Halohydrin dehalogenase mutants with improved dehalogenation efficiency and their applications

Abstract

The present invention discloses a halohydrin dehalogenase mutant with improved dehalogenation efficiency and its application. The wild-type halohydrin dehalogenase is from Agrobacterium radiobacter ( Agrobacterium radiobacter AD1). It is modified by means of directed evolution to screen out halohydrin dehalogenase mutants with improved dehalogenation efficiency. The mutations of the mutants include at least one of A83W, P84G, and F86W. The halohydrin dehalogenase mutant provided by the present invention can efficiently and accurately degrade halogenated alcohol pollutants 2,3-dichloro-1-propanol and 3-chloro-1,2-propanediol, reduce their toxicity, and produce high-value-added products. The dehalogenation efficiency of the mutant A83W / P84G / F86W for these two substances can reach 42.2% and 98.6% respectively within 1 hour, which are 6 times and 5.2 times that of the wild type. The halohydrin dehalogenase mutant provided by the present invention can achieve efficient dehalogenation at normal temperature in the aqueous phase without the addition of external temperature control equipment. It has the characteristics of high efficiency, low energy consumption, and high selectivity, and has the industrial application advantages of being green, low-carbon, economical, and convenient, which is conducive to the dehalogenation and resource utilization of pollutants.

Description

Technical Field

[0001] The present invention relates to a halohydrin dehalogenase, and particularly to a halohydrin dehalogenase mutant, belonging to the technical field of environmental remediation microorganisms. Background Art

[0002] Persistent organic pollutants (POPs) refer to organic pollutants that have environmental persistence, bioaccumulation potential, the potential for long-range environmental migration, and have adverse effects on human health or the ecological environment. China 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. Halohydrins are important intermediates for the synthesis of chiral drugs and pesticide chemicals, and are also important intermediate metabolites in the biodegradation of halogenated persistent organic pollutants. They are generated in large quantities during industrial production processes and accumulate continuously during biodegradation processes, and are ultimately discharged into the environment. Halogenated aliphatic alcohols in halohydrins belong to highly toxic compounds, which can cause nervous system and metabolic disorders and have obvious stimulatory effects, and are toxic organic pollutants in the environment. Typical halogenated aliphatic alcohols such as chloropropanol include four homologues such as 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 environmentally friendly products with high added value. For example, it can catalyze the degradation of DCP to produce epichlorohydrin (ECH), or catalyze the 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] The currently known types of halohydrin dehalogenases are relatively few, and the dehalogenation efficiency of natural halohydrin dehalogenases is insufficient, which restricts their application in the biological treatment of high-concentration halogenated alcohol pollutants. In addition, the research on halohydrin dehalogenases mainly focuses on the asymmetric synthesis of pharmaceutical intermediates, and there are few reports on the environmental application research to improve their dehalogenation and detoxification efficiency. For example, the halohydrin dehalogenase mutant and engineered bacterium 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 mutant has higher enantioselectivity, but no modification is carried out to improve the dehalogenation activity. Chinese Patent Application CN 110423740 A discloses single-point mutations or combined mutations of arginine at position 89, valine at position 137, proline at position 178, asparagine at position 179, and phenylalanine at position 187 of halohydrin dehalogenase to obtain mutants, and the enantioselectivity rates are significantly improved when preparing (S)-o-nitrophenyl glycidyl ether, (R)-benzyl glycidyl ether, and (R)-phenyl glycidyl ether, but the dehalogenation efficiency for other aliphatic halogenated alcohol substrates needs to be improved. Chinese Patent Application CN 118222538 A discloses a halohydrin dehalogenase HheC mutant T134M or P84L / W139A, using the halohydrin dehalogenase HheC from Agrobacterium radiobacter AD1 with the most stereoselective reaction activity as the mutant original enzyme, realizing the efficient enantioselective biosynthesis of the key pharmaceutical intermediate chiral tetrahydrofuran compound, but the substrate is δ-halohydrin, and other aliphatic halogenated alcohols are not mentioned. Halohydrin dehalogenase HheC can catalyze the dehalogenation reaction of halohydrin to generate the corresponding epoxide and halide ion, and has a detoxification and metabolism effect on aliphatic halogenated alcohol pollutants. However, the existing halohydrin dehalogenase HheC has a low catalytic dechlorination efficiency for DCP and CPD and does not meet the conditions for application in environmental pollution control or resource utilization, etc. Summary of the Invention

[0005] Object of the Invention: The object of the present invention is to provide a halohydrin dehalogenase mutant with improved dehalogenation efficiency, realizing the efficient and precise 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, as well as the preparation method and application of the mutant.

