CN114317508B

CN114317508B - Halohydrin dehalogenase mutant, engineering bacterium and application thereof

Info

Publication number: CN114317508B
Application number: CN202210007671.5A
Authority: CN
Inventors: 汤晓玲; 万欣雨; 郑仁朝; 郑裕国
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2020-05-22
Filing date: 2020-05-22
Publication date: 2023-09-05
Anticipated expiration: 2040-05-22
Also published as: CN111647588B; CN114317508A; CN111647588A

Abstract

The invention discloses a halohydrin dehalogenase mutant, engineering bacteria and application thereof, which are characterized in that the mutant is obtained by single mutation or multiple mutation of 105 th, 134 th, 136 th, 137 th, 178 th or 186 th of an amino acid sequence shown in SEQ ID NO. 2. The invention utilizes a water-organic biphase buffer solution system (ethyl acetate: sodium phosphate buffer solution=6:4) to ensure that the e.e. value of the product (S) -epichlorohydrin of the halohydrin dehalogenase mutant exceeds 99%, and the wet cell enzyme activity is respectively improved from 23.56U/mL to 100-600U/mL, and the yield is 15.9% -80.2%.

Description

Halohydrin dehalogenase mutant, engineering bacterium and application thereof

Field of the art

The invention relates to a halohydrin dehalogenase mutant and application thereof in preparation of (S) -epichlorohydrin.

(II) background art

The halohydrin compound is used as an important intermediate in the fields of pharmaceutical chemical industry and the like, has wide application in aspects of organic synthesis, medicine research and development and the like, but most of halides in nature have become one of main environmental pollutants due to the defects of poor degradation capability, high toxicity, potential carcinogenicity and the like, so that people pay more attention to degradation treatment of the halohydrin compound so as to reduce the pollution of the organic halides to the natural environment and the potential threat to human beings. The traditional physicochemical method is used, so that the reaction conditions are harsh, secondary pollution is likely to be brought, and the biodegradation method is environment-friendly and mild in reaction conditions; the method has the outstanding advantages of normal temperature, normal pressure, neutral or nearly neutral pH, simple reaction steps, easy separation of products, low requirement on equipment performance in the operation process, relatively low investment, safe production process and the like, and becomes the main development trend of the degradation of halogenated compounds at present.

A series of enzymes capable of degrading such toxic substances are found in microorganisms, one of the key enzymes being the halohydrin dehalogenase. The halohydrin dehalogenase (Halohydrin dehalogenase, EC 4.5.1.X, HHDH for short) is a dehalogenase which catalyzes the conversion of o-halohydrin into epoxide through an intramolecular nucleophilic substitution mechanism, can catalyze the conversion between epoxide and o-halohydrin with high efficiency and high selectivity, and can be used for synthesizing epoxide with optical purity, and has superiority which is incomparable with the traditional chemical synthesis method.

The halohydrin dehalogenase can catalyze the cleavage of carbon-halogen bond to form epoxide, can perform corresponding reverse reaction, catalyzes the ring opening of epoxide in the presence of non-natural nucleophile such as N3-, CN-, NO2-, SCN-, OCN-, HCOO-and the like to form a series of novel C-C, C-O and C-N bonds and the like, provides a high-efficiency, economical and environment-friendly method for preparing various optically pure beta-substituted alcohols and epoxides, and has higher application value in the aspects of drug development, organic synthesis and the like. The first time Castro was found in the flavobacterium (flavobacterium sp.) strain that survived 2, 3-dibromopropanol as the sole carbon source since 1968, a number of halohydrin dehalogenases were subsequently discovered by scientists. To date, some of the known halohydrin dehalogenases have been cloned, sequenced and subjected to in vitro recombinant expression studies. In recent years, with the development of bioinformatics, the research on halohydrin dehalogenase has been gradually advanced. In the process of synthesizing chiral Epichlorohydrin (ECH) by using halohydrin dehalogenase, the catalytic efficiency is low, the stereoselectivity is low, and in the process of catalyzing the halohydrin dehalogenase, the optical purity of the product is low due to racemization or degradation of the chiral ECH, so that the application and industrial production of the product are limited.

