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

Abstract

The invention belongs to the technical field of biology, and discloses a halohydrin dehalogenase mutant, a coding gene, a recombinant vector, a recombinant strain, an enzyme preparation and application of the halohydrin dehalogenase mutant, the coding gene, the recombinant vector, the recombinant strain and the enzyme preparation in synthesis of chiral (S)-5-phenoxymethyl-2-oxazolidinone compounds. The halohydrin dehalogenase mutant is obtained by performing combined mutation on an enzyme with an amino acid sequence as shown in SEQ ID NO.1 at the 15th site, the 137th site and the 179th site. The mutant can obviously improve the ee value of a chiral (S)-5-phenoxymethyl-2-oxazolidinone compound synthesized by catalyzing phenyl glycidyl ether, the purity of catalytic synthesis and resolution is higher, and industrial production is facilitated.

Description

Technical Field

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

[0002] Oxazolidinone compounds are a class of oxygen-containing and nitrogen-containing five-membered heterocyclic compounds with a variety of biological and pharmacological activities, such as antibacterial, antiviral and antitumor properties. They are also a class of highly valuable drug intermediates that can be used as precursors of β-amino acids and are widely used in organic synthesis and the medical field. They were first reported in 1981, and since then, more and more chiral 4-substituted and 4,5-substituted-2-oxazolidinone compounds have been clinically validated for their effectiveness in antibacterial, anti-asthmatic, and immunosuppressive effects. For example, linezolid is the first oxazolidinone antibiotic approved for use in humans, and has been shown to be effective in treating infections caused by multidrug-resistant Gram-positive bacteria. 5-Phenoxymethyl-2-oxazolidinone (Metaxalone) is a central muscle relaxant used to treat pain caused by muscle strain, sprain or other causes. It also has a sedative effect, as well as anticholinergic and antipyretic analgesic effects, and has great 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 the focus of organic synthesis. The traditional method for preparing 2-oxazolidinone and its derivatives is based on the enantiomeric raw materials of β-amino alcohols and their derivatives, and 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 is still relatively difficult. There are also problems such as poor selectivity, the need to provide excess donors, and cumbersome synthetic routes. The asymmetric hydrogenation reaction of unsaturated heterocyclic compounds involves expensive and polluting metal catalysts. The ring opening of epoxides by isocyanates / cyanates has the disadvantages of poor regioselectivity and stereoselectivity, high cost, strict reaction temperature, and long reaction time.

[0004] In contrast, biocatalysts have higher regioselectivity and enantioselectivity, and can obtain compounds with higher yields and optical purity. In biosynthesis, previous studies have included lipase, cytochrome P450 enzyme and halohydrin dehalogenase as catalysts for the synthesis of enantiomeric 2-oxazolidinone, but the synthesis of enantiomeric 2-oxazolidinone still has problems such as poor stereoselectivity and many by-products. Recently, Chen et al. synthesized (R) configuration and racemic metaxalone through halohydrin dehalogenase HHDHamb and its mutants. However, these works all focused on the study of (R) products, and no related enzymatic synthesis reports of (S) products, which are also of economic value, were found. Summary of the invention

[0005] The purpose of the present invention is to overcome the problem that there is no research related to the enzymatic synthesis of 2-oxazolidinone (S) type products in the prior art, and to provide a halohydrin dehalogenase mutant and encoding gene, recombinant vector, recombinant strain, enzyme preparation and phenyl glycidyl ether as substrate 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 purpose, 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 as shown in 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 as described above 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] The seventh aspect of 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 obtained 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 the wild-type halohydrin dehalogenase derived from Sneathiella limimaris through rational design technology, provides a new halohydrin dehalogenase mutant, takes phenyl glycidyl ether as a substrate, catalyzes the synthesis of chiral (S)-5-phenoxymethyl-2-oxazolidinone compounds, and the ee value of the synthesized chiral (S)-5-phenoxymethyl-2-oxazolidinone compounds is significantly improved, and solves the problem of low stereoselectivity of the wild halohydrin dehalogenase for substrates of (S) configuration; the present invention is easy to operate, 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 of the synthesis of 1b catalyzed by the mutant of halohydrin dehalogenase in Example 5;

[0023] Figure 4 This is a liquid chromatogram of the synthesis of 2b by 2a catalyzed by the 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 the 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 the wild-type halohydrin dehalogenase in Example 8;

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

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

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

[0031] The endpoints and any values ​​of the ranges disclosed in this article 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 endpoint values ​​of each range, the endpoint values ​​of each range and the 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 as specifically disclosed in this article.

