Halogen alcohol dehalogenase mutant and application thereof in asymmetric synthesis of epsilon-substituted alcohol

Abstract

This invention relates to the fields of enzyme engineering and biocatalysis, disclosing a halohydrin dehalogenase mutant and its catalytic enantioselective dehalogenation hydroxylation reaction of a series of racemic ε-halohydrins, preparing corresponding chiral ε-diols and chiral ε-halohydrins. The invention utilizes a fungicide derived from *Agrobacterium radiodurans* (…). Agrobacterium tumefaciens The wild-type haloalcohol dehalogenase HheC (amino acid sequence SEQ ID NO:2) of AD1 was used as the parent enzyme, and was obtained by combining 2 to 6 amino acid site mutations at L142, W249, P84, T134, N176, and F186. The mutant can be used to catalyze the kinetic resolution and dehalogenation reactions of racemic ε-haloalcohols, synthesizing a series of high-optical-purity chiral ε-diols and chiral ε-haloalcohols. These compounds are important chiral building blocks for the preparation of various pharmaceutical chemicals and have broad industrial application value.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering, enzyme engineering and biocatalysis, specifically involving halohydrin dehalogenase mutants obtained through protein engineering, and their application in catalyzing the enantioselective dehalogenation and hydroxylation of racemic ε-halohydrins, while simultaneously preparing chiral ε-diols and chiral ε-halohydrins. Background Technology

[0002] Chiral ε-substituted alcohols are an important class of chiral building blocks, characterized by three methylene groups (i.e., ω-chiral alcohols) separating the chiral center from the terminal hydroxyl group. These structures are widely found in the core skeletons of natural products, bioactive molecules, and drugs; for example, some lipid-lowering drugs (such as statin side chains), immunosuppressants, and insect pheromone molecules contain chiral ε-substituted alcohol fragments. Therefore, developing efficient and highly enantioselective asymmetric synthetic methods for ε-substituted alcohols has significant academic value and promising industrial applications.

[0003] Traditionally, the synthesis of chiral ε-substituted alcohols has primarily relied on chemocatalytic methods, such as the asymmetric hydrogenation of the corresponding ε-keto esters using chiral metal catalysts (e.g., Ru-BINAP complexes), or the asymmetric reduction of prochiral ketones using stoichiometric chiral reducing agents (e.g., Corey-Bakshi-Shibata reagents). However, these chemical methods face significant challenges when applied to ε-keto substrates: Low stereoinduction efficiency: Due to the long carbon chain between the carbonyl group and the reaction site, the stereoinduction effect of the chiral catalyst decreases sharply with increasing distance, resulting in an unsatisfactory enantiomeric excess value (ee value) of the product (often below 85%).

[0004] High substrate conformational flexibility: The conformational flexibility of long-chain substrates in organic solvents increases the entropy penalty effect, further reducing the selectivity of the reaction.

[0005] The reaction conditions are harsh: many precious metal catalysts are expensive, the reaction requires high-pressure hydrogen, anhydrous and oxygen-free environment, and the products may contain residual toxic metals, which limits their large-scale application in the production of pharmaceutical intermediates.

[0006] In the field of biocatalysis, halohydrin dehalogenases are a class of enzymes with unique catalytic heterogeneity. They can catalyze the dehalogenation and cyclization of β-, γ-, and δ-halohydrins to generate corresponding epoxides, oxetanes, and tetrahydrofurans. They can also reverse-catalyze the ring-opening reactions of epoxides and oxetanes with various nucleophiles. However, when the substrate carbon chain length increases to the point of generating ε-substituted alcohols (i.e., C5 and above), wild-type halohydrin dehalogenases often exhibit extremely low catalytic activity. This is mainly because the structure of the active pocket of natural enzymes evolved for shorter carbon chain substrates (C3-C4), making it difficult to form effective binding and recognition for longer chain substrates.

