FLUOROACETATE DEHALOGENASE MUTANT AND APPLICATION THEREOF

Abstract

Disclosed in the present invention is a fluoroacetate dehalogenase mutant, a sequence of the fluoroacetate dehalogenase mutant comprising a mutated sequence having an amino acid residue H at position 155 and/or an amino acid residue W at position 156, as shown in SEQ ID NO: 1; the fluoroacetate dehalogenase mutant has activity catalyzing bromination of a substrate, particularly a 2-bromobutyric acid substrate. Also provided in the present invention is an application of said fluoroacetate dehalogenase mutant in the preparation of (R)-2-bromobutyric acid and/or (R)-2-hydroxybutyric acid. When using the fluoroacetate dehalogenase mutant of the present invention to prepare (R)-2-bromobutyric acid, production costs are low and stereoselectivity is high, facilitating industrialized production.

Claims

1. A fluoroacetate dehalogenase mutant, wherein the fluoroacetate dehalogenase mutant comprises a sequence obtained by mutating amino acid residue H at position 155, and/or amino acid residue W at position 156 of SEQ ID NO. 1; the fluoroacetate dehalogenase mutant has activity catalyzing a brominated substrate, particularly a 2-bromobutyric acid substrate.

2. The fluoroacetate dehalogenase mutant of claim 1, wherein the fluoroacetate dehalogenase mutant comprises a sequence obtained by mutating amino acid residue H at position 155, and/or amino acid residue W at position 156 of SEQ ID NO. 1 to a natural amino acid residue; preferably, the fluoroacetate dehalogenase mutant further comprises a sequence obtained by mutating amino acid residue Y at position 219 of SEQ ID NO. 1 to a natural amino acid residue.

3. The fluoroacetate dehalogenase mutant of claim 2, wherein the amino acid residue H at position 155 is mutated to A, C, D, E, F, G, I, L, M, N, P, Q, S, T, V or W; and/or, the amino acid residue W at position 156 is mutated to A, C, D, F, G, I, L, M, P, R, S, T, V or Y; and/or, the amino acid residue Y at position 219 is mutated to a hydrophobic amino acid residue; preferably, the amino acid residue H at position 155 is mutated to I, N, V, F, L, Q, A, C, M, P, T or W; and/or, the amino acid residue W at position 156 is mutated to F, M, R, S, T, G, L, A, C, D, I or Y; and/or, the amino acid residue Y at position 219 is mutated to F, L or M; more preferably, the amino acid residue H at position 155 is mutated to I, N or V; and/or, the amino acid residue W at position 156 is mutated to F, M, R, S or T; and/or, the amino acid residue Y at position 219 is mutated to F, L or M; more preferably, the amino acid residue H at position 155 is mutated to V, or the amino acid residue W at position 156 is mutated to M, T, C, F, S, V, A or L.

4. (canceled)

5. The fluoroacetate dehalogenase mutant of claim 1, wherein an amino acid sequence of the fluoroacetate dehalogenase mutant is shown in SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129; preferably, a nucleotide sequence of the fluoroacetate dehalogenase mutant is shown in SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130.

6. An isolated nucleic acid, wherein the nucleic acid encodes the fluoroacetate dehalogenase mutant of claim 1.

7. A recombinant expression vector comprising the nucleic acid of claim 6.

8. A transformant comprising the nucleic acid of claim 6.

9. A dehalogenase combination catalyzing a halogenated substrate, wherein the dehalogenase combination catalyzing a halogenated substrate comprises two or more of fluoroacetate dehalogenases or mutants thereof, wherein the fluoroacetate dehalogenase mutants are the fluoroacetate dehalogenase mutant of claim 1; preferably, the halogenated substrate is a brominated substrate; more preferably a 2-bromobutyric acid, methyl 2-bromobutyrate or ethyl 2-bromobutyrate substrate.

10. A catalyst combination, wherein the catalyst combination comprises (a) the fluoroacetate dehalogenase mutant of claim 1, and (b) a lipase, wherein the lipase is preferably lipase B from Candida antartica.

