehalogenase from Parvibaculum lavamentivorans ZJB14001 (CCTCCM2014373), the nucleotide sequence of which is shown in sequence 1, and the amino acid sequence is shown in sequence 2, which has been published in the patent 201410552622.5 ( application number).

The term "halohydrin dehalogenase mutant" used in the text of this application refers to a kind of amino acid sequence shown in SEQ ID NO: 2 as a reference sequence, the 176th and 187th positions are subjected to single or double mutations, and (S)-CHBE is a substrate, and its activity is at least 1.6 times higher than that of the parent enzyme, and the catalytic activity is up to 3.1 times higher. Therefore, in the present application, the halohydrin dehalogenase mutant includes conservative substitutions, additions or deletions of one or several positions in the amino acid sequence shown in SEQ ID NO: 2 except positions 176 and 187. Amino acid forms, amino-terminal truncated forms, and carboxy-terminal truncated forms, these mutant forms are also included in the scope of the present invention.

The three-letter or one-letter expression of amino acids used in the text of this application adopts the amino acid code specified by IUPAC (Eur. J. Biochem., 138:9-37, 1984).

In the present invention, the halohydrin dehalogenase HHDH-PL from Leucophilus Leptophilum is used as the starting strain, and the random mutation library is constructed by using error-prone PCR and MEGAWHOP PCR technology; a high-throughput method for screening mutations based on azide ion detection is established library, obtain a series of excellent mutants; obtain excellent mutant enzymes through fermentation; and use the mutant enzymes in the synthesis of atorvastatin intermediate (R)-4-cyano-3-hydroxybutyrate ethyl ester.

Compared with the prior art, the beneficial effect of the present invention is mainly reflected in: the present invention obtains the mutant F176M/A187R through the establishment of a high-pass screening scheme for directional transformation of the halohydrin dehalogenase, whose activity is comparable to that of the wild-type halohydrin dehalogenase Increased by 3.11 times. Whole-cell mutant F176M/A187R catalyzes the synthesis of (R)-4-cyano-3-hydroxybutyrate ethyl from 100 g/L (S)-ethyl 4-chloro-3-hydroxybutyrate, and can obtain 86% mole Yield.

(4) Description of drawings

Fig. 1 Structural formula of atorvastatin calcium.

Fig. 2 The reaction process of preparing HN from (S)-CHBE catalyzed by halohydrin dehalogenase.

Figure 3 Mechanism of the high-throughput screening method for gene azide ion detection.

Figure 4 High-throughput screening of positive mutants in a 96-well plate, intuitive color changes in the figure: dark wells indicate low activity of halohydrin dehalogenase mutants, light wells indicate halohydrin dehalogenase mutations Physical activity is high.

Figure 5 SDS-PAGE analysis of the crude enzyme solution of halohydrin dehalogenase, Lane M: standard protein molecular weight; Lane1: HHDH-PL; Lane 2: mutant A187R; Lane 3: mutant A187S; Lane 4: mutant F176M: Lane 5: mutant F176M/A187S; Lane 6: mutant F176M/A187R.

(5) Specific implementation methods

The present invention is further described below in conjunction with specific embodiment, but protection scope of the present invention is not limited thereto:

The mutants prepared by the present invention and their properties are described below with specific examples. The following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. Those who do not indicate the conditions in the examples are carried out according to conventional conditions or the conditions suggested by the manufacturer.

Example 1: Construction of random mutation library of halohydrin dehalogenase

Random mutation technology reference (Current Protocols in Protein Science 26.6.1-26.6.10, 2011; Anal. Biochem. 2008, 375: 376-378) description. Primer P1 and primer P2 were designed according to GenBank ABS64560.1 gene sequence (see Table 1). First, an error-prone PCR experiment is performed on the parental HHDH-PL gene (the nucleotide sequence is shown in SEQ ID NO.1) with primers P1 and P2. 50μL error-prone PCR reaction system: 25μL 2×GoTaq MasterMix, 5 μL 50 mM MgCl ₂ buffer, 5 μL 1 mM MnCl ₂ , 0.5 μL P1 (50 μM) and P2 (50 μM), 1 μL (100-200 ng) HHDH-PL plasmid template, 13 μL deionized water. PCR program: pre-denaturation at 95°C for 3 minutes, 25 cycles: 94°C for 30s, 55°C for 30s, 72°C for 60s, and finally extension at 72°C for 10 minutes. After the PCR products were verified by agarose nucleic acid electrophoresis, the products were purified using PCR cleanup Kit. MEGAWHOP PCR was performed using the purified product as a primer. 50μL MEGAWHOP PCR system: 10μL 5×PS buffer, 4μL dNTPMix (0.8mM), 1μL (100-200ng) HHDH-PL plasmid, 0.5μL PrimerStar, 8μL (1μg) purified error-prone PCR product and 26.5μL deionized water. PCR program: 72°C for 10 min, 95°C for 5 min, 30 cycles: 98°C for 10 s, 55°C for 15 s, 72°C for 8 min and 72°C for 10 min. After PCR was positive by 0.9% agarose gel electrophoresis analysis, take 20 μL of PCR solution, add 1 μL DpnI, digest at 37°C for 2 hours to remove template plasmid DNA, inactivate at 65°C for 10 minutes, and transform into competent cells E.coli BL21(DE3) , Spread LB plates containing ampicillin (50 mg/L).

