Directional transformation method for improving enzyme activity of halohydrin dehalogenase

Abstract

The invention relates to a directional transformation method for improving enzyme activity of halohydrin dehalogenase, and belongs to the technical field of bioengineering. According to a halohydrin dehalogenase mutant, single-point mutation or combined mutation is carried out on 60th alanine and 179th histidine in a sequence shown in SEQ ID NO.1 to obtain the mutant. Compared with wild halohydrin dehalogenase, the mutant obtained in the invention has obviously improved enzyme activity and dehalogenation efficiency in catalytic preparation of 1-chloro-2-propanol and 2-chloro-1-ethanol, the enzyme activity of the mutant is improved by 1.78 times, the dehalogenation efficiency is improved by 77.8%, and the mutant has a good industrial application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and particularly relates to a directed transformation method for improving the enzymatic activity of a halohydrin dehalogenase. Background Art

[0002] Halohydrin dehalogenases have important application value in the field of biotechnology. They can catalyze the dehalogenation of halohydrins to produce epoxides or catalyze the ring-opening reaction of epoxides to produce chiral β-substituted alcohols. However, the enzymatic activity of wild-type halohydrin dehalogenases is often difficult to meet the needs of actual industrial production and scientific research. For example, in some processes where halohydrin dehalogenases are used to catalyze the preparation of high-value-added chiral compounds, the low enzyme activity leads to slow reaction rates and low substrate conversion rates, which in turn increases production costs and limits their large-scale application.

[0003] At present, although a variety of halohydrin dehalogenases have been discovered, the activity of most enzymes needs to be improved. Traditional chemical synthesis methods have problems such as harsh reaction conditions, many by-products, and severe environmental pollution when preparing related chiral compounds. Biocatalysis, relying on the action of halohydrin dehalogenases, has the advantages of mild reaction conditions, high stereoselectivity, and green environmental protection.

[0004] Prior art CN104745556B discloses a halohydrin dehalogenase mutant and an engineered bacterium that catalyzes the asymmetric dehalogenation of 1,3-dichloropropanol to synthesize (S)-epichlorohydrin and β-substituted alcohols, and obtains a halohydrin dehalogenase mutant with higher enantioselectivity, but does not improve the dehalogenation activity; Prior art CN119120418 A discloses a halohydrin dehalogenase mutant with improved dehalogenation efficiency and its application, and discloses that HheC is used as the mutant original enzyme, and any one of halohydrin dehalogenases A83W, P84G, and F86W is mutated to improve the dehalogenation efficiency. The substrate targeted is aliphatic halohydrins, and α-halohydrins are not mentioned; Prior art CN110423740 A discloses mutants obtained by single-point mutation or combined mutation 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, and the enantiomeric selectivity in the preparation of (S)-o-nitrophenyl glycidyl ether, (R)-benzyl glycidyl ether, and (R)-phenyl glycidyl ether is significantly improved, but α-halohydrins are not mentioned.

[0005] Therefore, there is an urgent need to develop a method that can effectively improve the activity of halohydrin dehalogenase and improve the catalytic dehalogenation efficiency of α-halohydrins. Summary of the Invention

[0006] The present application provides a directed modification method for improving the enzymatic activity of a halohydrin dehalogenase. Through the directed modification technology of amino acids, the dehalogenation activity and dehalogenation efficiency of HheC for α-halohydrins can be effectively improved. Site-directed saturation iterative saturation mutagenesis is performed on the 60th and 179th sites, and halohydrin dehalogenase mutants with improved dehalogenation efficiency for α-halohydrins: 1-chloro-2-propanol and 2-chloro-1-ethanol are screened, thereby enhancing the catalytic degradation of 1-chloro-2-propanol and 2-chloro-1-ethanol and converting them into propylene oxide and ethylene oxide.