11. A reaction system for a catalyst, wherein the reaction system comprises a substrate and a catalyst, wherein the substrate is 2-bromobutyric acid, methyl 2-bromobutyrate or ethyl 2-bromobutyrate, and the substrate is preferably one or more of racemate, R-form or S-form enriched enantiomer; wherein, when the substrate is 2-bromobutyric acid, the catalyst comprises the fluoroacetate dehalogenase mutant of claim 1; preferably, the 2-bromobutyric acid has a concentration of 0.02 g/ml-0.2 g/ml, preferably 0.1-0.15 g/ml, more preferably 0.1 g/ml; and/or, the fluoroacetate dehalogenase or mutant thereof has a concentration of 2-20 U/ml, preferably 10 U/ml. more preferably, the reaction system further comprises a reaction solvent, wherein the reaction solvent is preferably water; and/or, pH of the reaction system is 6-8, preferably 7.

12. (canceled)

13. A method for preparing (R)-2-bromobutyric acid and/or (R)-2-hydroxybutyric acid by use of the fluoroacetate dehalogenase mutant of claim 1.

14. A preparation method of (R)-2-bromobutyric acid or (R)-2-hydroxybutyric acid, wherein the preparation method comprises following steps: a catalytic reaction is performed in the presence of the reaction system of claim 11 to obtain (R)-2-bromobutyric acid or (R)-2-hydroxybutyric acid, respectively; wherein the catalytic reaction has a reaction system with a temperature of 20-30? C., preferably 30? C.; and/or, the catalytic reaction has a reaction time of 5-10 hours, preferably 8 hours; wherein the catalytic reaction comprises a dehalogenation reaction, or a dehalogenation reaction and an ester hydrolysis reaction.

15. (canceled)

16. A catalyst combination, wherein the catalyst combination comprises (a) the dehalogenase combination of claim 9 catalyzing a halogenated substrate, and (b) a lipase, wherein the lipase is preferably lipase B from Candida antartica.

17. A reaction system for a catalyst, wherein the reaction system comprises a substrate and a catalyst, wherein the substrate is 2-bromobutyric acid, methyl 2-bromobutyrate or ethyl 2-bromobutyrate, and the substrate is preferably one or more of racemate, R-form or S-form enriched enantiomer; wherein, when the substrate is 2-bromobutyric acid, the catalyst comprises the dehalogenase combination of claim 9 catalyzing a halogenated substrate; preferably, the 2-bromobutyric acid has a concentration of 0.02 g/ml-0.2 g/ml, preferably 0.1-0.15 g/ml, more preferably 0.1 g/ml; and/or, the fluoroacetate dehalogenase or mutant thereof has a concentration of 2-20 U/ml, preferably 10 U/ml; more preferably, wherein the reaction system further comprises a reaction solvent, wherein the reaction solvent is preferably water; and/or, pH of the reaction system is 6-8, preferably 7.

18. A reaction system for a catalyst, wherein the reaction system comprises a substrate and a catalyst, wherein the substrate is 2-bromobutyric acid, methyl 2-bromobutyrate or ethyl 2-bromobutyrate, and the substrate is preferably one or more of racemate, R-form or S-form enriched enantiomer; wherein, when the substrate is methyl 2-bromobutyrate or ethyl 2-bromobutyrate, the catalyst comprises the catalyst combination of claim 10; preferably, the methyl 2-bromobutyrate or ethyl 2-bromobutyrate has concentration of 0.02 g/ml-0.2 g/ml, preferably 0.1-0.15 g/ml, more preferably 0.15 g/ml; and/or, the fluoroacetate dehalogenase or mutant thereof has a concentration of 2-20 U/ml, preferably 10 U/ml; and/or, the lipase has a concentration of 1-20 g/L, preferably 10 g/L; more preferably, the reaction system further comprises methanol, wherein a volume ratio of the methanol to the reaction system is preferably 1-10%, more preferably 10%; more preferably, wherein the reaction system further comprises a reaction solvent, wherein the reaction solvent is preferably water; and/or, pH of the reaction system is 6-8, preferably 7.