[0007] Therefore, developing a halohydrin dehalogenase mutant that can efficiently and with high enantioselectivity catalyze the asymmetric dehalogenation of long-chain ε-halohydrins, and establishing a corresponding green preparation process, has become a technical challenge that urgently needs to be solved in this field. Summary of the Invention

[0008] This invention addresses the aforementioned technological gap by utilizing materials derived from *Agrobacterium radiodurans*. Agrobacterium tumefaciens Through rational design and directed evolution of the wild-type halool dehalogenase HheC of AD1, a series of mutants were obtained, which can efficiently catalyze the kinetic resolution dehalogenation and hydroxylation reaction of racemic ε-halools, while obtaining chiral ε-diols and chiral ε-halools with high optical purity.

[0009] To achieve the above objectives, this invention provides a halohydrin dehalogenase mutant with catalytic enantioselective dehalogenation hydroxylation of racemic ε-halohydrins, simultaneously preparing chiral ε-diols and chiral ε-halohydrins. The mutant is derived from... Agrobacterium tumefaciens Using wild-type halohydrin dehalogenase (HheC-WT) with the amino acid sequence shown in SEQ ID NO:2 (nucleotide sequence as shown in SEQ ID No:1) as the parent, site-directed saturation mutagenesis and combinatorial mutagenesis were performed on key residues of the active site to obtain 7 highly efficient mutants: HheC-TB1: Leucine at position 142 is mutated to alanine, and tryptophan at position 249 is mutated to proline. The resulting amino acid sequence is shown in SEQ ID NO:4. HheC-TB2: Leucine at position 142 is mutated to alanine, tryptophan at position 249 is mutated to proline, and proline at position 84 is mutated to glycine. The resulting amino acid sequence is shown in SEQ ID NO:6. HheC-TB3: Leucine at position 142 is mutated to alanine, tryptophan at position 249 is mutated to proline, and threonine at position 134 is mutated to leucine. The resulting amino acid sequence is shown in SEQ ID NO:8. HheC-TB4: ​​Leucine at position 142 is mutated to alanine, tryptophan at position 249 is mutated to proline, proline at position 84 is mutated to glycine, and threonine at position 134 is mutated to leucine. The resulting amino acid sequence is shown in SEQ ID NO:10. HheC-TB5: Leucine at position 142 is mutated to alanine, tryptophan at position 249 is mutated to proline, proline at position 84 is mutated to glycine, and asparagine at position 176 is mutated to glutamine. The resulting amino acid sequence is shown in SEQ ID NO:12. HheC-TB6: Leucine at position 142 is mutated to alanine, tryptophan at position 249 is mutated to proline, proline at position 84 is mutated to glycine, and phenylalanine at position 186 is mutated to tyrosine. The resulting amino acid sequence is shown in SEQ ID NO:14. HheC-TB7: Leucine at position 142 is mutated to alanine, tryptophan at position 249 is mutated to proline, proline at position 84 is mutated to glycine, asparagine at position 176 is mutated to glutamine, and phenylalanine at position 186 is mutated to tyrosine. The resulting amino acid sequence is shown in SEQ ID NO:16.

[0010] As shown in Table 1: Table 1: Halohydrin dehalogenase mutants and their corresponding amino acid sequences and coding gene base sequences

[0011] The three-letter or single-letter representation of amino acids in the application shall adopt the amino acid codes specified by IUPAC.

[0012] The halohydrin dehalogenase mutant described in this invention can be used in the form of recombinant Escherichia coli wet cells; if further application is required, it can also be used in the form of freeze-dried stem cells or broken enzyme solution, partially purified enzyme or fully purified enzyme, and can also be made into immobilized enzyme or immobilized cell form using immobilization techniques known in the art.

[0013] By transforming a recombinant plasmid containing a halohydrin dehalogenase mutant gene into host cells, followed by culturing in TB medium (containing 50 μg / mL kanamycin sulfate) and inducing expression with 0.2 mM IPTG, highly active positive recombinant bacteria were obtained through screening. In this invention, the expression vector is preferably pET-28b(+), and the host cell is preferably Escherichia coli BL21(DE3).

