CN117402920A - Ketoreductase BsSDR10 mutant and application thereof in asymmetric reduction of alpha-oxazolidinyl substituted acetophenone derivatives - Google Patents

Ketoreductase BsSDR10 mutant and application thereof in asymmetric reduction of alpha-oxazolidinyl substituted acetophenone derivatives Download PDF

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CN117402920A
CN117402920A CN202311050346.8A CN202311050346A CN117402920A CN 117402920 A CN117402920 A CN 117402920A CN 202311050346 A CN202311050346 A CN 202311050346A CN 117402920 A CN117402920 A CN 117402920A
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ketoreductase
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秦斌
方佳丽
徐一平
任汉文
游松
张文鹤
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Shenyang Pharmaceutical University
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Abstract

The invention discloses a ketoreductase BsSDR10 mutant and application thereof in asymmetrically reducing alpha-oxazolidinyl substituted acetophenone derivatives, belongs to the technical field of biology, and particularly discloses a high-activity and high-selectivity ketoreductase wild type and mutant and application of encoding genes thereof in asymmetrically reducing alpha-oxazolidinyl substituted acetophenone and derivatives thereof. Mutant enzymes with high stereoselectivity were obtained by mutating wild-type ketoreductase BsSDR10 from bacillus subtilis (Bacillus subtilis), and utilized for asymmetric reduction of α -oxazolidinyl substituted acetophenones and derivatives thereof. The related mutant enzyme has the advantages of stereospecificity, mild reaction conditions, simple and convenient operation and the like, and has good application prospect in the green manufacture of chiral drugs taking alpha-oxazolidinyl substituted phenethyl alcohol derivatives as synthetic building blocks.

Description

Ketoreductase BsSDR10 mutant and application thereof in asymmetric reduction of alpha-oxazolidinyl substituted acetophenone derivatives
Technical Field
The invention relates to the technical field of biology, in particular to a high-activity and high-selectivity ketoreductase wild type and mutant and application of a coding gene thereof in asymmetrically reducing alpha-oxazolidinyl substituted acetophenone and derivatives thereof.
Background
The biocatalysis technology is a method for carrying out chemical conversion by using enzyme or biological organism as a catalyst, is an important foundation for biological manufacture, and has the advantages of mild reaction conditions, high selectivity and the like. In recent years, biocatalysis gradually becomes a key technology for manufacturing chiral chemicals, medicines and intermediates thereof, and the establishment of a high-efficiency synthetic route with good economical efficiency and high selectivity through biocatalysis is an important technical means. Enzymes are widely used as biocatalysts in the fields of green chemistry and medicine.
Alpha-oxazolidinyl substituted phenylethanol is a key intermediate for the synthesis of various drugs and active ingredients, such as the antiepileptic drug benananate, the antifungal agents octreonazole, itraconazole, fluconazole, miconazole, ketoconazole, the fungicide imazalil and the Transient Receptor Potential Classical (TRPC) channel antagonist SKF 96365. In addition, there are many other bioactive compounds, such as antibacterial, anticonvulsant, antitumor, hepatocyte proliferation-accelerating, antiproliferative, etc., which also contain alpha-oxazolidinyl substituted phenethyl alcohol in their structures.
Among the existing studies for synthesizing these products, most have used chemical methods to obtain racemic or chiral α -oxazolidinyl substituted phenethyl alcohol, and only a few biocatalytic routes have been used for synthesizing α -oxazolidinyl substituted phenethyl alcohol. However, under more environmentally friendly, milder, simpler conditions, there is no general method to obtain both stereoisomers of α -oxazolidinyl substituted phenylethanol.
Asymmetric catalysis of α -oxazolidinyl substituted acetophenones by chemical methods has been previously reported to yield enantiomerically enriched alcohols, for example as chemical catalysts for Jonathan Barrios-river a et al: an N-functionalized [ (benzone) Ru (II) (TsDPEN) ] complex, performing an asymmetric hydrogen transfer reaction on a series of acetophenone derivatives to obtain (R) -8a, 9a of high enantiomer, wherein the yield of (R) -8a is 87%, and ee is 96% (R); the yield of (R) -9a was 74% and ee was 90% (R). (J.Barrios-river, Y.Xu, G.J.Clarkson and M.Wills, tetrahedron,2022,103,132562.). At present, a very general method for obtaining the alpha-oxazolidinyl substituted phenethyl alcohol in an environment-friendly, mild and efficient way is not available.
