Halohydrin dehalogenase mutant with improved dehalogenation efficiency and application thereof

Abstract

The invention discloses a halohydrin dehalogenase mutant with improved dehalogenation efficiency and application of the halohydrin dehalogenase mutant, wild halohydrin dehalogenase comes from radioactive agrobacterium tumefaciens AD1, the wild halohydrin dehalogenase is modified by means of directed evolution, the halohydrin dehalogenase mutant with improved dehalogenation efficiency is screened out, and mutations of the mutant comprise at least one of A83W, P84G and F86W. The halohydrin dehalogenase mutant provided by the invention can efficiently and accurately degrade halohydrin pollutants 2, 3-dichloro-1-propanol and 3-chloro-1, 2-propylene glycol, the toxicity of the halohydrin pollutants is reduced, a high-added-value product is generated, and the dehalogenation efficiencies of the mutant A83W / P84G / F86W on the two substances within one hour can respectively reach 42.2% and 98.6%, which are 6 times and 5.2 times of those of a wild type; the halohydrin dehalogenase mutant provided by the invention can realize efficient dehalogenation at normal temperature in a water phase, does not need additional temperature control equipment, has the characteristics of high efficiency, low energy consumption and high selectivity, has the industrial application advantages of greenness, low carbon, economy and convenience, and is beneficial to pollutant dehalogenation and resource utilization.

Description

Technical Field

[0001] The invention relates to a halohydrin dehalogenase, in particular to a halohydrin dehalogenase mutant, and belongs to the technical field of environmental remediation microorganisms. Background Art

[0002] Persistent organic pollutants (POPs) refer to organic pollutants that have the potential for environmental persistence, bioaccumulation, long-range environmental migration, and adverse effects on human health or the ecological environment. my country attaches great importance to POPs control and has carried out a lot of related basic research, technology development and industrial application. Halogenated organic pollutants are organic pollutants containing halogen atoms. Most halogenated organic pollutants have the same characteristics as POPs and are called halogenated persistent organic pollutants or persistent halogenated organic pollutants. Halogenated alcohols are important intermediates for the synthesis of chiral drugs and pesticide chemicals, and are also important intermediate metabolites for the biodegradation of halogenated persistent organic pollutants. They are produced in large quantities during industrial production processes, and continue to accumulate during the biodegradation process and are eventually discharged into the environment. Halogenated aliphatic alcohols in halogenated alcohols are highly toxic compounds that can cause nervous system and metabolic disorders and have obvious irritating effects. They are toxic organic pollutants in the environment. Typical halogenated aliphatic alcohols such as chloropropanols have four homologues, including 2,3-dichloro-1-propanol (DCP) and 3-chloro-1,2-propanediol (CPD). Among them, CPD may cause cancer, affect the kidneys and fertility, and many countries in the world have regulations on its content in food.

[0003] Methods for treating the above pollutants include physical adsorption, biodegradation, chemical degradation, etc. In the biodegradation method, halohydrin dehalogenase can catalyze the dehalogenation reaction of halogenated aliphatic alcohol pollutants through an intramolecular nucleophilic substitution mechanism, converting them into high value-added environmentally friendly products, such as catalytic degradation of DCP to produce epichlorohydrin (ECH), or catalytic degradation of CPD to produce glycidol (GDL). Therefore, halohydrin dehalogenase has important application prospects in the fields of environmental pollution control and resource utilization.

