CN114317508B - Halohydrin dehalogenase mutant, engineering bacterium and application thereof - Google Patents

Abstract

The invention discloses a halohydrin dehalogenase mutant, an engineering bacterium and applications thereof, which are characterized in that the mutant is the 105th, 134th, 136th, 136th, 136th, The 137th, 178th or 186th position is obtained by single or multiple mutations. The present invention utilizes water-organic biphasic buffer solution system (ethyl acetate: sodium phosphate buffer = 6:4) to make the e.e. value of the product (S)-epichlorohydrin of the halohydrin dehalogenase mutant exceed 99%, The wet cell enzyme activity increased from 23.56U/mL to 100-600U/mL, and the yield reached 15.9%-80.2%.

Description

A kind of halohydrin dehalogenase mutant, engineering bacteria and application thereof

(1) Technical field

The invention relates to a halohydrin dehalogenase mutant and its application in preparing (S)-epichlorohydrin.

(2) Background technology

As important intermediates in pharmaceutical and chemical industries, halohydrin compounds are widely used in organic synthesis and pharmaceutical research and development. However, most halides in nature have disadvantages such as poor degradation ability, high toxicity and potential carcinogenicity. It has become one of the main environmental pollutants, so people pay more and more attention to its degradation treatment in order to reduce the pollution of this kind of organic halides to the natural environment and the potential threat to human beings. The use of traditional physical and chemical methods not only has harsh reaction conditions, but also may cause secondary pollution, while the use of biodegradation methods has environmental pollution-free, mild reaction conditions; normal temperature, normal pressure, neutral, or close to neutral pH, etc., The advantages of simple reaction steps, easy separation of products, low requirements on equipment performance during operation, relatively small investment, and safe production process have become the main development trend of the degradation of halogenated compounds.

A series of enzymes that can degrade such toxic substances have been found in microorganisms, and one of the key enzymes is halohydrin dehalogenase. Halohydrin dehalogenase (EC 4.5.1.X, HHDH for short) is a class of dehalogenases that catalyze the conversion of ortho-halohydrins into epoxides through an intramolecular nucleophilic substitution mechanism, and can catalyze with high efficiency and high selectivity. The conversion between epoxides and o-halohydrins can therefore be used to synthesize optically pure epoxides, which has a sense of superiority that cannot be compared with traditional chemical synthesis methods.

Halohydrin dehalogenase can not only catalyze the cleavage of carbon-halogen bonds to form epoxides, but also carry out the corresponding reverse reaction, and in the presence of unnatural nucleophiles, such as N3-, CN-, NO2-, SCN -, OCN- and HCOO-, etc., catalyze the ring opening of epoxides to form a series of new C-C, C-O and C-N bonds, etc., which provides an efficient and economical method for the preparation of various optically pure β-substituted alcohols and epoxides The method is environmentally friendly and has high application value in drug development and organic synthesis. Since Castro was first discovered in 1968 in a strain of Flavobaterium sp. that uses 2,3-dibromopropanol as the sole carbon source, scientists have successively discovered a variety of halohydrin dehalogenases. So far, some known halohydrin dehalogenases have been cloned, sequenced and studied in vitro recombinant expression. In recent years, with the development of bioinformatics, the research on halohydrin dehalogenase has gradually deepened. In the synthesis process of chiral epichlorohydrin (ECH) catalyzed by halohydrin dehalogenase, because of its low catalytic efficiency and low stereoselectivity, and currently in the process of halohydrin dehalogenase catalyzed reaction, due to chiral Racemization or degradation of ECH leads to low optical purity of the product, which limits its application and industrial production.

(3) Contents of the invention

The problem to be solved by the present invention is to overcome the problems of low e.e. value and product racemization of halohydrin dehalogenase catalysis in the existing method, and provide a halohydrin dehalogenase mutant derived from radioactive Agrobacterium and the Application of mutants to prepare (S)-ECH with high yield and e.e. value in biphasic buffer solution. In order to better realize the industrial production and application of halohydrin dehalogenase, the present invention obtains the enzyme activity center and key amino acids near the Loop ring, which can be used in a biphasic buffer solution (organic phase: aqueous phase = 6:4 ) catalyzed the substrate 1,3-DCP to obtain mutants with high stereoselectivity and high yield.

The technical scheme adopted in the present invention is:

The present invention provides a halohydrin dehalogenase mutant, the mutant is the 105th, 134th, 136th, 137th, 178th or 186th amino acid sequence shown in SEQ ID NO.2 single or multiple mutations.

Further, the halohydrin dehalogenase mutant is mutated from leucine at position 105 to aspartic acid (L105N) and alanine at position 134 into leucine in the amino acid sequence shown in SEQ ID NO.2 (A134L), the 136th proline mutation to asparagine (P136N), the 137th phenylalanine mutation to serine (F137S), the 178th tyrosine mutation to methionine (Y178M), the 186th tyrosine mutation Amino acid was mutated to asparagine (Y186N), proline at position 136 was mutated to asparagine and phenylalanine at position 137 was mutated to serine (P136N/F137S).