[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] The inventors of the present invention discovered during the research process 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 is possible to effectively catalyze the synthesis of chiral 2-oxazolidinone compounds and have a higher ee value for the (S) product, effectively solving the problem of low stereoselectivity of wild halohydrin dehalogenase for substrates of (S) configuration.

[0034] According to the present invention, preferably, the enzyme having the amino acid sequence shown in SEQ ID NO.1 is mutated to tryptophan at phenylalanine at position 15, mutated to threonine at alanine at position 137, and mutated to leucine at asparagine at position 179. 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 names and abbreviations of the amino acids in the SEQ ID NO.1 sequence are shown in Table 1.

[0037] Table 1

[0038]

[0039]

[0040] The present invention provides a 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 as described above on specific sites in the amino acid sequence, 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 techniques known in the art. The embodiments of the present 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 among the 20 different amino acids that make up proteins, except for Met (ATG) or Trp (TGG) which are encoded by a single codon, the other 18 amino acids are encoded by 2-6 codons respectively (Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, second edition, 1989, see Appendix D on page 950). That is, due to the degeneracy of the genetic code, there are usually more than one codon to determine an amino acid, and the substitution of the third nucleotide in the triplet codon often does not change the amino acid composition, so the nucleotide sequence of the gene encoding the same protein can be different.

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

[0046] According to the present invention, preferably, when the amino acid sequence of the halohydrin dehalogenase mutant is as shown in SEQ ID NO. 3, the nucleotide sequence of the halohydrin dehalogenase mutant is as shown in SEQ ID NO. 4. At the same time, for the convenience of recording, the coding 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, which contains 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 various commercially available plasmids, cosmids, phages and retroviruses, etc. The preferred expression vector of the present invention is pET-28a(+) plasmid. At the same time, for the convenience of recording, the recombinant plasmid is represented as pET28a(+)-HHDHmut in the present embodiment.

[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, respectively, 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 endonuclease Nco I and Xho I in the examples of the present application are not particularly limited, for example, they can be obtained from Fermeten in a commercial manner, or synthesized according to genetic engineering methods known in the relevant field. A fourth aspect of the present invention provides a recombinant strain, the recombinant strain containing the gene as described above or the recombinant vector as described above.

[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 by calcium chloride method or high voltage electroporation transformation. 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 Escherichia coli BL21 (DE3).

[0052] The present invention transforms the recombinant plasmid pET28a(+)-HHDHmut into the E.co1i BL21(DE3) recipient bacteria, spreads it on an LB agar plate containing kanamycin (mass concentration of 40-60 mg / L), and then cultures it at a temperature of 30-40°C for 10-14 hours to grow a single colony on the plate: randomly pick a single colony and perform single cloning, then inoculate it into an LB liquid culture medium and culture it for 6-10 hours, extract the plasmid and perform sequencing, and screen out the recombinant strain according to the sequencing result, which is represented as the 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 inoculation 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 When the concentration of isopropyl-β-D-thiogalactopyranoside (IPTG) inducer is 0.05-0.15mM, and the induction culture is carried out at a temperature of 20-30°C and a rotation speed of 100-200rpm for 16-18h to obtain a fermentation broth: finally, centrifuge, discard the supernatant, collect the bacteria, and use a concentration of 45-55mM Tris-SO 4 The cells were resuspended in a buffer solution (pH = 7.0-8.0), and the wet cells were collected after centrifugation again and stored at -30 to -20°C for future use.

[0054] Tris-SO used in the present invention 4 The buffer solution (pH=7.0-8.0) was prepared by the laboratory. The preparation method was as follows: 1.52 g of Tris was weighed and dissolved in deionized water, and the pH was 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, there is no particular limitation on the conditions for fermenting the recombinant strain, as long as the recombinant strain can be proliferated in large quantities through the fermentation process. Preferably, the fermentation process comprises: firstly carrying out seed culture of the recombinant strain to obtain a seed liquid, and then inoculating the seed liquid into a fermentation medium containing kanamycin and culturing it until the bacterial cell concentration OD 600 The pH value is 0.6-0.8, and then an isopropyl-β-D-thiogalactoside (IPTG) inducer with a final concentration of 0.05-0.15 mM is added, and the culture is carried out 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 method for preparing the seed solution of the present invention comprises: picking 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 the freshly prepared recombinant strain or the recombinant strain frozen at low temperature.

