Halohydrin dehalogenase mutant and encoding gene, recombinant vector, recombinant strain, enzyme preparation and application

Abstract

The invention belongs to the field of biotechnology, and discloses a halohydrin dehalogenase mutant and encoding gene, recombinant vector, recombinant strain, enzyme preparation and their application in synthesizing chiral (S)-5-phenoxymethyl-2-oxazolidinone compounds. The halohydrin dehalogenase mutant is a combination mutation of an enzyme with the amino acid sequence shown in SEQ ID NO.1 at positions 15, 137 and 179. The mutant can significantly improve the ee value of catalyzing phenyl glycidyl ether to synthesize chiral (S)-5-phenoxymethyl-2-oxazolidinone compounds, and the purity of catalytic synthesis and splitting is higher, which is conducive to industrial production.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a halohydrin dehalogenase mutant and encoding gene, a recombinant vector, a recombinant strain, an enzyme preparation, and applications of the same in synthesizing chiral (S)-5-phenoxymethyl-2-oxazolidinone compounds. Background Art

[0002] Oxazolidinone compounds are a class of oxygen- and nitrogen-containing five-membered heterocyclic compounds with diverse biological and pharmacological activities, such as antibacterial, antiviral, and antitumor properties. They are also highly valuable pharmaceutical intermediates and can serve as precursors for β-amino acids, with widespread applications in organic synthesis and medicine. They were first reported in 1981, and since then, a growing number of chiral 4-substituted and 4,5-substituted 2-oxazolidinone compounds have been clinically validated for their effectiveness in antibacterial, anti-asthmatic, and immunosuppressant applications. For example, linezolid is the first oxazolidinone antibiotic approved for human use and has been shown to be effective in treating infections caused by multidrug-resistant Gram-positive bacteria. 5-Phenoxymethyl-2-oxazolidinone (Metaxalone) is a centrally acting muscle relaxant used to treat pain caused by muscle strains, sprains, or other causes. It also exhibits sedative, anticholinergic, and antipyretic analgesic effects, resulting in significant economic benefits.

[0003] Due to the importance of oxazolidinone in organic synthesis and medicine, finding an effective method to synthesize pure enantiomers of 2-oxazolidinone is a key focus of organic synthesis. Traditional methods for preparing 2-oxazolidinone and its derivatives are based on enantiomeric starting materials of β-amino alcohols and their derivatives, using electrophilic "C=O" reagents for carbonylation reactions. However, this method usually involves the use of highly toxic and dangerous carbonylation reagents, and the acquisition of chiral β-amino alcohols remains relatively difficult. It also suffers from problems such as poor selectivity, the need for excess donors, and cumbersome synthetic routes. Asymmetric hydrogenation reactions of unsaturated heterocyclic compounds involve expensive and polluting metal catalysts. Isocyanate / cyanate ester ring opening of epoxides has disadvantages such as poor regioselectivity and stereoselectivity, high cost, strict reaction temperature, and long reaction time.

[0004] In contrast, biocatalysts have higher regioselectivity and enantioselectivity, enabling the production of compounds with higher yields and optical purity. Previous studies have used lipases, cytochrome P450 enzymes, and halohydrin dehalogenases as catalysts for the synthesis of enantiomeric 2-oxazolidinones. However, these methods still suffer from poor stereoselectivity and the production of numerous byproducts. Recently, Chen et al. synthesized (R)- and racemic metaxalone using the halohydrin dehalogenase HHDHamb and its mutants. However, these studies focused on the (R) product, and no reports on the enzymatic synthesis of the equally economically valuable (S) product have been found. Summary of the Invention

[0005] The purpose of the present invention is to overcome the problem in the prior art that there is no research related to the enzymatic synthesis of 2-oxazolidinone (S) type products, and to provide a halohydrin dehalogenase mutant and its encoding gene, a recombinant vector, a recombinant strain, an enzyme preparation and a substrate of phenyl glycidyl ether and their applications. The halohydrin dehalogenase mutant catalyzes the synthesis of 2-oxazolidinone (S) type products with high optical purity.

[0006] In order to achieve the above-mentioned object, the first aspect of the present invention provides a halohydrin dehalogenase mutant, characterized in that the halohydrin dehalogenase mutant is an enzyme having an amino acid sequence shown in SEQ ID NO.1 with combined mutations at positions R1, R2 and R3, wherein the R1 position is phenylalanine at position 15, the R2 position is alanine at position 137, and the R3 position is asparagine at position 179.

[0007] Preferably, the halohydrin dehalogenase mutant has the amino acid sequence shown in SEQ ID NO.3.

[0008] The second aspect of the present invention provides a gene encoding a halohydrin dehalogenase mutant, characterized in that the gene has the nucleotide sequence of the halohydrin dehalogenase mutant as described above;

[0009] Preferably, the nucleotide sequence of the halohydrin dehalogenase mutant is shown as SEQ ID NO.4.

[0010] The third aspect of the present invention provides a recombinant vector, characterized in that the recombinant vector contains the gene as described above;

[0011] Preferably, the expression vector of the recombinant vector is pET-28a(+) plasmid.

[0012] A fourth aspect of the present invention provides a recombinant strain, characterized in that the recombinant strain contains the gene or the recombinant vector as described above.

[0013] A fifth aspect of the present invention provides a method for preparing a halohydrin dehalogenase mutant, characterized in that the preparation method comprises: inoculating the aforementioned recombinant strain into a culture medium for fermentation culture.

[0014] The sixth aspect of the present invention provides an enzyme preparation, characterized in that the enzyme preparation comprises the halohydrin dehalogenase mutant prepared by the method described above.

[0015] In a seventh aspect, the present invention provides the use of at least one of the aforementioned halohydrin dehalogenase mutant, the aforementioned gene, the aforementioned recombinant vector, the aforementioned recombinant strain, the halohydrin dehalogenase mutant prepared by the aforementioned method, and the aforementioned enzyme preparation in the synthesis of chiral (S)-5-phenoxymethyl-2-oxazolidinone compounds.