Disclosure of Invention
The invention obtains mutant enzyme with high stereoselectivity through mutation of wild ketoreductase BsR 10 from bacillus subtilis (Bacillus subtilis), and utilizes mutant enzyme protein for asymmetric reduction of alpha-oxazolidinyl substituted acetophenone and derivatives thereof.
In order to solve the problems, the invention firstly adopts the technology of site-directed mutagenesis to obtain the ketoreductase BsSDR10 mutant protein with improved stereoselectivity and catalytic activity.
In order to improve the activity and the stereoselectivity of ketoreductase, the invention mutates wild-type ketoreductase with the amino acid sequence of SEQ ID NO.2, and the amino acids of 88 th, 91 st, 138 th, 139 th, 142 th, 144 th, 184 th, 189 th, 190 th and 193 th are replaced by other amino acids, the 88 th mutation is V/S, the 91 st mutation is A/V/T, the 138 th mutation is G/V/I/L/S/T, the 139 th mutation is A/V/I/S/T/G, the 142 th mutation is G/V/I/L/M/F/Y/W, the 144 th mutation is W/R/K/V/A/G, the 184 th mutation is A/V/L, the 189 th mutation is V, and the 193 th mutation is G/V/L. The above mutations may be single mutations or combination mutations. It has also been found that when these site amino acids are replaced with less sterically hindered amino acids, the activity and selectivity are better, and the less sterically hindered amino acids are: glycine, alanine, valine, serine, preferably alanine and valine. Wherein the amino acid sequence and the nucleic acid sequence of the mutant are shown in SEQ ID NO. 1-SEQ ID NO.154, and the optimal amino acid sequence of the mutant is shown in SEQ ID NO. 131.
The ketoreductase BsSDR10 is obtained by NCBI database gene mining and is derived from bacillus subtilis. The wild type nucleic acid sequence SEQ ID NO.1 is used as a template to carry out site-directed mutagenesis, so that mutant ketoreductase with higher stereoselectivity and catalytic activity is obtained.
The invention provides application of ketoreductase and a mutant thereof in asymmetric catalysis of alpha-oxazolidinyl substituted acetophenone and derivatives thereof.
The invention predicts the space configuration of the action of protein and substrate by reasonable design mutation, constructs the mutant by overlapping extension PCR method, thus obtaining the mutant library of ketoreductase BsSDR10, and screens ketoreductase with high stereoselectivity and catalytic activity by detecting the selectivity and conversion rate of the product.
The invention also provides a recombinant expression transformant cell containing the expression vector, wherein the cell is escherichia coli.
The construction and culture expression of the recombinant engineering bacteria of the invention, the construction of E.coli Rosetta2 (DE 3) -pET28b_NhisMBP-BsSDR10 strain comprises the following steps:
in order to obtain mutants with higher stereoselectivity and catalytic activity, a wild ketoreductase BsSDR10 gene (SEQ TD No. 1) is used as a template, a mutation primer containing mutation points (15-20 bp bases on the upstream and downstream of the mutation points are selected, the bases at the mutation points are replaced by codons of amino acids after mutation to serve as PCR forward primers, and reverse complementary sequences of the codons serve as reverse primers) is used for obtaining mutant genes of mutants through PCR amplification.
Further, the mutant gene and the vector plasmid pET28b_NhisMBP are subjected to double digestion (37 ℃ C., reaction for 4-8 h) by endonuclease NdeI and XhoI, and the digested nucleic acid fragment is recovered by using a common DNA product gel recovery kit; by T 4 The DNA ligase connects the mutated gene fragments and the vector plasmid fragments which are subjected to double enzyme digestion (reaction at 16 ℃ for 2-6 h) to obtain recombinant plasmids; the recombinant plasmid is transformed into E.coli Rosetta2 (DE 3) competent cells, recombinant engineering bacteria are established, and the transformation method adopted is a heat shock method: the specific process is heat shock at 45 ℃ for 90s. The constructed recombinant transformant cells were cultured to express the mutant ketoreductase.