Example 2: Establishment of a high-throughput method for screening halohydrin dehalogenase mutants

Since the purpose of this experiment is to screen a halohydrin dehalogenase mutant that can efficiently catalyze (S)-CHBE to synthesize HN, establishing an efficient high-throughput screening method is a key step to improve the realization of this invention. The methods reported in the literature to detect the activity of halohydrin dehalogenases are based on protons and halide ions produced by the dehalogenation process, but cannot detect the ring-opening process, so these methods are not suitable for screening halohydrin dehalogenation of (S)-CHBE to HN enzyme. In order to establish a suitable screening method, the present invention establishes a new high-throughput screening method based on azide detection, as shown in FIG. 3 .

Pick a single colony clone (the mutant library constructed in Example 1) and culture it in a 2 mL deep 96-well plate containing 1 mL LB and 50 μg/mL ampicillin. At the same time, three wild-type HHDH-PL (HHDH-PL WT) were selected as controls. A 2 mL deep 96-well plate was cultured at 37°C for 5 hours, then 100 μL of the bacterial solution was transferred to another sterile 2 mL 96-well plate, and 100 μL of 30% (wt/vol) sterile glycerol was added to it. Add 100 μL LB medium containing 50 μg/mL ampicillin and 1 mM IPTG (isopropyl-β-D-thiogalactopyranoside) to the remaining 900 μL bacterial solution, and place at 28 °C for 12-14 h, after induction centrifuge at 3,000×g and 4°C for 30 min, discard the supernatant, and collect the cells. 500 μL of reaction system (50 mM Tri-H ₂ SO ₄ buffer, 20 mM (S)-CHBE and 20 mM NaN ₃ ) was added to each well, the cells were resuspended, and then reacted at 40° C. and 150 rpm for 30 min. Take 10 μL of the reaction solution and add it to a standard 96-well (300 μL) well plate, and add 190 μL of 50 mM ferric chloride solution (Figure 4). Colorimetric analysis was performed at a wavelength of 460 nm. The relative activity is calculated according to the equation (relative activity=20×(OD460(c)-OD460(r))/1.2173t), where OD460(c) represents the OD value after the reaction, and OD460(r) represents the initial OD of the reaction value, and t represents the reaction time. Using the high-throughput screening method, 2500 mutants were screened, and three excellent mutant strains were obtained, which were designated as mutant F176M, mutant A187R and mutant A187S.

Example 3: Construction of double mutants F176M/A187R and F176M/A187S

The plasmid of mutant F176M was extracted, and mutation primers P3 and P4 (see Table 1) were designed for A187R mutation, and mutation primers P5 and P6 (see Table 1) were designed for A187S mutation. Mutation system 50μL: 10μL 5×PS buffer, 4μL dNTPMix (0.8mM), 1μL (100-200ng) F176M plasmid, 0.5μL PrimerStar, 1μL (50μM) mutation primer and 32.5μL deionized water. PCR program: 72°C for 10 min, 95°C for 5 min, 30 cycles: 98°C for 10 s, 55°C for 15 s, 72°C for 8 min and 72°C for 10 min. After PCR was positive by 0.9% agarose gel electrophoresis analysis, take 20 μL of PCR solution, add 1 μL DpnI, digest at 37°C for 2 hours to remove template plasmid DNA, inactivate at 65°C for 10 minutes, and transform into competent cells E.coli BL21(DE3) , Spread LB plates containing ampicillin (50 mg/L). A single colony was picked and sequenced to analyze that the mutants F176M/A187R and F176M/A187S were successfully constructed.