The present invention also relates to the recombinant vector constructed by the gene encoding the halohydrin dehalogenase mutant and the recombinant genetically engineered bacteria prepared by transformation of the recombinant vector. The recombinant vector of the present invention is not limited, as long as it can maintain its replication or autonomous replication in various host cells of prokaryotic and/or eukaryotic cells, the vector can be various conventional vectors in the art, such as various plasmids, For phage or viral vectors, the pET28a(+) plasmid is preferably used as the expression vector, and Escherichia coli is used as the expression host (Escherichia coli BL21 cells or Escherichia coli DH5α).

The present invention also provides a kind of said halohydrin dehalogenase mutant in catalyzing 1,3-dichloro-2-propanol (1,3-DCP) to synthesize (S)-epichlorohydrin ((S)-ECH ), the application method is as follows: the wet thallus obtained by fermenting and culturing the recombinant genetically engineered bacteria containing the halohydrin dehalogenase mutant coding gene is used as a catalyst, and 1,3-dichloro-2-propane Alcohol is used as the substrate, and the water-organic solvent two-phase system with pH 7.0-10.0 (preferably pH 8.0) is used as the reaction medium to form the reaction system. The reaction is carried out at 300-700rpm (preferably 600rpm) and 37°C. After the reaction , to obtain a reaction solution containing (S)-epichlorohydrin, the reaction solution is separated and purified to obtain (S)-epichlorohydrin; the water-organic solvent two-phase system is composed of ethyl acetate with a volume ratio of 6:4 ester and 200mM sodium phosphate buffer, pH 8.0.

Further, the amount of the catalyst is 10-40g/L buffer solution (preferably 20g/L) based on the weight of wet bacteria, and the initial concentration of the substrate is 10-80mM (preferably 20mM).

Further, the wet bacterium was prepared as follows: Inoculate the recombinant engineered bacteria containing the gene encoding the halohydrin dehalogenase mutant into the LB culture solution containing the final concentration of 50 μg/mL kanamycin, and inoculate it at 37°C. Cultivate for 8 hours to obtain seed solution; then inoculate the seed solution with an inoculum volume concentration of 2% into sterile LB liquid medium containing a final concentration of 50 μg/mL kanamycin, and cultivate at 37°C for about 1.5 -2.5h, so that the bacterium concentration OD ₆₀₀ is 0.4-0.8, then add isopropylthio-β-D-galactoside (IPTG) with a final concentration of 0.1-1.0mM (preferably 0.1mM) ), after inducing expression at 28°C for 12h, centrifuge at 4°C and 4000rpm for 10-20min to collect the wet cells; LB liquid medium: peptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, solvent is deionized water, pH 8.0.

The halohydrin dehalogenase mutants of the present invention can be catalyzed in the form of whole cells, or can be catalyzed by the crude enzyme solution of broken cells or completely broken pure enzymes. In addition, the halohydrin dehalogenase can also be prepared as an immobilized enzyme or an enzyme in the form of immobilized cells by using a specific immobilization technique.

Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

The present invention uses directed evolution and semi-rational design methods to transform the halohydrin dehalogenase HheC derived from radioactive Agrobacterium, and finds the 105th, 134th, 136th, 137th, 176th, and 186th The site is the key site that affects the enzyme activity: the specific enzyme activities of L105N, A134L, P136N, F137S, Y178M, and Y186N were screened through 96-well plates for the three sites using the site-fixed saturation technique. 251.19U/mL, 176.86U/mL, 507.19U/mL, 613.45U/mL, 478.63U/mL, 109.37U/mL, 10.7 times, 7.5 times, 21.5 times, 26.0 times, 20.3 times of the original enzyme , 4.6 times.

In the present invention, the obtained 6 mutation sites are randomly combined in pairs, and the specific enzyme activity of the mutant P136N/F137S obtained by screening is 481.52 U/mL, which is 20.4 times of the original enzyme activity.

The present invention utilizes the water-organic biphasic buffer solution system (ethyl acetate: sodium phosphate buffer = 6:4) to make the e.e. value of the product (S)-epichlorohydrin of the halohydrin dehalogenase mutant exceed 99%, The wet cell enzyme activity increased from 23.56U/mL to 100-600U/mL, and the yield reached 15.9%-80.2%.

The halohydrin dehalogenase mutant of the present invention is modified on the basis of the amino acid of the wild-type halohydrin dehalogenase HheC shown in SEQ ID NO. Changes in structure and function, and then through the method of directional screening, obtain the halohydrin dehalogenase mutant with the above mutation, the halohydrin dehalogenase mutant of the present invention has the advantage of greatly improving the enzyme activity, and its enzyme activity is relatively The wild-type halohydrin dehalogenase HhecC is increased many times, which is more conducive to the realization of industrial production.

(4) Description of drawings

Figure 1 is a three-dimensional structure diagram of the substrate 1,3-DCP.

Figure 2 is a chromatogram of the catalytic product after gas phase detection.

(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:

LB liquid medium: peptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, solvent is deionized water, pH 8.0.

