Haloalkane dehalogenase mutants and their application in the degradation of 1,2,3-tribromopropane

Abstract

The present invention discloses a haloalkane dehalogenase mutant and its application in degrading 1,2,3-tribromopropane. Rhodococcus rhodochrous NCIMB 13064 strain dhaA The genetically encoded haloalkane dehalogenase DhaA31 was modified to obtain a haloalkane dehalogenase mutant F168W / I246C. The haloalkane dehalogenase mutant has a high degradation efficiency for the haloalkane pollutant 1,2,3-tribromopropane. Under normal temperature conditions in an aqueous phase, the haloalkane dehalogenase mutant has a degradation rate of 100% for 10 mM 1,2,3-tribromopropane within 1 hour; the haloalkane dehalogenase mutant has high stereoselectivity for (R)-2,3-dibromo-1-propanol, with an er value of 6:94. The haloalkane dehalogenase mutant provided by the present invention has high catalytic efficiency and high stereoselectivity, which is conducive to the recognition and further degradation of downstream enzymes, and has good application prospects in the bioremediation of emerging haloalkane pollutants.

Description

Technical Field

[0001] The invention relates to a haloalkane dehalogenase, in particular to a haloalkane dehalogenase with high degradation efficiency and high stereoselectivity, and belongs to the technical field of bioremediation and enzyme engineering. Background Art

[0002] Halogenated alkanes are commonly used as chemical solvents, organic synthesis raw materials, and intermediates in chemical production processes. They are also discharged into the environment with chemical waste liquids and wastewater. Halogenated alkanes are also disinfection byproducts generated during water treatment. Due to their high persistence and difficulty in biodegrading, they have been frequently detected in water treatment systems and natural water bodies in recent years. As an emerging class of difficult-to-biodegrade organic pollutants, they pose a serious threat to human health and ecological security. 1,2,3-Tribromopropane (1,2,3-TBP) is a halogenated alkane. As a widely used chemical solvent and industrial byproduct, it is produced in large quantities during chemical production processes and discharged into water bodies with sewage and wastewater, thus becoming an organic pollutant.

[0003] Microbial haloalkane dehalogenases play a crucial role in the biodegradation of haloalkane pollutants, converting toxic, recalcitrant haloalkanes into their corresponding harmless alcohols through hydrolysis. However, existing haloalkane dehalogenases have low efficiency for 1,2,3-tribromopropane (TBP), hindering their application in complex wastewater environments. Furthermore, the low stereoselectivity of existing haloalkane dehalogenases hinders the further transformation and complete degradation of 1,2,3-TBP within the metabolic pathway. Summary of the Invention

[0004] Purpose of the invention: The purpose of the present invention is to provide a haloalkane dehalogenase mutant that has high degradation efficiency for the substrate 1,2,3-tribromopropane and high stereoselectivity for the product (R)-2,3-dibromo-1-propanol ((R)-2,3-dibromopropan-1-ol, abbreviated as (R)-DBP).

[0005] Technical solution: The present invention provides a haloalkane dehalogenase mutant, the amino acid sequence of which is shown in SEQ ID NO.4.

[0006] The present invention is based on the Rhodococcus rhodochrous NCIMB 13064 strain dhaAThe gene encoding the haloalkane dehalogenase DhaA31 (PDB ID: 3RK4) is a mutant, native enzyme (wild type). The nucleotide sequence of this enzyme is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2. Its crystal structure and catalytic molecular mechanism have been fully elucidated. Haloalkane dehalogenases belong to the α / β hydrolase fold family and possess a core domain containing the Asp-His-Asp / Glu catalytic triad and a variable, predominantly helical cap domain that provides essential residues for selectivity. Haloalkane dehalogenase DhaA31 catalyzes the conversion of 1,2,3-tribromopropane to the racemic enantiomer (R,S)-2,3-dibromo-1-propanol. 1,2,3-Tribromopropane is a difficult-to-degrade halogenated hydrocarbon pollutant. The natural haloalkane dehalogenase DhaA31 has low degradation efficiency for 1,2,3-tribromopropane and poor stereoselectivity for (R)-2,3-dibromo-1-propanol.

