Epoxide hydrolase mutant capable of efficiently degrading epichlorohydrin and application of epoxide hydrolase mutant

Abstract

The invention discloses an epoxide hydrolase mutant capable of efficiently degrading epichlorohydrin and application of the epoxide hydrolase mutant, wild epoxide hydrolase comes from radioactive agrobacterium tumefaciens AD1, the wild epoxide hydrolase is modified by means of directed evolution, the epoxide hydrolase mutant capable of efficiently degrading epichlorohydrin is screened out, and the mutation comprises at least one of D131N, Q134M and C248M. The invention further discloses a preparation method of the epoxide hydrolase mutant capable of efficiently degrading epichlorohydrin and application of the epoxide hydrolase mutant capable of efficiently degrading epichlorohydrin and application of the epoxide hydrolase mutant capable of efficiently degrading epichlorohydrin and application of the epoxide hydrolase mutant capable of efficiently degrading epichlorohydrin. The epoxide hydrolase mutant provided by the invention can be used for efficiently and accurately degrading epoxy chloropropane and generating a high value-added product 3-chloro-1, 2-propylene glycol, and the degradation efficiency of the mutant D131N / Q134M / C248M can reach 50% and is two times that of a wild type; the epoxide hydrolase mutant provided by the invention can efficiently degrade epoxy chloropropane in a water phase at normal temperature, 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 further degradation and resource utilization of pollutants.

Description

Technical Field

[0001] The invention relates to an epoxide hydrolase, in particular to an epoxide hydrolase for degrading epichlorohydrin, and belongs to the technical field of environmental remediation microorganisms. Background Art

[0002] Halogenated epoxides are widely used in industry, for example, as solvents, surfactants, adhesives, synthetic resins, etc. They are also used as soil fumigants and fungicides. Halogenated epoxides are toxic to the central nervous system and kidneys, and have obvious irritating effects. Some halogenated epoxides have hormone-like activity. As potential endocrine disruptors, they have adverse effects on the ecological environment and human health. Epichlorohydrin (ECH) is a typical halogenated epoxide. It is not only an important organic chemical raw material and fine chemical product, but also a priority controlled pollutant for water quality monitoring. Reasonable disposal of ECH is an inevitable choice for effective resource utilization and pollution reduction.

[0003] Epoxide hydrolases are widely present in animals, plants and microorganisms. They can catalyze the hydrolysis of halogenated epoxide pollutants through intramolecular nucleophilic attack mechanisms, converting them into less toxic or non-toxic diols. Epoxide hydrolases do not require the assistance of coenzymes to catalyze this reaction, have a wide range of sources, and still have catalytic activity in non-aqueous solvents. Therefore, they have good application prospects in the field of toxic and harmful environmental pollutants. In addition to the above advantages, epoxide hydrolases from microorganisms also have the characteristics of a broad substrate spectrum, making them the best choice for screening and modifying epoxide hydrolases. For example, epoxide hydrolases from Agrobacterium radiobacter AD1 can catalyze the hydrolysis of halogenated epoxides to generate corresponding diols, including catalyzing ECH to 3-chloro-1,2-propanediol (CPD). They have a detoxification and metabolic effect on halogenated epoxide pollutants and have important environmental significance and application value.

