A sulfoxide reductase mutant and its application in preparation of chiral sulfoxide and sulfinic ester

Abstract

The scheme discloses a sulfoxide reductase mutant in the field of biotechnology, and the mutant has wider substrate universality compared with wild-type enzyme protein. E.coli is used as a host strain, and pET-28a(+) plasmid is used as a carrier to express the sulfoxide reductase mutant paMsrA-F59A, and the amino acid sequence of the mutant is shown as SEQ ID No.2. With the mutant as a catalyst, a series of kinetic resolutions of aromatic sulfoxide, heteroaromatic sulfoxide and sulfoxide derivative sulfinyl ester are carried out, and corresponding high-optical-purity R configuration sulfoxide and sulfinyl ester are obtained. The application provides an efficient strategy for green manufacturing of chiral sulfoxide and derivatives thereof.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and specifically relates to a sulfoxide reductase mutant and its application in the preparation of chiral sulfoxides and sulfinyl esters. Background Technology

[0002] Chiral sulfoxides and their derivatives are compounds with important biological activities and wide applications in organic and pharmaceutical synthesis. They can be used as chiral intermediates and excipients, chiral ligands, catalysts, and in clinical drugs. Currently, although some transition metal catalysts such as titanium, vanadium, and rhodium can effectively catalyze the asymmetric oxidation of thioether substrates or the kinetic resolution of racemic sulfoxides to generate chiral sulfoxides, most chemical catalysts suffer from problems such as over-oxidation, numerous byproducts, and harsh reaction conditions. Furthermore, these chemical catalytic reaction systems typically use heavy metal catalysts and strong oxidants such as peroxy acids, which do not meet the requirements of environmental protection and sustainable development. On the other hand, asymmetric reactions in biocatalysis are considered a new route for the synthesis of sulfoxide compounds. Among them, the asymmetric resolution reaction catalyzed by methionine sulfoxide reductase A (MsrA) holds promise as an effective means for the green production of chiral sulfoxides; however, wild-type MsrA exhibits strict substrate selectivity, limiting its application. Therefore, this technology aims to develop a MsrA mutant with a broader substrate spectrum for the efficient and green synthesis of more types of chiral sulfoxides and their derivatives. Summary of the Invention

[0003] The purpose of this invention is to overcome the shortcomings of the prior art, provide a sulfoxide reductase mutant with a broader substrate spectrum, and utilize the sulfoxide reductase variant to prepare chiral sulfoxides with high optical purity, providing a low-cost, safe and environmentally friendly biocatalytic preparation method for chiral sulfoxides and their derivatives.

[0004] One of the sulfoxide reductase mutants in this scheme, paMsrA, is obtained by mutating phenylalanine at position 59 to alanine based on the parent enzyme MsrA with the amino acid sequence shown in SEQ ID No. 1, and is named paMsrA-F59A, with its amino acid sequence shown in SEQ ID No. 2.

[0005] The method for constructing the sulfoxide reductase mutant described in this application is as follows: First, by using computer homology modeling and performing docking simulation with the substrate molecule, the proposed mutation site is determined; second, a site-directed saturated mutant library is constructed based on the determined site, and the library is subjected to high-throughput screening; thus, an enzyme mutant that can effectively catalyze complex sulfoxide substrates inactive by wild-type enzyme proteins is obtained.

[0006] This application also provides a gene encoding the sulfoxide reductase mutant; a recombinant vector carrying the gene; and recombinant cells expressing the sulfoxide reductase mutant, the gene, or the recombinant vector.

[0007] A genetically engineered bacterium containing a gene recombinant vector of the sulfoxide reductase mutant described in this application.

[0008] The genetically engineered bacteria of this invention are obtained by transforming the recombinant expression vector into a host microorganism using conventional methods in the art; preferably, the recombinant expression plasmid pET-28a-paMsrA-F59A mutant is transformed into *Escherichia coli* BL21(DE3). The host microorganism can be any conventional host microorganism in the art, as long as the recombinant expression vector can stably replicate spontaneously and the sulfoxide reductase gene it carries can be effectively expressed; preferably, it is *Escherichia coli* BL21(DE3).