[0007] Based on the molecular mechanism of haloalkane dehalogenase catalysis, the present invention transforms the key amino acids in the active center of the haloalkane dehalogenase through directed evolution and semi-rational design methods. By using site-directed saturation mutagenesis, iterative saturation mutagenesis and directed screening, it is found that when the phenylalanine at position 168 of the amino acid sequence shown in SEQ ID NO.2 is mutated to tryptophan (F168W) and the isoleucine at position 246 is mutated to cysteine ​​(I246C), the resulting haloalkane dehalogenase mutant F168W / I246C has high catalytic activity and high stereoselectivity, can efficiently biodegrade haloalkane pollutants represented by 1,2,3-tribromopropane, and simultaneously produce high-value (R)-2,3-dibromo-1-propanol.

[0008] The present invention also provides a nucleotide sequence encoding the haloalkane dehalogenase mutant, including SEQ ID NO.3.

[0009] The present invention also provides a recombinant vector comprising the nucleotide sequence.

[0010] The recombinant vector can maintain its replication or autonomous replication in various prokaryotic and / or eukaryotic host cells, and can be various conventional vectors in the art, such as various plasmids, phage or viral vectors, etc., preferably using pET22b(+) plasmid as the expression vector.

[0011] The present invention also provides a recombinant cell, which comprises the recombinant vector.

[0012] The recombinant cell is preferably Escherichia coli, such as Escherichia coli C43 or Escherichia coli BL21.

[0013] The present invention also provides a method for preparing the haloalkane dehalogenase mutant, comprising the following steps: (1) constructing a recombinant vector containing a nucleotide sequence encoding the haloalkane dehalogenase mutant; (2) transforming and preparing a recombinant cell; and (3) inducing the recombinant cell to express the haloalkane dehalogenase mutant.

[0014] Preferably, in step (3), after expression is completed, the cells are collected; or the cells are disrupted to collect the crude enzyme solution or pure enzyme.

[0015] Preferably, the collected cells or pure enzymes are prepared into immobilized cells or immobilized enzymes using immobilization technology.

[0016] The haloalkane dehalogenase mutant of the present invention can catalyze the reaction of 1,2,3-tribromopropane in various forms, including whole cells, crude enzyme solution, pure enzyme, and other immobilized forms.

[0017] The preparation method comprises: inoculating a recombinant engineered bacterium containing a gene encoding a haloalkane dehalogenase mutant into an 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 inoculating the seed solution into a sterile LB liquid culture medium containing ampicillin at a final concentration of 100 μg / mL at a volume concentration of 2%, and culturing at 37°C for about 8-12 hours to obtain a bacterial cell concentration of OD 0. 600 The pH value was 0.4-0.8, and then isopropylthio-β-D-galactoside (IPTG) was added to the culture medium at a final concentration of 0.1-1.0 mM (preferably 0.5 mM). After inducing expression at 20°C for 16 h, the culture medium was centrifuged at 4°C and 4000 rpm for 10-20 min to collect the wet cells.

[0018] The present invention also provides a product, which comprises the haloalkane dehalogenase mutant, or the nucleotide sequence, or the recombinant vector, or the recombinant cell, or the cell or crude enzyme solution or pure enzyme, or the immobilized cell or immobilized enzyme.

[0019] The present invention also provides application of the product in degrading 1,2,3-tribromopropane.

[0020] The present invention also provides application of the product in the preparation of (R)-2,3-dibromo-1-propanol.

[0021] The application method comprises: using wet bacteria obtained by fermentation culture of a recombinant genetically engineered bacterium containing a gene encoding a haloalkane dehalogenase mutant as a catalyst, using 1,2,3-tribromopropane as a substrate, and using a Tris-SO4 solution with a pH of 8.0-10.0 (preferably pH 8.5) to form a reaction system, carrying out the reaction at 300-500 rpm (preferably 400 rpm) and 25-37° C. (preferably 25° C.), obtaining a reaction solution containing (R)-2,3-dibromo-1-propanol after the reaction is completed, and separating and purifying the reaction solution to obtain (R)-2,3-dibromo-1-propanol.