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

[0007] In the present invention, the halohydrin dehalogenase HheC from Agrobacterium radiobacter AD1 is used as the mutant original enzyme (wild type), and its amino acid sequence is shown in SEQ ID NO.2. This enzyme can catalyze the degradation of DCP to produce ECH and can also catalyze the degradation of CPD to produce GDL. The present invention modifies the key amino acids at the active center of HheC through directed evolution technology and protein engineering, and finds that the 83rd, 84th, and 86th positions are the key sites affecting the activity and selectivity of HheC. Site-directed saturation and iterative saturation mutagenesis are respectively carried out on these three sites, and halohydrin dehalogenase mutants with improved dehalogenation efficiency for DCP and CPD are screened out. The mutants are obtained by mutating the aforementioned sites individually or in combination, specifically: alanine at the 83rd position is mutated to tryptophan, proline at the 84th position is mutated to glycine, and phenylalanine at the 86th position is mutated to tryptophan. The halohydrin dehalogenase mutants provided by the present invention can achieve green, efficient, and precise biotoxicity removal of haloalcohols and be converted into high-value-added epoxy products.

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

[0009] In the second aspect, the present invention provides a nucleic acid molecule that encodes the halohydrin dehalogenase mutant described in 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 from the sequence shown in SEQ ID NO.1.

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

[0012] The recombinant vector includes a cloning vector or an expression vector, and can maintain its replication or autonomous replication ability in various host cells such as prokaryotic and / or eukaryotic cells, so as to amplify or express the nucleotide sequence. The vector can be various conventional vectors in the art, such as various plasmids, phage or viral vectors, etc. Preferably, the pET22b(+) plasmid is used as the expression vector.

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

[0014] The recombinant cell includes the recombinant genetically engineered bacterium prepared by transformation with the recombinant vector. Preferably, Escherichia coli is used as the recombinant cell to express the haloalkane 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 haloalkane dehalogenase mutant described in the first aspect, including the following steps: (1) culturing the recombinant cell described in the fourth aspect; (2) inducing the expression of the haloalkane dehalogenase mutant.

[0016] In step (1), for the culture of the recombinant cell, an LB culture medium containing ampicillin with a final concentration of 100 μg / mL can be first used to culture at 37 °C for 8 h to obtain a seed solution; then the seed solution is inoculated into a sterile TB culture medium containing ampicillin with a final concentration of 100 μg / mL at an inoculation amount of 0.5% - 5% (preferably 1%) by volume, and cultured at 37 °C for about 8 h - 12 h until the cell concentration OD600 is 0.4 - 0.8.

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

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

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

[0020] The cells, namely the wet bacterial cells, can be collected by centrifugation at 4 °C and 4000 rpm for 10 min - 20 min. The collected cells, crude enzyme solution, and haloalkane dehalogenase mutant can all catalyze the efficient and precise dehalogenation degradation of DCP and CPD.

[0021] In a sixth aspect, the present invention provides a product, which includes the haloalkane 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 haloalkane dehalogenase mutant described in the fifth aspect.

[0022] The product can be used to prepare the haloalkane dehalogenase mutant described in the first aspect, or catalyze the efficient and precise dehalogenation degradation of DCP and CPD.

[0023] Furthermore, the product includes immobilized enzymes or immobilized cells prepared by using immobilization technology. The immobilization technology localizes the enzymes or cells in a defined area, which can increase their concentration, maintain high biological activity and be reused, facilitating their application in the field of pollution treatment such as sewage treatment.

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

[0025] The application includes using a halohydrin dehalogenase mutant as a catalyst, using DCP or CPD as a substrate, using a buffer solution with a pH of 7 to 10 (preferably a Tris-SO4 buffer solution with a pH of 8 to 8.5) to form a reaction system, and carrying out the reaction at 300 rpm to 500 rpm (preferably 400 rpm) and 20°C to 37°C (preferably 25°C). After the reaction is completed, it is extracted with ethyl acetate and detected by gas chromatography. The Tris-SO4 buffer solution system is a 50 mM Tris (tris(hydroxymethyl)aminomethane) buffer solution, and the pH is adjusted to 8 to 8.5 using 50% sulfuric acid.