19. A reaction system for a catalyst, wherein the reaction system comprises a substrate and a catalyst, wherein the substrate is 2-bromobutyric acid, methyl 2-bromobutyrate or ethyl 2-bromobutyrate, and the substrate is preferably one or more of racemate, R-form or S-form enriched enantiomer; wherein, when the substrate is methyl 2-bromobutyrate or ethyl 2-bromobutyrate, the catalyst comprises the catalyst combination of claim 16; preferably, the methyl 2-bromobutyrate or ethyl 2-bromobutyrate has concentration of 0.02 g/ml-0.2 g/ml, preferably 0.1-0.15 g/ml, more preferably 0.15 g/ml; and/or, the fluoroacetate dehalogenase or mutant thereof has a concentration of 2-20 U/ml, preferably 10 U/ml; and/or, the lipase has a concentration of 1-20 g/L, preferably 10 g/L; more preferably, the reaction system further comprises methanol, wherein a volume ratio of the methanol to the reaction system is preferably 1-10%, more preferably 10%; more preferably, wherein the reaction system further comprises a reaction solvent, wherein the reaction solvent is preferably water; and/or, pH of the reaction system is 6-8, preferably 7.

20. A preparation method of (R)-2-bromobutyric acid or (R)-2-hydroxybutyric acid, wherein the preparation method comprises following steps: a catalytic reaction is performed in the presence of the reaction system of claim 17 to obtain (R)-2-bromobutyric acid or (R)-2-hydroxybutyric acid, respectively; wherein the catalytic reaction has a reaction system with a temperature of 20-30? C., preferably 30? C.; and/or, the catalytic reaction has a reaction time of 5-10 hours, preferably 8 hours; wherein the catalytic reaction comprises a dehalogenation reaction, or a dehalogenation reaction and an ester hydrolysis reaction.

21. A preparation method of (R)-2-bromobutyric acid or (R)-2-hydroxybutyric acid, wherein the preparation method comprises following steps: a catalytic reaction is performed in the presence of the reaction system of claim 18 to obtain (R)-2-bromobutyric acid or (R)-2-hydroxybutyric acid, respectively; wherein the catalytic reaction has a reaction system with a temperature of 20-30? C., preferably 30? C.; and/or, the catalytic reaction has a reaction time of 5-10 hours, preferably 8 hours; wherein the catalytic reaction comprises a dehalogenation reaction, or a dehalogenation reaction and an ester hydrolysis reaction.

22. A preparation method of (R)-2-bromobutyric acid or (R)-2-hydroxybutyric acid, wherein the preparation method comprises following steps: a catalytic reaction is performed in the presence of the reaction system of claim 19 to obtain (R)-2-bromobutyric acid or (R)-2-hydroxybutyric acid, respectively; wherein the catalytic reaction has a reaction system with a temperature of 20-30? C., preferably 30? C.; and/or, the catalytic reaction has a reaction time of 5-10 hours, preferably 8 hours; wherein the catalytic reaction comprises a dehalogenation reaction, or a dehalogenation reaction and an ester hydrolysis reaction.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0047] FIG. 1 is the results of chiral HPLC analysis of the reaction feedstock 2-bromobutyric acid in Example 3.

[0048] FIG. 2 is the results of chiral HPLC analysis of (R)-2-bromobutyric acid after the reaction involving fluoroacetate dehydrogenase mutant 3 in Example 3.

[0049] FIG. 3 is the results of chiral HPLC analysis of (R)-2-bromobutyric acid standard.

[0050] FIG. 4 is the HPLC analysis spectrum of the reaction feedstock 2-bromobutyric acid in Example 3.

[0051] FIG. 5 is the HPLC analysis spectrum of the racemic 2-hydroxybutyric acid standard.

[0052] FIG. 6 is the HPLC analysis spectrum of the reaction solution after the reaction involving fluoroacetate dehydrogenase mutant 3 in Example 3.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0053] The following examples further illustrate the present disclosure, but the present disclosure is not limited thereto. Experimental methods for which specific conditions are not indicated in the following examples are selected according to conventional methods and conditions, or according to the trade description.

[0054] The experimental methods in the present invention are conventional unless otherwise specified, and the gene cloning operations can be specified in Molecular Cloning: A Laboratory Manual, edited by J Sambrook et al.

[0055] The 2-bromobutyric acid in the present disclosure can be one or more of 2-bromobutyric acid racemate, R-form-enriched 2-bromobutyric acid and S-form-enriched 2-bromobutyric acid unless otherwise specified, wherein the R-form-enriched means that the R-form coexists with the S-form and the molar ratio of R-form to S-form is more than 1:1, and the S-form-enriched means that the R-form coexists with the S-form and the molar ratio of S-form to R-form is more than 1:1.