[0004] At present, there are relatively few known types of halohydrin dehalogenases, and the dehalogenation efficiency of natural halohydrin dehalogenases is insufficient, which restricts their application in the biological treatment of high-concentration halohydrin pollutants. In addition, the research on halohydrin dehalogenases mainly focuses on the asymmetric synthesis of drug intermediates, while there are few reports on environmental application research on improving their dehalogenation and detoxification efficiency. For example, the halohydrin dehalogenase mutants and engineered bacteria disclosed in Chinese patent CN 104745556 B catalyze the asymmetric dehalogenation of 1,3-dichloropropanol to synthesize (S)-epichlorohydrin and β-substituted alcohols, and the obtained halohydrin dehalogenase mutants have higher enantioselectivity, but no modification to improve the dehalogenation activity has been performed. Chinese patent application CN 110423740A discloses that a mutant is obtained by performing single point mutation or combined mutation on arginine at position 89, valine at position 137, proline at position 178, asparagine at position 179, and phenylalanine at position 187 of a halohydrin dehalogenase, and the enantiomeric selectivity is significantly improved when preparing (S)-o-nitrophenyl glycidyl ether, (R)-benzyl glycidyl ether, and (R)-phenyl glycidyl ether, but the dehalogenation efficiency of other aliphatic halohydrin substrates needs to be improved. Chinese patent application CN 118222538A discloses a halohydrin dehalogenase HheC mutant T134M or P84L / W139A, using the halohydrin dehalogenase HheC from radioactive soil bacillus AgrobacteriumradiobacterAD1 with the most stereoselective reaction activity as the mutant original enzyme, realizing the efficient enantioselective biosynthesis of key drug intermediate chiral tetrahydrofuran compounds, but the substrate is δ-halohydrin, and other aliphatic halohydrins are not mentioned. Halohydrin dehalogenase HheC can catalyze the dehalogenation reaction of halohydrin to generate corresponding epoxides and halide ions, and has a detoxification and metabolic effect on aliphatic halohydrin pollutants. However, the existing halohydrin dehalogenase HheC has a low catalytic dechlorination efficiency for DCP and CPD, and does not have the conditions for application in environmental pollution control or resource utilization. Summary of the invention

[0005] Purpose of the invention: The purpose of the present invention is to provide a halohydrin dehalogenase mutant with improved dehalogenation efficiency, to achieve efficient and accurate biological dehalogenation of DCP and CPD, and to provide a nucleic acid molecule encoding the mutant, a vector or recombinant cell or product containing the mutant, and a preparation method and application of the mutant.

[0006] Technical solution: The first aspect of the present invention provides a halohydrin dehalogenase mutant with improved dehalogenation efficiency, wherein the amino acid sequence of the halohydrin dehalogenase mutant is obtained by mutation of the sequence shown in SEQ ID NO.2, and the mutation is at least one of A83W, P84G, and F86W.

[0007] The present invention uses the halohydrin dehalogenase HheC from radioactive soil bacteria (Agrobacterium radiobacterAD1) as a mutant original enzyme (wild type), and its amino acid sequence is shown in SEQ ID NO.2. The enzyme can catalyze the degradation of DCP to produce ECH, and can also catalyze the degradation of CPD to produce GDL. The present invention transforms the key amino acids in the HheC active center through directed evolution technology and protein engineering, and finds that the 83rd, 84th, and 86th positions are key sites that affect the activity and selectivity of HheC. The three sites are respectively subjected to site-directed saturation and iterative saturation mutations, and the halohydrin dehalogenase mutants with improved dehalogenation efficiency for DCP and CPD are screened out. The mutant is obtained by mutating the aforementioned sites alone or in combination, specifically: the 83rd alanine is mutated to tryptophan, the 84th proline is mutated to glycine, and the 86th phenylalanine is mutated to tryptophan. The halohydrin dehalogenase mutant provided by the present invention can realize the green, efficient and accurate biological detoxification of halogenated fatty alcohols, and convert them into high value-added epoxidation products.

[0008] The present invention uses the standard single-letter code for amino acids and the standard substitution notation, for example: A83W means that the alanine (A) at the 83rd position of the N-terminus is mutated to tryptophan (W); A83W / P84G means that the alanine (A) at the 83rd position of the N-terminus is mutated to tryptophan (W), and the proline (P) at the 84th position of the N-terminus is mutated to glycine (G).

[0009] In a second aspect, the present invention provides a nucleic acid molecule encoding the halohydrin dehalogenase mutant according to the first aspect. The nucleic acid molecule can express the halohydrin dehalogenase mutant with improved dehalogenation efficiency.

[0010] Furthermore, the nucleotide sequence of the nucleic acid molecule is obtained by base mutation of the sequence shown in SEQ ID NO.1.

[0011] In a third aspect, the present invention provides a recombinant vector comprising the nucleotide sequence described in the second aspect.

[0012] The recombinant vector includes a cloning vector or an expression vector, which can maintain its ability to replicate or autonomously replicate in various host cells such as prokaryotic and / or eukaryotic cells, thereby amplifying or expressing the nucleotide sequence. The vector can be various conventional vectors in the art, such as various plasmids, phages or viral vectors, etc. Preferably, pET22b (+) plasmid is used as the expression vector.