Table 1: Primers

Primer name Primer nucleotide sequence (5'-3') P1 ATGGCGCGCAGCATTTCTCATC P2 CTAGCGCGCGGTGGCCC P3 GACTACTTCCCCTCCTCGCTCCTTG P4 GATTTTCAAGGAGCGAGGAGGGGAAGTAG P5 GACTACTTCCCCCGGTCGCTCCTTG P6 GATTTTCAAGGAGCGACCGGGGGAAGTAG

Example 4: Induced expression of parental halohydrin dehalogenase and halohydrin dehalogenase mutant engineered bacteria

With parental HHDH-PL WT, mutant F187M, mutant A187R and mutant A187R (embodiment 2) and mutant F176M/A187R, mutant F176M/A187S (embodiment 3) were inoculated into respectively with containing 50mg/L ampicillin In the LB liquid medium of 37 ℃ for 12h, then inoculate it into fresh 100mL LB liquid medium containing 50mg/L kanamycin with 1% (v/v) inoculum, and cultivate to the cell concentration OD ₆₀₀ About 0.6, then add IPTG (isopropyl-β-D-thiogalactopyranoside) with a final concentration of 0.2mM to the LB liquid medium, induce culture at 24°C for 12h, centrifuge at 4°C, 5000rpm for 10min, The bacterial cells containing the recombinant halohydrin dehalogenase are collected, which can be used for enzyme activity determination, enzyme extraction and preparation of HN.

Example 5: Relative Activity Comparison of Parental Halohydrin Dehalogenase and Halohydrin Dehalogenase Mutants Catalyzing (S)-CHBE to Synthesize HN

Take the parental halohydrin dehalogenase and the five halohydrin dehalogenase mutants prepared in Example 4, 0.1 g wet cells, resuspend in 10 mL of 200 mM PBS, pH 7.5, and sonicate for 10 min at 50% power. Centrifuge at 4° C. and 15000 rpm for 10 min, and the supernatant is the prepared crude enzyme solution. SDS-PAGE analysis showed that the expression levels of the six halohydrin dehalogenases in the crude enzyme solutions were similar (Figure 5). The crude enzyme solutions were used to catalyze (S)-CHBE to synthesize HN, and their relative activities were compared. The reaction was carried out on a 902 Titrando system (Metrohm, Switzerland) potentiometric titration solution. Add 30mL of PBS (200mM, pH 7.5) and 400μL of 30% (wt/vol) NaCN solution (Note: NaCN is a highly toxic substance, and precautions should be taken during operation) into a 50mL three-necked flask, and then adjust the system with 50% dilute sulfuric acid pH to 7.5. Heat the reaction solution to 40°C, add 1.0g (S)-CHBE and 10mL crude enzyme solution (equivalent to 1.0g of wet bacteria crushed), and react at 500rpm and 40°C. During the dehalogenation reaction process, hydrogen ions will be released to reduce the pH of the reaction, 30% NaCN solution is added as the substrate, and the pH is controlled to 7.5 by a pH potentiometric titrator. After reacting for 30 minutes, 800 μL of the reaction solution was taken, extracted by adding 800 μL of ethyl acetate, dried over anhydrous sodium sulfate, and analyzed by gas phase. Gas phase analysis conditions: GC-14C and chiral G-TA chromatographic column, gas phase program is 90 ° C for 5 min, 5 ° C / min program temperature rise to 180 ° C for 2 min. The retention times are R _t (S)-CHBE = 13.9 min, R _t HN = 18.6 min. The amount of HN produced by HHDH-PL WT was used as the control, and the relative activity was 100%. The relative activities of several halohydrin dehalogenases are compared in Table 2.

Table 2 Comparison of relative activities of halohydrin dehalogenases to synthesize HN

halohydrin dehalogenase Corresponding amino acid sequence number Relative activity (%) HHDH-PL SEQ ID NO.2 100 F176M SEQ ID NO.3 183 A187S SEQ ID NO.4 164 A187R SEQ ID NO.5 210 F176M/A187S SEQ ID NO.6 232 F176M/A187R SEQ ID NO.7 311

Example 6: Application of whole cells of halohydrin dehalogenase mutant F176M/A187R in the preparation of HN

The transformation system composition and transformation operation are as follows: the addition amount of the substrate (S)-CHBE is 100 g/L, and the addition amount of halohydrin dehalogenase mutant F176M/A187R wet bacterial cells is 4% (W/V). The reaction was carried out in 200mM PBS with pH 7.5, the reaction temperature was controlled at about 40°C, and the pH value of the reaction solution was controlled at about 7.5 by adding 30% NaCN solution dropwise during the reaction. During the period, the reaction process was detected by gas chromatography until the end of the reaction (10h); after the reaction (conversion rate>98%), the bacterial cells were removed by centrifugation or filtration, extracted three times with an equal volume of ethyl acetate, and the organic phases were combined. The ethyl acetate organic phase was distilled off under reduced pressure to obtain the dark yellow target product HN with a molar yield of 86% (based on the amount of the substrate substance). See Example 5 for gas phase detection conditions.