[0056] The amino acid abbreviation symbols in the present disclosure are conventional in the art unless otherwise specified, and the amino acids corresponding to the specific abbreviation symbols are shown in Table 1.

TABLE-US-00001 TABLE 1 Amino Three Single Three Single acid letters letter Amino letters letter name symbol symbol acid name symbol symbol Alanine Ala A Leucine Leu L Arginine Arg R Lysine Lys K Asparagine Asn N Methionine Met M Aspartic acid Asp D Phenylalanine Phe F Cysteine Cys C Proline Pro P Glutanine Gln Q Serine Ser S Glutamic acid Glu E Threonine Thr T Glycine Gly G Tryptophan Trp W Histidine His H Tyrosine Tyr Y Isoleucine Ile I Valine Val V

[0057] The codons corresponding to the amino acids are also conventional in the art, and the corresponding relationships of specific amino acids with the codons are shown in Table 2.

TABLE-US-00002 TABLE 2 The first The second nucleotide The third nucleotide T C A G nucleotide T Phenylalanine (F) Serine (S) Tyrosine (Y) Cysteine (C) T Phenylalanine (F) Serine (S) Tyrosine (Y) Cysteine (C) C Leucine (L) Serine (S) Stop codon Stop codon A Leucine (L) Serine (S) Stop codon Tryptophan (W) G C Leucine (L) Proline (P) Histidine (H) Arginine (R) T Leucine (L) Proline (P) Histidine (H) Arginine (R) C Leucine (L) Proline (P) Glutamine (Q) Arginine (R) A Leucine (L) Proline (P) Glutamine (Q) Arginine (R) G A Isoleucine (I) Threonine (T) Asparagine (N) Serine (S) T Isoleucine (I) Threonine (T) Asparagine (N) Serine (S) C Isoleucine (I) Threonine (T) Lysine (K) Arginine (R) A Methionine (M) Threonine (T) Lysine (K) Arginine (R) G G Valine (V) Alanine (A) Aspartic acid (D) Glycine (G) T Valine (V) Alanine (A) Aspartic acid (D) Glycine (G) C Valine (V) Alanine ( A) Glutamic acid (E) Glycine (G) A Valine (V) Alanine (A) Glutamic acid (E) Glycine (G) G

[0058] pET28a and bugbuster protein extraction reagent were purchased from Novagen; enzyme DpnI was purchased from Invitrogen Shanghai Trading Co., Ltd; E. coli BL21(DE3) competent cell was purchased from Beijing Dingguo Changsheng Biotech Co., Ltd; 2-bromobutyric acid was purchased from Yancheng City Shengda Chemical Co., Ltd.

[0059] The method of chiral HPLC (High Performance Liquid Chromatography) analysis for the substrate 2-bromobutyric acid and the product 2-bromobutyric acid is as follows:

[0060] Chromatographic condition: Daicel Chiralpak IG, 4.6 mm*250 mm, 5 ?m; mobile phase: n-hexane/isopropanol/TFA=99:1:0.1; wavelength of detection: 230 nm; flow rate: 0.7 mL/min; temperature of column: 25? C.

[0061] The method of HPLC analysis for the substrate 2-bromobutyric acid and the product is as follows:

[0062] Chromatographic condition: Inertsil ODS-3, 4.6 mm*150 mm, 5 ?m; mobile phase A: 10 mmol sodium dihydrogen phosphate aqueous solution (pH=3.0), mobile phase B: acetonitrile; gradient elution; wavelength of detection: 205nm; flow rate: 0.7 mL/min; temperature of column: 35? C.

Conversion rate=(Reagent?Remaining Reagent)/Reagent?100% (Reagent: 2-bromobutyric acid).