[0014] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in: (1) Provides a high efficiency and high stereoselectivity halohydrin dehalogenase mutant, which solves the core bottleneck of existing technologies where wild-type halohydrin dehalogenases have extremely low catalytic activity for long-chain (C5 and above) ε-halohydrin substrates, poor selectivity of chemical catalysis and harsh conditions.

[0015] (2) For the first time, the enantioselective dehalogenation hydroxylation reaction of ε-haloalcohols catalyzed by haloalcohol dehalogenase was realized, and chiral ε-diols and chiral ε-haloalcohols were obtained simultaneously through a kinetic resolution strategy. The reaction conditions were mild (room temperature at 30℃, pH 9.5 Gly-NaOH buffer system), and no expensive metal catalysts or high-pressure hydrogen were required. The environmental friendliness and operational safety were significantly better than chemical methods.

[0016] (3) The enantiomeric excess (ee) of the chiral ε-diol product and the remaining chiral ε-halool substrate can reach up to 98% and 96% respectively, the reaction selectivity E value can reach up to 168, and the optical purity fully meets the stringent requirements of pharmaceutical intermediates.

[0017] (4) The haloalcohol dehalogenase mutant required for catalysis can be prepared by large-scale fermentation of recombinant Escherichia coli. The whole-cell catalyst can be used directly, is inexpensive and readily available, and is compatible with ε-haloalcohol substrates with various structures (such as aryl and heterocyclic substitutions). It has a wide range of applications and provides an efficient and practical technical route for the large-scale preparation of chiral ε-substituted alcohols. Attached Figure Description

[0018] Figure 1 : The 1H NMR spectrum of the synthesis of chiral (R)-5-chloro-1-phenyl-1-pentanol catalyzed by the HheC-TB6 mutant; Figure 2 : Carbon NMR spectrum of chiral (R)-5-chloro-1-phenyl-1-pentanol catalyzed by HheC-TB6 mutant; Figure 3 : The 1H NMR spectrum of the synthesis of chiral (S)-1-phenyl-1,5-pentanediol catalyzed by the HheC-TB6 mutant; Figure 4 : Carbon NMR spectrum of chiral (S)-1-phenyl-1,5-pentanediol synthesized by HheC-TB6 mutant; Figure 5 Chiral high-performance liquid chromatography (HPLC) chromatogram of the synthesis of chiral (R)-5-chloro-1-phenyl-1-pentanol catalyzed by the HheC-TB6 mutant; Figure 6 Chiral high-performance liquid chromatography (HPLC) chromatogram of chiral (S)-1-phenyl-1,5-pentanediol synthesized by HheC-TB6 mutant. Detailed Implementation

[0019] The following detailed description illustrates the specific implementation method: Example 1: Mutation and Screening of Haloalcohol Dehalogenases Site-directed saturation mutagenesis primers were designed based on the parental halohydrin dehalogenase gene sequence for the hot amino acid residues L142A, W249P, P84G, T134, F186, and N176Q. L142-F (5'- CGTGGAAAGAANNKTCCACTTATAC-3'), L142-R (5'-GTATAAGTGCAMNNTTCTTTCCACGC-3'); T134-F (5'- CCACGGACCGAATGG) MNNTGCAGAGGTAAT-3'), T134-R (5'- CGTGGAAAGAANNKTCCACTTATAC-3'); W249-F (5'- GATGATCGAACGTNNKCCGGGTATGC-3'), W249-R (5'-CATACCCGGMNNACGTTCGATCATCG-3'); P84-F (5'-CAACGATATTTTTCGCGNNKGAATTCCAGCCG-3'), P84-R (5'-TCGGCTGGAATTCMNNCGCGAAAATATCGTTGGAC-3'); N176-F (5'- TCGCGATCGGTCCGNNKTACCTGCAC-3'), N176-R (5'-TTCAGAGTGCAGGTA) MNN CGGACCGATC-3'). F186-F (5'-T GAAGACAGCCCGTACNNKTACCCGACCGAACCGT-3'), F186-R (5'-GTTCGGTCGGGTAMNNGTACGGGCTGTCTTCAGAGT-3').

