Halogenated alkane dehalogenase mutant and application thereof in degradation of 1, 2, 3-tribromopropane

Abstract

The invention discloses a haloalkane dehalogenase mutant and an application thereof in degradation of 1, 2, 3-tribromopropane, haloalkane dehalogenase DhaA31 coded by dhaA gene of a Rhodococcus rhodococcus NCIMB 13064 strain is modified to obtain the haloalkane dehalogenase mutant F168W / I246C, the haloalkane dehalogenase mutant has high degradation efficiency on haloalkane pollutant 1, 2, 3-tribromopropane, and the haloalkane dehalogenase mutant has high degradation efficiency on haloalkane pollutant 1, 2, 3-tribromopropane under the condition of water phase normal temperature. The degradation rate of the alkyl halide dehalogenase mutant to 10 mM of 1, 2, 3-tribromopropane within 1 h reaches 100%, and the degradation rate of the alkyl halide dehalogenase mutant to 10 mM of 1, 2, 3-tribromopropane reaches 100%. The haloalkane dehalogenase mutant has high stereoselectivity on (R)-2, 3-dibromo-1-propanol, and the e.r. Value of the haloalkane dehalogenase mutant reaches 6: 94. The haloalkane dehalogenase mutant provided by the invention has high catalytic efficiency and high stereoselectivity, is beneficial to recognition and further degradation of downstream enzymes, and has a good application prospect in bioremediation of emerging haloalkane pollutants.

Description

Technical Field

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

[0002] Haloalkanes are often used as chemical solvents, organic synthesis raw materials and intermediates, and are widely used in the chemical production process. They are discharged into the environment along with chemical waste liquids and wastewaters. At the same time, haloalkanes are also disinfection by-products generated during the water treatment process. Due to their strong persistence and difficult biodegradability, they have been frequently detected in water treatment systems and natural water bodies in recent years. As a new type of refractory organic pollutant, they pose a serious threat to human health and ecological safety. 1,2,3-Tribromopropane (1,2,3-tribromopropane, abbreviated as 1,2,3-TBP) is a haloalkane. As a widely used chemical solvent and industrial by-product, it is produced in large quantities during the chemical production process and is discharged into water bodies along with sewage and wastewater, thus becoming an organic pollutant.

[0003] Microbial haloalkane dehalogenases play an important role in the biodegradation of haloalkane pollutants and can convert toxic and refractory haloalkanes into corresponding harmless alcohols through hydrolysis reactions. However, the existing haloalkane dehalogenases have low degradation efficiency for 1,2,3-tribromopropane and are ineffective in the complex environment of sewage. In addition, the existing haloalkane dehalogenases have low stereoselectivity, which restricts the further conversion and complete degradation of 1,2,3-tribromopropane in the metabolic pathway. Summary of the Invention

[0004] Object of the Invention: The object of the present invention is to provide a mutant haloalkane dehalogenase with high degradation efficiency for the substrate 1,2,3-tribromopropane and high stereoselectivity for the product (R)-2,3-dibromopropan-1-ol ((R)-2,3-dibromopropan-1-ol, abbreviated as (R)-DBP).

[0005] Technical Solution: The present invention provides a mutant haloalkane dehalogenase, and its amino acid sequence is as shown in SEQ ID NO.4.