[0004] But the catalytic performance of epoxide hydrolase still needs to be further improved to meet the demand of large-scale application. Epoxide hydrolase can be transformed from aspects such as improving hydrolysis efficiency, shortening reaction time, reducing enzyme dosage, simplifying reaction conditions such as high temperature or cofactors. In addition, current research mainly focuses on improving the enantioselectivity of epoxide hydrolase, for the asymmetric synthesis of drug intermediates, and the research on improving the hydrolysis efficiency of epoxide hydrolase for environmental treatment is less. For example, Chinese patent CN 104531630A discloses a kind of epoxide hydrolase derived from Aspergillus iusami, and its mutant enzyme has the characteristics of high enantioselectivity and catalytic activity, but this research does not carry out the research of mutant enzyme practical application. Chinese patent CN 104745547 A discloses an epoxide hydrolase mutant cloned from soil mold, which mutates the tryptophan at position 182 to phenylalanine, the serine at position 207 to valine, or the asparagine at position 240 to aspartic acid, and the enantioselectivity is greatly improved, but its catalytic efficiency needs to be further improved. Zou Shuping et al. (DOI: 10.1007 / s00253-017-8634-5) mutated the epoxide hydrolase of radioactive soil bacteria, and the resulting mutant had high catalytic efficiency and enantioselectivity in the synthesis of (R)-epichlorohydrin, but did not involve application scenarios such as environmental governance such as pollutant degradation. Xu Jianhe's research group (Kong Xudong et al. Structural basis of epoxide hydrolase and development of new enzymes [J]) studied epoxide hydrolases from the aspects of screening, cloning expression, structural analysis and modification, and synthetic application, mainly focusing on its application in the synthesis of chiral compounds, but not in the degradation of pollutants. Summary of the invention

[0005] Purpose of the invention: The purpose of the present invention is to provide an epoxide hydrolase mutant capable of efficiently degrading the environmental pollutant epichlorohydrin into 3-chloro-1,2-propanediol, 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 an epoxide hydrolase mutant that efficiently degrades epichlorohydrin, wherein the amino acid sequence of the epoxide hydrolase mutant is obtained by mutation of the sequence shown in SEQ ID NO.2, and the mutation is at least one of D131N, Q134M, and C248M.

[0007] The present invention uses the epoxide hydrolase EchA 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 ECH to produce CPD. The present invention transforms the key amino acids in the active center of EchA by means of directed evolution, and finds that the 131st, 134th, and 248th are key sites that affect the activity and selectivity of EchA. These three sites are subjected to site-directed saturation and iterative saturation mutations, respectively, and epoxide hydrolase mutants that can efficiently degrade ECH are screened out. The mutant is obtained by mutating the aforementioned sites alone or in combination, specifically: the 131st aspartic acid is mutated to asparagine, the 134th glutamine is mutated to methionine, and the 248th cysteine ​​is mutated to methionine. The epoxide hydrolase mutant provided by the present invention can realize the biological efficient and accurate degradation of halogenated epoxy pollutants, and generate high value-added degradation products, which is conducive to the further degradation and resource utilization of pollutants.

[0008] The present invention uses the standard single-letter code for amino acids and the standard substitution notation, for example: D131N means that the aspartic acid (D) at position 131 at the N-terminus is mutated to asparagine (N); D131N / Q134M means that the aspartic acid (D) at position 131 at the N-terminus is mutated to asparagine (N), and the glutamine (Q) at position 134 at the N-terminus is mutated to methionine (M).

[0009] In a second aspect, the present invention provides a nucleic acid molecule encoding the epoxide hydrolase mutant described in the first aspect. The nucleic acid molecule can express the epoxide hydrolase that efficiently degrades epichlorohydrin.

[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 a recombinant cell to express the epoxide hydrolase 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 of the first aspect, comprising the following steps: (1) culturing the recombinant cell of the fourth aspect; and (2) inducing expression of the epoxide hydrolase 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 the expression is completed, the cells are collected, or the cells are disrupted and the crude enzyme solution is collected, or the cells are disrupted and the separated and purified epoxide hydrolase mutant is collected.

[0020] The cells, i.e. wet bacteria, can be collected by centrifugation at 4°C and 4000 rpm for 10-20 minutes. The collected cells, crude enzyme solution and epoxide hydrolase mutant can all efficiently catalyze and degrade ECH to produce CPD.

[0021] In a sixth aspect, the present invention provides a product, which comprises the epoxide hydrolase 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 epoxide hydrolase mutant described in the fifth aspect.

[0022] The product can be used to prepare the epoxide hydrolase mutant described in the first aspect, or catalyze the degradation of ECH.

[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 environmental management fields such as pollutant treatment.

[0024] In a seventh aspect, the present invention provides an application of the product described in the sixth aspect in catalyzing epichlorohydrin to produce 3-chloro-1,2-propylene glycol.

