A carbonyl reductase mutant and its use in enzymatic synthesis of chiral alcohol

Abstract

This invention relates to the field of biotechnology and discloses a carbonyl reductase mutant and its application in the enzymatic synthesis of chiral alcohols. The invention generates carbonyl reductase mutants with varying degrees of activity compared to the wild type by mutating the amino acid sequence shown in SEQ ID NO.2 by altering the 36th lysine to any one of aspartic acid, threonine, or valine, or / and the 125th histidine to any one of lysine, serine, or leucine, or the 170th histidine to any one of lysine, cysteine, or leucine. In particular, the single-point mutants Mut-T75K, Mut-T75D, Mut-E216M, and the combined mutant Mut-T75K-E216M exhibit excellent relative enzyme activity, thermostability, and substrate tolerance. Furthermore, they demonstrate high activity and stability against various chiral alcohols, possessing advantages such as high catalytic activity, high thermostability, strong substrate tolerance, and ease of fermentation.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a carbonyl reductase mutant and its application in the enzymatic synthesis of chiral alcohols. Background Technology

[0002] Chiral alcohols are a class of optically active alcohols whose molecular structures contain one or more chiral centers, preventing the molecule from overlapping with its mirror image. Chiral alcohols have wide applications in the asymmetric synthesis of pharmaceuticals, pesticides, fragrances, flavorings, and natural products. Carbonyl reductase-mediated biocatalytic asymmetric reduction reactions are an important pathway for the synthesis of chiral alcohols.

[0003] Carbonyl reductase (CaRbonyl Reductase Se, EC1.1.1.148), a member of the oxidoreductase family, uses the coenzyme NADPH (nicotinamide adenine dinucleotide phosphate) as a hydrogen donor to catalyze the reduction and hydrogenation of a series of prochiral ketones or aldehydes to the corresponding secondary alcohols. Under the condition of H-donated coenzyme, carbonyl reductase can catalyze the asymmetric reduction of a series of carbonyl compounds. Its catalytic mechanism is as follows: the carbonyl reductase first combines with the coenzyme to form a complex, then collides with the substrate, transferring hydrogen from the coenzyme to the carbonyl group of the oxidizing substrate to form the corresponding chiral alcohol. Simultaneously, the coenzyme NAD(P)H is oxidized to NAD(P)+, and finally the product is released from the enzyme-coenzyme complex, completing the catalytic process. The coenzyme NAD(P)H attacks the carbonyl carbon from different directions, generating products with different stereoconfigurations. When the [H] at the C4 position of the nicotinamide ring of NAD(P)H attacks the carbonyl carbon of the substrate from the Re-face, it follows the PRelog rule, generating... S The product is of the [H] type; while when [H] attacks the substrate carbonyl carbon from the Si-face, it follows the anti-PRelog rule, generating [H]-type products; R Type products. Most carbonyl reductase-catalyzed reactions follow the PRelog rule.

[0004] Florfenicol is a widely used chloramphenicol antibiotic for veterinary use. (2) S ,3 R ethyl p-methylsulfonylbenzylserine (D-ethyl ester) and its analogues, as key precursors for the synthesis of florfenicol, contain adjacent chiral centers, making their synthesis challenging. Currently, industrial production of D-ethyl ester both domestically and internationally mainly employs aldol condensation and L-(+)-tartaric acid resolution. This method generates copper ammonia wastewater and copper sulfide solid waste, and the chiral resolution yield is only 41%, resulting in poor atom economy and high production costs. An asymmetric synthesis of D-ethyl ester and its analogues using dynamic reduction kinetics resolution of p-methylsulfonylphenyl-α-amino-β-keto ester substrates via carbonyl reductase can overcome the shortcomings of traditional routes.

[0005] Viberon is the first β3-adrenergic receptor agonist (β3-AR agonist) drug used to treat overactive bladder (OAB). (2) S ,3 R methyl 2-((tert-butoxycarbonyl)amino)-3-hydroxy-3-phenylpropionate (methyl(2-) S ,3 R )-2-((te R t-butoxycaRbonyl)amino)-3-hyd R oxy-3-phenylpRopanoate, (2 S ,3 R (-Aminohydroxy ester) is a key chiral intermediate in the synthesis of viberone. In existing techniques, studies have constructed coupling systems between the NADPH-dependent carbonyl reductase KRED and the glucose dehydrogenase GDH. S methyl 2-((tert-butoxycarbonyl)amino)-3-oxo-3-phenylpropionate (methyl( S )-2-((teRt-butoxycaRbonyl)amino) -3-oxo-3-phenylp R opanoate, ( S The asymmetric reduction of )-amino ketone ester yields product (2) S ,3 R The reaction of α-aminohydroxy esters with a substrate concentration of 85 g / L and a co-substrate glucose concentration of 70 g / L yielded a product yield >95.0% and ee >99.9% after 4 h. However, the yield of chiral alcohols synthesized by this enzymatic method for asymmetric reduction reaction is relatively low.