[0020] Plasmids containing the parental halohydrin dehalogenase gene (gene sequence SEQ ID NO:1) or pET-28b(+) plasmids carrying a fragment of the earlier halohydrin dehalogenase mutant gene were extracted as DNA templates and PCR amplification was performed using the primers described above. PCR system (50 μL): 10 μL 5×Plus buffer, 0.2 μL each of 100 μM pre- and post-primer, 4 μL template plasmid, 4 μL dNTP reagent, 0.5 μL Prime STAR DNA polymerase, and 31.5 μL deionized water. PCR program: Step 1, 95℃ / 5 min; Step 2, 98℃ / 10 s, (Tm±5)℃ / 5 s, 72℃ / 7 min, for a total of 30 cycles; Step 3, 72℃ / 10 min. PCR products were used... Dpn After digestion, the cells were directly transformed into *E. coli* BL21(DE3) competent cells via heat shock (42°C, 90 s). After resuscitation culture (37°C, 1 h), the cells were evenly spread onto TB solid medium containing kanamycin sulfate and cultured overnight at 37°C. Single colonies were picked and subjected to fermentation culture and IPTG induction to obtain cells containing a haloalcohol dehalogenase mutant, which were used as a biocatalyst.

[0021] Using 5-chloro-1-phenyl-1-pentanol as the ε-haloalcohol template substrate, the catalytic activity and enantioselectivity of haloalcohol dehalogenase mutants were investigated. The reaction system (5 mL) contained 10 mM 5-chloro-1-phenyl-1-pentanol substrate and 10 g dcw / L whole cells of haloalcohol dehalogenase. The reaction solvent was 100 mM Gly-NaOH buffer at pH 9.5, the reaction temperature was 30 ℃, and the reaction time was 8 h. After the reaction, the mixture was extracted with an equal volume of ethyl acetate, and the optical purity of the corresponding chiral ε-diol product (1-phenyl-1,5-pentanediol) and the remaining 5-chloro-1-phenyl-1-pentanol substrate was analyzed by chiral high-performance liquid chromatography (HPLC). The enantiomeric excess values ​​of the two compounds were also considered. ee The reaction selectivity E value was calculated. Each reaction was repeated in triplicate. Mutants with enhanced catalytic activity or enantioselectivity were sent for gene sequencing to obtain accurate mutation information and amino acid sequences of superior halohydrin dehalogenase mutants. After multiple rounds of iterative mutation and screening, the finally obtained superior halohydrin dehalogenase mutants and their specific reaction details are shown in Table 2.

[0022] Table 2: Halohydrin dehalogenase mutants and their catalytic effects

[0023] Example 2: Preparation of whole-cell biocatalyst of halohydrin dehalogenase mutant Recombinant Escherichia coli genetically engineered bacteria containing the gene encoding a halohydrin dehalogenase mutant were inoculated into TB liquid medium (glycerol 5 g / L, tryptone 12 g / L, yeast extract 24 g / L) containing kanamycin sulfate to a final concentration of 50 μg / mL and cultured at 37 ℃ and 250 rpm for 8 h. Seed culture was inoculated at a volume concentration of 2% into fresh TB liquid medium containing kanamycin sulfate to a final concentration of 50 μg / mL and cultured at 37 ℃ and 250 rpm with shaking for approximately 2.5 h until the bacterial OD of the culture was reached. 600 After adding 0.6 to 0.8, IPTG was added to a final concentration of 0.2 mM, and the cells were induced and cultured at 28℃ and 250 rpm for 12-14 h. The induced culture solution was centrifuged at 4℃ and 8000 rpm for 3 min, and the wet cells were collected to obtain recombinant whole cells containing halohydrin dehalogenase mutant protein for subsequent biocatalytic reactions.