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

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

[0028] 1. The halohydrin dehalogenase mutant provided by the present invention can efficiently and precisely degrade two kinds of halogenated alcohol pollutants, DCP and CPD, reduce their toxicity, and produce high-value-added epoxidized products. The dehalogenation efficiency of mutant A83W / P84G / F86W for the two substances can reach 42.2% and 98.6% respectively within 1 hour, which is 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 normal temperature in the aqueous phase, without the need for external temperature control equipment, and has the characteristics of high efficiency, low energy consumption, and high selectivity. It has the industrial application advantages of being green, low-carbon, economical, and convenient, and is beneficial to the dehalogenation and resource utilization of substances in the fields such as sewage treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is a schematic diagram of the reaction of HheC catalyzing DCP to produce ECH;

[0031] Figure 2 It is a schematic diagram of the reaction of HheC catalyzing CPD to produce GDL;

[0032] Figure 3 Schematic diagram of the yields of wild-type HheC and its mutants catalyzing DCP to produce ECH;

[0033] Figure 4 Schematic diagram of the yields of wild-type HheC and its mutants catalyzing CPD to produce GDL;

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

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

[0036] Figure 7 Effect of pH on the catalysis of DCP by HheC and mutant A83W / P84G / F86W to produce ECH;

[0037] Figure 8 Effect of pH on the catalysis of CPD by HheC and mutant A83W / P84G / F86W to produce GDL;

[0038] Figure 9 Effect of temperature on the catalysis of DCP by HheC and mutant A83W / P84G / F86W to produce ECH;

[0039] Figure 10 Effect of temperature on the catalysis of CPD by HheC and mutant A83W / P84G / F86W to produce GDL. Detailed implementation manners

[0040] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0041] In Example 1, the halohydrin dehalogenase HheC (PDB ID: 1PWX) from Agrobacterium radiobacter AD1 was 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. Both wild-type HheC and its mutants can catalyze 2,3-dichloro-1-propanol (DCP) to produce epichlorohydrin (ECH), and the reaction process is as Figure 1 shown; both wild-type HheC and its mutants can also catalyze 3-chloro-1,2-propanediol (CPD) to produce glycidol (GDL), and the reaction process is as Figure 2 shown.

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

[0043] The halohydrin dehalogenase HheC gene was synthesized by Genewiz (Suzhou) Co., Ltd. and constructed on the pET22b(+) vector. The constructed plasmid was transformed into competent cells of E. coli DH5α, and the transformed mixture was evenly spread on a plate of LB solid medium and cultured upside down in an incubator at 37°C for 16 h. Single colonies were picked and inoculated into 5 mL of sterile LB liquid medium, and after culturing for 8 h to 12 h at 37°C and 150 rpm, the pET22b(+)-HheC plasmid was extracted from E. coli DH5α using a column plasmid extraction kit as a template for iterative saturation mutagenesis.

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

[0045] Based on 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 subjected to iterative saturation mutagenesis 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. Using pET22b(+) as the expression vector, mutant plasmids carrying the target gene were obtained respectively, and the mutant plasmids carrying the target gene were transformed into E. coli BL21(DE3) to obtain mutants of recombinant bacteria containing the HheC mutant gene respectively.

[0046] Table 1 Primers for constructing the HheC iterative saturation mutagenesis 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. Finally, extension was carried out at 72°C for 10 min, and the termination temperature was 4°C. After the PCR products were analyzed and verified by 1% agarose gel electrophoresis, 1 μL of DpnI was added to the PCR products and digested at 37°C for 2 h to remove the template plasmid DNA. The mixture was transformed into competent cells E. coli DH5α, and the transformed mixture was evenly spread on a plate of LB solid medium containing 100 μg / mL ampicillin and incubated inverted in a 37°C incubator for 16 h. Single colonies were picked and inoculated into 5 mL of sterile LB liquid medium containing 100 μg / mL ampicillin and cultured at 37°C under the condition of 150 rpm for 8 h to 12 h. Then, the saturated mutant library plasmid was extracted from E. coli DH5α using a column plasmid extraction kit, and then transformed into competent cells E. coli BL21(DE3). It was spread on an LB plate containing 100 μg / mL ampicillin and cultured overnight at 37°C to obtain the mutant library of HheC. At this time, many single colonies with different mutations appeared on the LB plate, and these single colonies were used for the subsequent screening of the mutant library.