The present invention is not limited by the specific written description above, and various changes can be made in the present invention within the scope outlined in the claims, and these modified bacteria are within the scope of the present invention.

Claims (6)

1. A halohydrin dehalogenase mutant derived from the food cleaner Coryne parvum, characterized in that the halohydrin dehalogenase mutant is one of the following: (1) the amino acid shown in SEQ ID NO: 2 The 176th phenylalanine of the sequence is mutated into methionine; (2) the 187th alanine of the amino acid sequence shown in SEQ ID NO: 2 is mutated into arginine; (3) the SEQ ID The 187th alanine in the amino acid sequence shown in NO: 2 is mutated into serine; (4) the 176th phenylalanine in the amino acid sequence shown in SEQ ID NO: 2 is mutated into methionine, and at the same time The 187th alanine in the amino acid sequence shown in SEQ ID NO: 2 is mutated into arginine; (5) the 176th phenylalanine in the amino acid sequence shown in SEQ ID NO: 2 is mutated into methionine acid, and at the same time, the 187th alanine in the amino acid sequence shown in SEQ ID NO: 2 is mutated into serine. 2. an application of the halohydrin dehalogenase mutant described in claim 1 in the preparation of (R)-4-cyano-3-hydroxybutyrate ethyl ester, characterized in that the application is: Alcohol dehalogenase mutant coding gene recombinant genetically engineered bacteria obtained through induced culture wet bacteria or wet bacteria supernatant obtained after ultrasonic disruption as a catalyst, with (S)-4-chloro-3-hydroxybutyrate Ethyl acetate was used as the substrate, and PBS buffer solution with pH 7.5 was used as the reaction medium. The pH value of the reaction solution was controlled by adding a 30% NaCN aqueous solution with a mass concentration of 7.5, and the reaction was carried out at 500 rpm and 40 ° C. After the reaction was completed, the The reaction solution was separated and purified to obtain ethyl (R)-4-cyano-3-hydroxybutyrate. 3. the application of the halohydrin dehalogenase mutant in the preparation of (R)-4-cyano-3-hydroxybutyrate ethyl ester as claimed in claim 2, is characterized in that the consumption of described catalyzer is by wet thalline weight Calculated as 70-100 g/L buffer, the initial concentration of the substrate is 100-120 g/L buffer. 4. The application of the halohydrin dehalogenase mutant in the preparation of (R)-4-cyano-3-hydroxybutyrate ethyl ester as claimed in claim 2, characterized in that the wet thallus is prepared as follows: Inoculate the recombinant genetically engineered bacteria containing the coding gene of the halohydrin dehalogenase mutant into LB liquid medium containing ampicillin at a final concentration of 50 mg/L, and cultivate at 37°C for 12 hours to obtain seed liquid; then inoculate with 1% volume concentration The seed solution was inoculated into fresh LB liquid medium containing a final concentration of 50 mg/L kanamycin, cultivated at 37°C until the cell concentration OD ₆₀₀ was 0.4-0.8, and then added to the culture solution with a final concentration of 0.2mM IPTG, 24°C induction culture for 12h, 4°C, 5000rpm centrifugation for 10min, collected wet cells. 5. the application of the halohydrin dehalogenase mutant in the preparation of (R)-4-cyano-3-hydroxybutyrate ethyl ester as claimed in claim 2, is characterized in that the condition of ultrasonic disruption of the wet thallus is : The wet cells were resuspended with 200mM PBS, pH 7.5, broken for 30min, and the breaking power was 50%. 6. The application of the halohydrin dehalogenase mutant in the preparation of (R)-4-cyano-3-hydroxybutyrate ethyl ester as claimed in claim 2, characterized in that the method for separating and purifying the reaction solution is: After the reaction, the reaction solution was filtered, the filtrate was extracted with ethyl acetate, the organic phase was taken, dried over anhydrous sodium sulfate, and the ethyl acetate was distilled off under reduced pressure to obtain the product (R)-4-cyano-3 - Ethyl hydroxybutyrate.