Example 1 Construction of the Fluoroacetate Dehalogenase Mutant Library

[0063] The primer sequences designed for the construction of the mutant library with mutations at positions 155, 156, and 219 in the sequence of the fluoroacetate dehalogenase FACD-RPA1163 (the source is Rhodopseudomonas palustris) (i.e., SEQ ID NO: 1 in the sequence listing) are shown in Table 3:

TABLE-US-00003 TABLE3 Mutationpositions SEQID No. andprimernames primersequence NO: 1 H155W156-Forward GCACTGAAAATCTATNNKNNKAGCTTTCTGGCACAG 3 primer C 2 H155W156-Reverse GCTGTGCCAGAAAGCTMNNMNNATAGATTTTCAGT 4 primer GC 3 Y219-Forwardprimer GTGATGTGTGAAGATNNKCGTGCGGGTGCGTATG 5 4 Y219-Reverseprimer CATACGCACCCGCACGMNNATCTTCACACATCAC 6

[0064] Wherein N represents any one of nucleotides A, G, C and T, M represents A or C, and K represents G or T; which are selected according to the coding nucleotide of the amino acid to be mutated at the position. For example, NNK in A166-Forward primer can represent AAG (Lysine), AAT (Aspartic acid), AGG (Arginine) or AGT (Serine), etc. The nucleotides corresponding to the specific amino acids can refer to Table 2.

[0065] The plasmid template pET28a-FACD-RPA1163 was constructed according to the method disclosed in J. Am. Chem. Soc., 2017,139(32), 11241-11247, and the target bands were amplified by PCR with pET28a-FACD-RPA1163 as the template. The amplification reaction system was:

[0066] 50 ?L PCR amplification system:

TABLE-US-00004 Reagents Dosage (?L) 2 ? PCR buffer ( with high fidelity enzyme) 25 Primer F 1 Primer R 1 Template 1 Deionized water 22

[0067] The amplification procedure was as follows:

TABLE-US-00005 95? C. 5 min 95? C. 30 s 50? C. 30 s {close oversize brace} 30 cycles 72? C. 5 min 72? C. 10 min 12? C. Hold

[0068] The PCR products were digested by DpnI at 37? C. for 2 hr, followed by being transformed into E. coli BL21 (DE3) competent cells after the reaction. The cells were coated on LB medium containing 100 ?g/mL Ampicillin, and incubated overnight at 37? C. to obtain transformants containing the mutant library.

Example 2 Screening the Mutant Library in High-Throughput

[0069] The screening was performed according to the following experimental steps:

[0070] The transformants obtained in Example 1 were inoculated in the 96-well plate and induced by IPTG overnight at 30? C. After that, the bacteria were collected, lysed by adding bugbuster protein extraction reagent and centrifuged to obtain the total enzyme solution.

[0071] 250 mg of substrate racemic 2-bromobutyric acid was taken to be dissolved in 45 mL of 1 mM Tris solution, and the pH was adjusted to 8.0-8.5 with dilute NaOH on ice, and the volume was set to 50 ml to prepare the substrate solution with the substrate 2-bromobutyric acid in a concentration of 30 mM. 100 ?l of the substrate solution (final concentration of 25 mM) and 20 ?L of the above total enzyme solution were added in each reaction. After the solution was reacted for a period of time, 20 ?l of the reaction solution was added into the 96-well ELISA plate, 30 ?l of saturated Hg(SCN).sub.2 ethanol solution and 150 ?L of 60 g/L FeNH.sub.4(SO.sub.4).sub.2.Math.12H.sub.2O (containing 1% nitric acid, w/v) aqueous solution were added sequentially. After 10 min at room temperature, the absorbance was detected at 500 nm by a Microplate Reader.

[0072] The wild type enzyme was used as the reference system (the activity of the wild type enzyme could not be detected, which shows that the wild type fluoroacetate dehalogenase FAcD-RPA1163 has no catalytic effect on 2-bromobutyric acid), and positive clones were selected, sequenced and the enzyme activity of which was detected. The sequences of the positive clones were determined by sequencing at Sangon Biotech (Shanghai) Co., Ltd, 698 Xiangmin Road, Songjiang District, Shanghai.

[0073] The positive clones were selected and cultured as follows:

[0074] 10% of the above positive clones were inoculated in 150 mL TB medium and incubated at 37? C. until the OD.sub.600 value reached about 0.8. Then IPTG was added until the final concentration thereof is 0.1 mM and cooled to 30? C. for induction overnight. After incubation, the culture medium was centrifuged at 4000 rpm for 30 min, the supernatant was discarded, and the bacteria were collected and stored in an ultra-low temperature refrigerator at ?80? C. for later use.