[0016] In an eighth aspect, the present invention provides a method for catalyzing the synthesis of at least one of the aforementioned halohydrin dehalogenase mutant, the aforementioned gene, the aforementioned recombinant vector, the aforementioned recombinant strain, the halohydrin dehalogenase mutant prepared by the aforementioned method, and the aforementioned enzyme preparation in a substrate.

[0017] Preferably, the substrate is at least one selected from phenyl glycidyl ether, 2-benzyl-phenyl glycidyl ether, 3-benzyl-phenyl glycidyl ether, 2-phenylethyl-phenyl glycidyl ether and 2-phenylnitro-phenyl glycidyl ether.

[0018] Preferably, the reaction conditions of the catalytic synthesis include: temperature of 25-30° C. and pH of 7-8.

[0019] Through the above technical scheme, the present invention mutates a wild-type halohydrin dehalogenase derived from Sneathiella limimaris through rational design technology, providing a new halohydrin dehalogenase mutant. Phenyl glycidyl ether is used as a substrate to catalyze the synthesis of chiral (S)-5-phenoxymethyl-2-oxazolidinone compounds. The ee value of the synthesized chiral (S)-5-phenoxymethyl-2-oxazolidinone compounds is significantly improved, and the problem of low stereoselectivity of the wild halohydrin dehalogenase for substrates with (S) configuration is solved. The present invention is easy to operate and highly economical, and has good industrial application prospects in the application of catalytic synthesis of 5-substituted 2-oxazolidinones. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the SDS-PAGE analysis pattern of the wild-type and mutant halohydrin dehalogenase proteins after purification in Example 4.

[0021] Figure 2 This is a liquid chromatogram of the synthesis of 1b by 1a catalyzed by the wild-type halohydrin dehalogenase in Example 5;

[0022] Figure 3 This is a liquid chromatogram showing the synthesis of 1b by 1a catalyzed by the halohydrin dehalogenase mutant in Example 5;

[0023] Figure 4 This is a liquid chromatogram of the synthesis of 2b from 2a catalyzed by wild-type halohydrin dehalogenase in Example 6;

[0024] Figure 5 This is a liquid chromatogram of the synthesis of 2b by 2a catalyzed by the halohydrin dehalogenase mutant in Example 6;

[0025] Figure 6 This is a liquid chromatogram of the synthesis of 3b by 3a catalyzed by wild-type halohydrin dehalogenase in Example 7;

[0026] Figure 7 This is a liquid chromatogram of the synthesis of 3b by 3a catalyzed by the halohydrin dehalogenase mutant in Example 7;

[0027] Figure 8 This is a liquid chromatogram of the synthesis of 4b by 4a catalyzed by wild-type halohydrin dehalogenase in Example 8;

[0028] Figure 9 This is a liquid chromatogram of the synthesis of 4b catalyzed by the halohydrin dehalogenase mutant in Example 8;

[0029] Figure 10 This is a liquid chromatogram of the synthesis of 5b from 5a catalyzed by wild-type halohydrin dehalogenase in Example 9;

[0030] Figure 11 This is a liquid chromatogram of the synthesis of 5b by 5a catalyzed by the halohydrin dehalogenase mutant in Example 9; DETAILED DESCRIPTION

[0031] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0032] The first aspect of the present invention provides a halohydrin dehalogenase mutant, wherein the enzyme having the amino acid sequence shown in SEQ ID NO.1 is subjected to combined mutations at positions 15, 137 and 179.

[0033] During the research process, the inventors of the present invention discovered that by using AI deep learning enzyme engineering technology to perform combinatorial mutations on the wild-type halohydrin dehalogenase from Sneathiella limimaris, whose amino acid sequence is shown in SEQ ID NO.1, it can effectively catalyze the synthesis of chiral 2-oxazolidinone compounds, and has a high ee value for the (S) product, effectively solving the problem of low stereoselectivity of wild halohydrin dehalogenase for substrates with (S) configuration.

[0034] According to the present invention, preferably, the enzyme having the amino acid sequence shown in SEQ ID NO. 1 is mutated from phenylalanine at position 15 to tryptophan, from alanine at position 137 to threonine, and from asparagine at position 179 to leucine. That is, the amino acid sequence of the halohydrin dehalogenase mutant is shown in SEQ ID NO. 3.

[0035] The inventors have found that under this preferred embodiment, it is beneficial to improve the ee value of the catalytic synthesis of chiral (S)-5-phenoxymethyl-2-oxazolidinone compounds in phenyl glycidyl ether.

[0036] In the present invention, the amino acid names and abbreviations in the SEQ ID NO. 1 sequence are shown in Table 1.

[0037] Table 1

[0038]

[0039]

[0040] The present invention provides the nucleotide sequence of the amino acid sequence of the wild halohydrin dehalogenase as described above (as shown in SEQ ID NO.1), and the nucleotide sequence of the wild halohydrin dehalogenase is shown in SEQ ID NO.2.

[0041] The present invention uses the amino acid sequence of the wild-type halohydrin dehalogenase shown in SEQ ID NO.1 as a vector, and after performing combined mutations on specific sites in the amino acid sequence as described above, the amino acid sequence of the obtained halohydrin dehalogenase mutant is shown in SEQ ID NO.3.

[0042] Those skilled in the art should understand that the above-mentioned combined mutation modification methods are all implemented in accordance with relevant genetic engineering technologies known in the art. The embodiments of this application do not particularly limit the specific gene mutation technology, and it is subject to the ability of those skilled in the art to obtain the target mutant through the above-mentioned combined mutation.

[0043] The second aspect of the present invention provides a gene encoding a halohydrin dehalogenase mutant, wherein the gene has a nucleotide sequence encoding the halohydrin dehalogenase mutant as described above.