(III) summary of the invention

The invention aims to solve the problems of low e.e. value, racemization of products and the like of the catalysis of halohydrin dehalogenase in the existing method, and provides a halohydrin dehalogenase mutant from radioactive agrobacterium and application of the mutant in preparing (S) -ECH with high yield and e.e. value in a biphase buffer solution. In order to better realize the industrial production and application of the halohydrin dehalogenase, the invention obtains the mutant with high stereoselectivity and high yield by modifying the key amino acid near the enzyme active center and Loop to catalyze the substrate 1,3-DCP in a biphasic buffer solution (organic phase: water phase=6:4).

The technical scheme adopted by the invention is as follows:

the invention provides a halohydrin dehalogenase mutant, which is obtained by single mutation or multiple mutation of 105 th, 134 th, 136 th, 137 th, 178 th or 186 th of an amino acid sequence shown in SEQ ID NO. 2.

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

The invention also relates to a recombinant vector constructed by the halohydrin dehalogenase mutant coding gene and recombinant genetic engineering bacteria prepared by the 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, and the vector may be various vectors conventional in the art, such as various plasmids, phage or viral vectors, etc., preferably a pET28a (+) plasmid is used as an expression vector, and escherichia coli is used as an expression host (escherichia coli BL21 cells or escherichia coli dh5α).

The invention also provides an application of the halohydrin dehalogenase mutant in catalyzing synthesis of (S) -epichlorohydrin ((S) -ECH) by 1, 3-dichloro-2-propanol (1, 3-DCP), wherein the application method comprises the following steps: taking wet thalli obtained by fermenting recombinant genetic engineering bacteria containing halogen alcohol dehalogenase mutant encoding genes as a catalyst, taking 1, 3-dichloro-2-propanol as a substrate, taking a water-organic solvent biphasic system with pH of 7.0-10.0 (preferably pH of 8.0) as a reaction medium to form a reaction system, reacting at 300-700rpm (preferably 600 rpm) and 37 ℃, obtaining a reaction solution containing (S) -epichlorohydrin after the reaction is finished, and separating and purifying the reaction solution to obtain (S) -epichlorohydrin; the water-organic solvent biphasic system consists of ethyl acetate and 200mM sodium phosphate buffer solution with the volume ratio of 6:4 and the pH value of 8.0.

Further, the catalyst is used in an amount of 10 to 40g/L buffer (preferably 20 g/L) based on the weight of the wet cells, and the substrate is initially added at a concentration of 10 to 80mM (preferably 20 mM).

Further, the wet cell is prepared as follows: inoculating recombinant engineering bacteria containing a halohydrin dehalogenase mutant coding gene into LB culture solution containing kanamycin with the final concentration of 50 mug/mL, and culturing for 8 hours at 37 ℃ to obtain seed solution; then inoculating the seed solution into sterile LB liquid medium containing kanamycin with final concentration of 50 μg/mL at 2% by volume, and culturing at 37deg.C for about 1.5-2.5 hr to obtain thallus concentration OD ₆₀₀ 0.4-0.8, adding isopropyl thio-beta-D-galactoside (IPTG) with a final concentration of 0.1-1.0mM (preferably 0.1 mM) into the culture solution, carrying out induced expression at 28 ℃ for 12h, centrifuging at 4 ℃ at 4000rpm for 10-20min, and collecting wet thalli; LB liquid medium: 10g/L peptone, 5g/L yeast extract, 10g/L sodium chloride, deionized water as solvent and pH 8.0.

The halohydrin dehalogenase mutant can be catalyzed in a whole cell form, or can be catalyzed by crude enzyme liquid of cell disruption or pure enzyme of complete disruption. In addition, the halohydrin dehalogenase may be prepared as an immobilized enzyme or as an immobilized cell form enzyme using specific immobilization techniques.