LB plate is added 15g/L agar in LB liquid medium.

Example 1: Homology Modeling and Molecular Dynamics Simulation of Halohydrin Dehalogenases

Using the three-dimensional structure of HheC (PDB ID: 1ZO8) derived from Agrobacterium radiobacterium (Agrobacterium radiobacterstrain AD1) in GenBank AAK92099 as a template, the three-dimensional structure of 1,3-dcp was drawn using ChemDraw 8.1, and the molecular docking program followed the AutoDock 4.6.2 program Docking of HheC and substrate 1,3-dcp. All the above structures were visually analyzed using the PyMOL program, and it was found that the 105th, 134th, 136th, 137th, 178th, and 186th positions are key sites that affect the enzyme activity (Figure 1).

Example 2: Construction of Halohydrin Dehalogenase Site-Directed Saturation Library

According to the conclusion of Example 1, design based on the gene sequence (nucleotide sequence shown in SEQ ID NO.1, amino acid sequence shown in SEQ ID NO.2) of the wild-type halohydrin dehalogenase HheC included in GenBank Primers (see Table 1). Respectively with primers L105X-F/L105X-R, A134X-F/A134X-R, P136X-F/P136X-R, F137X-F/F137X-R, Y178X-F/Y178X-R, T186X-F/T186X-R The parental HheC gene (nucleotide sequence is SEQ ID NO.1) was subjected to site-directed saturation mutation, and pET-28b(+) was used as the expression vector to obtain mutant plasmids with the target gene, and the target gene The mutant plasmid was transformed into E.coli BL21(DE3), and the mutants of recombinant bacteria containing the halohydrin dehalogenase mutant gene were respectively obtained, which were respectively E.coli BL21(DE3)-L105X (referred to as mutant L105X), E. .coli BL21(DE3)-A134X (referred to as mutant A134X), E.coli BL21(DE3)-P136X (referred to as mutant P136X), E.coli BL21(DE3)-F137X (referred to as mutant F137X), E. coli BL21(DE3)-Y178X (referred to as mutant Y178X), E. coli BL21(DE3)-Y186X (referred to as mutant Y186X).

SEQ ID NO.2:

MASTAIVTNVKHFGGMGSALRLSEAGHTVACHDESFKQKDELEAFEATYPQLKPMSEQEPAELIEAVTSAYGQVDVLVSNDIFAPEFQPIDKYAVEDYRGAVEALQIRPFALVNAVASQMKKRKSGHIFITSATPFGPWKELSTYTSARAGACTLANALSKELGEYNIPVFAIGSNYLHSEDSPY FYPTEPWKTNPEHVAHVKKVTALQRLGTQKELGELVAFLASGSCDYLTGQVFWLAGGFPMIERPPGMPELE.

Table 1: Primer design table for construction of halohydrin dehalogenase site-directed saturation mutation library

The PCR amplification system is: 50μL reaction system:

2×phantamax Buffer: 25μL;

dNTP Mix (10Mm each): 1μL;

Upstream primer (50 μM): 2 μL;

Downstream primer (50 μM): 2 μL;

Phanta Max super-Fidelity DNA Polymerase: 1μL;

Template DNA (plasmid): 0.5 μL;

_ddH2O : 18.5 μL;

The PCR reaction conditions were as follows: pre-denaturation at 95°C for 10 min, followed by a temperature cycle of 95°C for 30 s, 55°C for 30 s, and 72°C for 6 min, a total of 30 cycles, and finally an extension at 72°C for 10 min, with a termination temperature of 4°C. After the PCR product was verified by 1% agarose gel electrophoresis analysis, 1 μL DpnI and 5 μL buffer were added to the PCR product, digested at 37°C for 2 hours to remove the template plasmid DNA, and inactivated at 65°C for 10 minutes to purify the product with PCR cleanup Kit , transformed into E.coli BL21(DE3) competent cells, spread on LB plates containing kanamycin (50 μg/mL), and culture overnight at 37°C to obtain a mutant library of halohydrin dehalogenase, at this time on LB plates Single colonies exhibiting many different mutations were used for subsequent screening of mutant libraries.

The same method was used to construct the parental strain: E.coli BL21(DE3)-HheC.

Example 3: Screening of Halohydrin Dehalogenase Mutant Library

1. Screening of the halohydrin dehalogenase mutant library Taking wild-type HheC before mutation as a reference, pick a single colony clone (the mutant library constructed in Example 2) and culture it in a 2mL deep 96-well plate. 600 μL of LB culture solution with a concentration of 50 μg/mL kanamycin, and at the same time pick two parental strains in the last two wells of a 96-well plate as controls. Place a 2 mL 96-well plate at 37°C for 8 hours as a seed solution, then add 200 μL of the seed solution to a new sterile 600 μL LB culture solution containing a final concentration of 50 μg/mL kanamycin and 0.1 mM IPTG , after induction of expression at 28°C containing the seed solution for 12 hours, centrifuge at 4000 rpm for 20 minutes, discard the supernatant, collect the wet cells, and carry out the next step of high-throughput screening.