[0044] It is well known in the art that, of the 20 different amino acids that make up proteins, with the exception of Met (ATG) and Trp (TGG), which are each encoded by a single codon, the other 18 amino acids are encoded by 2-6 codons (Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, 2nd edition, 1989, see Appendix D on page 950). Because of the degeneracy of the genetic code, there is often more than one codon specifying an amino acid. Substitution of the third nucleotide in a triplet codon generally does not change the amino acid composition, and therefore, the nucleotide sequences of genes encoding the same protein can differ.

[0045] The present invention synthesizes the coding gene of the halohydrin dehalogenase mutant as described above by a total synthesis method.

[0046] According to the present invention, preferably, when the amino acid sequence of the halohydrin dehalogenase mutant is shown in SEQ ID NO. 3, the nucleotide sequence of the halohydrin dehalogenase mutant is shown in SEQ ID NO. 4. At the same time, for the convenience of recording, the encoding gene of the halohydrin dehalogenase mutant is represented by HHDHmut in the present embodiment.

[0047] The third aspect of the present invention provides a recombinant vector comprising the gene as described above.

[0048] In the present invention, the "vector" used in the recombinant vector can be selected from various vectors known in the art, such as commercially available plasmids, cosmids, phages, and retroviruses. The preferred expression vector of the present invention is the pET-28a(+) plasmid. For ease of description, the recombinant plasmid is referred to as pET28a(+)-HHDHmut in the examples of the present invention.

[0049] Exemplarily, the encoding gene of the halohydrin dehalogenase mutant and the expression vector pET28a(+) of Escherichia coli are double-digested with restriction endonucleases Nco I and Xho I for 3-6 hours, and the digestion products are recovered and connected with T4 DNA ligase at a temperature of 10-16°C for 15-20 hours to obtain a recombinant plasmid containing the encoding gene of the halohydrin dehalogenase mutant.

[0050] It should be noted that the specific sources of T4 DNA ligase, restriction endonucleases Nco I, and Xho I in the examples of this application are not particularly limited. For example, they can be commercially obtained from Fermeten or synthesized using genetic engineering methods known in the relevant art. A fourth aspect of the present invention provides a recombinant strain containing the aforementioned gene or recombinant vector.

[0051] In the present invention, the recombinant vector can be transformed, transduced, or transfected into a host cell (strain) by conventional methods in the art, such as chemical transformation using the calcium chloride method or high-voltage electroporation. The host cell can be a prokaryotic cell or a eukaryotic cell, preferably Escherichia coli or Bacillus subtilis. More preferably, the host cell is Escherichia coli, such as E. coli BL21 (DE3).

[0052] The present invention transforms the recombinant plasmid pET28a(+)-HHDHmut into E.co1i BL21(DE3) recipient bacteria, spreads the plasmid on an LB agar plate containing kanamycin (mass concentration of 40-60 mg / L), and then cultures the plate at 30-40°C for 10-14 hours to allow single colonies to grow on the plate. A single colony is randomly picked and cloned, then inoculated into an LB liquid culture medium and cultured for 6-10 hours. The plasmid is extracted and sequenced, and the recombinant strain is screened according to the sequencing results, which is represented as a positive clone E.co1iBL21(DE3) / pET28a(+)-HHDHmut.

[0053] The present invention can further induce expression of the recombinant strain, and the specific process can be: inoculating the recombinant strain into an LB culture medium containing kanamycin at a concentration of 40-60 mg / L, and initially culturing for 6-10 hours at a temperature of 30-40°C and a rotation speed of 180-250 rpm to obtain a seed solution; inoculating the seed solution into an LB culture medium containing kanamycin at a concentration of 40-60 mg / L at an inoculum amount of 0.8-1.2% (v / v), and culturing at a temperature of 30-40°C and a rotation speed of 180-250 rpm to an optical density OD 600 is 0.6-0.8; then, isopropyl-β-D-thiogalactopyranoside (IPTG) inducer is added to a final concentration of 0.05-0.15 mM, and the culture is induced at a temperature of 20-30°C and a rotation speed of 100-200 rpm for 16-18 hours to obtain a fermentation broth: finally, centrifugation is performed, the supernatant is discarded, and the bacteria are collected and resuspended in a Tris-SO4 buffer solution (pH = 7.0-8.0) with a concentration of 45-55 mM. After further centrifugation, the wet bacteria are collected and stored at a temperature of -30 to -20°C for later use.

[0054] The Tris-SO4 buffer solution (pH=7.0-8.0) used in the present invention is prepared by the laboratory. The preparation method is as follows: 1.52 g of Tris is weighed and dissolved in deionized water, and the pH is adjusted to 7.0-8.0 with 20% concentrated sulfuric acid.

[0055] A fifth aspect of the present invention provides a method for preparing a halohydrin dehalogenase mutant, the method comprising: inoculating the aforementioned recombinant strain into a fermentation medium for fermentation.

[0056] In the present invention, the fermentation conditions of the recombinant strain are not particularly limited, as long as the recombinant strain can be proliferated in large quantities through the fermentation process. Preferably, the fermentation process comprises: firstly, the recombinant strain is subjected to seed culture to obtain a seed liquid, and then the seed liquid is inoculated into a fermentation medium containing kanamycin and cultured until the bacterial cell concentration OD 600 The induction agent is 0.05-0.15 mM isopropyl-β-D-thiogalactopyranoside (IPTG), and the mixture is cultured at a temperature of 20-30° C. and a rotation speed of 100-200 rpm for 16-18 hours to obtain a fermentation broth.

[0057] The seed solution preparation method of the present invention comprises: selecting a single colony of the recombinant strain and inoculating it into a seed culture medium containing kanamycin for seed culture to obtain the seed solution. In the present invention, the single colony of the recombinant strain can be selected from a freshly prepared recombinant strain or a recombinant strain frozen at low temperature.

[0058] The present invention does not particularly limit the seed culture method, as long as the recombinant strain can be activated and proliferated by the method. Preferably, the kanamycin content in the seed culture medium is 40-60 mg / L; the temperature, pH, rotation speed, time, and other parameters used in the seed culture can be conventional settings in the art. Preferably, the seed culture conditions include: a temperature of 30-40°C, a rotation speed of 180-250 rpm, and a time of 6-10 hours.