[0058] The present invention has no particular restrictions on the seed culture method, as long as the recombinant strain can be activated and proliferated by the method. Preferably, the content of kanamycin 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: temperature of 30-40°C, rotation speed of 180-250rpm, and time of 6-10h.

[0059] The sixth aspect of 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 a solid, semi-solid or liquid form. The enzyme preparation can be prepared using conventional Ni + Column purification method for enzyme separation.

[0061] The seventh aspect of 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 aforementioned 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 catalytic synthesis of 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 the methods in the prior art.

[0064] The invention discloses a mutant of halohydrin dehalogenase, which uses phenyl glycidyl ether and its derivatives as substrates to catalyze the synthesis of chiral (S)-5-phenoxymethyl-2-oxazolidinone compounds, has high optical purity of the product, and solves the problem that the wild-type halohydrin dehalogenase has low stereoselectivity 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 the laboratory of Professor Xue Feng of 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, sulfuric acid, etc. were purchased from Beijing Solebow 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: tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L, pH adjusted to 6.5, steam sterilized under high pressure for 21min, and set aside;

[0068] LB agar plate medium: tryptone 10g / L, yeast extract 5g / L, NaCL 10g / L, agar 12g / L, pH adjusted to 7.0, steam sterilized under high pressure for 21min, and set aside;

[0069] Tris-SO 4 The buffer solution (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 fixed to 200 mL for later use.

[0070] Example 1

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

[0072] A halohydrin dehalogenase mutant, wherein 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 an enzyme having the amino acid sequence shown in SEQ ID NO.1, wherein the R1 position is the 15th phenylalanine, and the 15th phenylalanine is mutated to tryptophan; the R2 position is the 137th alanine, and the 137th alanine is mutated to threonine; the R3 position is the 179th asparagine, and the 179th asparagine 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 coding gene with the halohydrin dehalogenase mutant, specifically, SEQ ID NO.4 is synthesized by a total synthesis method through genetic engineering operation, that is, a coding gene with a mutant enzyme, and the halohydrin dehalogenase coding 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 digestion, the 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 the E. coli BL21(DE3) recipient bacteria and spread on an LB agar plate containing kanamycin (mass concentration of 50 mg / L), and then cultured at 37°C for 12 hours to allow single colonies to grow on the plate; a single colony was randomly picked and inoculated into LB liquid culture medium and cultured for 8 hours, and the plasmid was extracted for sequencing, and the genetically engineered bacteria were screened out according to the sequencing results, which were represented 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 LB medium containing 50 mg / L kanamycin at 37° C. and 200 rpm for 8 h to obtain a seed solution;

[0083] (2) Inoculate the seed solution at a 1% (v / v) inoculum into a conical flask containing 30 mL of LB liquid medium and culture at 37°C, 200 rpm for 2 h until the optical density OD 600 When the concentration of the inducing agent was 0.6, IPTG 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 liquid;

[0084] (3) The fermentation broth was centrifuged at 5000 × g for 5 min, the supernatant was discarded, and the mixture was washed with 50 mM Tris-SO at pH 7.5. 4 The culture medium was resuspended and washed with buffer, and the wet cells were collected after centrifugation again (5000×g, 5 min) to obtain 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 6Fast Flow (FF) filler, and the impurities were eluted with 10 column volumes of buffer A (10mM Tris-HCl pH 7.0, 250mMNaC1, 20mM imidazole), and then the target protein was eluted with buffer B (10mM Tris-HC1 pH 7.0, 250mM NaC1, 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 (the amino acid sequence is shown in SEQ ID NO.3, and the nucleotide sequence is 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 dehalogenase mutant enzyme, no impurity proteins are visible to the naked eye.

[0088] Example 5

[0089] This example illustrates the application of the halohydrin dehalogenase mutant in the synthesis of chiral (S)-5-phenoxymethyl-2-oxazolidinone compounds, that is, the halohydrin dehalogenase mutant enzyme solution prepared in Example 4 is 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 mutant enzyme solution of halohydrin dehalogenase is weighed and suspended in 1 mL of 75 mM NaOCN-Tris-SO 4 In the buffer system (pH7.5, 50mM), add phenyl glycidyl ether with a concentration of 50mM, shake at 30℃, 200rpm, take a sample after 12h, add 900μL of ethyl acetate, shake for 20min, centrifuge at 13000×g for 1min, take out 500μL of ethyl acetate, air-dry overnight, re-dissolve with 1ml mixed reagent (n-hexane: isopropanol = 1:1), filter with 0.22μm organic membrane, and perform HPLC detection. 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, and the same enzyme activity as the mutant halohydrin dehalogenase was used. The other reaction conditions were the same, and 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 high performance liquid chromatography analysis method of the product (S)-1b is as follows: using an Agilent-1220 system, chromatographic column type: Chiralcel OD-H column (DaicelCo, Japan; 4.6×250mmL, 5μm); chromatographic conditions: column temperature 30°C; mobile phase is n-hexane:isopropanol = 88:12 (v / v); flow rate is 0.8mL / min; column temperature 30°C; ultraviolet detection wavelength is 220nm.