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

the invention respectively utilizes directed evolution and semi-rational design methods to modify halohydrin dehalogenase HheC derived from radioactive agrobacterium, and finds that 105 th, 134 th, 136 th, 137 th, 176 th and 186 th are key sites influencing enzyme activity: the specific enzyme activities of the 6 single mutants screened through the 96-well plate by utilizing the site-specific saturation technology at the three sites are 251.19U/mL, 176.86U/mL, 507.19U/mL,613.45U/mL, 478.63U/mL and 109.37U/mL respectively, and the specific enzyme activities of the L105N, A L, P136N, F137S, Y178M, Y N are 10.7 times, 7.5 times, 21.5 times, 26.0 times, 20.3 times and 4.6 times of that of the original enzyme.

The invention also carries out random combination of every two of the obtained 6 mutation sites, and the specific enzyme activity of the mutant P136N/F137S obtained by screening is 481.52U/mL, which is 20.4 times of the original enzyme activity.

The invention utilizes a water-organic biphase buffer solution system (ethyl acetate: sodium phosphate buffer solution=6:4) to ensure that the e.e. value of the product (S) -epichlorohydrin of the halohydrin dehalogenase mutant exceeds 99%, and the wet cell enzyme activity is respectively improved from 23.56U/mL to 100-600U/mL, and the yield is 15.9% -80.2%.

The halohydrin dehalogenase mutant is obtained by modifying the amino acid sequence of the halohydrin dehalogenase HhecC shown in SEQ ID No.2 by a site-directed saturation mutation method based on the amino acid of the wild type halohydrin dehalogenase HhecC, and then by a directional screening method, the halohydrin dehalogenase mutant with the mutation has the advantage of greatly improving the enzyme activity, and the enzyme activity of the halohydrin dehalogenase mutant is improved by multiple times compared with that of the wild type halohydrin dehalogenase HhecC, so that the halohydrin dehalogenase mutant is more beneficial to realizing industrial production.

(IV) description of the drawings

FIG. 1 is a three-dimensional block diagram of a substrate 1, 3-DCP.

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

(fifth) detailed description of the invention

The invention will be further described with reference to the following specific examples, but the scope of the invention is not limited thereto:

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

LB plates were prepared by adding 15g/L agar to LB liquid medium.

Example 1: homology modeling and molecular dynamics simulation of halohydrin dehalogenase

Using the three-dimensional structure of HheC (PDB ID:1ZO 8) derived from Agrobacterium radiobacter (Agrobacterium radiobacterstrain AD 1) in GenBank AAK92099 as a template, the three-dimensional structure of 1,3-dcp was mapped using ChemDraw 8.1, and the HheC and substrate 1,3-dcp were docked according to Autodock 4.6.2 by the molecular docking procedure. All the structures are subjected to visual analysis by using a PyMOL program, and the 105 th, 134 th, 136 th, 137 th, 178 th and 186 th positions are found to be key sites influencing enzyme activity (figure 1).

Example 2: construction of site-directed saturation library of halohydrin dehalogenase

According to the conclusion of example 1, primers were designed based on the gene sequence of the wild-type halohydrin dehalogenase HheC (nucleotide sequence shown in SEQ ID NO.1 and amino acid sequence shown in SEQ ID NO. 2) as reported in GenBank (see Table 1). The primers L105X-F/L105X-R, A X-F/A134X-R, P X-F/P136X-R, F137X-F/F137X-R, Y X-F/Y178X-R, T X-F/T186X-R are used for site-directed saturation mutation of the parent HheC gene (nucleotide sequence SEQ ID NO. 1), pET-28b (+) is used as an expression vector, mutant plasmids with the target genes are obtained respectively, the mutant plasmids with the target genes are transformed into E.coli BL21 (DE 3), and mutants of recombinant bacteria containing the halogen-removing enzyme mutant genes are obtained respectively, namely E.coli BL21 (DE 3) -L105X (L105X), E.coli BL21 (DE 3) -A134X (DE 3) -P136X (DE 3) -E.i BL21 (DE 3) -F137X), E.BL 21 (DE 3) -F137X (DE 3) -E136X (DE 3) -BL 21 (DE 3) -L136X (DE 3) -E.BL 21 (DE 3) -L136X) and E.BL 21 (DE 3) -L.L 136X (DE 3).