[0009] This application also provides a series of biocatalytic preparation methods for R-configuration chiral sulfoxides and R-configuration chiral sulfinyl esters. Specifically, the genetically engineered bacteria described in this application are suspended in a buffer solution, racemic sulfoxide substrate or racemic sulfinyl ester substrate is added, the mixture is placed in a shaker for reaction, the organic phase is extracted and collected, dried with anhydrous sodium sulfate, the sodium sulfate is removed by centrifugation and filtered, the solvent is removed by pressure evaporation, and the mixture is separated by column chromatography. Based on the different substrates, R-configuration chiral sulfoxides and R-configuration chiral sulfinyl esters are obtained respectively.

[0010] The genetically engineered bacteria described in this invention can be resting cells containing the sulfoxide reductase, growing cells, immobilized cells, lysed crude enzyme solution, or purified paMsrA-F59A enzyme protein. Preferably, it is a crude enzyme solution containing the sulfoxide reductase.

[0011] The R-configuration chiral sulfoxides and R-configuration chiral sulfinyl esters prepared in this invention include, but are not limited to, the following 30 types:

[0012] R-configuration chiral sulfoxides: (R)-phenylmethyl sulfoxide, (R)-methyl-p-toluene sulfoxide, (R)-4-fluoro-phenylmethyl sulfoxide, (R)-3-fluoro-phenylmethyl sulfoxide, (R)-2-fluoro-phenylmethyl sulfoxide, (R)-4-chloro-phenylmethyl sulfoxide, (R)-3-chloro-phenylmethyl sulfoxide, (R)-2-chloro-phenylmethyl sulfoxide, (R)-2-bromo-phenylmethyl sulfoxide, (R)-3-bromo- Phenylacetyl sulfoxide, (R)-4-bromo-phenyl methyl sulfoxide, (R)-2-hydroxy-phenyl methyl sulfoxide, (R)-2-amino-phenyl methyl sulfoxide, (R)-phenylethyl sulfoxide, (R)-benzylmethyl sulfoxide, (R)-conylmethyl sulfoxide, (R)-phenyl-n-propyl sulfoxide, (R)-4-methyl-phenyl-n-propyl sulfoxide, (R)-phenyl-n-butyl sulfoxide, (R)-4-boronic acid-phenyl-n-butyl sulfoxide.

[0013] R-configuration chiral sulfinyl esters: (R)-4-methylsulfinyl-benzaldehyde, (R)-2-methylsulfinyl-pyridine, (R)-2-methylsulfinyl-cyclohexane, (R)-phenylsulfinyl acetate methyl ester, (R)-4-methyl-phenylsulfinyl acetate methyl ester, (R)-4-bromo-phenylsulfinyl acetate methyl ester, (R)-3-methyl-phenylsulfinyl acetate methyl ester, (R)-phenylsulfinyl ethyl acetate, (R)-4-methyl-phenylsulfinyl ethyl acetate, (R)-4-chlorophenylsulfinyl ethyl acetate.

[0014] The prepared R-configuration chiral sulfoxide and R-configuration chiral sulfinyl ester both had enantiomeric excess values ​​(ee) greater than 90%.

[0015] Furthermore, the genetically engineered bacteria described in this invention can be resting cells containing the sulfoxide reductase, growing cells, immobilized cells, lysed crude enzyme solution, or purified paMsrA-F59A enzyme protein. Preferably, it is a crude enzyme solution containing the sulfoxide reductase.

[0016] Furthermore, the buffer solution described in this invention is a conventional sodium phosphate buffer with a pH of 7.0–11.0, preferably a 50 mM Na₂HPO₄-NaH₂PO₄ solution with a pH of 9.0; the cell concentration is 10–50 g / L, preferably 30 g / L; the concentration of the sulfoxide reductase mutant is 1–5 g / L, preferably 3 g / L; the shaking speed is 250 rpm / min; the reaction temperature is 25–35 °C, preferably 25 °C; the reaction time is 2–8 hours; and the concentrations of the racemic sulfoxide substrate and the racemic sulfinyl ester substrate are both below 300 mM. The reaction process can be monitored using conventional testing methods in the art, such as gas chromatography (capillary column HP-5MS), HPLC, or NMR.

[0017] The extraction is preferably performed by repeatedly extracting the reaction solution with an organic solvent, combining the extracts, drying with anhydrous sodium sulfate overnight, and then removing the solvent by rotary evaporation. The organic solvent is a conventional organic solvent in the art; preferably, it is one or more selected from ethyl acetate, butyl acetate, and dichloromethane.