[0013] In a fourth aspect, the present invention provides a recombinant cell, wherein the recombinant cell comprises the recombinant vector described in the third aspect.

[0014] The recombinant cell includes a recombinant genetically engineered bacterium prepared by transforming the recombinant vector. Preferably, Escherichia coli is used as the recombinant cell to express the halohydrin dehalogenase mutant, such as Escherichia coli C43 (DE3) or Escherichia coli BL21 (DE3).

[0015] In a fifth aspect, the present invention provides a method for preparing the halohydrin dehalogenase mutant described in the first aspect, comprising the following steps: (1) culturing the recombinant cell described in the fourth aspect; and (2) inducing expression of the halohydrin dehalogenase mutant.

[0016] In step (1), the recombinant cells can be cultured by first using LB culture medium containing ampicillin at a final concentration of 100 μg / mL, and culturing at 37°C for 8 hours to obtain a seed solution; then the seed solution is inoculated into a sterile TB culture medium containing ampicillin at a final concentration of 100 μg / mL at a volume concentration of 0.5% to 5% (preferably 1%), and cultured at 37°C for about 8 hours to 12 hours, so that the bacterial concentration OD600 is 0.4 to 0.8.

[0017] The LB culture solution contains 10 g / L peptone, 5 g / L yeast extract, and 10 g / L sodium chloride, and the solvent is deionized water, with a pH of 7.0. The TB culture solution contains 2% tryptone, 2.4% yeast extract, 72 mM K2HPO4, 17 mM KH2PO4, and 0.4% glycerol.

[0018] In step (2), isopropylthio-β-D-galactoside (IPTG) is added to the culture medium at a final concentration of 0.2 mM to 0.8 mM (preferably 0.5 mM), and the expression is induced at 20°C for 12 h to 16 h.

[0019] Furthermore, in step (2), after expression is completed, the cells are collected, or the cells are crushed and the crude enzyme solution is collected, or the cells are crushed and the isolated and purified halohydrin dehalogenase mutant is collected.

[0020] The cells, i.e., wet bacteria, can be collected by centrifugation at 4°C and 4000 rpm for 10 to 20 minutes. The collected cells, crude enzyme solution, and halohydrin dehalogenase mutants can all catalyze efficient and accurate dehalogenation degradation of DCP and CPD.

[0021] In a sixth aspect, the present invention provides a product, comprising the halohydrin dehalogenase mutant described in the first aspect, or the nucleic acid molecule described in the second aspect, or the recombinant vector described in the third aspect, or the recombinant cell described in the fourth aspect, or the cell or crude enzyme solution or halohydrin dehalogenase mutant described in the fifth aspect.

[0022] The product can prepare the halohydrin dehalogenase mutant described in the first aspect, or catalyze the efficient and accurate dehalogenation degradation of DCP and CPD.

[0023] Furthermore, the product includes immobilized enzymes or immobilized cells made using immobilization technology. The immobilization technology positions the enzymes or cells in a limited area, which can increase their concentration, maintain high biological activity and allow for repeated use, facilitating their application in pollution control fields such as sewage treatment.

[0024] In a seventh aspect, the present invention provides an application of the product described in the sixth aspect in catalyzing the production of epichlorohydrin from 2,3-dichloro-1-propanol or in catalyzing the production of glycidol from 3-chloro-1,2-propylene glycol.

[0025] The application includes using a halohydrin dehalogenase mutant as a catalyst, using DCP or CPD as a substrate, and forming a reaction system with a buffer solution of pH 7 to 10 (preferably a Tris-SO4 buffer solution of pH 8 to 8.5), reacting at 300 rpm to 500 rpm (preferably 400 rpm) and 20° C. to 37° C. (preferably 25° C.), and after the reaction is completed, extracting with ethyl acetate and performing gas chromatography detection. The Tris-SO4 buffer system is a 50 mM Tris (trishydroxymethylaminomethane) buffer solution, and 50% sulfuric acid is used to adjust the pH to 8 to 8.5.

[0026] Furthermore, the dosage of the catalyst is 2 to 40 (preferably OD600=10) in terms of OD600, and the initial concentration of the substrate is 0.5 mM to 80 mM (preferably 20 mM).