[0059] In a sixth aspect, the present invention provides an enzyme preparation, which comprises the halohydrin dehalogenase mutant prepared by the method described above.

[0060] In the present invention, the halohydrin dehalogenase mutant can be prepared into a corresponding enzyme preparation. Specifically, the enzyme preparation can be in the form of solid, semi-solid or liquid. The enzyme preparation can be prepared using conventional Ni + Column purification method for enzyme separation.

[0061] In a seventh aspect, the present invention provides the use of at least one of the aforementioned halohydrin dehalogenase mutant, the aforementioned gene, the aforementioned recombinant vector, the aforementioned recombinant strain, the halohydrin dehalogenase mutant prepared by the aforementioned method, and the aforementioned enzyme preparation in the synthesis of chiral (S)-5-phenoxymethyl-2-oxazolidinone compounds.

[0062] In the present invention, the method for catalytically synthesizing chiral (S)-5-phenoxymethyl-2-oxazolidinone compounds may include: contacting at least one of the aforementioned halohydrin dehalogenase mutant, the aforementioned gene, the aforementioned recombinant vector, the aforementioned recombinant strain, the halohydrin dehalogenase mutant prepared by the aforementioned method, and the aforementioned enzyme preparation with a substrate; wherein the contact conditions include: a temperature of 25-30° C. and a pH of 7-8.

[0063] According to the present invention, preferably, the substrate is selected from at least one of phenyl glycidyl ether, 2-benzyl-phenyl glycidyl ether, 3-benzyl-phenyl glycidyl ether, 2-phenylethyl-phenyl glycidyl ether, and 2-phenylnitro-phenyl glycidyl ether. The above substrates can be commercially available or prepared by methods known in the art.

[0064] The invention discloses a mutant halohydrin dehalogenase, which uses phenyl glycidyl ether and its derivatives as substrates to catalyze the synthesis of chiral (S)-5-phenoxymethyl-2-oxazolidinone compounds. The product has high optical purity and solves the problem of low stereoselectivity of wild-type halohydrin dehalogenase for substrates with (S) configuration. The invention is easy to operate and highly economical, and has good industrial application prospects in the application of catalyzing the synthesis of chiral (S)-5-phenoxymethyl-2-oxazolidinone compounds.

[0065] The present invention will be described in detail below through examples.

[0066] In the following examples, Escherichia coli BL21 (DE3) and plasmid expression vector pET-28a (+) were purchased from Bao Biotechnology (Dalian) Co., Ltd., phenyl glycidyl ether (PGE), (S)-PGE, and (R)-PGE were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., and substrates 1a-5a were prepared and provided by Professor Xue Feng's laboratory at Nanjing Normal University. Peptone, yeast extract, and agar were purchased from Shanghai Sinopharm Chemical Reagent Co., Ltd., China. Isopropyl β-d-1-thiogalactopyranoside (IPTG), kanamycin, and sulfuric acid were purchased from Beijing Solebau Technology Co., Ltd., T4 DNA ligase, restriction endonucleases Nco I and Xho I were purchased from Fermetens, and other biological reagents were purchased from Sangon Biotechnology (Shanghai) Co., Ltd.

[0067] LB liquid medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, adjust the pH to 6.5, and sterilize under high pressure for 21 min.

[0068] LB agar plate medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, 12 g / L agar, adjust pH to 7.0, autoclave for 21 min, and set aside.

[0069] Tris-SO4 buffer (pH = 7.5) was prepared by the laboratory. The preparation method was as follows: 1.52 g of Tris was weighed and dissolved in deionized water. The pH was adjusted to 7.5 with 20% concentrated sulfuric acid and the volume was made up to 200 mL.

[0070] Example 1

[0071] This example illustrates the site-directed mutagenesis of halohydrin dehalogenase

[0072] A halohydrin dehalogenase mutant is disclosed. The halohydrin dehalogenase mutant is mutated based on a wild-type halohydrin dehalogenase, and the amino acid sequence of the wild-type halohydrin dehalogenase is shown in SEQ ID NO.1. The halohydrin dehalogenase mutant is obtained by combining mutations at positions R1, R2, and R3 of the enzyme having the amino acid sequence shown in SEQ ID NO.1, wherein the R1 position is phenylalanine at position 15, and the phenylalanine at position 15 is mutated to tryptophan; the R2 position is alanine at position 137, and the alanine at position 137 is mutated to threonine; and the R3 position is asparagine at position 179, and the asparagine at position 179 is mutated to leucine.

[0073] Example 2

[0074] The coding gene of the halohydrin dehalogenase mutant and the recombinant vector carrying the gene are obtained.

[0075] The halohydrin dehalogenase mutant in Example 1 is encoded to form a gene encoding the halohydrin dehalogenase mutant. Specifically, SEQ ID NO. 4 is synthesized by a total synthesis method through genetic engineering, that is, a gene encoding the mutant enzyme. The halohydrin dehalogenase encoding gene sequence is HHDHmut.

[0076] The halohydrin dehalogenase encoding gene and the Escherichia coli expression vector pET-28a(+) were double-digested with Nco I and Xho I, respectively. After 3-6 hours of enzyme digestion, the enzyme digestion products were recovered and ligated with T4 DNA ligase at 16° C. for 16 hours to obtain the recombinant plasmid pET28a(+)-HHDHmut.

[0077] Example 3

[0078] The recombinant plasmid pET28a(+)-HHDHmut was transformed into E. coli BL21(DE3) recipient bacteria and spread on LB agar plates containing kanamycin (mass concentration of 50 mg / L). The plates were then cultured at 37°C for 12 hours to allow single colonies to grow on the plates. Single colonies were randomly picked and inoculated into LB liquid culture medium and cultured for 8 hours. The plasmids were extracted and sequenced. The genetically engineered bacteria were screened based on the sequencing results and designated as positive clones, E. coli BL21(DE3) / pET28a(+)-HHDHmut.