[0075] The bacteria collected at the end of the culture were washed twice with 20 mM pH 8.0 phosphate buffer, after which the bacteria were resuspended in phosphate buffer with pH 8.0, homogenized and crushed under low temperature and high pressure, and the crushed solution was centrifuged to remove the precipitate, and the obtained supernatant was the mutant crude enzyme solution containing the recombinant dehalogenase.

[0076] The above methods were used for the preparation of the crude enzyme solution of the dehalogenase mutant in the following examples.

[0077] The method of the enzyme activity assay is: 2.5 g of bacteria were resuspended with 40 mL of 20 mM pH 8.0 phosphate buffer, homogenized and crushed under low temperature and high pressure, the precipitate was discarded by centrifugation, and the supernatant was left to obtain the crude enzyme solution. 900 ?L of the substrate solution (30 mM) was added at 30? C., and 100 ?L of the crude enzyme solution to be tested was added to obtain 1 mL of the reaction solution. 150 ?l of saturated Hg(SCN).sub.2 ethanol solution and 750 ?L of 60 g/L FeNH.sub.4(SO.sub.4).sub.2.Math.12H.sub.2O (containing 1% nitric acid, w/v) aqueous solution were added into 100 ?L of the reaction solution sequentially. After 10 min at room temperature, the absorbance value was detected at 500 nm with an UV spectrophotometer. The enzyme activity was calculated based on the production rate of bromine ion. The results are shown in Table 4.

[0078] The unit enzyme activity is defined as: the amount of enzyme required to produce 1 ?mol of bromine ion per minute under specific reaction conditions (30? C.)

TABLE-US-00006 TABLE 4 fluoroacetate Amino Nucleotide dehalogenase Enzyme acid SEQ SEQ mutant No. Mutation position activity ID NO: ID NO: 1 H155I, W156S, Y219F ***** 7 8 2 H155V, W156M, Y219L ***** 9 10 3 H155V, W156R, Y219M ***** 11 12 4 H155I, W156S ***** 13 14 5 H155N, W156F ***** 15 16 6 H155V, W156T ***** 17 18 7 W156M ***** 19 20 8 H155V, W156M ***** 21 22 9 H155V, W156R ***** 23 24 10 H155Q, W156F **** 25 26 11 H155L, W156S **** 27 28 12 H155F, W156G **** 29 30 13 H155F, W156L **** 31 32 14 H155Q, W156L *** 33 34 15 H155V, W156D *** 35 36 16 H155M, W156A *** 37 38 17 H155L, W156I *** 39 40 18 H155F, W156M *** 41 42 19 H155F, W156R *** 43 44 20 H155A, W156F *** 45 46 21 H155P, W156Y *** 47 48 22 W156T *** 49 50 23 H155V *** 51 52 24 W156C *** 53 54 25 H155T, W156F *** 55 56 26 H155W, W156I *** 57 58 27 H155L, W156L *** 59 60 28 H155P, W156T *** 61 62 29 H155C, W156M *** 63 64 30 H155I, W156A ** 65 66 31 H155L, W156A ** 67 68 32 W156F ** 69 70 33 H155T, W156L ** 71 72 34 H155V, W156Y ** 73 74 35 H155I, W156P ** 75 76 36 H155L, W156F ** 77 78 37 H155V, W156G ** 79 80 38 H155F, W156T ** 81 82 39 H155A, W156M ** 83 84 40 H155V, W156A ** 85 86 41 H155L, W156P ** 87 88 42 H155D, W156L ** 89 90 43 H155N, W156I ** 91 92 44 W156L ** 93 94 45 H155V, W156I ** 95 96 46 H155L, W156T ** 97 98 47 H155Q, W156I ** 99 100 48 H155I, W156C ** 101 102 49 H155S, W156M * 103 104 50 W156S * 105 106 51 W156V * 107 108 52 H155A, W156S * 109 110 53 H155M, W156F * 111 112 54 H155E, W156T * 113 114 55 W156A * 115 116 56 H155E, W156M * 117 118 57 H155L, W156G * 119 120 58 H155L, W156F * 121 122 59 H155E, W156P * 123 124 60 H155L, W156P * 125 126 61 H155G, W156F * 127 128 62 H155D, W156R * 129 130 63 WT 1 2

[0079] Wherein no enzyme activity could be detected in wild-type WT, *represents enzyme activity is below 0.1 U/ml; **represents enzyme activity is between 0.1-1 U/ml: *** represents enzyme activity is between 1-5 U/ml; ****represents enzyme activity is between 5-10 U/ml; *****represents enzyme activity is between 10 U/mL-20 U/mL.