[0022] The Tris-SO4 solution system is a 50 mM tris (hydroxymethyl)aminomethane (Tris) aqueous solution, and the pH is adjusted to 8.0-10.0 (preferably pH 8.5) with sulfuric acid (H2SO4).

[0023] Furthermore, the amount of the catalyst is 10-40 g / L buffer (preferably 20 g / L) based on the weight of the wet cells, and the initial concentration of the substrate is 1-20 mM (preferably 10 mM).

[0024] Beneficial Effects: Compared with the prior art, the present invention has the following significant advantages: The haloalkane dehalogenase mutant F168W / I246C provided by the present invention has high degradation efficiency for the haloalkane pollutant 1,2,3-tribromopropane. Under aqueous phase conditions at room temperature, the haloalkane dehalogenase mutant achieves a 100% degradation rate of 10 mM 1,2,3-tribromopropane within 1 hour. The haloalkane dehalogenase mutant also exhibits high stereoselectivity for (R)-2,3-dibromo-1-propanol, with an er value of 6:94. The haloalkane dehalogenase mutant provided by the present invention has high catalytic efficiency and high stereoselectivity, which facilitates recognition and further degradation by downstream enzymes, and has good application prospects in the bioremediation of emerging haloalkane pollutants. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The chemical reaction formula for the dehalogenation of 1,2,3-tribromopropane catalyzed by haloalkane dehalogenase DhaA31;

[0026] Figure 2 The yield and ee value of DhaA31 wild-type enzyme and mutants catalyzing the conversion of 1,2,3-tribromopropane to 2,3-dibromo-1-propanol;

[0027] Figure 3 The chromatogram of the racemic enantiomer (R,S)-2,3-dibromo-1-propanol standard was detected by gas chromatography;

[0028] Figure 4The chromatogram shows the gas phase detection of the racemic enantiomer (R,S)-2,3-dibromo-1-propanol produced from 1,2,3-tribromopropane catalyzed by the haloalkane dehalogenase DhaA31 mutant F168W / I246C.

[0029] Figure 5 The yield and ee value of the product 2,3-dibromo-1-propanol catalyzed by DhaA31 mutant F168W / I246C from 1,2,3-tribromopropane;

[0030] Figure 6 The yield of 2,3-dibromo-1-propanol from 1,2,3-tribromopropane catalyzed by DhaA31 mutant F168W / I246C at different pH values;

[0031] Figure 7 The yields of 2,3-dibromo-1-propanol from 1,2,3-tribromopropane catalyzed by DhaA31 mutant F168W / I246C at different temperatures. DETAILED DESCRIPTION

[0032] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0033] Example 1: Construction of pET22b(+)-DhaA31 plasmid

[0034] From Rhodococcus Rhodococcus rhodochrous The engineered haloalkane dehalogenase DhaA31 gene (PDB ID: 3RK4) of NCIMB 13064 was synthesized by GenWeiZhi (Suzhou) and constructed on the pET22b(+) vector. The constructed plasmid was transformed into competent cells. E.coli DH5α, the transformed mixture was evenly spread on a plate of LB solid medium, incubated upside down at 37°C for 16 h, a single colony was picked and inoculated into 5 mL of sterile LB liquid medium (peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, solvent: deionized water), incubated at 37°C and 150 rpm for 8-12 h, and then the plasmid was extracted from the plate using a column-type plasmid extraction kit. E.coli The pET22b(+)-DhaA31 plasmid was extracted from DH5α and used as a template for iterative saturation mutagenesis.

[0035] Example 2: Construction of a haloalkane dehalogenase site-directed saturation library