[0079] Example 4

[0080] This example illustrates the induced expression and purification of a halohydrin dehalogenase mutant.

[0081] a. Inducible expression of halohydrin dehalogenase mutants

[0082] (1) The recombinant strain was cultured in 50 mL of LB medium containing 50 mg / L kanamycin at 37°C and 200 rpm for 8 h to obtain seed solution;

[0083] (2) The seed solution was inoculated into a conical flask containing 30 mL of LB liquid medium at a 1% (v / v) inoculation rate and cultured at 37°C and 200 rpm for 2 h until the optical density OD 600 When the pH value was 0.6, IPTG inducer was added at a final concentration of 0.1 mM, and the expression was induced at 25°C and 200 rpm for 16 h to obtain the fermentation broth;

[0084] (3) The fermentation broth was centrifuged at 5000 × g for 5 min, the supernatant was discarded, and the broth was resuspended and washed with 50 mM Tris-SO4 buffer at pH 7.5. The broth was centrifuged again (5000 × g for 5 min) and the wet cells were collected as the crude enzyme solution.

[0085] b. Purification of halohydrin dehalogenase mutants

[0086] The protein was purified by Ni+ column purification: the collected crude enzyme solution of the halohydrin dehalogenase mutant was filtered and added to Nisepharose 6 Fast Flow (FF) filler, and impurities were eluted with 10 column volumes of buffer A (10mM Tris-HCl pH 7.0, 250mM NaCl, 20mM imidazole), and then the target protein was eluted with buffer B (10mM Tris-HCl pH 7.0, 250mM NaCl, 250mM imidazole) to obtain the halohydrin dehalogenase mutant enzyme solution, which was stored at -20°C.

[0087] The collected target protein was subjected to SDS-PAGE protein electrophoresis, wherein the protein electrophoresis of the halohydrin dehalogenase mutant (amino acid sequence as shown in SEQ ID NO.3, nucleotide sequence as shown in SEQ ID NO.4) was as follows: Figure 1 As shown, after purification, the wild-type halohydrin dehalogenase still has trace amounts of impurity proteins, while after purification of the halohydrin dehalogenation mutant enzyme, no impurity proteins are visible to the naked eye.

[0088] Example 5

[0089] This example illustrates the application of a halohydrin dehalogenase mutant in the synthesis of chiral (S)-5-phenoxymethyl-2-oxazolidinone compounds, namely, the halohydrin dehalogenase mutant enzyme solution prepared in Example 4 was used to catalyze the resolution of the substrate phenyl glycidyl ether (represented by 1a) to synthesize the product (S)-5-phenoxymethyl-2-oxazolidinone (represented by (S)-1b).

[0090] The reaction process is as follows:

[0091]

[0092] The specific method for catalytic resolution of substrate 1a is as follows: 50 mg of the halohydrin dehalogenase mutant enzyme solution was weighed and suspended in 1 mL of 75 mM NaOCN Tris-SO4 buffer system (pH 7.5, 50 mM), 50 mM phenyl glycidyl ether was added, and the reaction was shaken at 30°C and 200 rpm. After 12 hours, a sample was taken, 900 μL of ethyl acetate was added to the sample, and after shaking for 20 minutes, the sample was centrifuged at 13000 × g for 1 minute. 500 μL of ethyl acetate was taken out, air-dried overnight, and then re-dissolved in 1 ml of a mixed reagent (n-hexane: isopropanol = 1:1). After filtering with a 0.22 μm organic membrane, HPLC detection was performed. The results are as follows Figure 3 As shown, the results showed that the mutant halohydrin dehalogenase preferentially hydrolyzed the (S)-configuration phenyl glycidyl ether substrate, and the ee value of the catalytically generated product (S)-1b was 95%.

[0093] The same application method as above was used to replace the mutant enzyme solution of halohydrin dehalogenase with the wild-type enzyme of halohydrin dehalogenase. The same enzyme activity as that of the mutant halohydrin dehalogenase was used. The other reaction conditions were the same. The HPLC test results were as follows: Figure 2 As shown, the ee values ​​of the products catalyzed by wild-type halohydrin dehalogenase were all less than 5%.

[0094] The product (S)-1b was analyzed by high-performance liquid chromatography (HPLC) using an Agilent-1220 system and a Chiralcel OD-H column (Daicel Co., Japan; 4.6×250 mm L, 5 μm). The chromatographic conditions were: column temperature, 30°C; mobile phase, n-hexane:isopropanol = 88:12 (v / v); flow rate, 0.8 mL / min; column temperature, 30°C; and UV detection at 220 nm.

[0095] Substrate: ee1 = [(R1-S1) / (R1+S1)] × 100% or ee1 = [(S1-R1) / (S1+R1)] × 100%;

[0096] Product: ee2 = [(R2-S2) / (R2+S2)] × 100% or ee2 = [(S2-R2) / (S2+R2)] × 100%;

[0097] Where R1 and S1 are the peak areas of (R)- and (S)-substrate, and R2 and S2 are the peak areas of (R)- and (S)-product.

[0098] In this example, the wild-type halohydrin dehalogenase was used to catalyze the resolution of substrate 1a. The final resolution result is shown in FIG. Figure 2 As shown in the figure, (R)-1b peaks at 39.653 min and (S)-1b peaks at 50.89 min, with the peak area ratio of 51.581:48.419, which is relatively close, and the peak area of ​​the (S) configuration is smaller. Compared with the wild-type halohydrin dehalogenase, the enzyme catalytic resolution of the halohydrin dehalogenase mutant is better, such as Figure 3 As shown, (R)-1b peaked at 40.228 min, and (S)-1b peaked at 49.532 min. The peak area ratio of the two was 2.533:97.467. The peak area of ​​the (S) configuration was much larger than that of the (R) configuration.