2. According to the characteristics of the catalytic reaction of halohydrin dehalogenase, the high-throughput screening method can measure the activity of the enzyme by detecting the concentration of H+ in the reaction system. At the same time, a molecule of H+ will be released, and a pH indicator can be added to the reaction buffer system. According to the color change of the pH indicator, the activity of halohydrin dehalogenase can be qualitatively or quantitatively determined. Another method is the quantitative analysis of chloride ions generated in the halohydrin dehalogenase reaction.

Preferably, in this embodiment, according to the colorimetric reaction based on the pH index, it is easier to screen positive bacteria with high activity and enantioselectivity in the water-organic buffer system, specifically:

Add 200 μL of PB buffer (pH=8.0, 200 mM) to each well of the wet bacteria collected by centrifugation of the 96-well plate in step 1 to prepare a cell suspension. The colorimetric reaction was carried out in a 96-well quartz plate, and the reaction system was 200 μL: 80 μL of cell suspension, a final concentration of 20 mM 1,3-DCP and a final concentration of 0.09 mg/ml bromothymol blue, and acetic acid with a volume ratio of 6:4 Ethyl ester and sodium phosphate buffer (200mM, pH=8.0) were added to 200μL, after mixing, they were incubated at 37°C for 20min, and reacted for the same time, according to the change speed of the color of the reaction solution (from blue to yellow). Alcohol dehalogenase activity. In step 1, during the bacterial screening process, a total of about 400 single colonies were screened from the mutant library composed of each mutation site, and a total of 2,400 single colonies were screened by high-throughput screening in the mutant library composed of 6 mutation sites. In the same time period, according to the color change speed and depth of the pH indicator, compared with the parental HheC control group, it was found that the color of the reaction solution with higher enzyme activity than the parental reaction solution was more yellow than that of the parental reaction solution, thus initially The mutants of 7 highly active recombinant bacteria containing halohydrin dehalogenase mutant genes were obtained by screening, and obtained by sequencing were respectively E.coli BL21(DE3)-L105N (referred to as mutant L105N, and the nucleotide sequence is SEQ ID NO.3), E.coli BL21(DE3)-A134L (referred to as mutant A134L, nucleotide sequence shown in SEQID NO.4), E.coli BL21(DE3)-P136N (denoted as mutant Body P136N, the nucleotide sequence is shown in SEQ ID NO.5), E.coli BL21(DE3)-F137S (denoted as mutant F137S, the nucleotide sequence is shown in SEQ ID NO.6), E.coli BL21(DE3)-Y178M (referred to as mutant Y178M, the nucleotide sequence is shown in SEQ ID NO.7), E.coliBL21(DE3)-Y186N (referred to as mutant Y186N, the nucleotide sequence is shown in SEQ ID NO. .8).

Example 4: Construction of Halohydrin Dehalogenase Double Mutants

The six halohydrin dehalogenase mutants L105N, mutant A134L, mutant P136N, mutant F137S, mutant Y178M, and mutant Y186N obtained through high-throughput screening were combined in pairs to construct double mutants of halohydrin dehalogenase Libraries, 15 kinds of possible double mutation libraries were constructed in pairs. The PCR reaction conditions and construction method were the same as in Example 2. The primary screening method of the double mutation library was the same as in Example 3. In the screening process of Example 3, each A total of about 400 single colonies were screened from the double mutation library, and about 6,000 single colonies were screened from the 15 possible double mutation libraries constructed. In the same time period, according to the color change speed and depth of the pH indicator, it was compared with the parental HheC control group. After comparison, it was found that the color of the reaction solution with higher enzyme activity than that of the parent was more yellow than that of the parent reaction solution, thus obtaining a double mutant with higher activity, which was sequenced to obtain E.coli BL21(DE3)- P136N/F137S (denoted as mutant P136N/F137S, nucleotide sequence shown in SEQ ID NO.9).

Example 5: Preparation of recombinant bacterial cells containing halohydrin dehalogenase and its mutants

The mutant strains screened in Example 3 and Example 4 were inoculated into LB liquid medium containing kanamycin at a final concentration of 50 μg/mL, and cultivated at 37°C for 12 hours to obtain seed liquid; Inoculate fresh seed liquid into fresh LB liquid medium containing kanamycin with a final concentration of 50 μg/mL at an inoculum size of 2%, and cultivate at 37°C for about 1.5-2.5h, so that the cell concentration OD ₆₀₀ is 0.4-0.8, then add isopropylthio-β-D-galactoside (IPTG) at a final concentration of 0.1mM to the culture medium, induce expression at 28°C for 12h, then centrifuge at 4°C, 8000rpm for 10min , to collect the wet cells of the recombinant cells, which are used to catalyze the wet cells required for the asymmetric dehalogenation reaction of 1,3-DCP.

The preparation method and conditions of the parental wet cells are the same as those of the mutant wet cells.