[0007] The present application provides a directed modification method for improving the enzymatic activity of a halohydrin dehalogenase, wherein the directed modification method is to perform single-point mutations at positions 60 and 179 of the wild-type halohydrin dehalogenase HheC to obtain the corresponding halohydrin dehalogenase mutants: (1) Recombinant vector construction: Construction of halohydrin dehalogenase mutants; (2) Recombinant cells: Transform the constructed recombinant vector into host cells: The recombinant engineered bacteria containing the halohydrin dehalogenase gene were inoculated into 100 mL LB liquid culture medium containing 50 mg / L ampicillin at a mass concentration of 50 mg / L, and cultured at 37° C. and 200 rpm for 8 hours to obtain a bacterial liquid; then the bacterial liquid was inoculated into a new 50 mL LB medium containing 50 mg / L ampicillin at a final mass concentration of 50 mg / L at an inoculum size of 0.5-2%, and cultured at 37° C. and 200 rpm for 10 hours. When the optical density OD600 was 0.6-0.8, the next step was carried out; the LB culture formula was 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, and deionized water to a volume of 1 L, with a pH of 7.0-7.2, and the culture was autoclaved at 126° C. for 15 minutes; (3) Induced expression: Obtaining halohydrin dehalogenase mutant protein Add isopropylthio-β-D-galactoside (IPTG) to a final concentration of 0.2 mM to 0.6 mM to the culture medium in step (3), induce expression at 20 to 25°C for 12 to 14 hours, centrifuge the bacterial solution at 10,000 rpm / min for 2 minutes to collect the bacteria, resuspend and wash the bacteria in Tris-HCl buffer, and store the collected bacteria at -20°C for later use.

[0008] Preferably, the amino acid sequence of the halohydrin dehalogenase mutant is a mutation of the 60th alanine of the wild-type halohydrin dehalogenase HheC to tryptophan to obtain mutant A60W, the 179th histidine is mutated to valine to obtain mutant H179V, the 60th alanine is mutated to tryptophan and the 179th histidine is mutated to valine to obtain any one of mutants A60W-H179V.

[0009] Preferably, the wild-type sequence of the halohydrin dehalogenase is SEQ ID NO.1:

[0010] mstaivtnvk hfggmgsalr lseaghtvac hdesfkqkde leafaetypqlkpmseqepaelieavtsay

[0011] gqvdvlvsnd ifapefqpid kyavedyrga vealqirpfa lvnavasqmk

[0012] krksghiifi tsatpfgpwk elstytsara gactlanals kelgeynipv faigpnylhs

[0013] edspyfypte pwktnpehva hvkkvtalqr lgtqkelgel vaflasgscd yltgqvfwla

[0014] ggfpmierwp gmpe

[0015] Preferably, the corresponding nucleotide sequence encoding the wild-type halohydrin dehalogenase HheC is SEQ ID NO.5:

[0016] 1atgtcaaccg caattgtaac aaacgttaag cattttgggg gaatggggtc tgcacttcgt

[0017] 61ctctcggaag caggacatac agtggcttgc cacgatgaaa gcttcaaaca aaaggacgaa

[0018] 121cttgaagcctttgccgaaac ctatccacaa ctcaaaccaa tgtcggaaca agaaccagcg

[0019] 181gaactcatcg aggcagttac ctccgcttat ggtcaagttg atgtacttgt gagcaacgac

[0020] 241atattcgcac cagagttcca acccatagat aaatacgctg tagaggacta tcgcggtgcg

[0021] 301gtcgaggcgc tacaaattag accatttgca ctggtcaacg ccgttgcaag tcaaatgaag

[0022] 361aagcgcaaaa gcggacatat tatctttatt acctctgcaa cgcccttcgg gccttggaag

[0023] 421gaactttcta cctacacgtc agcccgagca ggtgcatgca ccttggcaaa tgccctttcg

[0024] 481aaggaactcg gtgaatacaa cattccggtg ttcgcaatag gacccaatta tcttcacagt

[0025] 541gaagatagtc cctacttcta ccccacagaa ccgtggaaaa cgaatccaga acacgttgcc

[0026] 601catgtcaaaa aagtcactgc gctccagcgg ttaggtacac agaaagaatt gggagaactc

[0027] 661gtcgcgtttc tcgcgtctgg tagttgtgac tatctgaccg gccaggtgtt ctggttggcc

[0028] 721ggcggattcc caatgatcga gcgttggcct ggtatgcccg ag

[0029] Preferably, the amino acid sequence of the halohydrin dehalogenase mutant A60W (alanine at position 60 is mutated to tryptophan) is SEQ ID NO. 2:

[0030] mstaivtnvk hfggmgsalr lseaghtvac hdesfkqkde leafaetypq lkpmseqepw

[0031] elieavtsay gqvdvlvsnd ifapefqpid kyavedyrga vealqirpfa lvnavasqmk

[0032] krksghiifi tsatpfgpwk elstytsara gactlanals kelgeynipv faigpnylhs

[0033] edspyfypte pwktnpehva hvkkvtalqr lgtqkelgel vaflasgscd yltgqvfwla

[0034] ggfpmierwp gmpe

[0035] Preferably, the nucleotide sequence corresponding to A60W (alanine at position 60 is mutated to tryptophan) is SEQ ID NO.6:

[0036] 1atgtcaaccg caattgtaac aaacgttaag cattttgggg gaatggggtc tgcacttcgt

[0037] 61ctctcggaag caggacatac agtggcttgc cacgatgaaa gcttcaaaca aaaggacgaa

[0038] 121cttgaagcctttgccgaaac ctatccacaa ctcaaaccaa tgtcggaaca agaaccatgg

[0039] 181gaactcatcg aggcagttac ctccgcttat ggtcaagttg atgtacttgt gagcaacgac

[0040] 241atattcgcac cagagttcca acccatagat aaatacgctg tagaggacta tcgcggtgcg

[0041] 301gtcgaggcgc tacaaattag accatttgca ctggtcaacg ccgttgcaag tcaaatgaag

[0042] 361aagcgcaaaa gcggacatat tatctttatt acctctgcaa cgcccttcgg gccttggaag

[0043] 421gaactttcta cctacacgtc agcccgagca ggtgcatgca ccttggcaaa tgccctttcg

[0044] 481aaggaactcg gtgaatacaa cattccggtg ttcgcaatag gacccaatta tcttcacagt

[0045] 541gaagatagtc cctacttcta ccccacagaa ccgtggaaaa cgaatccaga acacgttgcc

[0046] 601catgtcaaaa aagtcactgc gctccagcgg ttaggtacac agaaagaatt gggagaactc

[0047] 661gtcgcgtttc tcgcgtctgg tagttgtgac tatctgaccg gccaggtgtt ctggttggcc

[0048] 721ggcggattcc caatgatcga gcgttggcct ggtatgcccg ag

[0049] Preferably, the amino acid sequence of the halohydrin dehalogenase mutant H179V (histidine at position 179 is mutated to valine) is SEQ ID NO.3:

[0050] mstaivtnvk hfggmgsalr lseaghtvac hdesfkqkde leafaetypq lkpmseqepa

[0051] elieavtsay gqvdvlvsnd ifapefqpid kyavedyrga vealqirpfa lvnavasqmk

[0052] krksghiifi tsatpfgpwk elstytsara gactlanals kelgeynipv faigpnylvs

[0053] edspyfypte pwktnpehva hvkkvtalqr lgtqkelgel vaflasgscd yltgqvfwla

[0054] ggfpmierwp gmpe

[0055] Preferably, the nucleotide sequence corresponding to H179V (histidine at position 179 is mutated to valine) is SEQ ID NO.7:

[0056] 1atgtcaaccg caattgtaac aaacgttaag cattttgggg gaatggggtc tgcacttcgt

[0057] 61ctctcggaag caggacatac agtggcttgc cacgatgaaa gcttcaaaca aaaggacgaa

[0058] 121cttgaagcctttgccgaaac ctatccacaa ctcaaaccaa tgtcggaaca agaaccagcg

[0059] 181gaactcatcg aggcagttac ctccgcttat ggtcaagttg atgtacttgt gagcaacgac

[0060] 241atattcgcac cagagttcca acccatagat aaatacgctg tagaggacta tcgcggtgcg

[0061] 301gtcgaggcgc tacaaattag accatttgca ctggtcaacg ccgttgcaag tcaaatgaag

[0062] 361aagcgcaaaa gcggacatat tatctttatt acctctgcaa cgcccttcgg gccttggaag

[0063] 421gaactttcta cctacacgtc agcccgagca ggtgcatgca ccttggcaaa tgccctttcg

[0064] 481aaggaactcg gtgaatacaa cattccggtg ttcgcaatag gacccaatta tcttgtaagt

[0065] 541gaagatagtc cctacttcta ccccacagaa ccgtggaaaa cgaatccaga acacgttgcc

[0066] 601catgtcaaaa aagtcactgc gctccagcgg ttaggtacac agaaagaatt gggagaactc

[0067] 661gtcgcgtttc tcgcgtctgg tagttgtgac tatctgaccg gccaggtgtt ctggttggcc

[0068] 721ggcggattcc caatgatcga gcgttggcct ggtatgcccg ag

[0069] Preferably, the amino acid sequence of the halohydrin dehalogenase mutant A60W-H179V (alanine at position 60 is mutated to tryptophan and histidine at position 179 is mutated to valine) is SEQ ID NO.4: mstaivtnvk hfggmgsalrlseaghtvac hdesfkqkde leafaetypq lkpmseqepwelieavtsay gqvdvlvsnd ifapefqpidkyavedyrga vealqirpfa lvnavasqmk