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

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

[0053] The screening of the HheC mutant library was carried out with HheC WT as a reference. Single colony clones were picked from the mutant library constructed in Example 2 and transferred to a 1 mL deep 96-well plate for culture. 400 μL of LB culture medium containing ampicillin with a final concentration of 100 μg / mL was added in advance. At the same time, 3 parental strains were picked and used as controls in the last 3 wells of the 96-well plate. The 1 mL 96-well plate was incubated at 37°C for 8 h as the seed solution. Then, 100 μL of the seed solution was taken and added to a new 2 mL deep 48-well plate for culture. Sterile TB culture medium containing ampicillin with a final concentration of 100 μg / mL (formula: 2% tryptone, 2.4% yeast extract, 72 mM K2HPO4, 17 mM KH2PO4, 0.4% glycerol) was added in advance. After incubation at 37°C for 12 h, IPTG with a final concentration of 0.5 mM was added, and induction expression was carried out at 20°C for 16 h. Then, centrifugation was carried out at 4°C at 4000 rpm for 5 min, the supernatant was discarded, and the wet bacterial cells 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 DCP with a final concentration of 20 mM and CPD with a final concentration of 20 mM, and react at 25 °C and 400 rpm for 1 h. Extract with 500 μL of ethyl acetate, then centrifuge at 12000 rpm for 1 min, and take 300 μL of the supernatant for gas chromatography detection.

[0055] Gas analysis conditions: Agilent-8860 GC and Cyclosil-B chromatographic column. The gas chromatography program is to hold at 60 °C for 2 min, increase the temperature to 160 °C at a rate of 10 °C / min, and then increase the temperature to 240 °C at a rate of 50 °C / min. The retention times are Rt(DCP) = 11.7 min, Rt(CPD) = 12.5 min, Rt(ECH) = 5.8 min, and Rt(GDL) = 7.1 min.

[0056] The detection results are as Figure 3 and Figure 4 . Taking the amounts of ECH and GDL produced by the wild type as the control. The yield of ECH produced by the mutant A83W / P84G / F86W from the degradation of DCP within 1 h is 42.2%, which is 6 times that of the wild type HheC. The yield of GDL produced by the degradation of CPD is 98.6%, which is 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, inoculate the recombinant bacteria expressing the mutant A83W / P84G / F86W screened into a sterile test tube containing 10 mL of LB medium with a final concentration of 100 μg / mL ampicillin in advance, culture in a shaker at 37 °C and 150 rpm for 6 h - 8 h, then add it to a 2 L conical flask according to an inoculation amount of 1%, and add 1 L of sterile TB medium containing a final concentration of 100 μg / mL ampicillin in advance. After culturing at 37 °C for 12 h, add IPTG with a final concentration of 0.5 mM, induce expression at 20 °C for 16 h, then centrifuge at 4000 rpm for 30 min, discard the supernatant, and collect the wet cells. Add 20 mL of 50 mM Tris-SO4 (pH = 8.5) buffer to the wet cells to resuspend the cells, take 10 μL of the supernatant to measure the cell concentration OD600, and dilute with buffer to OD600 = 10.

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

[0060] Take 500 μL of each reaction solution, extract it with 500 μL of ethyl acetate, then centrifuge at 12000 rpm for 1 min, and take 300 μL of the supernatant for gas chromatography detection.

[0061] The degradation kinetic curves of DCP and CPD by the HheC mutant A83W / P84G / F86W are as Figure 5 and Figure 6 . As the reaction proceeds, DCP and CPD are degraded, and the concentrations of the dehalogenation products ECH and GDL gradually increase. The yield of ECH reaches 42.2% at 60 min, and the yield of GDL reaches 98.6%.

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

[0063] According to the conclusion of Example 4, explore the optimum pH for the degradation of DCP and CPD by the HheC mutant A83W / P84G / F86W. The method for preparing wet bacterial cells is the same as in Example 4, and resuspend to OD600 = 10 for standby.

[0064] Add 1 mL of the resuspended mixture to five 2-mL sterile centrifuge tubes respectively, centrifuge at 4000 rpm for 10 min at 4 °C, discard the supernatant, add 900 μL of buffers with different pH values (disodium hydrogen phosphate-citric acid buffer with pH = 6, disodium hydrogen phosphate-citric acid buffer with pH = 7.4, disodium hydrogen phosphate-citric acid buffer with pH = 8, Tris-SO4 buffer with pH = 8.5, borate buffer with pH = 9, borate buffer with pH = 10) to the five centrifuge tubes to resuspend the bacterial solution, take 500 μL of the supernatant and add it to five 5-mL glass reaction flasks respectively, then add DCP with a final concentration of 20 mM respectively, stir and mix well with a magnetic stirrer, and react for 1 h at 25 °C and 400 rpm. The extraction and detection methods of the reaction solution are the same as in Example 4.