[0024] Example 3: Kinetic resolution of 5-chloro-1-phenyl-1-pentanol catalyzed by HheC-TB6 engineered bacteria

[0025] Add 100 mL of Gly-NaOH buffer (100 mM, pH 9.5) containing recombinant cells (10 g dcw / L) of the halohydrin dehalogenase mutant HheC-TB6 to a 200 mL round-bottom flask, along with 1 mmol of the substrate (rac)-5-chloro-1-phenyl-1-pentanol. The reaction mixture was magnetically stirred at 30 °C for 10 h. The reaction mixture was centrifuged at 8000 rpm for 3 min at 4 °C to remove the cells, and the supernatant was collected. The supernatant was extracted three times with equal volumes of ethyl acetate (100 mL × 3), and the organic phases were combined. The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the concentrate was purified by silica gel column chromatography. The target components were collected and concentrated under reduced pressure to obtain chiral (S)-1-phenyl-1,5-pentanediol and (R)-5-chloro-1-phenyl-1-pentanol.

[0026] The separation yield of (S)-1-phenyl-1,5-pentanediol was 48%, with an optical purity of 96% ee, and optical rotation [α]. D 25 = -34.40 (c = 1.00, CH2Cl2). NMR characterization data: 1 H NMR (400 MHz, DMSO- d 6) d 1.65 – 1.49 (m, 2H), 1.44 – 1.30 (m, 3H), 1.25 – 1.18 (m, 1H). 13 C NMR (100 MHz, DMSO- d 6) d 146.5, 127.9, 126.6, 125.8, 72.4, 60.8, 39.4, 32.6, 22.0. The separation yield of (R)-5-chloro-1-phenyl-1-pentanol was 45%, with an optical purity of 96% ee, and optical rotation [α]. D 25 = +32.00 (c = 1.00, CH2Cl2). NMR characterization data: 1 H NMR (400 MHz, DMSO- d 6) d7.33 – 7.29 (m,4H), 7.23 – 7.19 (m, 1H), 5.22 – 5.17 (m, 1H), 4.54 – 4.48 (m, 1H), 3.62 –3.58 (m, 2H), 1.76 – 1.54 (m, 4H), 1.52 – 1.30 (m, 2H). 13 C NMR (100 MHz, DMSO- d 6) d 146.4, 128.0, 126.6, 125.8, 72.1, 45.5, 38.6, 32.1, 22.9. [α] D 25 = + 32.00 (c = 1.00, CH2Cl2). Example 5: Kinetic resolution of 5-chloro-1-(thien-2-yl)-1-pentanol catalyzed by HheC-TB6 engineered bacteria

[0027] Add 100 mL of Gly-NaOH buffer (100 mM, pH 9.5) containing recombinant cells (10 g dcw / L) of the haloalcohol dehalogenase mutant HheC-TB6 to a 200 mL round-bottom flask, along with 1 mmol of the substrate (rac)-5-chloro-1-(thien-2-yl)-1-pentanol. The reaction mixture was magnetically stirred at 30 °C for 12 h. The reaction solution was centrifuged at 4℃ and 8000 rpm for 3 min to remove the bacterial cells and collect the supernatant. The supernatant was extracted three times with an equal volume of ethyl acetate (100 mL × 3), and the organic phases were combined. The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the concentrate was purified by silica gel column chromatography. The target components were collected and concentrated under reduced pressure to obtain chiral (S)-1-(thien-2-yl)-1,5-pentanediol and (R)-5-chloro-1-(thien-2-yl)-1-pentanol.

[0028] The separation yield of (S)-1-(thiophen-2-yl)-1,5-pentanediol was 49%, with an optical purity of 96% ee, and optical rotation [α]. D 25 = – 13.40 (c = 1.00, CH2Cl2). NMR characterization data: 1 H NMR (400 MHz, DMSO- d 6) d 7.35(dd, J= 1.3, 5.0 Hz, 1H), 6.95 – 6.91 (m, 2H), 4.48 (d, J = 4.7 Hz, 1H), 4.76 –4.71 (m, 1H), 4.35 (t, J = 5.1 Hz, 1H), 3.38 – 3.37 (m, 1H), 3.35 – 3.34 (m,1H), 1.73 – 1.59 (m, 2H), 1.45 – 1.22 (m, 4H). 13 C NMR (100 MHz, DMSO- d 6) d 151.0, 126.4, 123.9, 122.8, 68.4, 60.8, 39.5, 32.4, 21.9. The separation yield of (R)-5-chloro-1-(thien-2-yl)-1-pentanol was 49%, with an optical purity of 91% ee and optical rotation [α]. D 25 = + 13.40 (c = 1.00, CH2Cl2). NMR characterization data: 1 H NMR (400 MHz, DMSO- d 6) d 7.37 –7.35 (m, 1H), 6.96 – 6.91 (m, 2H), 5.55 (s, 1H), 4.77 (q, J = 6.4 Hz, 1H), 3.63– 3.59 (m, 2H), 1.76 – 1.63 (m, 4H), 1.55 – 1.46 (m, 1H), 1.44 – 1.35 (m,1H). 13 C NMR (100 MHz, DMSO- d 6) d 150.9, 126.5, 123.9, 122.8, 68.3, 45.4, 38.7, 32.0, 22.8. Example 4: Kinetic resolution of 5-bromo-1-phenyl-1-pentanol catalyzed by HheC-TB6 engineered bacteria