[0070] krksghiifi tsatpfgpwk elstytsara gactlanals kelgeynipv faigpnylvs

[0071] edspyfypte pwktnpehva hvkkvtalqr lgtqkelgel vaflasgscd yltgqvfwla

[0072] ggfpmierwp gmpe

[0073] Preferably, the nucleotide sequence corresponding to A60W-H179V (the 60th alanine is mutated to tryptophan and the 179th histidine is mutated to valine) is SEQ ID NO.8:

[0074] 1atgtcaaccg caattgtaac aaacgttaag cattttgggg gaatggggtc tgcacttcgt

[0075] 61ctctcggaag caggacatac agtggcttgc cacgatgaaa gcttcaaaca aaaggacgaa

[0076] 121cttgaagcct ttgccgaaac ctatccacaa ctcaaaccaa tgtcggaaca agaaccatgg

[0077] 181gaactcatcg aggcagttac ctccgcttat ggtcaagttg atgtacttgt gagcaacgac

[0078] 241atattcgcac cagagttcca acccatagat aaatacgctg tagaggacta tcgcggtgcg

[0079] 301gtcgaggcgc tacaaattag accatttgca ctggtcaacg ccgttgcaag tcaaatgaag

[0080] 361aagcgcaaaa gcggacatat tatctttatt acctctgcaa cgcccttcgg gccttggaag

[0081] 421gaactttcta cctacacgtc agcccgagca ggtgcatgca ccttggcaaa tgccctttcg

[0082] 481aaggaactcg gtgaatacaa cattccggtg ttcgcaatag gacccaatta tcttgtaagt

[0083] 541gaagatagtc cctacttcta ccccacagaa ccgtggaaaa cgaatccaga acacgttgcc

[0084] 601catgtcaaaa aagtcactgc gctccagcgg ttaggtacac agaaagaatt gggagaactc

[0085] 661gtcgcgtttc tcgcgtctgg tagttgtgac tatctgaccg gccaggtgtt ctggttggcc

[0086] 721ggcggattcc caatgatcga gcgttggcct ggtatgcccg ag

[0087] Preferably, the HheC halohydrin dehalogenase is derived from any one of Rhodococcus equi, Agrobacterium and Mycobacterium; the HheC is a mutant original enzyme, and its amino acid sequence is shown in SEQ ID NO.1. The enzyme can catalyze the degradation of 1-chloro-2-propanol and 2-chloro-1-ethanol to produce propylene oxide and ethylene oxide. The present application uses directed evolution technology and protein engineering to modify the key amino acids in the active center of HheC, and finds that positions 60 and 179 are key sites affecting the activity and selectivity of HheC. These two sites are subjected to site-directed saturation and iterative saturation mutations, respectively, and screen out halohydrin dehalogenase mutants with improved dehalogenation efficiency for 1-chloro-2-propanol and 2-chloro-1-ethanol. The mutant is a single mutation or a combined mutation of the aforementioned sites, specifically: the 60th alanine is mutated to tryptophan, and the 179th histidine is mutated to valine. The halohydrin dehalogenase mutant provided in the present application can achieve green, efficient and precise dehalogenation of halogenated α-halohydrins, and convert them into high-value-added epoxidation products propylene oxide and ethylene oxide.

[0088] Preferably, 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, phage or viral vectors, etc.; preferably, the pET-21a(+) plasmid is used as the expression vector, and the expression host is Escherichia coli.

[0089] Preferably, the halohydrin dehalogenase mutant is used to catalyze the generation of epoxides from α-halohydrins.