Example 3 Preparation of (R)-2-Bromobutyric Acid Catalyzed by the Fluoroacetate Dehalogenase Mutant

[0080] 50 mL of tap water and 10 g of the substrate 2-bromobutyric acid were added into the reaction flask, and stirred to dissolve. The pH was adjusted to 7.0 with 30% NaOH solution. 20 mL of the mutant crude enzyme solution prepared according to the method in Example 2 (i.e., 20 mL of 100 mM pH 7.0 disodium hydrogen phosphate-sodium dihydrogen phosphate buffer was added into 4 g of the bacteria described in Example 2, stirred well, then homogenized and crushed under high pressure to obtain the mutant crude enzyme solution. Take fluoroacetate dehalogenase mutant 3 as an example, the concentration of which was 10 U/ml) was added, and the volume was set to 100 mL by tap water. The pH was controlled at about 7.0 with 2 mol/L sodium carbonate solution during the reaction. After reacted in water bath at 30? C. for 8 hours, the conversion rate of the substrate 2-bromobutyric acid was detected by sampling. The reaction solution has a pH of 1-2 adjusted with 40% aqueous sulfuric acid, filtered with diatomaceous earth, and the filtrate was extracted three times with an equal volume of toluene. The toluene was combined, and concentrated at 50? C. to obtain (R)-2-bromobutyric acid. The ee value of (R)-2-bromobutyric acid was detected by HPLC. The results of the conversion rate and the ee value are shown in Table 5. The aqueous phase has a pH of 0-1 adjusted with 40% aqueous sulfuric acid, then extracted three times with an equal volume of methyl tert-butyl ether. The methyl tert-butyl ether was combined, and concentrated at 50? C. to obtain 2-hydroxybutyric acid. The optical rotation of 2-hydroxybutyric acid was [a]25D=+4.2? (C=3.0, H.sub.2O). The product could be known as (R)-2-hydroxybutyric acid by referring to the content of Advanced Synthesis and Catalysis, 2016, vol. 358, #18, p. 2923-2928.

[0081] The results of the chiral HPLC analysis of 2-bromobutyric acid used as the reaction feedstock are shown in FIG. 1, wherein (R)-2-bromobutyric acid has a retention time of 17.272 min, and (S)-2-bromobutyric acid has a retention time of 15.972 min. The results of the chiral HPLC analysis of (R)-2-bromobutyric acid and (S)-2-bromobutyric acid in the post-reaction products are shown in FIG. 2 (the figures are all illustrated with fluoroacetate dehalogenase mutant 3 as an example), wherein (R)-2-bromobutyric acid has a retention time of 17.135 min and (S)-2-bromobutyric acid was almost undetectable. FIG. 3 is the chiral HPLC spectrum of (R)-2-bromobutyric acid standard, which was prepared in our laboratory according to CN101048402, and its HNMR identification data is .sup.1HNMR(400 MHZ, CDCl.sub.3) ? 4.209-4.173; (t, 1H), ?2.130-2.004; (m, 2H), ?1.081-1.044; (t, 3H). The component of the product prepared in this example has basically the same as the peak time of (R)-2-bromobutyric acid standard, indicating that almost all of the (S)-2-bromobutyric acid was reacted in this example, i.e., all of the (S)-2-bromobutyric acid was converted into (R)-2-hydroxybutyric acid.

[0082] The results of HPLC chromatogram analysis of 2-bromobutyric acid used in the reaction feedstock are shown in FIG. 4, wherein 6.652 min was the peak position of 2-bromobutyric acid. The HPLC chromatogram analysis results of the racemic 2-hydroxybutyric acid standard (purchased from Shanghai Titan Chemical Co., Ltd) are shown in FIG. 5, wherein the retention time of 2-hydroxybutyric acid standard was 1.930 min. The results of HPLC chromatogram analysis of the reaction solution after the reaction are shown in FIG. 6. It can be seen that the peak times of 2-bromobutyric acid and (R)-2-hydroxybutyric acid in the products after the reaction were basically the same as those of the respective standards.