[0018] The significant advantages of this invention are as follows: Genetically engineered bacteria containing the sulfoxide reductase mutant can effectively catalyze the asymmetric resolution of racemic sulfoxides and sulfinyl esters, yielding high-optical-purity R-configuration chiral sulfoxides and R-configuration chiral sulfinyl esters. Compared to wild-type enzyme proteins, this mutant exhibits broader substrate universality, capable of catalyzing complex substrates that wild-type enzyme proteins cannot catalyze. Furthermore, compared to other existing preparation methods, the method described above offers shorter reaction times, milder reaction conditions, environmental friendliness, and ease of operation. Attached Figure Description

[0019] Figure 1 For paMsrA protein structure modeling and molecular docking analysis;

[0020] Figure 2 SDS-PAGE gel electrophoresis image of the paMsrA-F59A mutant recombinant protein;

[0021] Figure 3 Results of the preparation of (R)-phenylsulfinyl ethyl acetate catalyzed from the paMsrA-F59A mutant recombinant protein;

[0022] Figure 4 Results of the preparation of a series of R-configuration chiral sulfoxides and R-configuration chiral sulfinyl esters catalyzed by the paMsrA-F59A mutant recombinant protein. Detailed Implementation

[0023] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0024] The sulfoxide reductase described in this invention is obtained from cells by adding an IPTG inducer to the genetically engineered bacteria. The culture method and conditions are conventional in the art and can be appropriately selected based on factors such as host type and culture method, as long as the genetically engineered bacteria can grow and produce the sulfoxide reductase. Preferably, the method is as follows: a single colony is picked and cultured in 2 mL of LB medium at 37°C and 180 rpm for 12 hours, then transferred to a 250 mL shake flask containing 50 mL of LB medium and cultured until OD... 600When the concentration of sulfoxide (SOD) reaches 0.6, IPTG is added to a final concentration of 0.2 μM. After culturing for 14 h, the supernatant is removed by centrifugation at 5000 rpm to obtain strain cells containing the sulfoxide reductase. The culture medium used for the culture is any culture medium in the art that can enable the genetically engineered bacteria to grow and produce the sulfoxide reductase. Preferably, it is LB medium: 10 g / L peptone, 5 g / L yeast extract, 5 g / L NaCl, pH 7.0.

[0025] Example 1: Homology modeling and molecular docking of the sulfoxide reductase mutant paMsrA:

[0026] Using bovine MsrA protein from the PDB database as a template, a homologous 3D model of paMsrA was constructed. The substrate molecule (S)-methyl phenylsulfinyl acetate was docked to the enzyme's active pocket using molecular docking. The results are as follows: Figure 1 The results show that the complex side chain groups in the substrate molecule affect the entry of the substrate molecule into the catalytic pocket, and the distance between the thionyl group of the substrate molecule and the catalytic residue of the enzyme (cysteine ​​residue C58) reaches [missing information]. The large distance between the sites prevents the wild-type enzyme from exhibiting catalytic activity. Based on the docking results, we believe that phenylalanine at position 59 (F59), tryptophan at position 60 (W60), and tyrosine at position 89 (Y89) may have the greatest impact, and therefore selected these three sites for saturation mutation studies.

[0027] Example 2: Construction and screening of a site-directed saturation mutant library of sulfoxide reductase mutant paMsrA

[0028] Use the following primers:

[0029] F59-F:5'-GGTGTTCGGTCTCGGCTGCNNKTGGGGCGCCGAACGACGC-3'

[0030] F59-R:5'-GCGTCGTTTCGGCGCCCCAMNNGCAGCCGAGACCGAACACC-3'

[0031] W60-F:5'-GTTCGGTCTCGGCTGCTTCNNKGGCGCCGAACGACGCTTC-3'

[0032] W60-R:5'-GAAGCGTCGTTCGGCGCCMNNGAAGCAGCCGAGACCGAAC-3'

[0033] Y89-F:5'-CCTCACGCCCAACCCCACCNNKGAGGAGGTCTGCTCGGGC-3'

[0034] Y89-R:5'-GCCCGAGCAGACCTCCTCMNNGGTGGGGTTGGGCGTGAGG-3'