[0095] Substrate: ee 1 =[(R 1 -S 1 ) / (R 1 +S 1 )]×100%or ee 1 =[(S 1 -R 1 ) / (S 1 +R 1 )]×100%;

[0096] Product: ee 2 =[(R 2 -S 2 ) / (R 2 +S 2 )]×100%or ee 2 =[(S 2 -R 2 ) / (S 2 +R 2 )]×100%;

[0097] Where R 1 and S 1 is the peak area of ​​(R)- and (S)-substrates, R 2 and S 2 are the peak areas of (R)- and (S)-products.

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

[0099] Example 6

[0100] This example illustrates the application of the halohydrin dehalogenase mutant in the synthesis of chiral (S)-5-phenoxymethyl-2-oxazolidinone compounds, that is, the halohydrin dehalogenase mutant prepared in Example 4 is used to enzymatically resolve the substrate 2-benzyl-phenyl glycidyl ether (represented by 2a) to synthesize the product (S)-5-phenoxymethyl-(2-benzyl)-2-oxazolidinone (represented by (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 mutant enzyme solution of halohydrin dehalogenase was weighed and suspended in 1 mL of 75 mM NaOCN in Tris-SO 4 In the buffer system (pH 7.5, 50mM), add 50mM phenyl glycidyl ether, shake at 30℃, 200rpm, take a sample after 12h, add 900μL ethyl acetate, shake for 20min, centrifuge at 13000×g for 1min, take out 500μL ethyl acetate, air-dry overnight, re-dissolve with 1ml mixed reagent (n-hexane: isopropanol = 1:1), filter with 0.22μm organic membrane, and perform HPLC detection. 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 enzyme of halohydrin dehalogenase with the wild-type enzyme of halohydrin dehalogenase, and the same enzyme activity as the mutant halohydrin dehalogenase was used. The other reaction conditions were the same, and 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 high performance liquid chromatography analysis method of the product (S)-2b is as follows: using an Agilent-1220 system, chromatographic column type: Chiralcel OD-H column (DaicelCo, Japan; 4.6×250mmL, 5μm); chromatographic conditions: column temperature 30°C; mobile phase n-hexane:isopropanol=90:10 (v / v); flow rate 0.6mL / min; column temperature 30°C; UV detection wavelength 220nm

[0106] Substrate: ee 1 =[(R 1 -S 1 ) / (R 1 +S 1 )]×100%or ee1 =[(S 1 -R 1 ) / (S 1 +R 1 )]×100%;

[0107] Product: ee 2 =[(R 2 -S 2 ) / (R 2 +S 2 )]×100%or ee 2 =[(S 2 -R 2 ) / (S 2 +R 2 )]×100%;

[0108] Where R 1 and S 1 is the peak area of ​​(R)- and (S)-substrates, R 2 and S 2 are the peak areas of (R)- and (S)-products.

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

[0110] Example 7

[0111] This example illustrates the application of the halohydrin dehalogenase mutant in the synthesis of chiral (S)-5-phenoxymethyl-2-oxazolidinone compounds, that is, the halohydrin dehalogenase mutant prepared in Example 4 is used to enzymatically resolve the substrate 3-benzyl-phenyl glycidyl ether (represented by 3a) to synthesize the product (S)-5-phenoxymethyl-(3-benzyl)-2-oxazolidinone (represented by (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 mutant halohydrin dehalogenase enzyme is weighed and suspended in 1 mL of 75 mM NaOCN in Tris-SO 4 In the buffer system (pH 7.5, 50mM), add 50mM phenyl glycidyl ether, shake at 30℃, 200rpm, take a sample after 12h, add 900μL ethyl acetate, shake for 20min, centrifuge at 13000×g for 1min, take out 500μL ethyl acetate, air-dry overnight, re-dissolve with 1ml mixed reagent (n-hexane: isopropanol = 1:1), filter with 0.22μm organic membrane, and perform HPLC 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 enzyme of halohydrin dehalogenase with the wild enzyme of halohydrin dehalogenase, and the same enzyme activity as the mutant halohydrin dehalogenase was used. The other reaction conditions were the same, and 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 high performance liquid chromatography analysis method of the product (S)-3b is as follows: using an Agilent-1220 system, chromatographic column type: Chiralcel OD-H column (DaicelCo, Japan; 4.6×250mmL, 5μm); chromatographic conditions: column temperature 30°C; mobile phase is n-hexane:isopropanol = 80:20 (v / v); flow rate is 0.8mL / min; column temperature 30°C; ultraviolet detection wavelength is 220nm.