[0036] According to the conclusion of Example 1, primers were designed based on the gene sequence of the engineered haloalkane dehalogenase DhaA31 included in GenBank (the nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2) (see Table 1). The parent DhaA31 gene (nucleotide sequence is SEQ ID NO.1) was subjected to site-directed saturation mutagenesis using primers C128X-F / C128X-R, F168X-F / F168X-R, F131X-F / F131X-R, A172X-F / A172X-R, Y176X-F / Y176X-R, and I246X-F / I246X-R, respectively. Mutant plasmids carrying the target gene were obtained using pET-22b(+) as the expression vector, and the mutant plasmids carrying the target gene were transformed into E .coli In BL21 (DE3), mutants of recombinant bacteria containing halogenated alkane dehalogenase mutant genes were obtained, namely E .coli BL21 (DE3) - C128X (denoted as mutant C128X), E .coli BL21 (DE3)-F168X (denoted as mutant F168X), E .coli BL21 (DE3)-F131X (denoted as mutant F131X), E .coli BL21 (DE3) - A172X (denoted as mutant A172X), E .coli BL21 (DE3) - Y176X (denoted as mutant Y176X), E .coli BL21(DE3)-I246X (denoted as mutant I246X).

[0037] Table 1: Primer design table for construction of haloalkane dehalogenase site-directed saturation mutagenesis library

[0038] Serial Number Primer Name Primer Sequence 5’-3’ SEQ ID NO.5 C128X-F GGCATTGCGNNKATGGAATTTATTC SEQ ID NO.6 C128X-R GAATAAATTCCATMNNCGCAATGCC SEQ ID NO.7 F131X-F GCGTGCATGGAANNKATTCGCCCGTTTCC SEQ ID NO.8 F131X-R GGAAACGGGCGAATMNNTTCCATGCACGC SEQ ID NO.9 F168X-F CAGAACGCCNNKATTGAAGGTG SEQ ID NO.10 F168X-R CACCTTCAATMNNGGCGTTCTG SEQ ID NO.11 A172X-F CTTTATTGAAGGTNNKCTGCCGAAATATG SEQ ID NO.12 A172X-R CATATTTCGGCAGMNNACCTTCAATAAAG SEQ ID NO.13 Y176X-F GTGCGCTGCCGAAANNKGTGGTGCGCCCGC SEQ ID NO.14 Y176X-R GCGGGCGCACCACMNNTTTCGGCAGCGCAC SEQ ID NO.15 I246X-F CCGGGCTTTNNKATTCCGCCG SEQ ID NO.16 I246X-R CGGCGGAATMNNAAAGCCCGG

[0039] The PCR amplification system was a 50 µL reaction system: 30 µL ddH2O, 5 µL 10× Buffer, 5 µL dNTPs, 3 µL MgSO4, 2 µL DMSO, 1.5 µL 50 µM upstream primer, 1.5 µL 50 µM downstream primer, 1 µL KOD enzyme, and 1 µL template DNA (plasmid).

[0040] The PCR reaction conditions were as follows: initial denaturation at 95°C for 3 min, followed by 30 cycles of temperature cycling at 95°C for 20 s, 55°C for 10 s, and 72°C for 30 s, with a final extension at 72°C for 10 min. The termination temperature was 4°C. After verification of the PCR product by 1% agarose gel electrophoresis, 1 µL DpnI and 5 µL buffer were added to the PCR product and digested at 37°C for 2 h to remove the template plasmid DNA. After inactivation at 65°C for 10 min, the product was purified using a PCR cleanup kit and transformed into E .coli BL21 (DE3) competent cells were coated on LB plates containing ampicillin (100 µg / mL) and cultured at 37°C overnight to obtain a mutation library of haloalkane dehalogenase. At this time, many single colonies with different mutations appeared on the LB plates, which were used for subsequent screening of the mutation library.

[0041] The parent strain was constructed in the same way: E .coli BL21(DE3)-DhaA31 WT.

[0042] Example 3: Screening of a Haloalkane Dehalogenase Mutant Library

[0043] Screening of the Haloalkane Dehalogenase Mutant Library Using the haloalkane dehalogenase DhaA31 as a reference, single colony clones (from the mutant library constructed in Example 2) were cultured in 1 mL deep 96-well plates. 400 µL of LB culture medium containing a final concentration of 100 µg / mL ampicillin was previously added. Two parent strains were simultaneously cultured in the last two wells of the 96-well plate as controls. The 1 mL 96-well plate was incubated at 37°C for 8 h as seed culture. Then, 100 µL of the seed culture was transferred to a new 2 mL deep 48-well plate and cultured in sterile TB culture medium containing a final concentration of 100 µg / mL ampicillin. After incubation at 37°C for 8 h, IPTG was added at a final concentration of 0.5 mM. Expression was induced at 20°C for 12 h, followed by centrifugation at 4000 rpm for 5 min. The supernatant was discarded, and the wet cells were collected for the next screening step.