[0029] Add 100 mL of Gly-NaOH buffer (100 mM, pH 9.5) containing recombinant cells (10 g dcw / L) of the halohydrin dehalogenase mutant HheC-TB6 to a 200 mL round-bottom flask, along with 2 mmol of the substrate (rac)-5-bromo-1-phenyl-1-pentanol. The reaction mixture was magnetically stirred at 30 °C for 12 h. The reaction mixture was centrifuged at 8000 rpm for 3 min at 4 °C to remove the cells, and the supernatant was collected. The supernatant was extracted three times with equal volumes of ethyl acetate (100 mL × 3), and the organic phases were combined. The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the concentrate was purified by silica gel column chromatography. The target components were collected and concentrated under reduced pressure to obtain chiral (S)-1-phenyl-1,5-pentanediol and (R)-5-bromo-1-phenyl-1-pentanol.

[0030] The separation yield of (S)-1-phenyl-1,5-pentanediol was 42%, with an optical purity of 90% ee, and optical rotation [α]. D 25 = -33.40 (c = 1.00, CH2Cl2). NMR characterization data: 1 H NMR (400 MHz, DMSO- d 6) d 1.62 – 1.51 (m, 2H), 1.40 – 1.36 (m, 3H), 1.25 – 1.16 (m, 1H). 13 C NMR (100 MHz, DMSO- d 6) d 146.5, 127.9, 126.5, 125.8, 72.4, 60.8, 39.3, 32.6, 22.0. The separation yield of (R)-5-bromo-1-phenyl-1-pentanol was 47%, with an optical purity of 98% ee, and optical rotation [α]. D 25 = +20.67 (c = 1.00, CH2Cl2). NMR characterization data: 1 H NMR (400 MHz, DMSO- d 6) d7.33 – 7.28 (m,4H), 7.24 – 7.19 (m, 1H), 4.68 – 4.49 (m, 2H), 3.52 – 3.48 (m, 2H), 1.83 –1.75 (m, 2H), 1.66 – 1.54 (m, 2H), 1.49 – 1.32 (m, 2H). 13 C NMR (100 MHz, DMSO- d 6) d 146.3, 127.9, 126.6, 125.8, 72.1, 38.4, 35.2, 32.3, 24.1. Example 5: Kinetic resolution of (rac)-5-bromo-1-(4-fluorophenyl)-1-pentanol catalyzed by HheC-TB7 engineered bacteria

[0031] Add 100 mL of Gly-NaOH buffer (100 mM, pH 9.5) containing recombinant HheC-TB7 halohydrin dehalogenase mutant cells (10 g dcw / L) to a 200 mL round-bottom flask, along with 2 mmol of the substrate (rac)-5-bromo-1-(4-fluorophenyl)-1-pentanol. The reaction mixture was magnetically stirred at 30 °C for 12 h. The reaction mixture was centrifuged at 8000 rpm for 3 min at 4 °C to remove the cells, and the supernatant was collected. The supernatant was extracted three times with equal volumes of ethyl acetate (100 mL × 3), and the organic phases were combined. The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the concentrate was purified by silica gel column chromatography. The target components were collected and concentrated under reduced pressure to obtain chiral (S)-1-(4-fluorophenyl)-1,5-pentanediol and (R)-5-bromo-1-(4-fluorophenyl)-1-pentanol.