[0065] The experimental results are as Figure 7 shown. The optimum pH for the dehalogenation of DCP by the HheC mutant A83W / P84G / F86W is 10, and it can maintain good activity in the pH range of 8 - 10, and the yield of ECH > 40%.

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

[0067] Example 6 Optimal temperature for the degradation of DCP and CPD by the HheC mutant A83W / P84G / F86W

[0068] According to the conclusion of Example 4, the optimal temperature for the degradation of DCP and CPD by the HheC mutant A83W / P84G / F86W was explored. The method for preparing wet cell bodies was the same as that in Example 4, and it was resuspended to OD600 = 10 for standby.

[0069] 1 mL of the diluted supernatant was taken and placed in 8 5 - mL glass reaction flasks respectively. To 4 of the reaction flasks, DCP with a final concentration of 20 mM was added, and to the remaining 4 reaction flasks, CPD with a final concentration of 20 mM was added. A magnetic stir bar was placed in each flask, and the mixture was stirred evenly using a magnetic stirrer. The reaction was carried out at 18°C, 25°C, 37°C, and 45°C under 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 Figure 9 and Figure 10 As shown. Different temperatures have a significant impact on the catalytic effect of the HheC mutant. The optimal temperature of the HheC mutant A83W / P84G / F86W for the substrate DCP is 45°C, and its catalytic activity increases with the increase of temperature. At room temperature, the catalytic activity > 40%; for the substrate CPD, it has the highest degradation activity at room temperature, and an increase or decrease in temperature will reduce the catalytic activity of the HheC mutant for CPD. Therefore, the HheC mutant can efficiently dehalogenate DCP and CPD at room temperature without the need for additional temperature control equipment, saving energy consumption and having the characteristics of green and low - carbon.

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

[0072] According to the conclusion of Example 4, the Michaelis - Menten kinetic parameters for the degradation of DCP and CPD by the HheC mutant A83W / P84G / F86W were explored. The method for preparing wet cell bodies was the same as that in Example 4, and it was resuspended for standby.

[0073] The mixed solution after resuspension was ultrasonically broken at 4°C for 10 min with an ultrasonic power of 65%, ultrasonic on for 2 s and off for 6 s. Then it was centrifuged at 12,000 rpm at 4°C for 15 min, and the supernatant was taken. The protein was purified by affinity chromatography. The absorbance was measured at A280 using an ultraviolet spectrophotometer, and the protein concentration was calculated. It was diluted to a protein concentration of 1 μM with 50 mM Tris-SO4 (pH = 8.5) buffer. 1 mL of the diluted protein solution was taken and placed in 10 5-mL glass reaction flasks respectively. Among them, 0.5 mM, 2 mM, 5 mM, 10 mM, and 50 mM DCP were added to 5 glass reaction flasks respectively, and 0.5 mM, 1 mM, 5 mM, 10 mM, and 25 mM CPD were added to the remaining 5 glass reaction flasks respectively. The reaction was magnetically stirred at 400 rpm at 25°C for 10 min. The extraction and detection methods of the reaction solution were the same as those in Example 4.

[0074] The enzymatic reaction rate was measured, and a double-reciprocal curve was made based on the reciprocal of the reaction rate and the substrate concentration to calculate the Michaelis kinetic parameters. The results showed that the Michaelis kinetic parameters Km and Kcat of the HheC mutant for DCP were 0.50 mM and 0.25 s -1 , and 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 haloalcohol dehalogenase mutant is obtained by mutating the sequence of PDB ID: 1PWX, and the mutants are F86W, P84G, A83W, A83W / P84G, A83W / P84G / F86W.

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

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

1.

4. A recombinant vector, characterized in that, Comprising the nucleotide sequence according to claim 3.

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

6. The preparation method of the halohydrin dehalogenase mutant according to claim 1, characterized in that Comprising the following steps: (1) culturing the recombinant cell according to claim 5; (2) inducing the expression of the haloalcohol 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 disrupted and the crude enzyme solution is collected, or the cells are disrupted and the isolated and purified haloalcohol dehalogenase mutant is collected.

8. A product for treating halogenated alcohol pollutants, characterized in that, The product comprises the haloalcohol 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 haloalcohol dehalogenase mutant according to claim 7.

9. The product according to claim 8, wherein, The product includes an immobilized enzyme or an immobilized cell prepared by using an immobilization technique.

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

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