[0027] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0028] 1. The halohydrin dehalogenase mutant provided by the present invention can efficiently and accurately degrade DCP and CPD, two halohydrin pollutants, to reduce their toxicity and produce high value-added epoxidation products. The mutant A83W / P84G / F86W can achieve a dehalogenation efficiency of 42.2% and 98.6% for the two substances within 1 hour, respectively, which are 6 times and 5.2 times that of the wild type;

[0029] 2. The halohydrin dehalogenase mutant provided by the present invention can achieve efficient dehalogenation at room temperature in the aqueous phase without the need for external temperature control equipment. It has the characteristics of high efficiency, low energy consumption, and high selectivity. It has the advantages of green, low-carbon, economical and convenient industrial application, and is beneficial to the dehalogenation and resource utilization of materials in the fields of sewage treatment and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Schematic diagram of the reaction of HheC catalyzing DCP to produce ECH;

[0031] Figure 2 Schematic diagram of the reaction of HheC catalyzing CPD to produce GDL;

[0032] Figure 3 Schematic diagram of the yield of ECH produced by DCP catalyzed by wild-type HheC and its mutants;

[0033] Figure 4 Schematic diagram of the yield of GDL produced by CPD catalyzed by wild-type HheC and its mutants;

[0034] Figure 5 The kinetic curves of wild-type HheC and mutant A83W / P84G / F86W catalyzing DCP to produce ECH;

[0035] Figure 6 The kinetic curves of wild-type HheC and mutant A83W / P84G / F86W catalyzing CPD to produce GDL;

[0036] Figure 7 The effect of pH on the production of ECH from DCP catalyzed by HheC and mutant A83W / P84G / F86W;

[0037] Figure 8 The effect of pH on the production of GDL by CPD catalyzed by HheC and mutant A83W / P84G / F86W;

[0038] Fig. 9 The effect of temperature on the production of ECH from DCP catalyzed by HheC and mutant A83W / P84G / F86W;

[0039] Fig.10 The effect of temperature on the production of GDL by CPD catalyzed by HheC and mutant A83W / P84G / F86W. DETAILED DESCRIPTION

[0040] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0041] In the embodiment, the halohydrin dehalogenase HheC (PDB ID: 1PWX) from Agrobacterium radiobacterAD1 is used as the mutant original enzyme (wild type), and its nucleic acid sequence is shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO.2. The wild type HheC and its mutants can catalyze 2,3-dichloro-1-propanol (DCP) to produce epichlorohydrin (ECH), and the reaction process is as follows: Figure 1 As shown; wild-type HheC and its mutants can also catalyze 3-chloro-1,2-propanediol (CPD) to produce glycidol (GDL), the reaction process is as follows Figure 2 shown.

[0042] Example 1 Construction of recombinant vector pET22b(+)-HheC plasmid

[0043] The halohydrin dehalogenase HheC gene was synthesized by Jinweizhi (Suzhou) Company and constructed on the pET22b(+) vector. The constructed plasmid was transformed into competent cells E. coli DH5α, and the transformed mixture was evenly spread on a plate of LB solid culture medium, incubated inverted in a 37°C incubator for 16 hours, and a single colony was picked and inoculated into 5mL of sterile LB liquid culture medium. After culturing at 37°C and 150rpm for 8h to 12h, a column plasmid extraction kit was used to extract the pET22b(+)-HheC plasmid from E. coli DH5α as a template for iterative saturation mutation.

[0044] Example 2 Construction of recombinant E. coli BL21 (DE3)-HheC strain

[0045] According to the conclusion of Example 1, primers were designed based on the gene sequence of wild-type halohydrin dehalogenase HheC (see Table 1). The parental HheC gene was iteratively saturated mutated using primers I81NNK-F / I81NNK-R, A83NNK-F / A83NNK-R, P84NNK-F / P84NNK-R, F86NNK-F / F86NNK-R, A133NNK-F / A133NNK-R, T134NNK-F / T134NNK-R, Y145NNK-F / Y145NNK-R, N176NNK-F / N176NNK-R, F186NNK-F / F186NNK-R, and F187NNK-F / F187NNK-R, respectively. pET22b(+) was used as the expression vector to obtain mutant plasmids carrying the target gene, and the mutant plasmids carrying the target gene were transformed into E. coli Mutants of recombinant bacteria containing the HheC mutant gene were obtained in BL21(DE3).