TABLE-US-00007 TABLE 5 Fluoroacetate ee value ((R)-2- dehalogenase Conversion bromobutyric mutant No. rate acid) 1 52% 99% 2 54% 60% 3 60% 100% 4 56% 100% 5 58% 100% 6 56% 99.12% 7 56% 98.32% 8 56% 99.72% 9 55% 97.1% 10 49% 93.67% 11 48% 93.56 12 48% 90.84 13 48% 90.63 14 47% 89.92 15 46% 88.87 16 46% 88.26 17 46% 88.22 18 46% 86.17 19 46% 85.18 20 46% 84.75 21 46% 84 22 46% 83.66 23 45% 81.91 24 45% 81.76 25 45% 81.64 26 45% 81.35 27 45% 80.93 28 45% 80.81 29 45% 80.53 30 44% 79.58 31 44% 78.61 32 44% 78.5 33 44% 78.44 34 44% 77.41 35 43% 76.89 36 43% 76.82 37 43% 76.77 38 43% 76.73 39 43% 76.69 40 43% 76.36 41 43% 75.39 42 43% 75.13 43 43% 74.98 44 42% 73.86 45 42% 73.79 46 42% 73.38 47 42% 72.32 48 42% 71.27 49 41% 69.72 50 41% 68.78 51 40% 67.39 52 40% 67.19 53 40% 66.94 54 40% 66.26 55 40% 66.23 56 40% 65.44 57 39% 65.12 58 39% 64.68 59 39% 63.54 60 39% 63.53 61 38% 62.25 62 37% 59.22

Example 4 Preparation of (R)-2-bromobutyric Acid Catalyzed by the Fluoroacetate Dehalogenase Mutant and Lipase by One-Pot Process

[0083] 40 mL of tap water, 10mL of methanol and 15 g of the substrate methyl 2-bromobutyrate were added into the reaction flask, stirred to dissolve, and the pH of which was controlled at 7.0. 20 mL of the mutant crude enzyme solution prepared according to the method in Example 2 (i.e., 20 mL of 100 mM pH 7.0 disodium hydrogen phosphate-sodium dihydrogen phosphate buffer was added into 4 g of the bacteria described in Example 2, stirred well, then homogenized and crushed under high pressure to obtain the mutant crude enzyme solution, take fluoroacetate dehalogenase mutant 3 as an example, the concentration of which was 10 U/ml) next 1 g of lipase Novozyme? 435 (the lipase B from Candida antartica (CALB), which was immobilized in macroporous polymethylmethacrylate resin (Lewatit), and its concentration in this reaction could be 1-20 g/L) were added, and finally the volume of the reaction system was set as 100 mL by tap water. The pH was controlled at about 7.0 with 2 mol/L sodium carbonate solution during the reaction. After reacted in water bath at 30? C. for 8 hours, the conversion rate of the reaction was detected by sampling.

[0084] The reaction solution has a pH of 1-2 adjusted with 40% aqueous sulfuric acid, filtered with diatomaceous earth, and the filtrate was extracted three times with an equal volume of toluene. The toluene was combined, and concentrated at 50? C. to obtain 3.5 g of (R)-2-bromobutyric acid. The ee value of (R)-2-bromobutyric acid was detected by HPLC and the ee value was 98%. The aqueous phase has a pH of 0-1 adjusted with 40% aqueous sulfuric acid, then extracted three times with an equal volume of methyl tert-butyl ether. The methyl tert-butyl ether was combined and concentrated at 50? C. to obtain 2-hydroxybutyric acid. The optical rotation of 2-hydroxybutyric acid was determined as [a]25D=+4.1? (C=3.0, H.sub.2O). The product could be known as (R)-2-hydroxybutyric acid by referring to the content of Advanced Synthesis and Catalysis, 2016, vol. 358, #18, p. 2923-2928.

[0085] Although the above describes specific embodiments of the present disclosure, it should be understood by a person skilled in the art that these are merely illustrative examples and that a variety of changes or modifications can be made to these embodiments without departing from the principles and substance of the present invention. Therefore, the scope of protection of the present disclosure is limited by the appended claims.