Example 6: Activity Comparison of Halohydrin Dehalogenase Asymmetric Catalytic Synthesis of (S)-ECH

Halohydrin dehalogenase enzyme activity unit (U) definition: under the conditions of 37°C and pH 8.0, the definition of the amount of bacteria required to catalyze the substrate (1,3-DCP) to generate 1mmoL product chiral epichlorohydrin within 1min 1U.

Reaction system: final concentration of 20mM 1,3-DCP, wet bacteria prepared in Example 5 with a final concentration of 20g/L, 0.2M, pH8.0 phosphate buffer and ethyl acetate at a volume ratio of 6:4 10 mL of the reaction medium constitutes the reaction system. React at 37°C and 600rpm for 30min, take 500μL of the reaction solution and add 1mL of ethyl acetate for extraction, centrifuge at 12000rpm for 1min, take the organic phase and dry it with anhydrous sodium sulfate, and detect the peak area of the chiral epichlorohydrin product and the residual bottom by gas phase Product 1,3-dcp peak area (Figure 2), product production and substrate consumption were calculated by the standard curve of product and substrate, and enzyme activity was calculated according to the definition of enzyme activity. The product (S)-ECH standard curve equation is: Y=15.103X-0.5779 (R ² =0.9993); (R)-ECH standard curve equation is: Y=15.334X-0.9597 (R ² =0.9984); substrate 1 , The equation of the 3-dcp standard curve is: Y=14.869X+9.6286 (R ² =0.9919).

The detection method of chiral epichlorohydrin: adopt Agilent GC-7890A system, column type: BGB-175 capillary column, chromatographic conditions: column temperature 90°C, injection chamber temperature 220°C, FID detector 220°C.

The enantiomeric excess (e.e.) of (S)-ECH was calculated as [e.e.(%)=(S-R)/(S+R)×100], where S was the yield of (S)-ECH and R was (R )-ECH yield. All experimental data are from triplicate experiments.

The results are shown in Table 2. The enzyme activities were increased from 23.56U/mL to 100-600U/mL, and the e.e. value of (S)-ECH was increased from 70% to >99%.

Table 2: Enzyme activity and e.e. value comparison of halohydrin dehalogenase mutants catalyzing the synthesis of (S)-ECH

Example 7: Asymmetric Catalytic Synthesis of (S)-ECH by Halohydrin Dehalogenase

Since the halohydrin dehalogenase catalyzes the reaction in the aqueous phase, racemization and hydrolysis reactions are likely to occur. In this example, a two-phase system with a volume ratio of ethyl acetate and sodium phosphate buffer (200mM, pH=8.0) of 6:4 was used as the In the reaction medium, 1,3-dichloro-2-propanol is used as a substrate, and the wet bacteria prepared by the method in Example 5 is used as a catalyst to carry out an asymmetric reaction of (S)-ECH biosynthesis, as follows:

1) Reaction system: 10 mL of reaction medium, add substrate with a final concentration of 10 mM, and a catalyst with a final concentration of 10 g/L to form a reaction system. React for 30 min at 37°C and 600 rpm, take 500 μL of the reaction solution and add 1 mL of ethyl acetate to dilute, Centrifuge at 12000rpm for 3min, collect the supernatant, and dry it with anhydrous sodium sulfate for gas phase analysis (the detection method is the same as in Example 6). The results are shown in Table 3.

Table 3: Comparison of yields of (S)-ECH catalyzed by halohydrin dehalogenase mutants

2) Reaction system: 10 mL of reaction medium, adding substrate with a final concentration of 20 mM, and catalyst with a final concentration of 10 g/L to form a reaction system, reacted at 37°C and 600 rpm for 30 min; take 500 μL of reaction solution and add 1 mL of ethyl acetate to dilute, Centrifuge at 12000rpm for 3min, collect the supernatant, and dry it with anhydrous sodium sulfate for gas phase analysis (the detection method is the same as in Example 6). The results are shown in Table 4.

Table 4: Comparison of yields of (S)-ECH catalyzed by halohydrin dehalogenase mutants

3) Reaction system: 10 mL of reaction medium, adding substrate with a final concentration of 20 mM, and a catalyst with a final concentration of 20 g/L to form a reaction system, reacted at 37°C and 600 rpm for 30 min; take 500 μL of the reaction solution and add 1 mL of ethyl acetate to dilute, Centrifuge at 12000rpm for 3min, collect the supernatant, and dry it with anhydrous sodium sulfate for gas phase analysis (the detection method is the same as in Example 6). The results are shown in Table 5.

Table 5: Comparison of yields of (S)-ECH catalyzed by halohydrin dehalogenase mutants

4) Reaction system: 10 mL of reaction medium, adding substrate with a final concentration of 30 mM, and a catalyst with a final concentration of 20 g/L to form a reaction system, reacted at 37°C and 600 rpm for 30 min; take 500 μL of the reaction solution and add 1 mL of ethyl acetate to dilute, Centrifuge at 12000rpm for 3min, collect the supernatant, and dry it with anhydrous sodium sulfate for gas phase analysis (the detection method is the same as in Example 6). The results are shown in Table 6.