[0044] Screening is based on the reaction yield and ee value of 1,2,3-tribromopropane dehalogenation catalyzed by haloalkane dehalogenase DhaA31. The chemical reaction formula is shown in Figure 1 500 µL of the reaction system (50 mM Tris-SO4 buffer, 10 mM 1,2,3-tribromopropane) was added to each well, the cells were resuspended, and then incubated at 25°C and 250 rpm for 30 min. The cells were extracted with 500 µL of dichloromethane and centrifuged at 1,2000 rpm for 1 min. 300 µL of the organic phase was analyzed by gas chromatography.

[0045] Gas chromatography analysis conditions: Agilent-8860GC and chiral Hydrodex-β-TBDAc column, GC program: 140°C isothermal, 1.5 mL / min, 20 min. Retention times: Rt(S)-DBP = 11.0 min, Rt(R)-DBP = 12.3 min. The amount of DBP produced by the parent strain and the er value were used as a control. The results are shown in Figure 2 The superior mutant strain was obtained, which was named mutant F168W / I246C, with an er value of 6:94. Figure 4 . Figure 3 This is the chromatographic result of (R,S)-2,3-dibromo-1-propanol standard.

[0046] Example 4: Degradation Kinetics of 1,2,3-Tribromopropane by DhaA31 Mutant F168W / I246C

[0047] According to the conclusion of Example 3, the mutants obtained by the above screening were E .coli BL21 (DE3)-DhaA31F168W / I246C (nucleotide sequence shown in SEQ ID NO. 3, amino acid sequence shown in SEQ ID NO. 4) was inoculated into a 10 mL sterile test tube containing LB medium containing a final concentration of 100 µg / mL ampicillin. The tube was shaken at 37°C and incubated at 150 rpm for 6-8 h. Then, a 1% inoculum volume was added to a 2 L conical flask containing 1 L of sterile TB medium containing a final concentration of 100 µg / mL ampicillin. The tube was incubated at 37°C for 12 h, and IPTG was added to a final concentration of 0.5 mM. Expression was induced at 20°C for 16 h, and the tube was centrifuged at 4000 rpm for 30 min. The supernatant was discarded and the wet cells were collected. Add 20 mL of 50 mM Tris-SO4 (pH = 8.5) buffer to the wet cells and resuspend them. Take 10 μL of the supernatant to determine the cell concentration (OD600), and dilute with 50 mM Tris-SO4 (pH = 8.5) buffer to an OD600 of 10. Add 5 mL of the diluted supernatant to each 10 mL glass reaction vial, add 1,2,3-tribromopropane to a final concentration of 10 mM, place a magnetic stir bar, and stir using a magnetic stirrer. Incubate at 25°C and 400 rpm for 60 min. At 3, 5, 10, 30, and 60 min, take 500 μL of the supernatant and extract with 500 μL of dichloromethane. Centrifuge at 12,000 rpm for 1 min, and collect 300 μL of the organic phase for gas chromatography.

[0048] The degradation kinetic curve of 1,2,3-tribromopropane by DhaA31 mutant F168W / I246C was obtained ( Figure 5 ), as the reaction proceeded, 1,2,3-tribromopropane was degraded and the concentration of the dehalogenation product 2,3-dibromo-1-propanol gradually increased. At 60 min, the yield of the dehalogenation product 2,3-dibromo-1-propanol reached 100%.