[0006] The present invention is derived from Rhodococcus Rhodococcus rhodochrous strain NCIMB 13064 dhaAThe gene-encoded haloalkane dehalogenase DhaA31 (PDB ID: 3RK4) is a mutant of the original 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 now been fully resolved. Haloalkane dehalogenases belong to the α / β hydrolase fold family and have a core domain carrying the Asp-His-Asp / Glu catalytic triad and a variable, mainly helical cap domain that provides the essential residues determining selectivity. Haloalkane dehalogenase DhaA31 can catalyze 1,2,3-tribromopropane to form the racemic enantiomers (R,S)-2,3-dibromo-1-propanol. 1,2,3-Tribromopropane is a halogenated alkane pollutant that is difficult to degrade. 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 catalytic molecular mechanism of haloalkane dehalogenase, the present invention modifies the key amino acids in the active center of haloalkane dehalogenase by directed evolution and semi-rational design methods. By site-directed saturation mutagenesis, iterative saturation mutagenesis and directed screening, it is found that when phenylalanine at position 168 of the amino acid sequence shown in SEQ ID NO.2 is mutated to tryptophan (F168W) and isoleucine at position 246 is mutated to cysteine (I246C), the obtained haloalkane dehalogenase mutant F168W / I246C has high catalytic activity and high stereoselectivity, and can efficiently biodegrade halogenated alkane pollutants represented by 1,2,3-tribromopropane, while generating high-value (R)-2,3-dibromo-1-propanol.

[0008] The present invention also provides a nucleotide sequence that encodes the haloalkane dehalogenase mutant. The nucleotide sequence includes SEQ ID NO.3.

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

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

[0011] The present invention also provides a recombinant cell that contains 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, which includes the following steps: (1) constructing a recombinant vector containing the nucleotide sequence encoding the haloalkane dehalogenase mutant; (2) transforming to prepare recombinant cells; (3) inducing the recombinant cells to express the haloalkane dehalogenase mutant.

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

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

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

[0017] The preparation method includes: inoculating a recombinant engineering bacterium containing the encoding gene of the haloalkane dehalogenase mutant into an LB culture medium containing ampicillin with a final concentration of 100 μg / mL, culturing at 37 °C for 8 h to obtain a seed solution; then inoculating the seed solution into a sterile LB liquid medium containing ampicillin with a final concentration of 100 μg / mL at an inoculation amount of 2% (v / v), culturing at 37 °C for about 8 - 12 h until the cell concentration OD 600 is 0.4 - 0.8, then adding isopropyl β-D-thiogalactoside (IPTG) with a final concentration of 0.1 - 1.0 mM (preferably 0.5 mM) to the culture medium, inducing expression at 20 °C for 16 h, and then centrifuging at 4 °C and 4000 rpm for 10 - 20 min to collect the wet cells.

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

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

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

[0021] The method of the application includes: using the wet bacterial cells obtained by fermentation and culture of a recombinant genetic engineering 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. The reaction is carried out at 300 - 500 rpm (preferably 400 rpm) and 25 - 37 °C (preferably 25 °C). After the reaction, a reaction solution containing (R)-2,3-dibromo-1-propanol is obtained, and the reaction solution is separated and purified to obtain (R)-2,3-dibromo-1-propanol.

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

[0023] Furthermore, the dosage of the catalyst is 10 - 40 g / L buffer (preferably 20 g / L) based on the weight of the wet bacterial cells, and the initial addition 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 remarkable advantages: The haloalkane dehalogenase mutant F168W / I246C provided by the present invention has a high degradation efficiency for the haloalkane pollutant 1,2,3-tribromopropane. Under the conditions of normal temperature in the aqueous phase, the degradation rate of the haloalkane dehalogenase mutant for 10 mM 1,2,3-tribromopropane reaches 100% within 1 h; the haloalkane dehalogenase mutant has high stereoselectivity for (R)-2,3-dibromo-1-propanol, and the e.r. value reaches 6:94. The haloalkane dehalogenase mutant provided by the present invention has high catalytic efficiency and high stereoselectivity, which is beneficial for the recognition and further degradation of downstream enzymes, and has good application prospects in the bioremediation of emerging haloalkane pollutants. Description of the Drawings