[0046] Table 1 Primers for constructing HheC iterative saturation mutation library

[0047]

[0048]

[0049] The PCR amplification system was: 50 μL reaction system (30 μL ddH2O, 5 μL 10×Buffer, 5 μL dNTP, 3 μL MgSO4, 2 μL DMSO, 1.5 μL 50 μM upstream primer, 1.5 μL 50 μM downstream primer, 1 μL KOD enzyme, 1 μL template DNA (plasmid)).

[0050] The PCR reaction conditions were as follows: pre-denaturation at 95°C for 3 min, followed by temperature cycling at 95°C for 20 s, 55°C for 10 s, and 72°C for 30 s for a total of 30 cycles, and finally 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, 1 μL of DpnI was added to the PCR product and digested at 37°C for 2 h to remove the template plasmid DNA. Transformed into competent cells E. coli DH5α, the transformed mixture was evenly spread on a plate containing 100 μg / mL ampicillin on an LB solid medium, inverted and cultured in a 37°C incubator for 16 h, a single colony was picked and inoculated into 5 mL of sterile LB liquid medium containing 100 μg / mL ampicillin, and cultured at 37°C and 150 rpm for 8 h to 12 h. The saturated mutation library plasmid was extracted from E. coli DH5α using a column plasmid extraction kit, and then transformed into E. coli BL21 (DE3) competent cells, coated with 100 μg / mL ampicillin on an LB plate, and cultured at 37°C overnight to obtain the HheC mutation library. At this time, many single colonies with different mutations appeared on the LB plate, and these single colonies were used for subsequent screening of the mutation library.

[0051] The parent strain E. coli BL21(DE3)-HheC WT was constructed using the same method.

[0052] Example 3 Screening of HheC mutants with improved dehalogenation efficiency

[0053] Screening of HheC mutant library Using HheC WT as a reference, single colony clones were picked from the mutant library constructed in Example 2 and cultured in a 1 mL deep 96-well plate. 400 μL of LB culture medium containing a final concentration of 100 μg / mL ampicillin was added in advance, and three parent strains were picked and placed in the last three wells of the 96-well plate as controls. A 1mL 96-well plate was cultured at 37°C for 8h as the seed solution, and then 100μL of the seed solution was added to a new 2mL deep 48-well plate for culture. Sterile TB culture medium (formula: 2% tryptone, 2.4% yeast extract, 72mM K2HPO4, 17mM KH2PO4, 0.4% glycerol) containing a final concentration of 100μg / mL ampicillin was added in advance. After culturing at 37°C for 12h, IPTG was added at a final concentration of 0.5mM. After inducing expression at 20°C for 16h, centrifuged at 4°C 4000rpm for 5min, the supernatant was discarded, and the wet bacteria were collected for the next step of screening.

[0054] Add 500 μL of 50 mM Tris-SO4 (pH=8.5) buffer to each well, resuspend the cells, add 20 mM DCP and 20 mM CPD at a final concentration, and react at 25°C and 400 rpm for 1 hour. Extract with 500 μL of ethyl acetate, centrifuge at 12000 rpm for 1 minute, and take 300 μL of supernatant for gas chromatography.

[0055] Gas phase analysis conditions: Agilent-8860GC and Cyclosil-B column, gas phase program is 60°C for 2 min, 10°C / min program to 160°C, and then 50°C / min program to 240°C. Retention time is Rt(DCP)=11.7min, Rt(CPD)=12.5min, Rt(ECH)=5.8min, Rt(GDL)=7.1min.

[0056] Test results such as Figure 3 and Figure 4 The amount of ECH and GDL produced by the wild type was used as a control. The mutant A83W / P84G / F86W degraded DCP to produce ECH with a yield of 42.2% within 1 hour, which was 6 times that of the wild type HheC, and degraded CPD to produce GDL with a yield of 98.6%, which was 5.2 times that of the wild type HheC.