[0025] The application includes using the epoxide hydrolase mutant as a catalyst, ECH as a substrate, and a pH 7.4-8.5 buffer (preferably a pH 8-8.5 Tris-SO4 buffer) to form a reaction system, reacting at 300rpm-500rpm (preferably 400rpm) and 20°C-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 50mM Tris (trishydroxymethylaminomethane) buffer solution, and 50% sulfuric acid is used to adjust the pH to 8-8.5.

[0026] Furthermore, the dosage of the catalyst is 1 to 40 (preferably OD600=1 to 10) in terms of OD600, and the initial concentration of the substrate is 10 mM to 80 mM (preferably 40 mM).

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

[0028] 1. The epoxide hydrolase mutant provided by the present invention can efficiently and accurately degrade epichlorohydrin and produce a high value-added product 3-chloro-1,2-propanediol. The degradation efficiency of the mutant D131N / Q134M / C248M can reach 50%, which is twice that of the wild type;

[0029] 2. The epoxide hydrolase mutant provided by the present invention can efficiently degrade epichlorohydrin 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, which is conducive to the further degradation and resource utilization of pollutants.

[0030] 3. The present invention improves the catalytic performance of epoxide hydrolase, further expands its application in the biological treatment of toxic organic wastewater, provides an effective means for achieving efficient and precise biological degradation of halogenated epoxide pollutants, and provides a reference for the efficient biological degradation of pollutants with similar structures. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a schematic diagram of the reaction of EchA catalyzing ECH to produce CPD;

[0032] Figure 2 Schematic diagram of the yield of CPD produced by ECH catalyzed by wild-type EchA and its mutants;

[0033] Figure 3 The kinetic curves of wild-type EchA and mutant D131N / Q134M / C248M catalyzing ECH to produce CPD;

[0034] Figure 4 The effect of pH on the production of CPD by ECH catalyzed by EchA and mutant D131N / Q134M / C248M;

[0035] Figure 5 The effect of temperature on the production of CPD by EchA and its mutant D131N / Q134M / C248M. DETAILED DESCRIPTION

[0036] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0037] In the embodiment, the epoxide hydrolase EchA (PDB ID: 1EHY) 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 EchA and its mutants can catalyze the degradation of epichlorohydrin (ECH) to produce 3-chloro-1,2-propanediol (CPD), and the reaction process is as follows: Figure 1 shown.

[0038] Example 1 Construction of recombinant vector pET22b(+)-EchA plasmid

[0039] The epoxide hydrolase EchA 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 in an incubator at 37°C 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(+)-EchA plasmid from E. coli DH5α as a template for iterative saturation mutation.

[0040] Example 2 Construction of recombinant E. coli BL21 (DE3)-EchA strain

[0041] According to the conclusion of Example 1, primers were designed based on the gene sequence of wild-type epoxide hydrolase EchA (see Table 1). Primers W45NNK-F / W45NNK-R, H106NNK-F / H106NNK-R, F108NNK-F / F108NNK-R, D131NNK-F / D131NNK-R, I133NNK-F / I133NNK-R, Q134NNK-F / Q134NNK-R, Y152NNK-F / Y152NNK-R, W183 The parental EchA gene was iteratively saturated mutated by NNK-F / W183NNK-R, Y215NNK-F / Y215NNK-R, C248NNK-F / C248NNK-R, and G274NNK-F / G274NNK-R, and mutant plasmids carrying the target gene were obtained using pET22b(+) as the expression vector. The mutant plasmids carrying the target gene were transformed into E.coli BL21(DE3) to obtain mutants of recombinant bacteria containing the EchA mutant gene.