SEQ ID NO.2：

MASTAIVTNVKHFGGMGSALRLSEAGHTVACHDESFKQKDELEAFAETYPQLKPMSEQEPAELIEAVTSAYGQVDVLVSNDIFAPEFQPIDKYAVEDYRGAVEALQIRPFALVNAVASQMKKRKSGHIIFITSATPFGPWKELSTYTSARAGACTLANALSKELGEYNIPVFAIGSNYLHSEDSPYFYPTEPWKTNPEHVAHVKKVTALQRLGTQKELGELVAFLASGSCDYLTGQVFWLAGGFPMIERPPGMPELE。

Table 1: primer design table constructed by halohydrin dehalogenase site-directed saturation mutation library

The PCR amplification system is as follows: 50. Mu.L of reaction system:

2×phantamax Buffera:25μL；

dNTP Mix(10Mm each):1μL；

upstream primer (50. Mu.M): 2. Mu.L;

downstream primer (50. Mu.M): 2. Mu.L;

Phanta Max super-Fidelity DNA Polymerase:1μL；

template DNA (plasmid): 0.5. Mu.L;

ddH ₂ O：18.5μL；

the PCR reaction conditions were: pre-denaturation at 95℃for 10min, then temperature cycling at 95℃for 30s,55℃for 30s, and 72℃for 6min for 30 cycles, and final extension at 72℃for 10min with a termination temperature of 4 ℃. After the PCR product is verified by 1% agarose gel electrophoresis analysis, 1 mu L of DpnI and 5 mu L of buffer are added into the PCR product, template plasmid DNA is removed by digestion for 2 hours at 37 ℃, after the PCR product is inactivated for 10 minutes at 65 ℃, the PCR product is purified by using a PCR clean Kit and then is transformed into E.coli BL21 (DE 3) competent cells, an LB plate containing kanamycin (50 mu g/mL) is coated, and the culture is carried out at 37 ℃ overnight, so that a mutant library of the halohydrin dehalogenase is obtained, and a plurality of single colonies with different mutations are displayed on the LB plate, and are used for the subsequent screening of the mutant library.

The parent strain was constructed in the same way: coli BL21 (DE 3) -HheC.

Example 3: screening of a halohydrin dehalogenase mutant library

1. Screening of the halohydrin dehalogenase mutation library with wild-type HheC before mutation as a reference, shan Junla clones (mutation library constructed in example 2) were picked up to 2mL deep 96-well plates for culture, 600 μl of LB broth containing kanamycin at a final concentration of 50 μg/mL was added in advance, and 2 parental strains were picked up in the last 2 wells of 96-well plates as controls. 2mL of 96-well plate was cultured at 37℃for 8 hours as a seed solution, then 200. Mu.L of the seed solution was added to a new sterile 600. Mu.L of LB medium containing 50. Mu.g/mL kanamycin and 0.1mM IPTG, the seed solution was induced to express at 28℃for 12 hours, and then centrifuged at 4000rpm for 20 minutes, the supernatant was discarded, and wet cells were collected for the next high-throughput screening.

2. According to the characteristics of the catalytic reaction of the halohydrin dehalogenase, the activity of the enzyme can be measured by detecting the concentration of H+ in a reaction system, the halohydrin dehalogenase can release 1 molecule of H+ while catalyzing o-halohydrin to form epoxide, a pH indicator can be added into a reaction buffer liquid system, and the activity of the halohydrin dehalogenase can be qualitatively or quantitatively determined according to the color change of the pH indicator. Another method is to quantitatively analyze chloride ions generated in the halohydrin dehalogenase reaction.