[0099] Example 6

[0100] This example illustrates the use of a halohydrin dehalogenase mutant in the synthesis of chiral (S)-5-phenoxymethyl-2-oxazolidinone compounds. Specifically, the halohydrin dehalogenase mutant prepared in Example 4 was used to enzymatically resolve the substrate 2-benzyl-phenyl glycidyl ether (represented as 2a) to synthesize the product (S)-5-phenoxymethyl-(2-benzyl)-2-oxazolidinone (represented as (S)-2b).

[0101] The reaction process is as follows:

[0102]

[0103] The specific method for catalytic resolution of substrate 2a is as follows: 50 mg of the halohydrin dehalogenase mutant enzyme solution was weighed and suspended in 1 mL of 75 mM NaOCN Tris-SO4 buffer system (pH 7.5, 50 mM), 50 mM phenyl glycidyl ether was added, and the reaction was shaken at 30°C and 200 rpm. After 12 hours, a sample was taken, 900 μL of ethyl acetate was added to the sample, and after shaking for 20 minutes, the sample was centrifuged at 13000 × g for 1 minute. 500 μL of ethyl acetate was taken out, air-dried overnight, and then re-dissolved in 1 ml of a mixed reagent (n-hexane: isopropanol = 1:1). After filtering with a 0.22 μm organic membrane, the sample was detected by HPLC. The results are as follows: Figure 5 As shown, the results showed that the mutant enzyme solution of halohydrin dehalogenase preferentially hydrolyzed the (S)-configuration phenyl glycidyl ether substrate, and the ee value of the catalytically generated product (S)-2b was 96%.

[0104] The same application method as above was used to replace the mutant halohydrin dehalogenase enzyme with the wild-type halohydrin dehalogenase enzyme. The same enzyme activity as that of the mutant halohydrin dehalogenase was used, and the other reaction conditions were the same. The HPLC test results were as follows: Figure 4 As shown, the ee values ​​of the products catalyzed by wild-type halohydrin dehalogenase were all less than 5%.

[0105] The product (S)-2b was analyzed by high performance liquid chromatography using an Agilent-1220 system, a Chiralcel OD-H column (Daicel Co., Japan; 4.6×250 mm L, 5 μm); chromatographic conditions: column temperature 30°C; mobile phase n-hexane:isopropanol = 90:10 (v / v); flow rate 0.6 mL / min; column temperature 30°C; UV detection wavelength 220 nm.

[0106] Substrate: ee1 = [(R1-S1) / (R1+S1)] × 100% or ee1 = [(S1-R1) / (S1+R1)] × 100%;

[0107] Product: ee2 = [(R2-S2) / (R2+S2)] × 100% or ee2 = [(S2-R2) / (S2+R2)] × 100%;

[0108] Where R1 and S1 are the peak areas of (R)- and (S)-substrate, and R2 and S2 are the peak areas of (R)- and (S)-product.

[0109] In Example 6, the wild-type halohydrin dehalogenase was used to catalyze the resolution of substrate 2a. The final resolution result was as follows: Figure 4As shown in the figure, (S)-2b peaks at 79.425 min and (R)-2b peaks at 83.588 min, with the peak area ratio of 42.309:57.691, which is relatively close, and the peak area of ​​the (S) configuration is smaller. Compared with the wild-type halohydrin dehalogenase, the enzyme catalytic resolution of the halohydrin dehalogenase mutant is better, such as Figure 5 As shown, (S)-2b peaked at 75.937 min and (R)-2b peaked at 88.467 min, with the peak area ratio of the two being 98.139:1.861. The peak area of ​​the (S) configuration was much larger than that of the (R) configuration.

[0110] Example 7

[0111] This example illustrates the use of a halohydrin dehalogenase mutant in the synthesis of chiral (S)-5-phenoxymethyl-2-oxazolidinone compounds. Specifically, the halohydrin dehalogenase mutant prepared in Example 4 was used to enzymatically resolve the substrate 3-benzyl-phenyl glycidyl ether (represented as 3a) to synthesize the product (S)-5-phenoxymethyl-(3-benzyl)-2-oxazolidinone (represented as (S)-3b).

[0112] The reaction process is as follows:

[0113]

[0114] The specific method for catalytic resolution of substrate 3a is as follows: 50 mg of the halohydrin dehalogenase mutant enzyme was weighed and suspended in 1 mL of 75 mM NaOCN Tris-SO4 buffer system (pH 7.5, 50 mM), 50 mM phenyl glycidyl ether was added, and the reaction was shaken at 30°C and 200 rpm. After 12 hours, a sample was taken, 900 μL of ethyl acetate was added to the sample, and after shaking for 20 minutes, the sample was centrifuged at 13000×g for 1 minute. 500 μL of ethyl acetate was taken out, air-dried overnight, and then re-dissolved in 1 mL of a mixed reagent (n-hexane: isopropanol = 1:1). After filtering with a 0.22 μm organic membrane, HPLC was performed for detection. The results are as follows: Figure 7 As shown, the results showed that the mutant halohydrin dehalogenase preferentially hydrolyzed the (S)-configuration phenyl glycidyl ether substrate, and the ee value of the catalytically generated product (S)-3b was 96%.

[0115] The same application method as above was used to replace the mutant halohydrin dehalogenase enzyme with the wild type halohydrin dehalogenase enzyme. The same enzyme activity as that of the mutant halohydrin dehalogenase was used, and the other reaction conditions were the same. The HPLC test results were as follows: Figure 6 As shown, the ee values ​​of the products catalyzed by wild-type halohydrin dehalogenase were all less than 5%.

[0116] The product (S)-3b was analyzed by high-performance liquid chromatography (HPLC) using an Agilent-1220 system and a Chiralcel OD-H column (Daicel Co., Japan; 4.6×250 mm L, 5 μm). The chromatographic conditions were: column temperature, 30°C; mobile phase, n-hexane:isopropanol = 80:20 (v / v); flow rate, 0.8 mL / min; column temperature, 30°C; and UV detection at 220 nm.