[0090] The beneficial effects of the embodiments of the present application are:

[0091] (1) The halohydrin dehalogenase mutants provided in this application can efficiently and accurately degrade two halohydrin pollutants, α-halohydrins: 1-chloro-2-propanol and 2-chloro-1-ethanol, reduce their toxicity, and produce high-value-added propylene oxide and ethylene oxide. Mutants A60W and H179V have high dehalogenation efficiency for these two substances;

[0092] (2) The halohydrin dehalogenase mutant provided in this application can achieve efficient dehalogenation at room temperature in aqueous phase without the need for external temperature control equipment. It has the characteristics of high efficiency, low energy consumption, and high selectivity, and has the advantages of green, low-carbon, economical and convenient industrial application, which is beneficial to the dehalogenation and resource utilization of substances in the fields of sewage treatment and so on.

[0093] (3) The method of the present application is targeted and operational. By precisely mutating key sites, it avoids the extensive screening work caused by blind mutations, saves time and costs, and can reduce production costs, improve product quality and output in industrial production by obtaining highly active halohydrin dehalogenase mutants. In the field of scientific research, it provides a powerful tool for further studying the structure-function relationship of halohydrin dehalogenases and expanding their application in the field of biocatalysis.

[0094] Therefore, the mutant obtained by the present invention is more conducive to the production demand of epoxides and has strong industrial application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0095] Figure 1 This is a structural diagram of the conversion of 1-chloro-2-propanol into propylene oxide catalyzed by a mutant of a halohydrin dehalogenase;

[0096] Figure 2 This is a structural diagram of the conversion of 2-chloro-1-ethanol to ethylene oxide catalyzed by a mutant of a halohydrin dehalogenase;

[0097] Figure 3 This is a schematic diagram of the enzyme activity curve at different final concentrations of isopropylthio-β-D-galactoside (IPTG) in Example 4;

[0098] Figure 4 Schematic diagram of the product of wild-type HheC in Example 5, with the abscissa representing retention time and the ordinate representing absorbance;

[0099] Figure 5 Schematic diagram of the product of mutant A60W-H179V in Example 5, with the abscissa representing retention time and the ordinate representing absorbance;

[0100] Figure 6 Schematic diagram of the enantioselectivity E value of Example 5. DETAILED DESCRIPTION

[0101] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0102] For a better understanding of the present invention and not to limit the scope of the present invention, all numerals expressing amounts, percentages, and other numerical values ​​used in this application should be understood as being modified by the word "about" in all cases. Each numerical parameter should at least be considered to be obtained in light of the number of reported significant digits and by conventional rounding methods.

[0103] The present invention is further described below by way of examples, which, however, do not limit the present invention in any way.

[0104] Example 1

[0105] Construction of recombinant vector pET-21a(+)-HheC plasmid

[0106] The halohydrin dehalogenase HheC gene was synthesized by Shanghai Sangon Biotechnology Co., Ltd. and constructed on the pET-21a(+) vector; the halohydrin dehalogenase was derived from Rhodococcus equi, and the vector was extracted using a plasmid extraction kit. The plasmid DNA was used as the template DNA. The plasmid extraction kit was QIAprep Spin Miniprep Kit.

[0107] Example 2

[0108] Site-directed saturation mutagenesis experiments

[0109] (1) Using the recombinant plasmid pET-21a(+)-HheC as a mutation template, appropriate mutation primers were designed. The primers used for site-directed mutagenesis are shown in Table 1:

[0110] Table 1 Primers for site-directed mutagenesis

[0111] Site Primer name Primer sequence 5'→3' 60 A60W-F CAGGACATACAGTGGCTTGC 60 A60W-R TCCCATGGTTCTTGTTCCGA 179 H179V-F AACACGTTGCCCATGTCAAA 179 H179V-R GCTCGATCATTGGGAATCCG

[0112] (2) The PCR amplification system is as shown in Table 2:

[0113] Table 2 PCR amplification system

[0114] Element Volume (μL) 5×Buffer 10 Prime STAR DNA Polymerase (8 U / μL) 0.5 dNTP (2.5 mM) 4 Forward primer-F (10 μM) 1 Reverse primer-R (10 μM) 1 Template (plasmid) (10 ng / μL) 5 Nuclease-free water Make up to 50 μL