[0025] Figure 1 It is the chemical reaction formula for the dehalogenation of 1,2,3-tribromopropane catalyzed by the haloalkane dehalogenase DhaA31; Figure 2 It is the yield and ee value of the product 2,3-dibromo-1-propanol formed by the wild-type enzyme and mutant of DhaA31 catalyzing 1,2,3-tribromopropane; Figure 3 It is the chromatogram of the racemic enantiomer (R,S)-2,3-dibromo-1-propanol standard detected by gas phase; Figure 4Chromatogram of the racemic enantiomers (R,S)-2,3-dibromo-1-propanol produced by the gas-phase detection of the DhaA31 mutant F168W / I246C of haloalkane dehalogenase catalyzing 1,2,3-tribromopropane; Figure 5 Yield and ee value of the product 2,3-dibromo-1-propanol produced by the DhaA31 mutant F168W / I246C catalyzing 1,2,3-tribromopropane; Figure 6 Yield of 2,3-dibromo-1-propanol produced by the DhaA31 mutant F168W / I246C catalyzing 1,2,3-tribromopropane at different pH values; Figure 7 Yield of 2,3-dibromo-1-propanol produced by the DhaA31 mutant F168W / I246C catalyzing 1,2,3-tribromopropane at different temperatures. Detailed implementation mode

[0026] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0027] Example 1: Construction of pET22b(+)-DhaA31 plasmid From Rhodococcus Rhodococcus rhodochrous The gene of the engineered haloalkane dehalogenase DhaA31 (PDB ID: 3RK4) from NCIMB 13064 was synthesized by Genewiz (Suzhou) Co., Ltd. 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 the plate of LB solid medium and cultured upside down in a 37°C incubator for 16 h. Single colonies were picked and inoculated into 5 mL of sterile LB liquid medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, and the solvent was deionized water). After culturing at 37°C and 150 rpm for 8-12 h, the pET22b(+)-DhaA31 plasmid was extracted from E.coli DH5α as the template for iterative saturation mutagenesis.

[0028] Example 2: Construction of a site-directed saturation library of haloalkane dehalogenase According to the conclusion of Example 1, primers were designed based on the gene sequence of the engineered halogenated alkane 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. The 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, respectively. 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).

[0029] Table 1: Primer design table for construction of haloalkane dehalogenase site-directed saturation mutation library 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 The PCR amplification system was a 50 µL reaction system: 30 µL ddH2O, 5 µL 10×Buffer, 5 µL dNTP, 3 µLMgSO4, 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).

[0030] The PCR reaction conditions were as follows: pre-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, and finally extension at 72°C for 10 min, with the termination temperature at 4°C. After verification by 1% agarose gel electrophoresis analysis of the PCR products, 1 μL of DpnI and 5 μL of buffer were added to the PCR products, and the template plasmid DNA was digested at 37°C for 2 h. After inactivating at 65°C for 10 min, the products were purified using a PCR cleanup Kit and then transformed into E .coli BL21(DE3) competent cells, and spread on an LB plate containing ampicillin (100 μg / mL). The plate was incubated at 37°C overnight to obtain a mutant library of haloalkane dehalogenase. At this time, many different mutant single colonies appeared on the LB plate, and these single colonies were used for subsequent screening of the mutant library.

[0031] The parental strain was constructed in the same method: E .coli BL21(DE3)-DhaA31 WT.

[0032] Example 3: Screening of the mutant library of haloalkane dehalogenase The screening of the mutant library of haloalkane dehalogenase was carried out with haloalkane dehalogenase DhaA31 as a reference. Single colony clones (from the mutant library constructed in Example 2) were picked into a 1 mL deep 96-well plate for cultivation. 400 μL of LB culture medium containing ampicillin with a final concentration of 100 μg / mL was added in advance. At the same time, 2 parental strains were picked as controls in the last 2 wells of the 96-well plate. The 1 mL 96-well plate was incubated at 37°C for 8 h as the seed solution, and then 100 μL of the seed solution was added to a new 2 mL deep 48-well plate for cultivation. Sterile TB culture medium containing ampicillin with a final concentration of 100 μg / mL was added in advance. After incubation at 37°C for 8 h, IPTG with a final concentration of 0.5 mM was added, and after induction at 20°C for 12 h, centrifugation was carried out at 4000 rpm for 5 min, the supernatant was discarded, and the wet cells were collected for the next step of screening.