[0117] Substrate: ee1 = [(R1-S1) / (R1+S1)] × 100% or ee1 = [(S1-R1) / (S1+R1)] × 100%;

[0118] Product: ee2 = [(R2-S2) / (R2+S2)] × 100% or ee2 = [(S2-R2) / (S2+R2)] × 100%;

[0119] Where R1 and S1 are the peak areas of (R)- and (S)-substrate, and R2 and S2 are the peak areas of (R)- and (S)-product.

[0120] In Example 7, the substrate 3a was catalyzed by the wild-type halohydrin dehalogenase, and the final resolution result was as follows: Figure 6 As shown, (S)-3b peaks at 22.996 min and (R)-3b peaks at 28.811 min, with the peak area ratio of the two being 50.147:49.853, which is relatively close. The peak area of ​​the (S) configuration is slightly larger. Compared with the wild-type halohydrin dehalogenase, the genetically engineered bacteria with the halohydrin dehalogenase mutant have better catalytic resolution effects. Figure 7 As shown, (S)-3b peaked at 22.933 min and (R)-3b peaked at 30.673 min, with the peak area ratio of the two being 98.203:1.797. The peak area of ​​the (S) configuration is much larger than that of the (R) configuration.

[0121] Example 8

[0122] This example illustrates the use of a halohydrin dehalogenase mutant in the synthesis of chiral (S)-5-phenoxymethyl-2-oxazolidinone compounds, namely, the halohydrin dehalogenase mutant prepared in Example 4 was used to enzymatically resolve the substrate 2-phenethyl-phenyl glycidyl ether (represented by 4a) to synthesize the product (S)-5-phenoxymethyl-(2-phenethyl)-2-oxazolidinone (represented by (S)-4b).

[0123] The reaction process is as follows:

[0124]

[0125] The specific method for catalytic resolution of substrate 4a is as follows: 50 mg of the halohydrin dehalogenase mutant enzyme was weighed and suspended in 1 mL of 75 mM NaOCN Tris-SO4 buffer system (pH 7.5, 50 mM), 50 mM phenyl glycidyl ether was added, and the reaction was shaken at 30°C and 200 rpm. After 12 hours, a sample was taken, 900 μL of ethyl acetate was added to the sample, and after shaking for 20 minutes, the sample was centrifuged at 13000×g for 1 minute. 500 μL of ethyl acetate was taken out, air-dried overnight, and then re-dissolved in 1 mL of a mixed reagent (n-hexane: isopropanol = 1:1). After filtering with a 0.22 μm organic membrane, the sample was detected by HPLC. The results are as follows: Figure 9 As shown, the results showed that the mutant halohydrin dehalogenase preferentially hydrolyzed the (S)-configuration phenyl glycidyl ether substrate, and the ee value of the catalytically generated product (S)-4b was 95%.

[0126] The same application method as above was used to replace the mutant halohydrin dehalogenase enzyme with the wild type halohydrin dehalogenase enzyme. The same enzyme activity as that of the mutant halohydrin dehalogenase was used, and the other reaction conditions were the same. The HPLC test results were as follows: Figure 8 As shown, the ee values ​​of the products catalyzed by wild-type halohydrin dehalogenase were all less than 5%.

[0127] Substrate: ee1 = [(R1-S1) / (R1+S1)] × 100% or ee1 = [(S1-R1) / (S1+R1)] × 100%;

[0128] Product: ee2 = [(R2-S2) / (R2+S2)] × 100% or ee2 = [(S2-R2) / (S2+R2)] × 100%;

[0129] Where R1 and S1 are the peak areas of (R)- and (S)-substrate, and R2 and S2 are the peak areas of (R)- and (S)-product.

[0130] The product (S)-4b was analyzed by high-performance liquid chromatography (HPLC) using an Agilent-1220 system and a Chiralcel OD-H column (Daicel Co., Japan; 4.6×250 mm L, 5 μm). The chromatographic conditions were: column temperature, 30°C; mobile phase, n-hexane:isopropanol = 90:10 (v / v); flow rate, 0.6 mL / min; column temperature, 30°C; and UV detection at 220 nm.

[0131] In Example 8, the wild-type halohydrin dehalogenase was used to catalyze the resolution of substrate 4a. The final resolution result is shown in FIG. Figure 8As shown in the figure, (R)-4b peaks at 62.364 min and (S)-4b peaks at 71.184 min, with the peak area ratio of the two being 49.715:50.285, which is relatively close. The peak area of ​​the (S) configuration is slightly larger. Compared with the wild-type halohydrin dehalogenase, the genetically engineered bacteria with the halohydrin dehalogenase mutant have better catalytic resolution effects. Figure 9 As shown, (R)-4b peaked at 66.748 min, and (S)-4b peaked at 71.517 min, with the peak area ratio of the two being 2.519:97.481. The peak area of ​​the (S) configuration is much larger than that of the (R) configuration.

[0132] Example 9

[0133] This example illustrates the application of a halohydrin dehalogenase mutant in the synthesis of chiral (S)-5-phenoxymethyl-2-oxazolidinone compounds. Specifically, the halohydrin dehalogenase mutant prepared in Example 4 was used to enzymatically resolve the substrate 2-phenylnitro-phenyl glycidyl ether (denoted as 5a) to synthesize the product (S)-5-phenoxymethyl-(2-phenylnitro)-2-oxazolidinone (denoted as (S)-5b).

[0134] The reaction process is as follows:

[0135]

[0136] The specific method for catalytic resolution of substrate 5a is as follows: 50 mg of the halohydrin dehalogenase mutant enzyme was weighed and suspended in 1 mL of 75 mM NaOCN Tris-SO4 buffer system (pH 7.5, 50 mM), 50 mM phenyl glycidyl ether was added, and the reaction was shaken at 30°C and 200 rpm. After 12 hours, a sample was taken, 900 μL of ethyl acetate was added to the sample, and after shaking for 20 minutes, the sample was centrifuged at 13000×g for 1 minute. 500 μL of ethyl acetate was taken out, air-dried overnight, and then re-dissolved in 1 mL of a mixed reagent (n-hexane: isopropanol = 1:1). After filtering with a 0.22 μm organic membrane, the sample was detected by HPLC. The results are as follows: Figure 11 As shown, the results showed that the mutant halohydrin dehalogenase preferentially hydrolyzed the (S)-configuration phenyl glycidyl ether substrate, and the ee value of the catalytically generated product (S)-5b was 96%.