[0117] Substrate: ee 1 =[(R 1 -S 1 ) / (R 1 +S 1 )]×100%or ee 1 =[(S 1 -R 1 ) / (S 1 +R 1 )]×100%;

[0118] Product: ee 2 =[(R 2 -S 2 ) / (R 2 +S 2 )]×100%or ee 2 =[(S 2 -R 2 ) / (S2 +R 2 )]×100%;

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

[0120] In Example 7, the wild-type halohydrin dehalogenase was used to catalyze the resolution of substrate 3a. The final resolution result is as follows: Figure 6 As shown in the figure, (S)-3b peaks at 22.996 min, and (R)-3b peaks at 28.811 min. The peak area ratios of the two are 50.147:49.853, which are relatively close. The peak area of ​​the (S) configuration is slightly larger. Compared with the catalytic resolution of the wild-type halohydrin dehalogenase, the catalytic resolution effect of the genetically engineered bacteria with the halohydrin dehalogenase mutant is better, such as Figure 7 As shown, (S)-3b peaks at 22.933 min and (R)-3b peaks at 30.673 min, with their peak area ratios 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 application 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 is 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 mutant halohydrin dehalogenase enzyme was weighed and suspended in 1 mL of 75 mM NaOCN in Tris-SO 4 In the buffer system (pH 7.5, 50mM), add 50mM phenyl glycidyl ether, shake at 30℃, 200rpm, take a sample after 12h, add 900μL ethyl acetate, shake for 20min, centrifuge at 13000×g for 1min, take out 500μL ethyl acetate, air-dry overnight, re-dissolve with 1ml mixed reagent (n-hexane: isopropanol = 1:1), filter with 0.22μm organic membrane, and perform HPLC detection. The results are as follows Fig. 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 enzyme of halohydrin dehalogenase with the wild enzyme of halohydrin dehalogenase, and the same enzyme activity as the mutant halohydrin dehalogenase was used. The other reaction conditions were the same, and 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: ee 1 =[(R 1 -S 1 ) / (R 1 +S 1 )]×100%or ee 1 =[(S 1 -R 1 ) / (S 1 +R 1 )]×100%;

[0128] Product: ee 2 =[(R 2 -S 2 ) / (R 2 +S 2 )]×100%or ee 2 =[(S 2 -R 2 ) / (S 2 +R 2 )]×100%;

[0129] Where R 1 and S 1 is the peak area of ​​(R)- and (S)-substrates, R 2 and S 2 are the peak areas of (R)- and (S)-products.

[0130] The high performance liquid chromatography analysis method of the product (S)-4b is as follows: using an Agilent-1220 system, chromatographic column type: Chiralcel OD-H column (DaicelCo, Japan; 4.6×250mmL, 5μm); chromatographic conditions: column temperature 30°C; mobile phase is n-hexane:isopropanol = 90:10 (v / v); flow rate is 0.6mL / min; column temperature 30°C; ultraviolet detection wavelength is 220nm.

