阅读说明：本技术 一种氟乙酸脱卤酶突变体及其应用 (Fluoroacetic acid dehalogenase mutant and application thereof ) 是由 瞿旭东 程占冰 丁少南 徐艳冰 黄瑶 于 2018-07-05 设计创作，主要内容包括：本发明公开了一种氟乙酸脱卤酶突变体,所述氟乙酸脱卤酶突变体的序列包括将SEQ ID NO.1所示的第155位氨基酸残基H、和/或第156位氨基酸残基W进行突变后的序列；所述氟乙酸脱卤酶突变体具有催化溴化底物、尤其是2-溴丁酸底物的活性。本发明还提供了一种所述氟乙酸脱卤酶突变体在制备(R)-2-溴丁酸和/或(R)-2-羟基丁酸中的应用。利用本发明的氟乙酸脱卤酶突变体制备(R)-2-溴丁酸时,生产成本低,立体选择性高,利于工业化生产。(The invention discloses a mutant of a fluoroacetate dehalogenase, wherein the sequence of the mutant of the fluoroacetate dehalogenase comprises a sequence obtained by mutating 155 th amino acid residue H and/or 156 th amino acid residue W shown in SEQ ID NO. 1; the mutant fluoroacetate dehalogenase has activity in catalyzing a brominated substrate, particularly a 2-bromobutyric acid substrate. The invention also provides application of the mutant of the fluoroacetate dehalogenase in preparation of (R) -2-bromobutyric acid and/or (R) -2-hydroxybutyric acid. When the (R) -2-bromobutyric acid is prepared by using the mutant of the fluoroacetate dehalogenase, the production cost is low, the stereoselectivity is high, and the industrial production is facilitated.)

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

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

3. the mutant fluoroacetate dehalogenase of claim 1 or 2 wherein amino acid residue 155, H, is mutated to A, C, D, E, F, G, I, L, M, N, P, Q, S, T, V or W, and/or amino acid residue 156, W, is mutated to A, C, D, F, G, I, L, M, P, R, S, T, V or Y, and/or amino acid residue 219, Y, is mutated to a hydrophobic amino acid residue;

Preferably, the 155 th amino acid residue H is mutated into I, N, V, F, L, Q, A, C, M, P, T or W, and/or the 156 th amino acid residue W is mutated into F, M, R, S, T, G, L, A, C, D, I or Y, and/or the 219 th amino acid residue Y is mutated into F, L or M;

More preferably, the 155 th amino acid residue H is mutated into I, N or V, and/or the 156 th amino acid residue W is mutated into F, M, R, S or T, and/or the 219 th amino acid residue Y is mutated into F, L or M.

4. The mutant fluoroacetate dehalogenase of any one of claims 1 to 3, wherein the 155 th amino acid residue H is mutated to V,

Or amino acid residue W at position 156 is mutated to M, T, C, F, S, V, A or L.

5. The mutant of the fluoroacetate dehalogenase of any one of claims 1 to 4, wherein the amino acid sequence of the mutant of the fluoroacetate dehalogenase is as set forth 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.69, SEQ ID NO.67, SEQ ID NO.73, SEQ ID NO.75, SEQ ID NO.71, SEQ ID NO.73, SEQ ID NO.75, SEQ ID NO.7, SEQ ID, 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, the nucleotide sequence of the mutant fluoroacetate dehalogenase is as 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.80, SEQ ID NO.82, SEQ ID NO.80, 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 encoding the mutant fluoroacetate dehalogenase of any one of claims 1 to 5.

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

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

9. Use of the mutant of fluoroacetate dehalogenase of any one of claims 1 to 5 in the preparation of (R) -2-bromobutyric acid and/or (R) -2-hydroxybutyric acid.

10. A preparation method of (R) -2-bromobutyric acid is characterized by comprising the following steps: carrying out catalytic reaction on the 2-bromobutyric acid in the presence of a reaction solvent and a fluoroacetate dehalogenase mutant to obtain (R) -2-bromobutyric acid; wherein the mutant of the fluoroacetate dehalogenase is the mutant of the fluoroacetate dehalogenase of any one of claims 1 to 5; the 2-bromobutyric acid is one or more of 2-bromobutyric acid racemate, R-type enriched 2-bromobutyric acid and S-type enriched 2-bromobutyric acid; preferably:

The concentration of the mutant of the fluoroacetate dehalogenase is 2-20U/ml, preferably 10U/ml;