[0115] (3) The PCR amplification program was as follows: pre-denaturation: 95°C for 1 min; 95°C for 10 s, 58°C for 15 s, 70°C for 30 s, 40 cycles; after the program was completed, enzyme digestion was performed, 30 μL of the PCR solution after amplification was aspirated with a pipette, 1.5 μL of Dpn I was added for enzyme digestion, the enzyme digestion program was set to 37°C for 1 to 2 h to remove the plasmid DNA used as the template, inactivated at 65°C for 10 min, and immediately transformed into competent cells E. coli C41 (DE3) (E. coli C41 (DE3) was purchased from Novozymes (China) Biotechnology Co., Ltd.) coated with LB plates containing ampicillin (50 mg / L), cultured at 37°C, and positive transformants were picked for verification and sent to the laboratory for sequencing; the recombinant plasmid was transformed into E. coli C41 (DE3) to obtain the corresponding halohydrin dehalogenase mutants A60W, H179V and A60W-H179V strains.

[0116] Example 3

[0117] Inducible expression

[0118] The recombinant engineered strain prepared in Example 2 was cultured in 100 mL LB medium containing 50 mg / L ampicillin at a final concentration of 50 mg / L at 37°C and 200 rpm / min for 8 h to obtain a fungus solution; the fungus solution was then inoculated into a new 50 mL LB medium containing 50 mg / L ampicillin at a final concentration of 50 mg / L at a 0.5-1.5% inoculum size. The culture was continued at 37°C and 200 rpm in LB medium for 10 h. When the optical density OD600 was 0.6-0.8, isopropylthio-β-D-galactoside (IPTG) was added at a final concentration of 0.2 mM to 0.6 mM, and expression was induced at 25°C for 12 h to 14 h. The bacterial liquid was centrifuged at 10,000 rpm / min for 2 min to collect the bacteria, resuspended and washed with Tris-HCl buffer, and the target protein was lysed by ultrasound. The culture was centrifuged at 4°C and 12,000 rpm / min for 30 min, and the precipitate was discarded to obtain the crude enzyme solutions of mutants A60W, H179V and A60W-H179V, which were stored at -20°C for later use.

[0119] Example 4 Halohydrin dehalogenase activity assay: using 1-chloro-2-propanol and 2-chloro-1-ethanol as substrates (1) Reagent preparation: 50 mM Tris-HCl buffer (pH 8.0), 100 mM 1-chloro-2-propanol and 2-chloro-1-ethanol mixed substrate solution, 10% trichloroacetic acid (TCA) stop solution, chloride ion detection reagents: Reagent A: 0.5 mM Hg (SCN)2 (dissolved in anhydrous ethanol), Reagent B: 0.2 mM Fe (NO3)3·9H2O (dissolved in 1.5 M HNO3); (2) Prepare four reagent tubes, label them as wild-type HheC, A60W, H179V, and A60W-H179V. Add 850 μL of buffer and 100 μL of 100 mM substrate to each tube, preheat at 30°C for 5 min, add 0.025 mg of the corresponding enzyme solution in Example 3, start the reaction for 5 min, add 100 μL of 10% TCA to terminate the reaction, centrifuge at 12,000 rpm / min for 5 min, and collect the supernatant. (3) Chloride ion detection: Take 200 μL supernatant + 1 mL ddH2O + 0.1 mL reagent A + 0.1 mL reagent B, react at room temperature in the dark for 10 minutes, and measure A 485 The experimental results are shown in Tables 3 to 5. The mutant catalyzes 1-chloro-2-propanol to generate the product structure shown in FIG. Figure 1 As shown, the variant catalyzes 2-chloro-1-ethanol to produce the product structure shown in FIG. Figure 2 The enzyme activity curve is shown in Figure 3 As shown; Table 3: Enzyme activity test results (final concentration of isopropylthio-β-D-galactopyranoside (IPTG) is 0.2 mM)

[0120] Table 4: Enzyme activity test results (final concentration of isopropylthio-β-D-galactopyranoside (IPTG) is 0.4 mM)

[0121] Table 5: Enzyme activity test results (final concentration of isopropylthio-β-D-galactopyranoside (IPTG) is 0.6 mM)

[0122] Calculate the amount of chloride ion generated:

[0123]

[0124] The dilution factor refers to the dilution factor of the sample from the reaction system to the detection system; Enzyme activity calculation:

[0125]

[0126] Calculation of relative enzyme activity:

[0127]