[0137] The same application method as above was used to replace the mutant halohydrin dehalogenase enzyme with the wild type halohydrin dehalogenase enzyme. The same enzyme activity as that of the mutant halohydrin dehalogenase was used, and the other reaction conditions were the same. The HPLC test results were as follows: Figure 10 As shown, the ee values ​​of the products catalyzed by wild-type halohydrin dehalogenase were all less than 5%.

[0138] The product (S)-5b was analyzed by high-performance liquid chromatography (HPLC) using an Agilent-1220 system and a Chiralcel OD-H column (Daicel Co., Japan; 4.6×250 mm L, 5 μm). The chromatographic conditions were: column temperature, 30°C; mobile phase, n-hexane:isopropanol = 70:30 (v / v); flow rate, 0.8 mL / min; column temperature, 30°C; and UV detection at 220 nm.

[0139] Substrate: ee1 = [(R1-S1) / (R1+S1)] × 100% or ee1 = [(S1-R1) / (S1+R1)] × 100%;

[0140] Product: ee2 = [(R2-S2) / (R2+S2)] × 100% or ee2 = [(S2-R2) / (S2+R2)] × 100%;

[0141] Where R1 and S1 are the peak areas of (R)- and (S)-substrate, and R2 and S2 are the peak areas of (R)- and (S)-product.

[0142] In Example 9, the substrate 5a was catalyzed by the wild-type halohydrin dehalogenase, and the final resolution result was as follows: Figure 10 As shown in the figure, (S)-5b peaks at 53.928 min and (R)-5b peaks at 60.248 min, with the peak area ratio of 51.079:48.921, which is relatively close. The peak area of ​​(S) configuration is slightly larger. Compared with the wild-type halohydrin dehalogenase, the genetically engineered bacteria with halohydrin dehalogenase mutants have better catalytic resolution. Figure 11 As shown, (S)-5b peaked at 53.234 min, and (R)-5b peaked at 62.099 min, with the peak area ratio of the two being 97.919:2.081. The peak area of ​​the (S) configuration is much larger than that of the (R) configuration.

[0143] The present invention provides a novel green biosynthetic route for the catalytic synthesis of chiral (S)-5-phenoxymethyl-2-oxazolidinone compounds using phenyl glycidyl ether and its derivatives as substrates. The product ee value is increased from less than 20% to 95%-96%. A mutant halohydrin dehalogenase preferentially reacts with the (S)-type substrate to produce the desired (S)-type product, resulting in enhanced stereoselectivity. The present invention is easy to operate, has high atom economy, and has promising industrial application prospects in the biocatalytic preparation of chiral (S)-5-phenoxymethyl-2-oxazolidinone compounds.

[0144] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, various simple variations of the technical solution of the present invention may be made, including combining the various technical features in any other appropriate manner. These simple variations and combinations should also be regarded as disclosed in the present invention and fall within the scope of protection of the present invention.

Claims

1. A halohydrin dehalogenase mutant, characterized in that The amino acid sequence of the halohydrin dehalogenase mutant is shown in SEQ ID NO.

3.

2. A gene encoding a halohydrin dehalogenase mutant, characterized in that The nucleotide sequence of the gene is shown in SEQ ID NO.

4.

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

4. The recombinant vector according to claim 3, characterized in that The expression vector of the recombinant vector is pET-28a(+) plasmid.

5. A recombinant strain, characterized in that The recombinant strain contains the gene according to claim 2 or the recombinant vector according to claim 3 or 4.

6. A method for preparing a halohydrin dehalogenase mutant, characterized in that: The preparation method comprises: inoculating the recombinant strain according to claim 5 into a culture medium for fermentation culture.

7. An enzyme preparation, characterized in that The enzyme preparation comprises the halohydrin dehalogenase mutant prepared by the method according to claim 6.

8. At least one of the halohydrin dehalogenase mutant according to claim 1, the gene according to claim 2, the recombinant vector according to claim 3 or 4, the recombinant strain according to claim 5, the halohydrin dehalogenase mutant prepared by the method according to claim 6, and the enzyme preparation according to claim 7 is used in the synthesis of chiral ( S )-5-phenoxymethyl-2-oxazolidinone compounds; the chiral ( S )-5-phenoxymethyl-2-oxazolidinone compounds are ( S )-5-phenoxymethyl-2-oxazolidinone, ( S )-5-phenoxymethyl-(2-phenylmethyl)-2-oxazolidinone, ( S )-5-phenoxymethyl-(3-phenylmethyl)-2-oxazolidinone, ( S )-5-phenoxymethyl-(2-phenethyl)-2-oxazolidinone and ( S )-5-phenoxymethyl-(2-phenylnitro)-2-oxazolidinone.

9. A chirality ( S )-5-phenoxymethyl-2-oxazolidinone compound catalytic synthesis method, characterized in that, The method comprises: contacting at least one of the halohydrin dehalogenase mutant according to claim 1, the gene according to claim 2, the recombinant vector according to claim 3 or 4, the recombinant strain according to claim 5, the halohydrin dehalogenase mutant prepared by the method according to claim 6, and the enzyme preparation according to claim 7 with a substrate to react: The substrate is selected from one of phenyl glycidyl ether, 2-benzyl-phenyl glycidyl ether, 3-benzyl-phenyl glycidyl ether, 2-phenylethyl-phenyl glycidyl ether and 2-phenylnitro-phenyl glycidyl ether.

10. The catalytic synthesis method according to claim 9, characterized in that: The reaction conditions include: temperature of 25-30° C. and pH of 7-8.

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