[0033] The screening was based on the reaction yield and ee value of the dehalogenation of 1,2,3-tribromopropane catalyzed by haloalkane dehalogenase DhaA31. The chemical reaction formula is shown in Figure 1 . A 500 μL reaction system (50 mM Tris-SO4 buffer, 1,2,3-tribromopropane with a final concentration of 10 mM) was added to each well, the cells were resuspended, and then the reaction was carried out at 25°C and 250 rpm for 30 min. Extraction was carried out with 500 μL of dichloromethane, followed by centrifugation at 12000 rpm for 1 min, and 300 μL of the organic phase solution was taken for gas chromatography detection.

[0034] Gas phase analysis conditions: Agilent-8860 GC and chiral Hydrodex-β-TBDAc chromatographic column. The gas phase program was isothermal at 140 °C, running at 1.5 mL / min for 20 min. The retention times were Rt(S)-DBP = 11.0 min and Rt(R)-DBP = 12.3 min. Taking the amount and e.r. value of DBP produced by the parent as the control, the results are shown in Figure 2 . An excellent mutant strain was obtained, designated as mutant F168W / I246C, with an e.r. value of 6:94, as shown in Figure 4 . Figure 3 Chromatographic results of (R,S)-2,3-dibromo-1-propanol standard.

[0035] Example 4: Degradation kinetics of 1,2,3-tribromopropane by DhaA31 mutant F168W / I246C According to the conclusion of Example 3, the above-screened mutant 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 sterile test tube containing 10 mL of LB medium with a final concentration of 100 μg / mL ampicillin, cultured in a shaker at 37 °C and 150 rpm for 6 - 8 h, and then added to a 2 L conical flask for culture according to an inoculation amount of 1%. A sterile TB medium containing a final concentration of 100 μg / mL ampicillin was added in advance. After culturing at 37 °C for 12 h, IPTG with a final concentration of 0.5 mM was added, induced to express at 20 °C for 16 h, centrifuged at 4000 rpm for 30 min, the supernatant was discarded, and the wet cells were collected. 20 mL of 50 mM 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 diluted to OD600 = 10 with 50 mM Tris-SO4 (pH = 8.5) buffer. 5 mL of the diluted supernatant was added to a 10 mL glass reaction flask, 1,2,3-tribromopropane with a final concentration of 10 mM was added, a magnetic stir bar was placed, and the mixture was stirred and mixed evenly with a magnetic stirrer. The reaction was carried out at 25 °C and 400 rpm for 60 min. 500 μL of the supernatant after reaction was taken at 3 min, 5 min, 10 min, 30 min, and 60 min, extracted with 500 μL of dichloromethane, centrifuged at 12000 rpm for 1 min, and 300 μL of the organic phase solution was detected by gas chromatography.

[0036] The degradation kinetic curve of DhaA31 mutant F168W / I246C against 1,2,3-tribromopropane was obtained ( Figure 5 ). As the reaction proceeded, 1,2,3-tribromopropane was degraded, and the concentration of the dehalogenated product 2,3-dibromo-1-propanol gradually increased. The yield of the dehalogenated product 2,3-dibromo-1-propanol reached 100% at 60 min.