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

the wet cells collected by centrifugation in the 96-well plate in step 1 were suspended in 200. Mu.L of PB buffer (pH=8.0, 200 mM) per well. The colorimetric reaction was carried out in a 96-well quartz plate with a reaction system of 200. Mu.L: 80. Mu.L of the cell suspension, 20mM 1,3-DCP and 0.09mg/ml bromothymol blue were supplemented to 200. Mu.L with ethyl acetate and sodium phosphate buffer (200 mM, pH=8.0) in a volume ratio of 6:4, and after mixing, the mixture was incubated at 37℃for 20 minutes, and the reaction was carried out for the same period of time, and the enzyme activity of the halohydrin dehalogenase was determined according to the rate of change in the color of the reaction solution (from blue to yellow). In the screening process of step 1, about 400 single colonies are screened in a mutation library formed by each mutation site, 2400 single colonies are screened in a mutation library formed by 6 mutation sites in a high-throughput screening method, and in the same time, according to the color change speed and the depth of a pH indicator, the color of a reaction liquid with higher enzyme activity than that of a parent HheC control group is found to be more yellow compared with that of the parent reaction liquid, so that mutants of 7 recombinant bacteria with higher-activity halogen-containing dehalogenase mutant genes are obtained through primary screening, and the mutants are obtained through sequencing, wherein the mutants are respectively E.coli BL21 (DE 3) -L105N (shown as mutant L105N, the nucleotide sequence is shown as SEQ ID NO. 3), E.coli BL21 (DE 3) -A134L (shown as mutant A134L, the nucleotide sequence is shown as SEQ ID NO. 4), E.coli BL21 (DE 3) -P136N (shown as mutant P136N, the nucleotide sequence is shown as SEQ ID NO. 5), E.coli BL21 (DE 3) -L (shown as mutant F136N, the nucleotide sequence is shown as SEQ ID NO. 3) -L (DE 3) -L134L (shown as mutant Y.L, the nucleotide sequence is shown as SEQ ID NO. 186).

Example 4: construction of halohydrin dehalogenase double mutants

The 6 halohydrin dehalogenase mutants L105N, mutant A134L, mutant P136N, mutant F137S, mutant Y178M and mutant Y186N obtained through high-throughput screening are combined in pairs to construct a halohydrin dehalogenase double-mutation library, 15 possible double-mutation libraries are constructed in pairs, PCR reaction conditions and construction methods are the same as in example 2, the initial screening method of the double-mutation library is the same as in example 3, in the bacterial screening process of example 3, about 400 single colonies are screened for each double-mutation library, about 6000 single colonies are screened for in the constructed 15 possible double-mutation libraries, and in the same time, according to the color change speed and the depth of a pH indicator, the color of a reaction solution with higher enzyme activity than that of a parent HheC control group is found to be more yellow than that of the color presented by the parent reaction solution, so that 1 double-mutation library with higher activity is obtained, and E.coliBL 21 (DE 3) -P N/F137 (with nucleotide sequence shown as P136N/F137S) is obtained through sequencing.

Example 5: preparation of recombinant bacterial cells containing halogen alcohol dehalogenase and mutant thereof

The mutant strains selected in example 3 and example 4 were inoculated into LB liquid medium containing kanamycin at a final concentration of 50. Mu.g/mL, and cultured at 37℃for 12 hours, respectively, to obtain seed solutions; inoculating fresh seed solution into fresh LB liquid medium containing kanamycin with final concentration of 50 μg/mL at 2% by volume, culturing at 37deg.C for about 1.5-2.5 hr to obtain thallus concentration OD ₆₀₀ 0.4-0.8, and adding the final product into the culture solutionIsopropyl thio-beta-D-galactoside (IPTG) with the concentration of 0.1mM is subjected to induction expression at 28 ℃ for 12 hours, and then is centrifuged at 8000rpm at 4 ℃ for 10 minutes, and recombinant cell wet thalli are collected and used for catalyzing wet thalli required by asymmetric dehalogenation reaction of 1, 3-DCP.