And/or the concentration of the 2-bromobutyric acid is 0.02g/ml to 0.2g/ml, preferably 0.1 g/ml;

And/or, the reaction solvent is water;

And/or the pH value of the reaction system for catalyzing the reaction is 6-8, preferably 7;

and/or the temperature of the reaction system of the catalytic reaction is 20-30 ℃, preferably 30 ℃;

And/or the reaction time of the catalytic reaction is 5 to 10 hours, preferably 8 hours;

11. A method for preparing (R) -2-hydroxybutyric acid, comprising the steps of: carrying out dehalogenation reaction on 2-bromobutyric acid in the presence of a reaction solvent and a fluoroacetic acid dehalogenase mutant to obtain (R) -2-hydroxybutyric acid; wherein the mutant of the fluoroacetate dehalogenase is the mutant of the fluoroacetate dehalogenase of any one of claims 1 to 5; the 2-bromobutyric acid is one or more of 2-bromobutyric acid racemate, R-type enriched 2-bromobutyric acid and S-type enriched 2-bromobutyric acid; preferably:

The concentration of the mutant of the fluoroacetate dehalogenase is 2-20U/ml, preferably 10U/ml;

and/or the concentration of the (S) -2-bromobutyric acid is 0.02g/ml to 0.2g/ml, preferably 0.1 g/ml;

And/or, the reaction solvent is water;

and/or the pH value of the reaction system of the dehalogenation reaction is 6-8, preferably 7;

And/or the temperature of the reaction system of the dehalogenation reaction is 20-30 ℃, preferably 30 ℃;

And/or the reaction time of the dehalogenation reaction is 5 to 10 hours, preferably 8 hours;

Technical Field

the invention belongs to the technical field of biology, and particularly relates to a fluoroacetate dehalogenase mutant and application thereof.

Background

2-oxo-1-pyrrolidine derivatives such as levetiracetam, brivaracetam and seletracetam are a novel antiepileptic drug developed by UCB company of Belgium. EP1806339 discloses a process for the preparation thereof by substitution of (R) -2-bromobutyric acid with the corresponding 2-oxo-1-pyrrolidine compound followed by reaction with triethylamine to give the corresponding product. Wherein (R) -2-bromobutyric acid is an important raw material and plays a decisive role in the production of antiepileptic 2-oxo-1-pyrrolidine derivatives, so that the search for a preparation method of (R) -2-bromobutyric acid with low cost and high optical purity is very important.

At present, the preparation method of (R) -2-bromobutyric acid includes a method for resolving racemic 2-bromobutyric acid, such as Journal of Biological Chemistry,1927(75), 337-ion 365, and brucine is adopted as a chiral resolving agent, and the method needs multiple recrystallization steps and expensive chiral resolving agent, and is not beneficial to industrial mass production. Or using chiral prosthetic group to resolve racemic 2-bromobutyric acid, such as Tetrhedron Asymmetry,16(2005), 3739-Ascenolide 3745 using (S) -N-phenylpantolactone as chiral prosthetic group, the (S) -N-phenylpantolactone reacts with 2-bromobutyric acid to generate ester compound, and then the ester compound is hydrolyzed into (R) -2-bromobutyric acid. The method has the advantages that the esterification yield is only 67 percent, the ee value is not ideal, and the industrial production cannot be realized by adopting silica gel column chromatography for separation.

In addition, chemical synthesis method is available, such as CN101048402, which uses (R) -2-aminobutyric acid as raw material, and reacts with nitrous acid and potassium bromide to obtain (R) -2-bromobutyric acid. However, the method has low yield, and the raw material is optical pure raw material, so the cost is high.

Dehalogenase is a catalytically active hydrolase that catalyzes the cleavage of carbon-halogen bonds in organohalogen compounds to release halogen atoms from the compound. There have been reports on the use of dehalogenases for the resolution of halogenated compounds, such as j.am. chem. soc.,2017, shuuguang Yuan and Manfred t.reetz that fluoroacetate dehalogenase (FAcD) RPA1163 can obtain (R) -2-hydroxy-2-phenylacetic acid or (R) -2-hydroxy-3-phenylpropionic acid by defluorinating the S-form of the racemic substrate 2-fluoro-2-phenylacetic acid or 2-fluoro-3-phenylpropionic acid, while leaving unreacted (R) -2-fluoro-2-phenylacetic acid or (R) -2-fluoro-3-phenylpropionic acid, thereby achieving kinetic resolution. Although the enzyme is dehalogenase, only the enzyme acts on fluorinated substrates, and no report that the fluoroacetate dehalogenase can be used for catalyzing other halogenated substrates such as chlorination and bromination is found.