The ketoreductase wild type and mutant thereof, or the cells containing the ketoreductase of the invention can be used as catalysts for catalyzing asymmetric reduction of alpha-oxazolidinyl substituted acetophenone (1 b) and derivatives thereof, and can be used for preparing optically pure (R) configuration products.
Particularly preferred, for enzyme catalysis of the substrate 1b and derivatives thereof, the invention provides a ketoreductase mutant A138V/L139A/Y144V/M184A with improved stereoselectivity, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 131.
The structural formula of the alpha-oxazolidinyl substituted acetophenone (1 b) and the derivative thereof is as follows:
chiral HPLC detection diagrams of the corresponding alcohols 6a-10j obtained by the asymmetric reduction 1b and the derivatives thereof are shown in FIG. 1-FIG. 50.
The invention has the beneficial effects that:
in recent years, a method for carrying out chemical conversion by using a biocatalysis technology is becoming a foundation of biological manufacture, not only because of being milder and more green compared with other synthetic methods, but also because of high selectivity and low cost, and is more and more suitable for industrial production. Enzymes are also increasingly being used in pharmaceutical and medical development as a means of biocatalysis. The invention obtains mutant enzyme with high stereoselectivity by mutating wild type ketoreductase BsR 10 from bacillus subtilis (Bacillus subtilis), and uses the mutant enzyme for asymmetric reduction of alpha-oxazolidinyl substituted acetophenone and derivatives thereof. The related mutant enzyme has the advantages of stereospecificity, mild reaction conditions, simple and convenient operation and the like, and has good application prospect in the green manufacture of chiral drugs taking alpha-oxazolidinyl substituted phenethyl alcohol derivatives as synthetic building blocks.
Drawings
FIGS. 1-50 are chiral HPLC detection diagrams of corresponding alcohols 6a-10j obtained by applying the ketoreductase of the invention to asymmetric reduction 1b and derivatives thereof.
Detailed Description
Example 1
Discovery of BsSDR10 ketoreductase
The amino acid sequence of known ketoreductase LfSDR1 is used as a template to obtain the potential ketoreductase sequence SEQ ID NO.1 through NCBI-BLAST on-line comparison, and the homology of the amino acid sequence is 26%. Through reasonable primer design, the two ends of the target gene are introduced into NDe I and XhoI cleavage sites and pass through T 4 The ligase inserts the target gene into pET28b_NhisMBP plasmid, and transfers the connected plasmid into E.coli Rosetta2 (DE 3) competent cells to establish recombinant engineering bacteria.
Example 2
Reasonable design of mutant and construction of recombinant escherichia coli
The site of wild-type ketoreductase with the amino acid sequence of SEQ ID NO.2 is reasonably designed, site-directed mutagenesis is carried out on 88 th, 91 st, 138 th, 139 th, 142 th, 144 th, 184 th, 189 th, 190 th and 193 th, 88 th mutation site I88S, 91 st mutation site S91T, 138 th mutation site A138 138 138 138 138 138 138T, 139 th mutation site L139 139 139 139 139 139 139 139G, 142 th mutation site A142 142 142 142 142 142W, 144 th mutation site Y144 144 144 144G, 184 th mutation site M184 184 184 184I, 189 189 189W, 190 th mutation site A190L, 193 rd mutation site A193 193L is designed on a primer, recombinant plasmid pET28b_NhisMBP with a target gene is used as a template, and the mutation site is introduced into the template sequence by an overlapping extension PCR technology. The PCR amplification system is shown in Table 1:
TABLE 1 PCR amplification System
PCR amplification procedure: (1) pre-denaturation at 95℃for 10min; (2) denaturation at 94℃for 30s; (3) annealing at 50 ℃ for 30s; (4) extending at 72 ℃ for 1min; (5) subjecting steps (2) - (4) to 35 cycles; (6) extension at 72℃for 10min.