[0042] Table 1 Primers for constructing EchA iterative saturation mutation library

[0043] Primer Name Primer Sequence 5’-3’ W45NNK-F CTTTTGGTGGGAANNKAGCAAAGTG W45NNK-R CACTTTGCTMNNTTCCCACCAAAAG H106NNK-F GTGGTGGGCNNKGATTTTGCG H106NNK-R CGCAAAATCMNNGCCCACCAC F108NNK-F GTGGGCCATGATNNKGCGGCGATTGTGCTG F108NNK-R CAGCACAATCGCCGCMNNATCATGGCCCAC D131NNK-F GCGATTTTTNNKCCGATTCAG D131NNK-R CTGAATCGGMNNAAAAATCGC I133NNK-F GATTTTTGATCCGNNKCAGCCGGATTTTG I133NNK-R CAAAATCCGGCTGMNNCGGATCAAAAATC Q134NNK-F GATCCGATTNNKCCGGATTTTG Q134NNK-R CAAAATCCGGMNNAATCGGATC Y152NNK-F GAAAGCTGGNNKAGTCAGTTTC Y152NNK-R GAAACTGACTMNNCCAGCTTTC W183NNK-F CATTTTTTTGATCATNNKAGCTATCGC W183NNK-R GCGATAGCTMNNATGATCAAAAAAATG Y215NNK-F CTTTAACTATNNKCGCGCGAACATTCGCCCGGATG Y215NNK-R CATCCGGGCGAATGTTCGCGCGMNNATAGTTAAAG C248NNK-F GGCGATACCNNKGTGCCGTATG C248NNK-R CATACGGCACMNNGGTATCGCC G274NNK-F GAAGATTGCNNKCATTTTCTG G274NNK-R CAGAAAATGMNNGCAATCTTC

[0044] 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)).

[0045] 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 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 EchA 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.

[0046] The parent strain E. coli BL21(DE3)-EchAWT was constructed in the same way.

[0047] Example 3 Screening of epoxide hydrolase mutants that efficiently degrade epichlorohydrin

[0048] Screening of EchA mutant library Using EchA 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 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.

[0049] Add 500 μL of 50 mM Tris-SO4 (pH=8.5) buffer to each well, resuspend the cells to OD600=2, add epichlorohydrin at a final concentration of 150 mM, 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.

[0050] Gas phase analysis conditions: Agilent-8860GC and Cyclosil-B column, gas phase program is 60℃ for 2min, 10℃ / min program to 160℃, and then 50℃ / min program to 240℃. Retention time is Rt(epichlorohydrin)=5.8min, Rt(3-chloro-1,2-propylene glycol)=12.5min.

[0051] Test results such as Figure 2 The amount of 3-chloro-1,2-propanediol produced by the wild type was used as a control. The mutant D131N / C248M / Q134M degraded epichlorohydrin to produce 3-chloro-1,2-propanediol with a yield of 50% within 1 hour, which was twice that of the wild type EchA.

[0052] Example 4 Degradation Kinetics of Epichlorohydrin by EchA Mutant D131N / C248M / Q134M

[0053] 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=1.

[0054] Add 5 mL of the diluted supernatant and epichlorohydrin at a final concentration of 40 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 0 min, 5 min, 10 min, 20 min, and 30 min.

[0055] 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.

[0056] The degradation kinetic curve of epichlorohydrin by EchA mutant D131N / Q134M / C248M is shown in Figure 3 As the reaction proceeded, epichlorohydrin was degraded, and the concentration of the hydrolysis product 3-chloro-1,2-propylene glycol gradually increased. The yield of 3-chloro-1,2-propylene glycol reached 30% at 30 minutes.

[0057] Example 5 Optimal pH for degradation of epichlorohydrin by EchA mutant D131N / Q134M / C248M

[0058] Based on the conclusion of Example 4, the optimal pH for degradation of epichlorohydrin by EchA mutant D131N / Q134M / C248M was investigated. The wet cell preparation method was the same as that of Example 4, and the cells were resuspended to OD600=1 for later use.