Parent wet bacteria preparation method and conditions are the same as those of mutant wet bacteria.

Example 6: comparison of the Activity of the asymmetric catalytic Synthesis of (S) -ECH by halohydrin dehalogenase

Definition of the halohydrin dehalogenase enzyme activity unit (U): the amount of cells required to catalyze the formation of 1mmoL of chiral epichlorohydrin from the substrate (1, 3-DCP) at 37℃and pH 8.0 in 1min was defined as 1U.

The reaction system: the wet cell prepared in example 5 at a final concentration of 20mM 1,3-DCP and a final concentration of 20g/L, and a mixture of 0.2. 0.2M, pH 8.0.0 phosphate buffer and ethyl acetate at a volume ratio of 6:4 were used as 10mL of a reaction medium to construct a reaction system. At 37 ℃,600rpm for 30min, adding 1mL of ethyl acetate into 500 mu L of reaction liquid for extraction, centrifuging at 12000rpm for 1min, taking organic phase, drying by anhydrous sodium sulfate, detecting chiral epichlorohydrin product peak area and residual substrate 1,3-dcp peak area (figure 2), calculating product throughput and substrate consumption by standard curves of the product and the substrate, and calculating enzyme activity according to enzyme activity definition. The product (S) -ECH standard curve equation is: y= 15.103X-0.5779 (R ² =0.9993); the (R) -ECH standard curve equation is: y= 15.334X-0.9597 (R ² =0.9984); the substrate 1,3-dcp standard curve equation is: y=14.869x+9.6286 (R ² ＝0.9919)。

The method for detecting chiral epichlorohydrin comprises the following steps: column type using Agilent GC-7890A system: BGB-175 capillary column, chromatographic conditions: column temperature 90 ℃, sample injection chamber temperature 220 ℃, FID detector 220 ℃.

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

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

Table 2: enzymatic activity and e.e. value comparison of (S) -ECH synthesized by halohydrin dehalogenase mutant

Example 7: asymmetric catalytic synthesis of (S) -ECH by halohydrin dehalogenase

As the halohydrin dehalogenase is easy to racemize and hydrolyze in the water phase, the two-phase system of ethyl acetate and sodium phosphate buffer (200 mM, pH=8.0) with the volume ratio of 6:4 is adopted as a reaction medium, 1, 3-dichloro-2-propanol is adopted as a substrate, and wet thalli prepared by the method of the example 5 is adopted as a catalyst to carry out asymmetric reaction of (S) -ECH biosynthesis, and the asymmetric reaction is as follows:

1) The reaction system: 10mL of reaction medium, 10g/L of substrate with final concentration of 10mM and catalyst with final concentration of 10g/L are added to form a reaction system, the reaction is carried out for 30min at 37 ℃ and 600rpm, 500 mu L of reaction solution is diluted by adding 1mL of ethyl acetate, the reaction solution is centrifuged at 12000rpm for 3min, the supernatant is collected, and the supernatant is dried by anhydrous sodium sulfate and subjected to gas phase analysis (the detection method is the same as in example 6), and the result is shown in Table 3.

Table 3: comparison of yields of (S) -ECH synthesized by catalytic synthesis of halohydrin dehalogenase mutants

2) The reaction system: 10mL of reaction medium, adding a substrate with a final concentration of 20mM, and a catalyst with a final concentration of 10g/L to form a reaction system, and reacting for 30min at 37 ℃ and 600 rpm; 500. Mu.L of the reaction mixture was diluted with 1mL of ethyl acetate, centrifuged at 12000rpm for 3min, and the supernatant was collected and dried over anhydrous sodium sulfate and analyzed in a gas phase (the detection method was the same as in example 6), and the results are shown in Table 4.