Disclosure of Invention

The invention aims to solve the technical problem that the existing wild-type fluoroacetate dehalogenase cannot be used for catalyzing a brominated substrate, particularly a 2-bromobutyric acid substrate, so that the invention provides a fluoroacetate dehalogenase mutant and application thereof in preparation of (R) -2-bromobutyric acid. When the (R) -2-bromobutyric acid is prepared by using the mutant of the fluoroacetate dehalogenase, the production cost is low, the stereoselectivity is high, and the industrial production is facilitated.

The source of the wild-type fluoroacetate dehalogenase used in the invention is Rhodopseudomonas palustris, and the specific sequence is shown as SEQ ID NO.1 in the sequence table. The wild-type fluoroacetate dehalogenase consists of 304 amino acid residues, and the applicant finds out through experiments that the wild-type fluoroacetate dehalogenase cannot be used for catalyzing 2-bromobutyric acid. The inventor conducts a large amount of screening of saturation mutation on different amino acid positions of the wild enzyme aiming at the substrate, and finds that partial mutants of H155, W156 and Y219 positions can be used for catalyzing the substrate 2-bromobutyric acid. And performing combined mutation on the sites, constructing a mutant library, and screening the wild-type fluoroacetate dehalogenase disclosed by the invention.

one of the technical solutions for solving the above technical problems of the present invention is: a mutant of a fluoroacetate dehalogenase, wherein a sequence of the mutant of the fluoroacetate dehalogenase comprises a sequence obtained by mutating amino acid residue H at position 155 and/or amino acid residue W at position 156 as shown in SEQ ID No. 1; the mutant fluoroacetate dehalogenase has activity in catalyzing a brominated substrate, particularly a 2-bromobutyric acid substrate.

Preferably, the sequence of the mutant of the fluoroacetate dehalogenase comprises a sequence obtained by mutating amino acid residue H at position 155 and/or amino acid residue W at position 156 shown in SEQ ID NO.1 to natural amino acid residues.

More preferably, the sequence of the mutant of the fluoroacetate dehalogenase further comprises a sequence obtained by mutating the 219 th amino acid residue Y shown in SEQ ID NO.1 into a natural amino acid residue.

Even more preferably, the 155 th amino acid residue H is mutated to A, C, D, E, F, G, I, L, M, N, P, Q, S, T, V or W, preferably to I, N, V, F, L, Q, A, C, M, P, T or W, more preferably to I, N or V; and/or, the 156 th amino acid residue W is mutated to A, C, D, F, G, I, L, M, P, R, S, T, V or Y, preferably to A, C, D, F, G, I, L, M, R, S, T or Y, more preferably to F, M, R, S or T; and/or the amino acid residue Y at position 219 is mutated to a hydrophobic amino acid residue, preferably to F, L or M.

Preferably, the 155 th amino acid residue H is mutated to V, or the 156 th amino acid residue W is mutated to M, T, C, F, S, V, A or L.

The capital English letters mentioned above represent amino acids as are well known to those skilled in the art, and in accordance with the present invention, represent the corresponding amino acid residues herein.

Preferably, the amino acid sequence of the mutant of the fluoroacetate dehalogenase is as 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.85, SEQ ID NO.83, SEQ ID NO.85, SEQ ID NO.87, SEQ ID NO.81, SEQ ID, 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; more preferably, the nucleotide sequence encoding the mutant fluoroacetate dehalogenase is as 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.80, SEQ ID NO.78, SEQ ID NO.82, SEQ ID NO.88, SEQ ID NO.82, SEQ ID NO.80, 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.

The second technical scheme for solving the technical problems is as follows: an isolated nucleic acid, wherein said nucleic acid encodes a mutant of the above-described fluoroacetate dehalogenase; preferably, the nucleotide sequence of the nucleic acid is as set forth 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.82, SEQ ID NO.88, SEQ ID NO.82, SEQ ID NO.88, SEQ ID NO.80, SEQ ID NO.82, SEQ ID NO., 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.