[0131] In Example 8, the wild-type halohydrin dehalogenase was used to catalyze the resolution of substrate 4a. The final resolution result is as follows: Figure 8As shown in the figure, (R)-4b peaks at 62.364min and (S)-4b peaks at 71.184min. The peak area ratios of the two are 49.715:50.285, which are relatively close. The peak area of ​​the (S) configuration is slightly larger. Compared with the catalytic resolution of the wild-type halohydrin dehalogenase, the catalytic resolution effect of the genetically engineered bacteria with the halohydrin dehalogenase mutant is better. Fig. 9 As shown, (R)-4b peaks at 66.748 min and (S)-4b peaks at 71.517 min, with their peak area ratios 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 the halohydrin dehalogenase mutant in the synthesis of chiral (S)-5-phenoxymethyl-2-oxazolidinone compounds, namely, the halohydrin dehalogenase mutant prepared in Example 4 is used to enzymatically resolve the substrate 2-phenylnitro-phenyl glycidyl ether (represented by 5a) to synthesize the product (S)-5-phenoxymethyl-(2-phenylnitro)-2-oxazolidinone (represented by (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 mutant halohydrin dehalogenase enzyme is weighed and suspended in 1 mL of 75 mM NaOCN in Tris-SO 4 In the buffer system (pH 7.5, 50mM), add 50mM phenyl glycidyl ether, shake at 30℃, 200rpm, take a sample after 12h, add 900μL ethyl acetate, shake for 20min, centrifuge at 13000×g for 1min, take out 500μL ethyl acetate, air-dry overnight, re-dissolve with 1ml mixed reagent (n-hexane: isopropanol = 1:1), filter with 0.22μm organic membrane, and perform HPLC detection. The results are as follows Fig.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 enzyme of halohydrin dehalogenase with the wild enzyme of halohydrin dehalogenase, and the same enzyme activity as the mutant halohydrin dehalogenase was used. The other reaction conditions were the same, and the HPLC test results were as follows: Fig.10 As shown, the ee values ​​of the products catalyzed by wild-type halohydrin dehalogenase were all less than 5%.

[0138] The high performance liquid chromatography analysis method of the product (S)-5b is as follows: using an Agilent-1220 system, chromatographic column type: Chiralcel OD-H column (DaicelCo, Japan; 4.6×250mmL, 5μm); chromatographic conditions: column temperature 30°C; mobile phase is n-hexane:isopropanol = 70:30 (v / v); flow rate is 0.8mL / min; column temperature 30°C; ultraviolet detection wavelength is 220nm.

[0139] Substrate: ee 1 =[(R 1 -S 1 ) / (R 1 +S 1 )]×100%or ee 1 =[(S 1 -R 1 ) / (S 1 +R 1 )]×100%;

[0140] Product: ee 2 =[(R 2 -S 2 ) / (R 2 +S 2 )]×100%or ee 2 =[(S 2 -R 2 ) / (S 2 +R 2 )]×100%;

[0141] Where R 1 and S 1 is the peak area of ​​(R)- and (S)-substrates, R 2 and S 2 are the peak areas of (R)- and (S)-products.

[0142] In Example 9, the wild-type halohydrin dehalogenase was used to catalyze the resolution of substrate 5a. The final resolution result is as follows: Fig.10 As shown in the figure, (S)-5b peaks at 53.928min and (R)-5b peaks at 60.248min, 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 catalytic resolution of wild-type halohydrin dehalogenase, the catalytic resolution effect of genetically engineered bacteria with halohydrin dehalogenase mutants is better, such as Fig.11 As shown, (S)-5b peaks at 53.234 min and (R)-5b peaks at 62.099 min, with their peak area ratios being 97.919:2.081. The peak area of ​​the (S) configuration is much larger than that of the (R) configuration.

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

[0144] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A halohydrin dehalogenase mutant, characterized in that The halohydrin dehalogenase mutant is obtained by combining mutations at positions R1, R2 and R3 of an enzyme having an amino acid sequence shown in SEQ ID NO.1, 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.

2. The halohydrin dehalogenase mutant according to claim 1, characterized in that The halohydrin dehalogenase mutant has an amino acid sequence shown in SEQ ID NO.

3.

3. A gene encoding a halohydrin dehalogenase mutant, characterized in that: The gene has a nucleotide sequence encoding the halohydrin dehalogenase mutant according to claim 1 or 2; Preferably, the nucleotide sequence of the halohydrin dehalogenase mutant is shown as SEQ ID NO.

4.

4. A recombinant vector, characterized in that: The recombinant vector contains the gene according to claim 4; Preferably, 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 3 or the recombinant vector according to claim 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. Use of at least one of the halohydrin dehalogenase mutant according to claim 1 or 2, the gene according to claim 3, the recombinant vector according to claim 4, the recombinant strain according to claim 5, the halohydrin dehalogenase mutant obtained by the method according to claim 6, and the enzyme preparation according to claim 7 in synthesizing chiral (S)-5-phenoxymethyl-2-oxazolidinone compounds.

9. A catalytic synthesis method of chiral (S)-5-phenoxymethyl-2-oxazolidinone compounds, characterized in that: The method comprises: contacting at least one of the halohydrin dehalogenase mutant according to claim 1 or 2, the gene according to claim 3, the recombinant vector according to claim 4, the recombinant strain according to claim 5, the halohydrin dehalogenase mutant obtained by the method according to claim 6, and the enzyme preparation according to claim 7 with a substrate for reaction: 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.

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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