[0035] Using the paMsrA gene sequence as a template, PCR amplification was performed to construct three saturated mutant libraries: F59, W60, and Y89. High-throughput screening of the mutant libraries was conducted using racemic phenylsulfinyl acetate as a substrate. Transformants from the mutant libraries were inoculated into 96-well deep-layer plates for culture and protein expression induction. After 24 hours of induction, the bacteria in the 96 wells were centrifuged and lysed to obtain crude enzyme. The crude enzyme solution was transferred to a new 96-well deep-layer plate, and 200 μL / well of the reaction solution (prepared with 10 mM each of racemic phenyl-n-propyl and DTT) was rapidly transferred using a multipipe to the 96-well plate. The reaction was incubated at 250 rpm for 15 min at 30°C in a shaker. 5 μL of the reaction solution was added to a microplate, followed by 195 μL of DTNB reaction solution, and incubated at 37°C for 10 min. The absorbance at 415 nm was measured using a microplate reader to calculate the catalytic activity of the mutants. Through multiple rounds of screening, one mutant with significantly enhanced catalytic activity was obtained. Sequencing identified it as paMsrA-F59A (sequence shown in SEQ ID No. 2), a mutant with a phenylalanine at position 59 mutated to alanine. Using this mutant enzyme as a catalyst, a single enantiomer of (R)-phenylsulfinyl acetate with an ee value greater than 99% and a yield of 49% was successfully obtained, effectively achieving kinetic resolution of the racemic substrate. In contrast, under the same conditions, wild-type paMsrA produced a product with an ee value less than 1% after the reaction, failing to achieve kinetic resolution to obtain a single-configuration chiral sulfinyl ester.

[0036] Example 3: Catalytic results of the sulfoxide reductase mutant paMsrA-F59A on a series of R-configuration chiral sulfoxides and R-configuration chiral sulfinyl esters

[0037] Using the crude enzyme solution of the sulfoxide reductase mutant paMsrA-F59A obtained in Example 2 as a catalyst and DDT as a cofactor, its catalytic activity on racemic sulfoxide substrates with other complex groups and racemic sulfinyl ester substrates was studied. In a 5 mL reaction system, crude enzyme solution containing 30 μM enzyme protein, 10 mM racemic substrate, and 10 mM cofactor DTT were added. After reacting at 25 °C for 4 h, the substrate conversion rate was detected by gas chromatography, and the ee value of the product was detected by high-performance liquid chromatography. The catalytic activity of this enzyme on 30 racemic sulfoxides and sulfinyl esters was investigated, including phenylmethyl sulfoxide, methyl-p-toluene sulfoxide, 4-fluoro-phenylmethyl sulfoxide, 3-fluoro-phenylmethyl sulfoxide, 2-fluoro-phenylmethyl sulfoxide, 4-chloro-phenylmethyl sulfoxide, 3-chloro-phenylmethyl sulfoxide, 2-chloro-phenylmethyl sulfoxide, 2-bromo-phenylmethyl sulfoxide, 3-bromo-phenylmethyl sulfoxide, 4-bromo-phenylmethyl sulfoxide, 2-hydroxy-phenylmethyl sulfoxide, 4-methylsulfinyl-benzaldehyde, 2-amino-phenylmethyl sulfoxide, and phenylethyl sulfoxide. 2-Methylsulfinyl-pyridine, methylsulfinyl-cyclohexane, furfural methyl sulfinyl, phenyl-n-propyl sulfinyl, 4-methyl-phenyl-n-propyl sulfinyl, phenyl-n-butyl sulfinyl, 4-boronic acid-phenyl-n-butyl sulfinyl, methyl phenylsulfinyl acetate, methyl 4-methyl-phenylsulfinyl acetate, methyl 4-bromo-phenylsulfinyl acetate, methyl 3-methyl-phenylsulfinyl acetate, ethyl phenylsulfinyl acetate, ethyl 4-methyl-phenylsulfinyl acetate, ethyl 4-chlorophenylsulfinyl acetate. Catalytic results are as follows... Figure 4 As shown, efficient resolution of all racemic substrates was achieved, yielding optically pure sulfoxides and sulfinyl esters with an ee value greater than 90%, and a preparation yield exceeding 40%. In contrast, the wild-type paMsrA enzyme protein, in a completely identical reaction system, only catalyzes substrates containing a methyl / ethyl group on the thionyl functional group side. Therefore, the mutant paMsrA-F59A exhibits a significantly improved substrate spectrum compared to the wild-type enzyme protein, demonstrating better application prospects. Furthermore, the reaction time was consistently within 4 hours, exhibiting extremely high catalytic efficiency.