[0032] The separation yield of (S)-1-(4-fluorophenyl)-1,5-pentanediol was 48%, with an optical purity of 89% ee, and optical rotation [α]. D 25 = – 17.63 (c = 1.00, CH2Cl2). NMR characterization data: 1 H NMR (400 MHz, DMSO- d 6) δ 7.35– 7.31 (m, 2H), 7.14 – 7.08 (m, 2H), 5.16 (d, J = 4.4 Hz, 1H), 4.51 – 4.47 (m,1H), 4.33 (t, J= 5.1 Hz, 1H), 3.36 – 3.32 (m, 2H), 1.63 – 1.47 (m, 2H), 1.43 –1.36 (m, 2H), 1.34 – 1.14 (m, 2H). 13 C NMR (100 MHz, DMSO- d 6) d 161.0 (d, J = 240.0Hz, 1C), 142.7 (d, J = 3.0 Hz, 1C), 127.7 (d, J = 7.0 Hz, 2C), 114.7,114.5,71.7,60.7, 32.5, 21.9. The separation yield of (R)-5-bromo-1-(4-fluorophenyl)-1-pentanol was 46%, with an optical purity of 90% ee, and optical rotation [α]. D 25 = + 10.30 (c = 1.00, CH2Cl2). NMR characterization data: 1 H NMR (400 MHz, DMSO- d 6) d 7.37 –7.33 (m, 2H), 7.15 – 7.09 (m, 2H), 4.96 – 4.50 (m, 2H), 3.49 (t, J = 6.7 Hz,2H), 1.82 – 1.73 (m, 2H), 1.65 – 1.53 (m, 2H), 1.49 – 1.29 (m, 2H). 13 C NMR (100 MHz, DMSO-) d 6) d 161.0 (d, J = 240.0 Hz, 1C), 142.5 (d, J = 3.0 Hz, 1C), 127.6(d, J = 8.0 Hz, 2C), 114.6 (d, J =21.0 Hz, 2C), 71.4, 38.4, 35.3, 32.3, 24.1. Example 6: Kinetic resolution of (rac)-5-bromo-1-(p-tolyl)-1-pentanol and (S)-1-(p-tolyl)-1,5-pentanediol catalyzed by HheC-TB5 engineered bacteria.

[0033] 100 mL of Gly-NaOH buffer (100 mM, pH 9.5) containing recombinant HheC-TB5 halohydrin dehalogenase mutant cells (10 g dcw / L) was added to a 200 mL round-bottom flask, along with 2 mmol of the substrate (rac)-5-bromo-1-(p-tolyl)-1-pentanol. The reaction mixture was magnetically stirred at 30 °C for 12 h. The reaction mixture was centrifuged at 8000 rpm for 3 min at 4 °C to remove the cells, and the supernatant was collected. The supernatant was extracted three times with equal volumes of ethyl acetate (100 mL × 3), and the organic phases were combined. The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the concentrate was purified by silica gel column chromatography. The target components were collected and concentrated under reduced pressure to obtain chiral (S)-1-(p-tolyl)-1,5-pentanediol and ((R)-5-bromo-1-(p-tolyl)-1-pentanol.