And (3) carrying out agarose gel electrophoresis verification on 5 mu L of the first PCR product, and after the correct size of the strip is verified, recovering the residual DNA by agarose gel electrophoresis gel, and carrying out a second PCR on the recovered product. The products of the two rounds of PCR are DNA purified to obtain DNA products containing mutation sites.
The purified product and pET28b_NhisMBP are subjected to double digestion by NdeI and XhoI, and the same sticky end product is obtained after recovery by agarose gel electrophoresis. Using T 4 The DNA ligase connects the target gene with the plasmid to construct the recombinant plasmid.
The recombinant plasmid after ligation was transformed into E.coli Rosetta2 (DE 3) competent cells. The recombinant colony is coated on LB solid culture medium containing kanamycin sulfate for overnight culture, monoclonal colony is selected for colony PCR verification, positive clone strain is selected for culture expression, and the obtained target protein is ketoreductase BsSDR10 and mutants thereof. The amino acid and nucleic acid sequences are shown in SEQ ID NO. 1-SEQ ID NO. 154.
Example 3
Use of wild-type and mutant ketoreductases for asymmetric reduction of 1b on analytical scale
The wild strain and the mutant strain obtained in the example 2 are fermented, expanded, cultured, induced and expressed, and then the collected thalli are subjected to a ketocarbonyl reduction reaction by using whole cell bacterial liquid as a catalyst. The reaction system is as follows: 0.55mg of 1b (dissolved in 50. Mu.L of dimethyl sulfoxide), 0.2mg of NADP + 9mg glucose, pH=6.5 phosphate buffer, 0.1g BsSDR10 or mutant thereof wet mass and 0.02g Glucose Dehydrogenase (GDH) wet mass, in a total volume of 1mL. The reaction condition is that the temperature is 37 ℃, the shaking is carried out at 200rpm/min for 12 hours, and the reaction product is extracted by ethyl acetate to obtain the optical pure target product.
TABLE 2 asymmetric reduction of ketoreductase 1b
Wild BsSDR10 catalyzes 1b (0.55 mg,2.5 mM), after 12h conversion reaches 53.6%, (R) -6b has an ee value of 95.61%; the conversion after 12h of mutant A138V/L139A/Y144V/M184A obtained according to the invention was 99.13%, the ee value of (R) -6b was >99%, and the conversion and ee value obtained for wild-type BsR 10 and the mutant catalytic substrate 1b obtained according to the invention are shown in Table 2.
Example 4: asymmetric reduction of 1b at preparation scale by mutant ketoreductase
The mutant strain obtained in example 2 was subjected to fermentation, amplification culture, induction and expression, and then the obtained cells were collected, and ketone carbonyl reduction reaction was carried out using whole cell bacterial liquid as a catalyst. The reaction system is as follows: 48mg of 1b (dissolved in 1.5mL of dimethyl sulfoxide), 2mg of NADP + 225mg glucose, pH=6.5 phosphate buffer (e.g. KH 2 PO 4 -K 2 HPO 4 ) 0.2g of BsSDR10 or mutant thereof and 0.04g of Glucose Dehydrogenase (GDH) in a total volume of 10mL. The reaction conditions were 37℃and 200rpm/min were allowed to oscillate for 12h. The mutant A138V/L139A/Y144V/M184A obtained by the invention catalyzes 1b (48 mg), the conversion rate after 12 hours is 99 percent, and the ee value of (R) -6b>99％。
Example 5: use of wild-type and mutant ketoreductases for asymmetric reduction of 1b derivatives
The whole cell strain obtained by culturing the wild-type strain and the mutant strain obtained in example 1 and example 2 was subjected to an enzyme-catalyzed reaction. Reverse-rotationThe reaction system is as follows: 0.55mg of 1a-5j (dissolved in 50. Mu.L of dimethyl sulfoxide), 0.2mg of NADP + 9mg glucose, pH=6.5 phosphate buffer, 0.2g BsSDR 10A 138V/L139A/Y144V/M184A mutant and 0.04g Glucose Dehydrogenase (GDH) wet, in a total volume of 1mL. The reaction conditions were 37℃and 200rpm/min were allowed to oscillate for 12h.