Table 6: Comparison of yields of (S)-ECH catalyzed by halohydrin dehalogenase mutants

5) Reaction system: 10 mL of reaction medium, adding substrate with a final concentration of 40 mM, and catalyst with a final concentration of 20 g/L to form a reaction system, reacted at 37°C and 600 rpm for 30 min; take 500 μL of reaction solution and add 1 mL of ethyl acetate to dilute, Centrifuge at 12000rpm for 3min, collect the supernatant, and dry it with anhydrous sodium sulfate for gas phase analysis (the detection method is the same as in Example 6). The results are shown in Table 7.

Table 7: Comparison of yields of (S)-ECH catalyzed by halohydrin dehalogenase mutants

6) Reaction system: 10 mL of reaction medium, adding substrate with a final concentration of 80 mM, and catalyst with a final concentration of 40 g/L to form a reaction system, reacted at 37°C and 600 rpm for 30 min; take 500 μL of reaction solution and add 1 mL of ethyl acetate to dilute, Centrifuge at 12000rpm for 3min, collect the supernatant, and dry it with anhydrous sodium sulfate for gas phase analysis (the detection method is the same as in Example 6). The results are shown in Table 8.

Table 8: Comparison of yields of (S)-ECH catalyzed by halohydrin dehalogenase mutants

Finally, the preferred substrate concentration is 20mM, the catalyst dosage is 20g/L wet bacteria, and the preferred mutants are P136N, F137S, Y178M, and P136N/F137S.

Since in the water phase, during the synthesis of (S)-ECH catalyzed by halohydrin dehalogenase, the product self-degradation and the racemization of HHDH are prone to occur. Considering the application of industrial production, the mutants obtained through screening were obtained by using water -The organic biphasic buffer solution is used as the reaction medium, on the one hand, it solves the problem of self-hydrolysis of the product in the aqueous phase, and on the other hand, it eliminates the side reaction of racemization by modifying the designed enzyme in the water-organic biphasic buffer solution.

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

sequence listing

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<213> Agrobacterium radiobacter