[0057] Example 4 Degradation Kinetics of DCP and CPD by HheC Mutant A83W / P84G / F86W

[0058] According to the conclusion of Example 3, the recombinant bacteria expressing mutant A83W / P84G / F86W obtained by screening were inoculated into a sterile test tube containing 100 μg / mL ampicillin LB medium in advance, and cultured at 37°C 150rpm shaking for 6h-8h, and then added to a 2L conical flask according to the inoculum of 1%, and 1L of sterile TB medium containing 100 μg / mL ampicillin was added in advance, and cultured at 37°C for 12h, and then IPTG with a final concentration of 0.5mM was added, and after induced expression at 20°C for 16h, the supernatant was discarded and the wet cells were collected. 20mL 50mM Tris-SO4 (pH=8.5) buffer was added to the wet cells to resuspend the cells, 10 μL of the supernatant was taken to measure the cell concentration OD600, and the buffer was added to dilute to OD600=10.

[0059] Add 5 mL of the diluted supernatant and DCP and CPD with a final concentration of 20 mM to two 10 mL glass reaction bottles, respectively, put in a magnetic stirrer, stir and mix using a magnetic stirrer, react at 25 °C and 400 rpm for 60 min, and take the supernatant after the reaction at 5 min, 10 min, 20 min, 30 min, and 60 min.

[0060] 500 μL of the reaction solution was taken from each sample, extracted with 500 μL of ethyl acetate, and then centrifuged at 12000 rpm for 1 min. 300 μL of the supernatant was taken and detected by gas chromatography.

[0061] The degradation kinetics of DCP and CPD by HheC mutant A83W / P84G / F86W are shown in Figure 2 Figure 5 and Figure 6 As the reaction proceeded, DCP and CPD were degraded, and the concentrations of the dehalogenated products ECH and GDL gradually increased. At 60 min, the yield of ECH reached 42.2%, and the yield of GDL reached 98.6%.

[0062] Example 5 Optimal pH for the degradation of DCP and CPD by HheC mutant A83W / P84G / F86W

[0063] Based on the conclusion of Example 4, the optimal pH for HheC mutant A83W / P84G / F86W to degrade DCP and CPD was explored. The wet cell preparation method was the same as that of Example 4, and the cells were resuspended to OD600=10 for later use.

[0064] 1 mL of the resuspended mixed solution was added to 5 sterile centrifuge tubes of 2 mL, centrifuged at 4 ° C 4000 rpm for 10 min, the supernatant was discarded, 900 μL of buffers of different pH (pH = 6 sodium hydrogen phosphate-citric acid buffer, pH = 7.4 sodium hydrogen phosphate-citric acid buffer, pH = 8 sodium hydrogen phosphate-citric acid buffer, pH = 8.5 Tris-SO4 buffer, pH = 9 sodium borate buffer, pH = 10 borate buffer) were added to 5 centrifuge tubes to resuspend the bacterial solution, 500 μL of the supernatant was added to 5 5 mL glass reaction bottles, and then DCP was added to a final concentration of 20 mM, and a magnetic stirrer was used to stir and mix, and the reaction was carried out at 25 ° C 400 rpm for 1 h. The extraction and detection methods of the reaction solution are the same as those in Example 4.

[0065] The experimental results are as follows Figure 7 As shown, the optimal pH for the HheC mutant A83W / P84G / F86W to dehalogenate DCP is 10, and it can maintain good activity in the pH range of 8 to 10, with an ECH yield of >40%.

[0066] The optimal pH results for CPD dehalogenation of HheC mutant A83W / P84G / F86W were obtained by referring to the above method. Figure 8 As shown, its optimum pH is 8, and it can maintain good activity in the pH range of 8-9, with a GDL yield of >98%.

[0067] Example 6 Optimal Temperature for Degrading DCP and CPD by HheC Mutant A83W / P84G / F86W

[0068] Based on the conclusion of Example 4, the optimal temperature for HheC mutant A83W / P84G / F86W to degrade DCP and CPD was explored. The wet cell preparation method was the same as that of Example 4, and the cells were resuspended to OD600=10 for later use.

[0069] 1 mL of the diluted supernatant was taken into 8 5 mL glass reaction bottles, and DCP with a final concentration of 20 mM was added to 4 of the reaction bottles, and CPD with a final concentration of 20 mM was added to the remaining 4 reaction bottles, and a magnetic stirrer was placed, and the mixture was stirred and mixed using a magnetic stirrer, and the mixture was reacted at 18 ° C, 25 ° C, 37 ° C, and 45 ° C at 400 rpm for 1 h. The extraction and detection methods of the reaction solution were the same as those in Example 4.