The third technical scheme for solving the technical problems is as follows: a recombinant expression vector comprising the nucleic acid.

the fourth technical scheme for solving the technical problems is as follows: a transformant comprising the above nucleic acid or the above recombinant expression vector.

the fifth technical scheme for solving the technical problems is as follows: an application of the mutant of the fluoroacetate dehalogenase in preparing (R) -2-bromobutyric acid and/or (R) -2-hydroxybutyric acid.

The sixth technical scheme for solving the technical problems of the invention is as follows: a method for preparing (R) -2-bromobutyric acid, said method comprising the steps of: and (R) -2-bromobutyric acid is obtained by carrying out catalytic reaction on 2-bromobutyric acid in the presence of a reaction solvent and the fluoroacetate dehalogenase mutant.

the 2-bromobutyric acid is one or more of 2-bromobutyric acid racemate, R-type enriched 2-bromobutyric acid and S-type enriched 2-bromobutyric acid, wherein the R-type enrichment refers to coexistence of R type and S type and a molar ratio of R type to S type of more than 1:1, and the S-type enrichment refers to coexistence of R type and S type and a molar ratio of S type to R type of more than 1: 1.

Preferably, the concentration of the mutant of the fluoroacetate dehalogenase is 2-20U/ml, and preferably 10U/ml;

And/or the concentration of the 2-bromobutyric acid is 0.02g/ml to 0.2g/ml, preferably 0.1 g/ml;

And/or, the reaction solvent is water;

And/or the pH value of the reaction system of the catalytic reaction is 6-8, preferably 7, and when the pH value is too high, the raw materials can be spontaneously decomposed, so that the yield is influenced;

And/or the temperature of the reaction system of the catalytic reaction is 20-30 ℃, preferably 30 ℃, and when the temperature is too high, the raw materials can be spontaneously decomposed, so that the yield is influenced;

and/or the reaction time of the catalytic reaction is 5 to 10 hours, preferably 8 hours;

The seventh technical scheme for solving the technical problems of the invention is as follows: a method for preparing (R) -2-hydroxybutyric acid, comprising the steps of: carrying out dehalogenation reaction on the 2-bromobutyric acid in the presence of a reaction solvent and the fluoroacetate dehalogenase mutant to obtain the (R) -2-hydroxybutyric acid.

The 2-bromobutyric acid is one or more of 2-bromobutyric acid racemate, R-type enriched 2-bromobutyric acid and S-type enriched 2-bromobutyric acid, wherein the R-type enrichment refers to coexistence of R type and S type and a molar ratio of R type to S type of more than 1:1, and the S-type enrichment refers to coexistence of R type and S type and a molar ratio of S type to R type of more than 1: 1.

In the dehalogenation reaction, the dehalogenase has high selectivity on (S) -2-bromobutyric acid in 2-bromobutyric acid (namely, preferentially reacts with (S) -2-bromobutyric acid, and after the reaction is completed, (R) -2-bromobutyric acid participates in the reaction to generate (R) -2-hydroxybutyric acid, so that the (R) -2-hydroxybutyric acid is subjected to dehalogenation reaction and configuration inversion to obtain (R) -2-hydroxybutyric acid, and unreacted or unreacted (R) -2-bromobutyric acid in the 2-bromobutyric acid is retained. The physical and chemical properties of (R) -2-bromobutyric acid and (R) -2-hydroxybutyric acid in the product are greatly different, so that the two are easy to separate.

preferably, the concentration of the mutant of the fluoroacetate dehalogenase is 2-20U/ml, and preferably 10U/ml;

And/or the concentration of the (S) -2-bromobutyric acid is 0.02g/ml to 0.2g/ml, preferably 0.1 g/ml;

And/or, the reaction solvent is water;

And/or the pH value of the reaction system of the dehalogenation reaction is 6-8, preferably 7, and when the pH value is too high, the raw materials can be spontaneously decomposed, so that the yield is influenced;

And/or the temperature of the reaction system of the dehalogenation reaction is 20-30 ℃, preferably 30 ℃, and when the temperature is too high, the raw materials can be spontaneously decomposed, so that the yield is influenced;

And/or the reaction time of the dehalogenation reaction is 5 to 10 hours, preferably 8 hours;

The concentrations of the above compounds are, unless otherwise specified, the concentrations of the above compounds in the whole reaction system before the reaction.

On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.

The reagents and starting materials used in the present invention are commercially available.