[0034] The separation yield of (S)-1-(p-Tolyl)-1,5-pentanediol was 43%, with an optical purity of 88% ee, and optical rotation [α]. D 25 = – 15.80 (c = 1.00, CH2Cl2). NMR characterization data: 1 H NMR (400 MHz, DMSO- d 6) d 7.18(d, J = 7.9 Hz, 2H), 7.10 (d, J = 7.8 Hz, 2H), 5.02 (d, J = 4.4 Hz, 1H), 4.46 –4.42 (m, 1H), 4.33 (t, J = 5.1 Hz, 1H), 3.37 – 3.32 (m, 2H), 2.27 (s, 3H), 1.62 – 1.47 (m, 2H), 1.43 – 1.36 (m, 2H), 1.34 – 1.15 (m, 2H). 13 C NMR (100 MHz, DMSO- d 6) d 143.5, 135.5, 128.5, 125.8, 72.2, 60.8, 39.4, 32.6, 22.0, 20.7. The separation yield of (R)-5-bromo-1-(p-tolyl)-1-pentanol was 47%, with an optical purity of 90% ee. Optical rotation [α] D25 = + 5.30 (c = 1.00, CH2Cl2). NMR characterization data. 1 H NMR (400 MHz, DMSO- d 6) d 7.19 (d, J = 8.0 Hz, 2H), 7.10 (d, J = 7.9 Hz, 2H), 4.88 (s, 1H), 4.48 – 4.45 (m, 1H), 3.49 (t, J = 6.7 Hz, 2H), 2.27 (s, 3H), 1.82 – 1.74 (m, 2H), 1.65 – 1.50 (m, 2H), 1.49 – 1.28 (m, 2H). 13 C NMR (100 MHz, DMSO- d 6) d 143.3, 135.5, 128.5, 125.8, 71.9, 38.4, 35.2, 32.3, 24.1, 20.7. The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A halohydrin dehalogenase mutant, characterized in that: From Agrobacterium tumefaciens The wild-type halohydrin dehalogenase HheC with the amino acid sequence shown in SEQ ID NO:2, as the parent, was obtained through any of the following combined mutations: HheC-TB1: Leucine at position 142 is mutated to alanine, and tryptophan at position 249 is mutated to proline. The resulting amino acid sequence is shown in SEQ ID NO:

4. HheC-TB2: Leucine at position 142 is mutated to alanine, tryptophan at position 249 is mutated to proline, and proline at position 84 is mutated to glycine. The resulting amino acid sequence is shown in SEQ ID NO:

6. HheC-TB3: Leucine at position 142 is mutated to alanine, tryptophan at position 249 is mutated to proline, and threonine at position 134 is mutated to leucine. The resulting amino acid sequence is shown in SEQ ID NO:

8. HheC-TB4: ​​Leucine at position 142 is mutated to alanine, tryptophan at position 249 is mutated to proline, proline at position 84 is mutated to glycine, and threonine at position 134 is mutated to leucine. The resulting amino acid sequence is shown in SEQ ID NO:

10. HheC-TB5: Leucine at position 142 is mutated to alanine, tryptophan at position 249 is mutated to proline, proline at position 84 is mutated to glycine, and asparagine at position 176 is mutated to glutamine. The resulting amino acid sequence is shown in SEQ ID NO:

12. HheC-TB6: Leucine at position 142 is mutated to alanine, tryptophan at position 249 is mutated to proline, proline at position 84 is mutated to glycine, and phenylalanine at position 186 is mutated to tyrosine. The resulting amino acid sequence is shown in SEQ ID NO:

14. HheC-TB7: Leucine at position 142 is mutated to alanine, tryptophan at position 249 is mutated to proline, proline at position 84 is mutated to glycine, asparagine at position 176 is mutated to glutamine, and phenylalanine at position 186 is mutated to tyrosine. The resulting amino acid sequence is shown in SEQ ID NO:

16.

2. A genetically engineered bacterium with a halohydrin dehalogenase mutant, characterized in that: The genetically engineered bacteria uses Escherichia coli as a host to express any one of the halohydrin dehalogenase mutants in claim 1.

3. A class of biocatalysts, characterized in that: The biocatalyst is a whole cell, cell lysate, or enzyme solution of a microorganism containing a halohydrin dehalogenase mutant obtained by fermentation of the halohydrin dehalogenase mutant genetically engineered bacteria as described in claim 2.

4. The application of the biocatalyst according to claim 3, characterized in that: It is used to catalyze the enantioselective dehalogenation and hydroxylation of racemic ε-halools.

5. The application of the biocatalyst according to claim 4, characterized in that: The biocatalyst catalyzes the kinetic resolution and dehalogenation hydroxylation of racemic ε-halools, simultaneously yielding chiral ε-diol compounds and chiral ε-halool compounds.

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