[0128] From Tables 3 to 5 above, it can be seen that the experimental data in Table 5 are better than those in Table 4, which are better than those in Table 3. The best induction condition is when the final concentration of isopropylthio-β-D-galactoside (IPTG) is 0.6 mM; From Table 5, we can conclude that: 485 : reflects the intensity of the color reaction related to the amount of chloride ions generated. The higher the absorbance, the more chloride ions are generated in the system. 485 is 0.450, and the A of mutant A60W 485 Slightly higher than the wild type, at 0.465; A 485 A60W-H179V's A 485 The value is 0.8, the highest among the enzymes. This preliminarily indicates that the mutant enzymes have different degrees of changes in the catalytic reaction to generate chloride ions, and the changes of A60W-H179V and H179V are more obvious. Cl - Production: directly reflects the amount of chloride ions generated by the enzyme-catalyzed reaction. The Cl - The production amount was 0.72 μmol, and the production amount of A60W mutant was 0.74 μmol, which was slightly increased compared with the wild type; the production amount of H179V mutant was significantly increased to 1.19 μmol; the Cl - The amount of production reached 1.28 μmol, which was the largest amount produced, further indicating that these mutants had different performances in catalyzing the production of chloride ions from substrates, and that some mutants had enhanced catalytic abilities. Enzyme activity: The wild-type enzyme had an activity of 2.88, while the A60W mutant had an activity of 2.96, slightly higher than the wild-type. The H179V mutant had an activity of 4.76, and the A60W-H179V mutant had an activity of 5.12, the highest among the enzymes. The activity was 1.781 times that of the original enzyme, indicating that the mutant enzymes had an effect on their catalytic activity after changing their structure, and some mutants had improved the catalytic activity of the enzymes. Relative enzyme activity: Taking the wild-type enzyme activity as the benchmark (set as 100%), the ratio of the enzyme activity of each mutant relative to the wild-type was calculated. The relative enzyme activity of the A60W mutant was 102.3%, indicating that its enzyme activity was slightly higher than that of the wild-type; the relative enzyme activity of the H179V mutant reached 165.3%, a significant increase in enzyme activity; the relative enzyme activity of A60W-H179V was 177.8%, the highest relative enzyme activity, reflecting the significant effect of the double mutant in improving enzyme activity and the synergistic effect between the mutation sites.

[0129] Based on Table 5, the dehalogenation efficiency is calculated as follows: wild type: 0.072, A60W: 0.074; H179V: 0.119; A60W-H179V: 0.128; the dehalogenation efficiency of the mutants is improved to: A60W: 2.8%; H179V: 65.3; A60W-H179V: 77.8%; the calculation formula is: ; .

[0130] Example 5 Enantioselectivity (1) The induction condition obtained in Example 4 is that the final concentration of isopropylthio-β-D-galactopyranoside (IPTG) is 0.6 mM, and the enzyme activity is the best. Therefore, the dehalogenation efficiency is tested when the final concentration of isopropylthio-β-D-galactopyranoside (IPTG) is 0.6 mM; (2) Add isopropylthio-β-D-galactoside (IPTG) to a final concentration of 0.6 mM in Example 3, induce expression at 25°C for 12 to 14 hours, centrifuge the bacterial solution at 10,000 rpm / min, collect the bacteria by centrifugation for 2 minutes, resuspend and wash the bacteria with Tris-HCl buffer, lyse the target protein by ultrasonication, centrifuge at 4°C and 12,000 rpm / min for 30 minutes, discard the precipitate, and obtain the crude enzyme solutions of mutants A60W, H179V, and A60W-H179V, which are stored at -20°C for later use; (3) Take 0.025 mg of crude enzyme solution of wild-type HheC and mutants A60W, H179V and A60W-H179V; the pH of PBS buffer is 7.4, and it is sterilized at 126℃ for 15 min; 1-chloro-2-propanol and 2-chloro-1-ethanol are mixed to make 100 mM, the chromatographic column is: Chiralcel OD-H column, the mobile phase is: n-hexane / ethanol (95:5, v / v), the flow rate is 1.0 mL / min, the column temperature is 30℃, the detection wavelength is 210 nm, and the expected retention time is: R-isomer (6.8 min), S-isomer (8.3 min); the results show that the peak area of ​​R-product is: wild-type HheC: 380, A60W: 400, H179V: 700, A60W-H179V: 750; the peak area of ​​S-product is: H HeC: 120, A60W: 100, H179V: 50, A60W-H179V: 30; R-substrate residual peak area: HheC: 280, A60W: 270, H179V: 150, A60W-H179V: 120; S-substrate residual peak area: HheC: 520, A60W: 530, H179V: 650, A60W-H179V: 680; The product of wild-type HheC is shown in the figure. Figure 4As shown, the product of mutant A60W-H179V is shown in FIG. Figure 5 As shown; (4) The enantioselectivity results are shown in Table 6, and the E value diagram is shown in Figure 6 As shown; Table 6 Enantioselectivity analysis