Table 4: comparison of yields of (S) -ECH synthesized by catalytic synthesis of halohydrin dehalogenase mutants

3) The reaction system: 10mL of reaction medium, adding a substrate with a final concentration of 20mM, and a catalyst with a final concentration of 20g/L to form a reaction system, and reacting for 30min at 37 ℃ and 600 rpm; 500. Mu.L of the reaction mixture was diluted with 1mL of ethyl acetate, centrifuged at 12000rpm for 3min, and the supernatant was collected and dried over anhydrous sodium sulfate and analyzed in a gas phase (the detection method was the same as in example 6), and the results are shown in Table 5.

Table 5: comparison of yields of (S) -ECH synthesized by catalytic synthesis of halohydrin dehalogenase mutants

4) The reaction system: 10mL of reaction medium, adding a substrate with a final concentration of 30mM, and a catalyst with a final concentration of 20g/L to form a reaction system, and reacting for 30min at 37 ℃ and 600 rpm; 500. Mu.L of the reaction mixture was diluted with 1mL of ethyl acetate, centrifuged at 12000rpm for 3min, and the supernatant was collected and dried over anhydrous sodium sulfate and analyzed in a gas phase (the detection method was the same as in example 6), and the results are shown in Table 6.

Table 6: comparison of yields of (S) -ECH synthesized by catalytic synthesis of halohydrin dehalogenase mutants

5) The reaction system: 10mL of reaction medium, adding a substrate with a final concentration of 40mM, and forming a reaction system by a catalyst with a final concentration of 20g/L, and reacting for 30min at 37 ℃ and 600 rpm; 500. Mu.L of the reaction mixture was diluted with 1mL of ethyl acetate, centrifuged at 12000rpm for 3min, and the supernatant was collected and dried over anhydrous sodium sulfate and analyzed in a gas phase (the detection method was the same as in example 6), and the results are shown in Table 7.

Table 7: comparison of yields of (S) -ECH synthesized by catalytic synthesis of halohydrin dehalogenase mutants

6) The reaction system: 10mL of reaction medium, adding a substrate with a final concentration of 80mM, and a catalyst with a final concentration of 40g/L to form a reaction system, and reacting for 30min at 37 ℃ and 600 rpm; 500. Mu.L of the reaction mixture was diluted with 1mL of ethyl acetate, centrifuged at 12000rpm for 3min, and the supernatant was collected and dried over anhydrous sodium sulfate and analyzed in a gas phase (the detection method was the same as in example 6), and the results are shown in Table 8.

Table 8: comparison of yields of (S) -ECH synthesized by catalytic synthesis of halohydrin dehalogenase mutants

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

In the process of synthesizing (S) -ECH by using halohydrin dehalogenase in water phase, the problems of self-degradation of products and racemization of HHDH are easy to occur, and the mutants obtained by screening are taken into consideration of industrial production application, and a water-organic biphasic buffer solution is used as a reflecting medium, so that on one hand, the problem of self-hydrolysis of products in the water phase is solved, and on the other hand, racemization side reactions are eliminated by modifying the designed enzyme in the water-organic biphasic buffer solution.

The invention is not limited by the specific literal description above. The invention is susceptible of various modifications within the scope of the claims, which modifications are all intended to be within the scope of the invention.

Sequence listing

<110> Zhejiang university of industry

<120> a halohydrin dehalogenase mutant, engineering bacterium and application thereof

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Met Ala Ser Thr Ala Ile Val Thr Asn Val Lys His Phe Gly Gly Met

1 5 10 15

Gly Ser Ala Leu Arg Leu Ser Glu Ala Gly His Thr Val Ala Cys His

20 25 30

Asp Glu Ser Phe Lys Gln Lys Asp Glu Leu Glu Ala Phe Ala Glu Thr

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Glu Ala Val Thr Ser Ala Tyr Gly Gln Val Asp Val Leu Val Ser Asn