[0059] 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, 400 μL of buffers of different pH (sodium hydrogen phosphate-citric acid buffer of pH = 6, sodium hydrogen phosphate-citric acid buffer of pH = 7.4, sodium hydrogen phosphate-citric acid buffer of pH = 8, Tris-SO4 buffer of pH = 8.5, sodium borate buffer of pH = 9, borate buffer of pH = 10) were added to resuspend the bacterial solution, 500 μL of the supernatant was added to 5 5 mL glass reaction bottles, and then epichlorohydrin with a final concentration of 40 mM was added, and a magnetic stirrer was used to stir and mix, and the reaction was carried out at 25 ° C 400 rpm for 30 min. The extraction and detection methods of the reaction solution are the same as those in Example 4.

[0060] The experimental results are as follows Figure 4 As shown, the optimal pH for the hydrolysis of epichlorohydrin by the EchA mutant D131N / Q134M / C248M is 8, and it can maintain good activity in the pH range of 7.4-8.5.

[0061] Example 6 Optimum temperature for degradation of epichlorohydrin by EchA mutant D131N / Q134M / C248M

[0062] Based on the conclusion of Example 4, the optimal temperature for degradation of epichlorohydrin by EchA mutant D131N / Q134M / C248M was investigated. The wet cell preparation method was the same as that of Example 4, and the cells were resuspended to OD600=1 for later use.

[0063] 1 mL of the diluted supernatant was taken into 4 5 mL glass reaction bottles, and epichlorohydrin with a final concentration of 40 mM was added, 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 30 min. The extraction and detection methods of the reaction solution were the same as those in Example 4.

[0064] The experimental results are as follows Figure 5 As shown. Different temperatures have a significant effect on the catalytic effect of EchA mutant D131N / Q134M / C248M. For the substrate epichlorohydrin, the optimum temperature of EchA mutant D131N / Q134M / C248M is 25°C, that is, it has the highest degradation activity at room temperature. Increasing or decreasing the temperature will reduce the catalytic activity of EchA mutant for the substrate epichlorohydrin. Therefore, EchA mutant can efficiently hydrolyze epichlorohydrin at room temperature without the need for external temperature control equipment, saving energy consumption, and has the characteristics of green and low carbon.

[0065] Example 7 Michaelis-Menten kinetic parameters for degradation of epichlorohydrin by EchA mutant D131N / Q134M / C248M

[0066] Based on the conclusion of Example 4, the Michaelis-Menten kinetic parameters of the degradation of epichlorohydrin by the EchA mutant D131N / Q134M / C248M were investigated. The wet cell preparation method was the same as that of Example 4, and the cells were resuspended for later use.

[0067] The resuspended mixed solution was ultrasonically broken at 4°C for 10 min, with an ultrasonic power of 65%, with ultrasound on for 2 s and off for 6 s. Centrifuge at 4°C 12000 rpm for 15 min, take the supernatant, and purify the protein using affinity chromatography. Use a UV spectrophotometer to measure the absorbance at A280 and calculate the protein concentration. Dilute with 50mM Tris-SO4 (pH=8.5) buffer to a protein concentration of 10 μM. Take 1 mL of the diluted protein solution in 5 5 mL glass reaction bottles, add epichlorohydrin with a final concentration of 35mM, 40mM, 45mM, 50mM, and 55mM, respectively, and react with magnetic stirring at 25°C 400rpm for 10 min. The extraction and detection methods of the reaction solution are the same as those in Example 4.

[0068] 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 EchA mutant D131N / Q134M / C248M for epichlorohydrin were 0.22M and 46.33s, respectively. -1 .

Claims

1. A mutant of an epoxide hydrolase that efficiently degrades epichlorohydrin, characterized in that: The amino acid sequence of the epoxide hydrolase mutant is obtained by mutation of the sequence shown in SEQ ID NO.2, and the mutation is at least one of D131N, Q134M, and C248M.

2. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the epoxide hydrolase 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 the epoxide hydrolase 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 epoxide hydrolase 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 disrupted and the crude enzyme solution is collected, or the cells are disrupted and the isolated and purified epoxide hydrolase mutant is collected.

8. A product, characterized in that The product comprises the epoxide hydrolase 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 epoxide hydrolase 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 epichlorohydrin to produce 3-chloro-1,2-propylene glycol.

Images