[0131] The calculation formula is:

[0132]

[0133] Among them, ee p is the enantiomeric excess of the product, C is the ratio of substrate converted to product, and E is the enantioselectivity factor;

[0134]

[0135] (5) Experimental results: The enantioselectivity E value of A60W-H179V increased from 3.4 to 23, which was significantly better than that of mutants A60W and H179V. It had extremely strong selectivity and the enzyme activity was also the highest, with an enzyme activity of 5.12, which was 1.78 times that of the wild enzyme.

[0136] In summary, the enzyme activity, dehalogenation efficiency and enantioselectivity of mutants A60W, H179V and A60W-H179V were significantly higher than those of the wild type, and mutant A60W-H179V had the best effect.

[0137] Although the embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations may be made to these embodiments without departing from the principles and spirit of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the patent coverage of the present invention.

Claims

1. A directed modification method for improving the activity of a halohydrin dehalogenase, characterized in that: The directed transformation method is to perform single-point or combined mutations at positions 60 and 179 of the wild-type halohydrin dehalogenase HheC to obtain the corresponding halohydrin dehalogenase mutants, and the steps are as follows: (1) Recombinant vector construction: Construction of halohydrin dehalogenase mutants; (2) Recombinant cells: Transform the constructed recombinant vector into host cells; (3) Induced expression: Obtaining the mutant protein of halohydrin dehalogenase.

2. A directed modification method for improving the activity of a halohydrin dehalogenase according to claim 1, characterized in that: In the step (1), the amino acid sequence of the halohydrin dehalogenase mutant is obtained by mutating the 60th alanine of the wild-type halohydrin dehalogenase HheC to tryptophan to obtain mutant A60W, mutating the 179th histidine to valine to obtain mutant H179V, and mutating the 60th alanine to tryptophan and the 179th histidine to valine to obtain any one of mutants A60W-H179V.

3. A directed modification method for improving the activity of a halohydrin dehalogenase as claimed in claim 2, characterized in that: The wild-type sequence of the halohydrin dehalogenase is shown in SEQ ID NO.1, and the corresponding nucleotide sequence encoding the wild-type halohydrin dehalogenase HheC is shown in SEQ ID NO.

5.

4. A directed modification method for improving the activity of a halohydrin dehalogenase as claimed in claim 2, characterized in that: The amino acid sequence of the halohydrin dehalogenase mutant A60W is shown in SEQ ID NO.2, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.

6.

5. A directed modification method for improving the activity of a halohydrin dehalogenase according to claim 2, characterized in that: The amino acid sequence of the halohydrin dehalogenase mutant H179V is shown in SEQ ID NO.3, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.

7.

6. A directed modification method for improving the activity of a halohydrin dehalogenase according to claim 2, characterized in that: The amino acid sequence of the halohydrin dehalogenase mutant A60W-H179V is shown in SEQ ID NO.4, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.

8.

7. A directed modification method for improving the activity of a halohydrin dehalogenase according to claim 2, characterized in that: The halohydrin dehalogenase is derived from any one of Rhodococcus equi, Agrobacterium and Mycobacterium.

8. A directed modification method for improving the activity of a halohydrin dehalogenase according to claim 1, characterized in that: The recombinant vector in step (1) contains the nucleotide sequence of the halohydrin dehalogenase mutant according to claims 4 to 6.

9. A directed modification method for improving the activity of a halohydrin dehalogenase according to claim 1, characterized in that: The recombinant cell in step (2) is characterized in that the recombinant cell contains the recombinant vector according to claim 8.

10. A directed modification method for improving the activity of a halohydrin dehalogenase according to claim 1, characterized in that: The halohydrin dehalogenase mutant is used in catalyzing α-halohydrin to generate epoxide.

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