[0037] Example 5: Screening of the Optimal pH for the Degradation of 1,2,3-Tribromopropane by DhaA31 Mutant F168W / I246C According to the conclusion of Example 3, the mutant E .coli BL21(DE3)-F168W / I246C (with the nucleotide sequence shown in SEQ ID NO.3 and the amino acid sequence shown in SEQ ID NO.4) was inoculated into a sterile test tube containing 10 mL of LB medium with a final concentration of 100 μg / mL ampicillin, and cultured in a shaker at 37 °C and 150 rpm for 6 - 8 h. Then, it was added to a 2 L conical flask according to an inoculation amount of 1% and cultured. 1 L of sterile TB medium with a final concentration of 100 μg / mL ampicillin was added in advance, and after culturing at 37 °C for 12 h, IPTG with a final concentration of 0.5 mM was added. After inducing expression at 20 °C for 16 h, it was centrifuged at 4 °C and 4000 rpm for 30 min, the supernatant was discarded, and the wet bacterial cells were collected. 20 mL of 50 mM Tris-SO4 (pH = 8.5) buffer was added to the wet bacterial cells to resuspend the cells, the OD600 of the resuspended cells was measured, and it was adjusted to OD600 = 10 using 50 mM Tris-SO4 (pH = 8.5) buffer. 1 mL of the resuspended mixture was added to each of the 5 sterile 2 mL centrifuge tubes, centrifuged at 4 °C and 4000 rpm for 10 min, the supernatant was discarded, and the bacterial suspension was resuspended in 900 μL of buffers with different pH values (sodium hydrogen phosphate-citric acid buffer with pH = 6, sodium phosphate buffer with pH = 7, sodium hydrogen phosphate-citric acid buffer with pH = 8, Tris-SO4 buffer with pH = 8.5, borate buffer with pH = 10) in the 5 centrifuge tubes. 500 μL of the supernatant was taken and added to each of the 5 5 mL glass reaction flasks, and then 1,2,3-tribromopropane with a final concentration of 10 mM was added respectively. The mixture was stirred and mixed evenly using a magnetic stirrer, and the reaction was carried out at 25 °C and 400 rpm for 30 min. 500 μL of the reaction solution was taken from each reaction flask, extracted with 500 μL of dichloromethane, centrifuged at 12000 rpm for 1 min, and 300 μL of the organic phase solution was detected by gas chromatography.

[0038] The experimental results are as Figure 6As shown, the DhaA31 mutant F168W / I246C can maintain good activity in the dehalogenation of 1,2,3-tribromopropane within the pH range of 8 - 10, and the yield of 2,3-dibromo-1-propanol is >65% after reacting for 30 min.

[0039] Example 6: Screening of the Optimal Temperature for the Degradation of 1,2,3-Tribromopropane by DhaA31 Mutant F168W / I246C According to the conclusion of Example 3, the mutant obtained by the above screening E .coli BL21(DE3)-F168W / I246C (the nucleotide sequence is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4) was inoculated into a sterile test tube containing 10 mL of LB medium with a final concentration of 100 μg / mL ampicillin, and cultured in a shaker at 37°C and 150 rpm for 6 - 8 h. Then, it was added to a 2 L conical flask for culture according to an inoculation amount of 1%. A sterile TB medium containing a final concentration of 100 μg / mL ampicillin was added in advance, and after culturing at 37°C for 12 h, IPTG with a final concentration of 0.5 mM was added. After inducing expression at 20°C for 16 h, it was centrifuged at 4°C and 4000 rpm for 30 min, the supernatant was discarded, and the wet cells were collected. 20 mL of 50 mM Tris-SO4 (pH = 8.5) buffer was added to the wet cells to resuspend the cells, the OD600 of the resuspended cells was measured, and it was adjusted to OD600 = 10 using 50 mM Tris-SO4 (pH = 8.5) buffer. 1 mL of the diluted supernatant was taken and placed into 5 5 mL glass reaction flasks respectively. 1,2,3-Tribromopropane with a final concentration of 20 mM was added to the 5 reaction flasks, a magnetic stir bar was added, and the mixture was stirred evenly using a magnetic stirrer. The reaction was carried out at 20°C, 25°C, 37°C, 45°C, and 55°C at 400 rpm for 30 min respectively. 500 μL of the reaction solution was taken, extracted with 500 μL of dichloromethane, then centrifuged at 12000 rpm for 1 min, and 300 μL of the organic phase solution was detected by gas chromatography.