[0049] Example 5: Screening of the optimal pH for degradation of 1,2,3-tribromopropane by the DhaA31 mutant F168W / I246C

[0050] According to the conclusion of Example 3, the mutants obtained by the above screening were E .coli BL21 (DE3)-F168W / I246C (nucleotide sequence shown in SEQ ID NO. 3, amino acid sequence shown in SEQ ID NO. 4) was inoculated into a 10 mL sterile test tube containing LB medium containing a final concentration of 100 µg / mL ampicillin. The tube was shaken at 37°C and incubated at 150 rpm for 6-8 h. Then, a 1% inoculum volume was added to a 2 L conical flask containing 1 L of sterile TB medium containing a final concentration of 100 µg / mL ampicillin. The tube was incubated at 37°C for 12 h, and IPTG was added to a final concentration of 0.5 mM. Expression was induced at 20°C for 16 h, and the tube was centrifuged at 4000 rpm at 4°C for 30 min. The supernatant was discarded and the wet cells were collected. Add 20 mL of 50 mM Tris-SO4 (pH = 8.5) buffer to the wet cells and resuspend the cells. Measure the OD600 of the resuspended cells and adjust the OD600 to 10 using 50 mM Tris-SO4 (pH = 8.5) buffer. 1 mL of the resuspended mixture was added to each of five 2 mL sterile centrifuge tubes. The mixture was centrifuged at 4000 rpm for 10 min at 4°C. The supernatant was discarded. 900 μL of buffers of different pH values ​​(sodium hydrogen phosphate-citrate buffer, pH 6; sodium phosphate buffer, pH 7; sodium hydrogen phosphate-citrate buffer, pH 8; Tris-SO4 buffer, pH 8.5; and borate buffer, pH 10) were added to each of the five centrifuge tubes to resuspend the bacteria. 500 μL of the supernatant was then added to each of five 5 mL glass reaction vials. 1,2,3-Tribromopropane was then added to a final concentration of 10 mM. The mixture was mixed using a magnetic stirrer and incubated at 400 rpm at 25°C for 30 min. 500 μL of the reaction solution was extracted with 500 μL of dichloromethane and centrifuged at 12,000 rpm for 1 min. 300 μL of the organic phase was collected and analyzed by gas chromatography.

[0051] The experimental results are as follows ​As shown, the DhaA31 mutant F168W / I246C maintained good activity for the dehalogenation of 1,2,3-tribromopropane in the pH range of 8-10, and the yield of 2,3-dibromo-1-propanol was >65% after 30 min of reaction.

[0052] Example 6: Screening of the optimal temperature for degradation of 1,2,3-tribromopropane by the DhaA31 mutant F168W / I246C

[0053] According to the conclusion of Example 3, the mutants obtained by the above screening were ​ BL21 (DE3)-F168W / I246C (nucleotide sequence shown in SEQ ID NO. 3, amino acid sequence shown in SEQ ID NO. 4) was inoculated into a 10 mL sterile test tube containing LB medium containing a final concentration of 100 µg / mL ampicillin. The tube was shaken at 37°C and incubated at 150 rpm for 6-8 h. Then, a 1% inoculum volume was added to a 2 L conical flask containing 1 L of sterile TB medium containing a final concentration of 100 µg / mL ampicillin. The tube was incubated at 37°C for 12 h, and IPTG was added to a final concentration of 0.5 mM. Expression was induced at 20°C for 16 h, and the tube was centrifuged at 4000 rpm at 4°C for 30 min. The supernatant was discarded and the wet cells were collected. Add 20 mL of 50 mM Tris-SO4 (pH = 8.5) buffer to the wet cells and resuspend the cells. Measure the OD600 of the resuspended cells and adjust the OD600 to 10 using 50 mM Tris-SO4 (pH = 8.5) buffer. Transfer 1 mL of the diluted supernatant to five 5 mL glass reaction vials. Add 1,2,3-tribromopropane to a final concentration of 20 mM to each of the five reaction vials. Stir the mixture using a magnetic stirrer and incubate at 20°C, 25°C, 37°C, 45°C, and 55°C at 400 rpm for 30 min. Take 500 μL of the reaction solution and extract it with 500 μL of dichloromethane. Centrifuge at 12,000 rpm for 1 min. Collect 300 μL of the organic phase and analyze it by gas chromatography.

[0054] The experimental results are as follows ​ As shown, the DhaA31 mutant F168W / I246C exhibits excellent activity at temperatures ranging from 20°C to 55°C, encompassing a wide temperature range. Therefore, the DhaA31 mutant can efficiently dehalogenate at both room and moderate temperatures, eliminating the need for external temperature control equipment, saving energy, and demonstrating its environmental and low-carbon potential.