[0038] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, equivalent changes, and alterations made to the above embodiments without departing from the technical essence of the present invention shall still fall within the scope of the present invention.

Claims

1. A sulfoxide reductase mutant, characterized in that: The sulfoxide reductase mutant is obtained by mutating phenylalanine at position 59 to alanine based on the parent enzyme whose amino acid sequence is shown in SEQ ID No.

1. The amino acid sequence of the mutant is shown in SEQ ID No.

2.

2. The gene encoding the sulfoxide reductase mutant as described in claim 1.

3. A recombinant vector carrying the gene of claim 2.

4. Recombinant cells expressing the sulfoxide reductase mutant of claim 1, the gene of claim 2, or the recombinant vector of claim 3.

5. A genetically engineered bacillus, characterized in that, The genetically engineered bacillus expressed the sulfoxide reductase mutant of claim 1.

6. A kind R Configurational chiral sulfoxides and R A biocatalytic method for preparing chiral sulfinyl esters, characterized in that, Using the genetically engineered bacillus as described in claim 5, it is suspended in a buffer solution, the substrate is added to the buffer solution, and after reaction on a shaker, the organic phase is extracted and collected, and separated by column chromatography. The substrate is obtained when it is a racemic sulfoxide substrate. R The configuration of chiral sulfoxides is obtained when the substrate is a racemic sulfinyl ester substrate. R Configuration: Chiral sulfinyl ester; the substrate includes any one of the following: phenylmethyl sulfoxide, methyl-p-toluene sulfoxide, 4-fluoro-phenylmethyl sulfoxide, 3-fluoro-phenylmethyl sulfoxide, 2-fluoro-phenylmethyl sulfoxide, 4-chloro-phenylmethyl sulfoxide, 3-chloro-phenylmethyl sulfoxide, 2-chloro-phenylmethyl sulfoxide, 2-bromo-phenylmethyl sulfoxide, 3-bromo-phenylmethyl sulfoxide, 4-bromo-phenylmethyl sulfoxide, 2-hydroxy-phenylmethyl sulfoxide, 4-methylsulfinyl-benzaldehyde, 2-amino-phenylmethyl sulfoxide, phenylethyl sulfoxide Benzyl methyl sulfoxide, 2-methylsulfinyl-pyridine, methylsulfinyl-cyclohexane, furfuryl methyl sulfoxide, phenyl n-propyl sulfoxide, 4-methyl-phenyl n-propyl sulfoxide, phenyl n-butyl sulfoxide, 4-boronic acid-phenyl n-butyl sulfoxide, methyl phenylsulfinyl acetate, methyl 4-methyl-phenylsulfinyl acetate, methyl 4-bromo-phenylsulfinyl acetate, methyl 3-methyl-phenylsulfinyl acetate, ethyl phenylsulfinyl acetate, ethyl 4-methyl-phenylsulfinyl acetate, ethyl 4-chlorophenylsulfinyl acetate.

7. The method as described in claim 6, characterized in that, The buffer solution is a phosphate buffer with a pH of 7.0 to 10.0; the shaking speed is 150 to 300 rpm / min; the reaction temperature is 25 to 35 ℃; and the reaction time is 2 to 8 hours.

8. The method as described in claim 6 or 7, characterized in that, The genetically engineered bacteria are selected from the following: resting cells, growing cells, immobilized cells, lysed crude enzyme solution, and recombinant enzyme protein isolated and extracted from the genetically engineered bacteria containing the sulfoxide reductase mutant.

9. The method as described in claim 8, characterized in that, The concentrations of the racemic sulfoxide substrate and the racemic sulfinyl ester substrate are both less than 300 mM. The buffer solution is a Na2HPO4-NaH2PO4 solution with a concentration of 10-200 mM and a pH of 7.0-11.0, and the cell concentration is not less than 10 g / L. The concentration of the sulfoxide reductase mutant is not less than 1 g / L.

10. The method as described in claim 9, characterized in that, The concentration of the cells was 30 g / L, and the concentration of the sulfoxide reductase was 3 g / L.

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