[0040] The experimental results are as Figure 7 shown, the DhaA31 mutant F168W / I246C has good activity at 20°C - 55°C and has a relatively wide suitable temperature range. Therefore, the DhaA31 mutant can efficiently dehalogenate at normal and medium temperatures without the need for additional temperature control equipment, saving energy consumption and having the characteristics of being green and low-carbon.

[0041] Example 7: Determination of the Michaelis-Menten Kinetic Parameters for the Degradation of 1,2,3-Tribromopropane by DhaA31 Mutant F168W / I246C According to the conclusion of Example 3, the mutant obtained by the above screeningE .coli BL21(DE3)-F168W / I246C (with the nucleotide sequence shown in SEQ ID NO.3 and the amino acid sequence shown in SEQ ID NO.4) was inoculated into a sterile test tube containing 10 mL of LB medium supplemented with 100 μg / mL ampicillin at a final concentration. The culture was incubated on a shaker at 37°C and 150 rpm for 6 - 8 h, and then transferred to a 2 L conical flask for culture at an inoculation amount of 1%. 1 L of sterile TB medium supplemented with 100 μg / mL ampicillin at a final concentration was added in advance. After culturing at 37°C for 12 h, IPTG at a final concentration of 0.5 mM was added, and induction expression was carried out at 20°C for 16 h. Then, the cells were centrifuged at 4°C and 4000 rpm for 30 min. The supernatant was discarded, and the wet cells were collected. 20 mL of 50 mM Tris-SO4 (pH = 8.5) buffer was added to the wet cells to resuspend the cells. The cells were sonicated at 4°C for 10 min with a sonication power of 65%, sonication on for 2 s and off for 6 s. Then, the cells were centrifuged at 4°C and 12000 rpm for 15 min, and the supernatant was taken. The protein was purified by affinity chromatography. The absorbance was measured at A280 using a UV spectrophotometer to calculate the protein concentration, and the protein was diluted to a protein concentration of 2 μM with 50 mM Tris-SO4 (pH = 8.5) buffer. 1 mL of the diluted protein solution was taken and placed into 10 5 mL glass reaction flasks respectively. 1,2,3-tribromopropane at final concentrations of 0.5 mM, 2 mM, 5 mM, 10 mM, and 50 mM was added to 5 of the glass reaction flasks respectively. The reaction was carried out with magnetic stirring at 25°C and 400 rpm for 5 min. 500 μL of the reaction solution was taken from each reaction flask, extracted with 500 μL of dichloromethane, and then centrifuged at 12000 rpm for 1 min. 300 μL of the organic phase solution was taken for gas chromatography detection. The enzymatic reaction rate was measured, and a double-reciprocal curve was made based on the reaction rate and the reciprocal of the substrate concentration to calculate the Michaelis kinetic parameters. The results showed that the Michaelis kinetic parameters K m and K cat of the DhaA31 mutant F168W / I246C for 1,2,3-tribromopropane were 4.8 mM and 6.2 s -1 respectively.

Claims

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

4.

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

3. A recombinant vector, characterized in that, The recombinant vector contains the nucleotide sequence according to claim 2.

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

5. The preparation method of the haloalkane dehalogenase mutant according to claim 1, characterized in that, It includes the following steps: (1) constructing a recombinant vector containing the nucleotide sequence encoding the haloalkane dehalogenase mutant; (2) transforming to prepare a recombinant cell; (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 the expression is completed, the cells are collected; or the cells are disrupted to collect the crude enzyme solution or pure enzyme.

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

8. A product, characterized in that, The product contains the haloalkane dehalogenase mutant according to claim 1, or the nucleotide sequence according to claim 2, or the recombinant vector according to claim 3, or the recombinant cell according to claim 4, or the cells or crude enzyme solution or pure enzyme according to claim 6, or the immobilized cells or immobilized enzymes according to claim 7.

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

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

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