[0055] Example 7: Determination of Michaelis-Menten kinetic parameters for the degradation of 1,2,3-tribromopropane by the DhaA31 mutant F168W / I246C

[0056] According to the conclusion of Example 3, the mutants obtained by the above screening were ​ BL21(DE3)-F168W / I246C (nucleotide sequence shown in SEQ ID NO. 3, amino acid sequence shown in SEQ ID NO. 4) was inoculated into a 10 mL sterile tube containing LB medium containing a final concentration of 100 µg / mL ampicillin. The tube was shaken at 37°C at 150 rpm for 6-8 h. A 1% inoculum was then added to a 2 L Erlenmeyer flask containing 1 L of sterile TB medium containing a final concentration of 100 µg / mL ampicillin. The tube was incubated at 37°C for 12 h. IPTG was then added to a final concentration of 0.5 mM. Expression was induced at 20°C for 16 h. The tube was centrifuged at 4000 rpm for 30 min at 4°C, and the supernatant was discarded to collect the wet cells. The wet cells were resuspended in 20 mL of 50 mM Tris-SO4 (pH 8.5) buffer. The cells were disrupted by sonication at 4°C for 10 min at 65% power (2 s on, 6 s off). The supernatant was then centrifuged at 12,000 rpm for 15 min at 4°C. The protein was purified by affinity chromatography, and the absorbance was measured at A280 using a UV spectrophotometer to calculate the protein concentration. The protein was then diluted with 50 mM Tris-SO4 (pH 8.5) buffer to a protein concentration of 2 µM. 1 mL of the diluted protein solution was transferred to ten 5 mL glass reaction vials. Five of these glass reaction vials were then added with 1,2,3-tribromopropane at final concentrations of 0.5 mM, 2 mM, 5 mM, 10 mM, and 50 mM, respectively. The reaction was incubated at 25°C with magnetic stirring at 400 rpm for 5 min. 500 µL of the reaction solution was extracted with 500 µL of dichloromethane and centrifuged at 12,000 rpm for 1 min. 300 µL of the organic phase was collected and analyzed by gas chromatography. The enzyme reaction rate was measured, and a double reciprocal curve was drawn based on the reciprocal of the reaction rate and substrate concentration to calculate the Michaelis-Menten kinetic parameter. The results showed that the Michaelis-Menten kinetic parameter K of DhaA31 mutant F168W / I246C for 1,2,3-tribromopropane was m and K cat 4.8 mM and 6.2 s, respectively -1 .

Claims

1. A haloalkane dehalogenase mutant, characterized in that Its amino acid sequence is shown in SEQ ID NO.

4.

2. A gene, characterized in that The gene encodes the haloalkane dehalogenase mutant according to claim 1.

3. A recombinant vector, characterized in that The recombinant vector comprises the gene according to claim 2.

4. A recombinant cell, characterized in that The recombinant cell comprises the recombinant vector according to claim 3.

5. The method for preparing a haloalkane dehalogenase mutant according to claim 1, wherein: The method comprises the following steps: (1) constructing a recombinant vector containing a gene encoding the haloalkane dehalogenase mutant; (2) transforming and preparing a recombinant cell; and (3) inducing the recombinant cell to express the haloalkane dehalogenase mutant.

6. The preparation method according to claim 5, characterized in that In step (3), after expression is completed, the cells are collected; or the cells are broken and the crude enzyme solution or pure enzyme is collected.

7. The preparation method according to claim 6, characterized in that The collected cells or pure enzymes are prepared into immobilized cells or immobilized enzymes using immobilization technology.

8. A product for degrading 1,2,3-tribromopropane, characterized in that: The product comprises the haloalkane dehalogenase mutant according to claim 1, or the gene according to claim 2, or the recombinant vector according to claim 3, or the recombinant cell according to claim 4.

9. Use of the product according to claim 8 in the degradation of 1,2,3-tribromopropane.

10. Use of the product according to claim 8 in the preparation of (R)-2,3-dibromo-1-propanol.

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