The positive progress effects of the invention are as follows:

The mutant of the fluoroacetate dehalogenase can be used for catalyzing a brominated substrate, in particular 2-bromobutyric acid. In a preferred embodiment of the invention, the substrate concentration catalyzed by the mutant fluoroacetate dehalogenase is up to 0.1 g/ml. When the (R) -2-bromobutyric acid is prepared by using the mutant of the fluoroacetate dehalogenase, the production cost is low, the stereoselectivity is high, and the industrial production is facilitated.

drawings

FIG. 1 shows the results of chiral HPLC analysis of 2-bromobutyric acid as the reaction starting material in example 3.

FIG. 2 shows the results of chiral HPLC analysis of (R) -2-bromobutyric acid after reaction using fluoroacetate dehydrogenase mutant 3 in example 3.

FIG. 3 shows the results of chiral HPLC analysis of (R) -2-bromobutyric acid standard.

FIG. 4 is an HPLC analysis chart of 2-bromobutyric acid as a reaction raw material in example 3.

FIG. 5 is an HPLC analysis chart of racemate 2-hydroxybutyric acid standard substance.

FIG. 6 is an HPLC analysis chart of the reaction mixture after the reaction in example 3 in which fluoroacetate dehydrogenase mutant 3 participates.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

The experimental methods in the invention are conventional methods unless otherwise specified, and the gene cloning operation can be specifically referred to in molecular cloning experimental guidelines compiled by J. SammBruk et al.

The 2-bromobutyric acid in the present invention may be one or more of 2-bromobutyric acid racemate, R-type enriched 2-bromobutyric acid and S-type enriched 2-bromobutyric acid, wherein R-type enrichment means that R-type and S-type coexist and the molar ratio of R-type to S-type is more than 1:1, and S-type enrichment means that R-type and S-type coexist and the molar ratio of S-type to R-type is more than 1:1, unless otherwise specified.

The abbreviations for the amino acids in the present invention are those conventional in the art unless otherwise specified, and the amino acids corresponding to the specific abbreviations are shown in Table 1.

TABLE 1

Name of amino acid	Three letter symbol	Single letter symbols	Name of amino acid	Three letter symbol	Single letter symbols
						Alanine (alanine)	Ala	A	Leucine (Leucine)	Leu	L
Arginine (arginin)	Arg	R	lysine (lysine)	Lys	K
						Asparagine (asparagine)	Asn	N	Methionine (methionine)	Met	M
Aspartic acid (aspartic acid)	Asp	D	Phenylalanine (phenylalanine)	Phe	F
						cysteine (cysteine)	Cys	C	proline (proline)	Pro	P
Glutamine (glutamine)	Gln	Q	Serine (serine)	Ser	S
						Glutamic acid (glutamic acid)	Glu	E	Threonine (threoninine)	Thr	T
Glycine (Glicine)	Gly	G	Tryptophan (tryptophan)	Trp	W
						Histidine (histidine)	His	H	Tyrosine (tyrosine)	Tyr	Y
Isoleucine (isoleucine)	Ile	I	Aspartic acid (valine)	Val	V

Codons corresponding to the amino acids are also conventional in the art, and the correspondence between specific amino acids and codons is shown in table 2.

TABLE 2

pET28a and bugbuster protein extraction reagent were purchased from Novagen; the DpnI enzyme Purchase from England Weiji (Shanghai) trade, Inc.; coli BL21(DE3) competent cells were purchased from Changsheng biotechnology, LLC of Beijing ancient cooking; 2-Bromobutyric acid was purchased from Dudada chemical Co., Ltd.

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

chromatographic conditions are as follows: daicel Chiralpak IG, 4.6mm 250mm, 5 μm; mobile phase: n-hexane/isopropanol/TFA 99:1: 0.1; detection wavelength: 230 nm; flow rate: 0.7 ml/min; column temperature: at 25 ℃.

The HPLC analysis method of the substrate 2-bromobutyric acid and the product comprises the following steps:

Chromatographic conditions are as follows: inertsil ODS-3, 4.6mm 150mm, 5 μm; mobile phase A: 10mmol sodium dihydrogen phosphate in water (pH 3.0), mobile phase B: acetonitrile; gradient elution; detection wavelength: 205 nm; flow rate: 0.7 ml/min; column temperature: 35 ℃ is carried out.

Conversion ═ reactant-remaining reactant/reactant × 100% (reactant: 2-bromobutyric acid).