<400> 3

atggcttcta ccgctattgt gactaacgta aagcatttcg gtggcatggg ctctgcgctg 60

cgtctgtctg aagctggtca cactgttgct tgccatgacg aaagcttcaa acagaaagat 120

gaactggaag ctttcgcgga aacttatcct cagctgaaac cgatgtctga acaggaaccg 180

gctgaactga ttgaagctgt gacctctgcc tacggccaag ttgacgtcct ggtgtccaac 240

gatattttcg cgccggaatt ccagccgatc gataaatatg ctgtggaaga ttaccgtggt 300

gctgtcgaag ctaatcagat ccgcccattt gcactggtta acgcggtggc ttcccagatg 360

aagaaacgta aatctggcca catcatcttc attacctctg caactccat cggtccgtgg 420

aaagaactgt ccacttatac ttccgcccgt gctggcgctt gcactctggc aaacgcgctg 480

tccaaagagc tgggcgaata caacattccg gttttcgcga tcggttcgaa ctacctgcac 540

tctgaagaca gcccgtactt ctacccgacc gaaccgtgga aaactaaccc ggaacacgtg 600

gcgcacgtaa aaaaggttac cgcactgcag cgtctgggta cccaaaaaga actgggcgaa 660

ctggttgcgt tcctggcatc tggttcctgt gattacctga ccggtcaagt cttttggctg 720

gcaggtggct tcccgatgat cgaacgtccg ccgggtatgc cggaactcga gtga 774

<210> 4

<211> 774

<212>DNA

<213> Agrobacterium radiobacter

<400> 4

atggcttcta ccgctattgt gactaacgta aagcatttcg gtggcatggg ctctgcgctg 60

cgtctgtctg aagctggtca cactgttgct tgccatgacg aaagcttcaa acagaaagat 120

gaactggaag ctttcgcgga aacttatcct cagctgaaac cgatgtctga acaggaaccg 180

gctgaactga ttgaagctgt gacctctgcc tacggccaag ttgacgtcct ggtgtccaac 240

gatattttcg cgccggaatt ccagccgatc gataaatatg ctgtggaaga ttaccgtggt 300

gctgtcgaag ctctgcagat ccgcccattt gcactggtta acgcggtggc ttcccagatg 360

aagaaacgta aatctggcca catcatcttc attacctctc tgactccatt cggtccgtgg 420

aaagaactgt ccacttatac ttccgcccgt gctggcgctt gcactctggc aaacgcgctg 480

tccaaagagc tgggcgaata caacattccg gttttcgcga tcggttcgaa ctacctgcac 540

tctgaagaca gcccgtactt ctacccgacc gaaccgtgga aaactaaccc ggaacacgtg 600

gcgcacgtaa aaaaggttac cgcactgcag cgtctgggta cccaaaaaga actgggcgaa 660

ctggttgcgt tcctggcatc tggttcctgt gattacctga ccggtcaagt cttttggctg 720

gcaggtggct tcccgatgat cgaacgtccg ccgggtatgc cggaactcga gtga 774

<210> 5

<211> 774

<212>DNA

<213> Agrobacterium radiobacter

<400> 5

atggcttcta ccgctattgt gactaacgta aagcatttcg gtggcatggg ctctgcgctg 60

cgtctgtctg aagctggtca cactgttgct tgccatgacg aaagcttcaa acagaaagat 120

gaactggaag ctttcgcgga aacttatcct cagctgaaac cgatgtctga acaggaaccg 180

gctgaactga ttgaagctgt gacctctgcc tacggccaag ttgacgtcct ggtgtccaac 240

gatattttcg cgccggaatt ccagccgatc gataaatatg ctgtggaaga ttaccgtggt 300

gctgtcgaag ctctgcagat ccgcccattt gcactggtta acgcggtggc ttcccagatg 360

aagaaacgta aatctggcca catcatcttc attacctctg caactaattt cggtccgtgg 420

aaagaactgt ccacttatac ttccgcccgt gctggcgctt gcactctggc aaacgcgctg 480

tccaaagagc tgggcgaata caacattccg gttttcgcga tcggttcgaa ctacctgcac 540

tctgaagaca gcccgtactt ctacccgacc gaaccgtgga aaactaaccc ggaacacgtg 600

gcgcacgtaa aaaaggttac cgcactgcag cgtctgggta cccaaaaaga actgggcgaa 660

ctggttgcgt tcctggcatc tggttcctgt gattacctga ccggtcaagt cttttggctg 720

gcaggtggct tcccgatgat cgaacgtccg ccgggtatgc cggaactcga gtga 774

<210> 6

<211> 774

<212>DNA

<213> Agrobacterium radiobacter

<400> 6

atggcttcta ccgctattgt gactaacgta aagcatttcg gtggcatggg ctctgcgctg 60

cgtctgtctg aagctggtca cactgttgct tgccatgacg aaagcttcaa acagaaagat 120

gaactggaag ctttcgcgga aacttatcct cagctgaaac cgatgtctga acaggaaccg 180

gctgaactga ttgaagctgt gacctctgcc tacggccaag ttgacgtcct ggtgtccaac 240

gatattttcg cgccggaatt ccagccgatc gataaatatg ctgtggaaga ttaccgtggt 300

gctgtcgaag ctctgcagat ccgcccattt gcactggtta acgcggtggc ttcccagatg 360

aagaaacgta aatctggcca catcatcttc attacctctg caactccaag cggtccgtgg 420

aaagaactgt ccacttatac ttccgcccgt gctggcgctt gcactctggc aaacgcgctg 480

tccaaagagc tgggcgaata caacattccg gttttcgcga tcggttcgaa ctacctgcac 540

tctgaagaca gcccgtactt ctacccgacc gaaccgtgga aaactaaccc ggaacacgtg 600

gcgcacgtaa aaaaggttac cgcactgcag cgtctgggta cccaaaaaga actgggcgaa 660

ctggttgcgt tcctggcatc tggttcctgt gattacctga ccggtcaagt cttttggctg 720

gcaggtggct tcccgatgat cgaacgtccg ccgggtatgc cggaactcga gtga 774

<210> 7

<211> 774

<212>DNA

<213> Agrobacterium radiobacter

<400> 7

atggcttcta ccgctattgt gactaacgta aagcatttcg gtggcatggg ctctgcgctg 60

cgtctgtctg aagctggtca cactgttgct tgccatgacg aaagcttcaa acagaaagat 120

gaactggaag ctttcgcgga aacttatcct cagctgaaac cgatgtctga acaggaaccg 180

gctgaactga ttgaagctgt gacctctgcc tacggccaag ttgacgtcct ggtgtccaac 240

gatattttcg cgccggaatt ccagccgatc gataaatatg ctgtggaaga ttaccgtggt 300

gctgtcgaag ctctgcagat ccgcccattt gcactggtta acgcggtggc ttcccagatg 360

aagaaacgta aatctggcca catcatcttc attacctctg caactccat cggtccgtgg 420

aaagaactgt ccacttatac ttccgcccgt gctggcgctt gcactctggc aaacgcgctg 480

tccaaagagc tgggcgaata caacattccg gttttcgcga tcggttcgaa catgctgcac 540