65 70 75 80

Asp Ile Phe Ala Pro Glu Phe Gln Pro Ile Asp Lys Tyr Ala Val Glu

85 90 95

Asp Tyr Arg Gly Ala Val Glu Ala Leu Gln Ile Arg Pro Phe Ala Leu

100 105 110

Val Asn Ala Val Ala Ser Gln Met Lys Lys Arg Lys Ser Gly His Ile

115 120 125

Ile Phe Ile Thr Ser Ala Thr Pro Phe Gly Pro Trp Lys Glu Leu Ser

130 135 140

Thr Tyr Thr Ser Ala Arg Ala Gly Ala Cys Thr Leu Ala Asn Ala Leu

145 150 155 160

Ser Lys Glu Leu Gly Glu Tyr Asn Ile Pro Val Phe Ala Ile Gly Ser

165 170 175

Asn Tyr Leu His Ser Glu Asp Ser Pro Tyr Phe Tyr Pro Thr Glu Pro

180 185 190

Trp Lys Thr Asn Pro Glu His Val Ala His Val Lys Lys Val Thr Ala

195 200 205

Leu Gln Arg Leu Gly Thr Gln Lys Glu Leu Gly Glu Leu Val Ala Phe

210 215 220

Leu Ala Ser Gly Ser Cys Asp Tyr Leu Thr Gly Gln Val Phe Trp Leu

225 230 235 240

Ala Gly Gly Phe Pro Met Ile Glu Arg Pro Pro Gly Met Pro Glu Leu

245 250 255

Glu

<210> 3

<211> 774

<212> DNA

<213> radioactive Agrobacterium (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 caactccatt 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> radioactive Agrobacterium (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> radioactive Agrobacterium (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> radioactive Agrobacterium (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> radioactive Agrobacterium (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 caactccatt 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> radioactive Agrobacterium (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 caactccatt 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> radioactive Agrobacterium (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

1. A halohydrin dehalogenase mutant is characterized in that the mutant is obtained by mutating tyrosine 178 of an amino acid sequence shown in SEQ ID NO.2 into methionine.

2. A recombinant genetically engineered bacterium constructed from the gene encoding the halohydrin dehalogenase mutant of claim 1.

3. Use of a halohydrin dehalogenase mutant according to claim 1 for catalyzing the synthesis of (S) -epichlorohydrin from 1, 3-dichloro-2-propanol, characterized in that the method of use comprises: taking wet thalli obtained by fermenting recombinant genetic engineering bacteria containing halogen alcohol dehalogenase mutant encoding genes as a catalyst, taking 1, 3-dichloro-2-propanol as a substrate, taking a water-organic solvent biphasic system with pH of 7.0-10.0 as a reaction medium to form a reaction system, reacting at 300-700rpm and 37 ℃, obtaining a reaction solution containing (S) -epichlorohydrin after the reaction is finished, and separating and purifying the reaction solution to obtain (S) -epichlorohydrin; the water-organic solvent biphasic system consists of ethyl acetate and 200mM in a volume ratio of 6:4 and a pH 8.0 sodium phosphate buffer solution; the catalyst is used in an amount of 10-40g/L buffer solution based on the weight of wet thalli, and the initial concentration of the substrate is 10-80 mM.

4. The use according to claim 3, wherein the wet cells are prepared as follows: inoculating recombinant engineering bacteria containing a halohydrin dehalogenase mutant coding gene into LB culture solution containing kanamycin with the final concentration of 50 mg/mL, and culturing at 37 ℃ for 8h to obtain seed solution; then inoculating the seed solution into sterile LB liquid medium containing kanamycin with final concentration of 50 mg/mL, and culturing at 37deg.C for 1.5-2.5h to obtain thallus concentration OD ₆₀₀ Adding isopropyl thio-beta-D-galactoside with final concentration of 0.1-1.0mM into the culture solution, inducing and expressing at 28deg.C for 12-h, centrifuging at 4deg.C and 4000rpm for 10-20min, and collecting wet thallus; LB liquid medium: peptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, deionized water as solvent, pH 8.0.