TABLE 3 stereoselective reduction of 1b derivatives by ketoreductase
The conversion rate of the obtained mutant A138V/L139A/Y144V/M184A catalytic reduction 1b derivative 1a-5j after (R) -6a-10j is generated for 12h can reach 99% mostly, and the ee value of the obtained mutant A138V/L139A/Y144V/M184A catalytic reduction 1b derivative is more than 99, as shown in Table 3.
The foregoing is a preferred embodiment of the present invention. It should be noted that, the scope of the present invention is not limited by the above description, and all the equivalents and simple changes of the features and principles described in the claims and the description are included in the scope of the present invention.

Claims (9)

1. The application of ketoreductase BsR 10 and mutants thereof in asymmetrically reducing alpha-oxazolidinyl substituted acetophenone and derivatives thereof is characterized in that the wild type amino acid sequence of the ketoreductase BsR 10 is shown as SEQ ID NO. 2.
2. The use according to claim 1, wherein the nucleotide sequence of the gene encoding the ketoreductase BsSDR10 is shown in SEQ ID No. 1.
3. The use according to claim 1, wherein the mutant of ketoreductase BsSDR10 is designed from the wild-type ketoreductase BsSDR10 shown in SEQ ID No.1 as a template for the following amino acid sequences: 88 th, 91 st, 138 th, 139 th, 142 th, 144 th, 184 th, 189 th, 190 th, 193 th, 88 th, V/S, 91 st, A/V/T, 138 th, G/V/I/L/S/T, 139 th, A/V/I/S/T/G, 142 th, G/V/I/L/M/F/Y/W, 144 th, W/R/K/V/A/G, 184 th, A/V/L, 189 th, 190 th, G/V/L, 193 rd; the above mutations are single mutations or combination mutations.
4. The use according to claim 3, wherein the amino acid sequence and the nucleic acid sequence of the mutant are shown in SEQ ID NO.1 to SEQ ID NO. 154.
5. The use according to claim 4, wherein the mutant amino acid sequence is shown as SEQ ID NO. 131.
6. The coding gene of the ketoreductase BsR 10 and the mutant thereof according to claim 1, wherein escherichia coli is used as host bacteria for induction expression, and engineering bacteria containing the ketoreductase coding gene are inoculated into a liquid culture medium for culture expression.
7. The use according to claim 1, characterized in that said use comprises the asymmetric reduction of α -oxazolidinyl substituted acetophenone derivatives 1a-5j by ketoreductase genetic engineering bacteria and mutations thereof, obtaining optically pure α -oxazolidinyl substituted phenethyl alcohol derivatives (R) -6a-10j;
8. the use according to claim 7, wherein the ketoreductase BsSDR10 wild and mutant genes are introduced into E.coli for cloning and expression induction, cell collection, direct whole-cell enzyme solution as catalyst and NADP addition + Glucose and glucose dehydrogenase constitute a reaction hydrogen transfer system providing NADPH, KH at ph=6.5 with 1b as substrate 2 PO 4 -K 2 HPO 4 And (3) carrying out reaction under the reaction condition of shaking for 12 hours at 37 ℃ and 200rpm/min under the buffer condition, and extracting the reaction product by ethyl acetate to obtain the final optical purity target product (R) -6b.
9. The use according to claim 7, wherein the final reaction mixture obtained by using the combined mutant strain as biocatalyst and 1a-5j as substrate is subjected to shaking reaction at 37℃and 200rpm/min for 12h under hydrogen transfer system is extracted with ethyl acetate to obtain the product (R) -6a-10j.
CN202311050346.8A 2023-08-21 2023-08-21 Ketoreductase BsSDR10 mutant and application thereof in asymmetric reduction of alpha-oxazolidinyl substituted acetophenone derivatives Pending CN117402920A (en)

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