tctgaagaca gcccgtactt ctacccgacc gaaccgtgga aaactaaccc ggaacacgtg 600

gcgcacgtaa aaaaggttac cgcactgcag cgtctgggta cccaaaaaga actgggcgaa 660

ctggttgcgt tcctggcatc tggttcctgt gattacctga ccggtcaagt cttttggctg 720

gcaggtggct tcccgatgat cgaacgtccg ccgggtatgc cggaactcga gtga 774

<210> 8

<211> 774

<212>DNA

<213> Agrobacterium radiobacter

<400> 8

atggcttcta ccgctattgt gactaacgta aagcatttcg gtggcatggg ctctgcgctg 60

cgtctgtctg aagctggtca cactgttgct tgccatgacg aaagcttcaa acagaaagat 120

gaactggaag ctttcgcgga aacttatcct cagctgaaac cgatgtctga acaggaaccg 180

gctgaactga ttgaagctgt gacctctgcc tacggccaag ttgacgtcct ggtgtccaac 240

gatattttcg cgccggaatt ccagccgatc gataaatatg ctgtggaaga ttaccgtggt 300

gctgtcgaag ctctgcagat ccgcccattt gcactggtta acgcggtggc ttcccagatg 360

aagaaacgta aatctggcca catcatcttc attacctctg caactccat cggtccgtgg 420

aaagaactgt ccacttatac ttccgcccgt gctggcgctt gcactctggc aaacgcgctg 480

tccaaagagc tgggcgaata caacattccg gttttcgcga tcggttcgaa ctacctgcac 540

tctgaagaca gcccgaattt ctacccgacc gaaccgtgga aaactaaccc ggaacacgtg 600

gcgcacgtaa aaaaggttac cgcactgcag cgtctgggta cccaaaaaga actgggcgaa 660

ctggttgcgt tcctggcatc tggttcctgt gattacctga ccggtcaagt cttttggctg 720

gcaggtggct tcccgatgat cgaacgtccg ccgggtatgc cggaactcga gtga 774

<210> 9

<211> 774

<212>DNA

<213> Agrobacterium radiobacter

<400> 9

atggcttcta ccgctattgt gactaacgta aagcatttcg gtggcatggg ctctgcgctg 60

cgtctgtctg aagctggtca cactgttgct tgccatgacg aaagcttcaa acagaaagat 120

gaactggaag ctttcgcgga aacttatcct cagctgaaac cgatgtctga acaggaaccg 180

gctgaactga ttgaagctgt gacctctgcc tacggccaag ttgacgtcct ggtgtccaac 240

gatattttcg cgccggaatt ccagccgatc gataaatatg ctgtggaaga ttaccgtggt 300

gctgtcgaag ctctgcagat ccgcccattt gcactggtta acgcggtggc ttcccagatg 360

aagaaacgta aatctggcca catcatcttc attacctctg caactaatag cggtccgtgg 420

aaagaactgt ccacttatac ttccgcccgt gctggcgctt gcactctggc aaacgcgctg 480

tccaaagagc tgggcgaata caacattccg gttttcgcga tcggttcgaa ctacctgcac 540

tctgaagaca gcccgtactt ctacccgacc gaaccgtgga aaactaaccc ggaacacgtg 600

gcgcacgtaa aaaaggttac cgcactgcag cgtctgggta cccaaaaaga actgggcgaa 660

ctggttgcgt tcctggcatc tggttcctgt gattacctga ccggtcaagt cttttggctg 720

gcaggtggct tcccgatgat cgaacgtccg ccgggtatgc cggaactcga gtga 774

Claims (4)

1. A halohydrin dehalogenase mutant, characterized in that the mutant is mutated from tyrosine at position 178 of the amino acid sequence shown in SEQ ID NO.2 to methionine. 2. A recombinant genetically engineered bacterium constructed from the coding gene of the halohydrin dehalogenase mutant described in claim 1. 3. the application of a halohydrin dehalogenase mutant described in claim 1 in catalyzing 1,3-dichloro-2-propanol synthesis (S)-epichlorohydrin, is characterized in that the method of described application For: the wet thallus obtained by fermenting the recombinant genetically engineered bacteria containing the coding gene of the halohydrin dehalogenase mutant is used as the catalyst, 1,3-dichloro-2-propanol is used as the substrate, and the pH is 7.0-10.0 The water-organic solvent two-phase system is used as the reaction medium to form the reaction system, and the reaction is carried out at 300-700 rpm and 37 °C. After the reaction is completed, the reaction liquid containing (S)-epichlorohydrin is obtained, and the reaction liquid is separated. Purify to obtain (S)-epichlorohydrin; the water-organic solvent biphasic system is composed of ethyl acetate and 200 mM, pH 8.0 sodium phosphate buffer at a volume ratio of 6:4; the catalyst consumption is expressed as wet The weight of the bacterium is 10-40 g/L buffer solution, and the initial concentration of the substrate is 10-80 mM. 4. The application according to claim 3, characterized in that the wet thallus is prepared as follows: inoculate the recombinant engineered bacterium containing the gene encoding the halohydrin dehalogenase mutant into a bacterium containing a final concentration of 50 mg/mL In the LB culture solution of namycin, cultivate it at 37 °C for 8 h to obtain the seed liquid; then inoculate the seed liquid with an inoculum volume concentration of 2% to a sterile culture medium containing a final concentration of 50 mg/mL kanamycin. In the LB liquid medium, cultured at 37 ℃ for 1.5-2.5 h, so that the OD ₆₀₀ of the bacterial cell concentration was 0.4-0.8, and then added isopropylthio- β-D-galactoside, induced expression at 28 ℃ for 12 h, centrifuged at 4 ℃, 4000 rpm for 10-20 min, collected wet bacteria; LB liquid medium: peptone 10 g/L, yeast extract 5 g/L, Sodium chloride 10 g/L, solvent is deionized water, pH 8.0.