[0070] The experimental results are as follows Fig. 9 and Fig.10 As shown. Different temperatures have a significant effect on the catalytic effect of HheC mutants. The optimum temperature of HheC mutant A83W / P84G / F86W for substrate DCP is 45°C, and the catalytic activity increases with increasing temperature. At room temperature, the catalytic activity is >40%; for substrate CPD, it has the highest degradation activity at room temperature, and the catalytic activity of HheC mutant for CPD will decrease with increasing or decreasing temperature. Therefore, HheC mutants can efficiently dehalogenate DCP and CPD at room temperature without the need for external temperature control equipment, saving energy consumption, and have the characteristics of green and low carbon.

[0071] Example 7 Michaelis-Menten kinetic parameters for degradation of DCP and CPD by HheC mutant A83W / P84G / F86W

[0072] Based on the conclusion of Example 4, the Michaelis-Menten kinetic parameters of the degradation of DCP and CPD by the HheC mutant A83W / P84G / F86W were investigated. The wet bacterial cells were prepared in the same manner as in Example 4 and resuspended for later use.

[0073] The mixed solution after resuspending was ultrasonically broken at 4°C for 10 min, the ultrasonic power was 65%, the ultrasound was turned on for 2s and turned off for 6s. Then centrifuged at 4°C 12000rpm for 15min, the supernatant was taken, and the protein was purified by affinity chromatography. The absorbance was measured at A280 using a UV spectrophotometer to calculate the protein concentration. Diluted with 50mM Tris-SO4 (pH=8.5) buffer to a protein concentration of 1μM. Take 1mL of the diluted protein solution in 10 5mL glass reaction bottles, of which 5 glass reaction bottles were respectively added with DCP at a final concentration of 0.5mM, 2mM, 5mM, 10mM, and 50mM, and the remaining 5 glass reaction bottles were respectively added with CPD at a final concentration of 0.5mM, 1mM, 5mM, 10mM, and 25mM, and the reaction was carried out at 25°C 400rpm with magnetic stirring for 10min. The extraction and detection methods of the reaction solution are the same as those in Example 4.

[0074] The enzyme catalyzed reaction rate was determined, and a double reciprocal curve was drawn based on the reciprocal of the reaction rate and substrate concentration to calculate the Michaelis-Menten kinetic parameters. The results showed that the Michaelis-Menten kinetic parameters Km and Kcat of the HheC mutant for DCP were 0.50 mM and 0.25 s, respectively. -1 The Km and Kcat for CPD were 1.84 mM and 2.87 s -1 .

Claims

1. A halohydrin dehalogenase mutant with improved dehalogenation efficiency, characterized in that: The amino acid sequence of the halohydrin dehalogenase mutant is obtained by mutation of the sequence shown in SEQ ID NO.2, and the mutation is at least one of A83W, P84G, and F86W.

2. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the halohydrin dehalogenase mutant according to claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that The nucleotide sequence of the nucleic acid molecule is obtained by base mutation of the sequence shown in SEQ ID NO.

1.

4. A recombinant vector, characterized in that: Comprising the nucleotide sequence of claim 3.

5. A recombinant cell, characterized in that The recombinant cell comprises the recombinant vector according to claim 4.

6. The method for preparing a halohydrin dehalogenase mutant according to claim 1, characterized in that: The method comprises the following steps: (1) culturing the recombinant cell according to claim 5; and (2) inducing expression of the halohydrin dehalogenase mutant.

7. The preparation method according to claim 6, characterized in that: In step (2), after the expression is completed, the cells are collected, or the cells are crushed and the crude enzyme solution is collected, or the cells are crushed and the isolated and purified halohydrin dehalogenase mutant is collected.

8. A product, characterized in that The product comprises the halohydrin dehalogenase mutant according to claim 1, or the nucleic acid molecule according to any one of claims 2 to 3, or the recombinant vector according to claim 4, or the recombinant cell according to claim 5, or the cell or crude enzyme solution or halohydrin dehalogenase mutant according to claim 7.

9. The product according to claim 8, characterized in that The products include immobilized enzymes or immobilized cells produced by using immobilization technology.

10. Use of the product according to claim 8 in catalyzing the production of epichlorohydrin from 2,3-dichloro-1-propanol or in catalyzing the production of glycidol from 3-chloro-1,2-propanediol.

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