[0006] In addition, the key chiral intermediate of the 5-hydroxytryptamine-norepinephrine re-extraction inhibitor duloxetine ( S Ethyl 3-hydroxy-3-(2-thienyl)propionate S )-3-hydRoxy-3-(thiophen-2-yl)pRopanoate, ( S )-HEES), a key chiral intermediate (3) of the selective HMG-CoA reductase inhibitor rosuvastatin. R 5 S 6-Chloro-3,5-dihydroxyhexanoate tert-butyl ester (teRt-Butyl (3) R5 S )-6-chloRo-3,5-dihydRoxyh-exanoate, (3 R 5 S )-CDHH), the key chiral intermediate of the lipid-lowering drug atorvastatin (3 R 5 R Carbonyl reductase catalyzes (3) cyano-3,5-dihydroxyhexanoate tert-butyl ester, etc. R 5 S The asymmetric synthesis of )-CDHH is also a significant challenge in the field of green biomanufacturing of pharmaceutical chemicals.

[0007] Currently, the yields of the key chiral intermediates synthesized by carbonyl reductase-mediated biocatalytic asymmetric reduction reactions are low. Carbonyl reductases are multifunctional biocatalysts with a broad substrate activity spectrum, but their low thermal stability limits their applications. Summary of the Invention

[0008] To address the technical problem of low yield in the synthesis of chiral alcohols mediated by carbonyl reductase, this invention provides a carbonyl reductase mutant and its application in the enzymatic synthesis of chiral alcohols.

[0009] The specific technical solution of this invention is as follows:

[0010] In a first aspect, the present invention provides a recombinant carbonyl reductase mutant, obtained by performing single-point or multi-point combination mutations at the following sites on the amino acid sequence shown in SEQ ID NO.2:

[0011] The threonine at position 75 is mutated to any one of lysine, leucine, or aspartic acid.

[0012] The glutamic acid at position 216 is mutated to any one of methionine, serine, or lysine.

[0013] Secondly, the present invention provides the encoding gene of the above-mentioned carbonyl reductase mutant.

[0014] Thirdly, the present invention provides an expression vector containing the above-mentioned coding gene.

[0015] Preferably, the expression vector is a plasmid, bacteriophage, or viral vector.

[0016] Fourthly, the present invention provides a cloning vector for the above-mentioned encoded gene.

[0017] Fifthly, the present invention provides the above-mentioned carbonyl reductase mutant or the host cell for the above-mentioned encoding gene.

[0018] Preferably, the cells are Escherichia coli.

[0019] In a sixth aspect, the present invention provides the application of the above-mentioned carbonyl reductase mutant in the synthesis of chiral alcohols.

[0020] Using a prochiral ketone as a substrate, and with wet bacterial cells obtained by fermentation culture of genetically engineered bacteria containing the above-mentioned recombinant carbonyl reductase mutant encoding gene, or with crude or pure enzyme obtained by ultrasonic disruption of the wet bacterial cells as a catalyst, the corresponding chiral alcohol can be prepared by biocatalysis.

[0021] Preferably, the chiral alcohol is a duloxetine intermediate, florfenicol intermediate, rosuvastatin intermediate, atorvastatin intermediate, or veberon intermediate. That is, the corresponding prochiral ketone may include: ( S 2-((tert-butoxycarbonyl)amino)-3-oxo-3-phenylpropionate methyl ester, 2-acetamido-3-(4-(methylsulfonyl)phenyl)-3-oxopropionate methyl ester, 3-oxo-3-(2-thienyl)propionate ethyl ester, ( S 6-chloro-5-hydroxy-3-carbonylhexanoate tert-butyl ester S )-CHOH), 6-cyano-5( R )-hydroxy-3-oxohexanoate tert-butyl ester.

[0022] More specifically:

[0023] The application includes: using wet bacterial cells obtained by fermentation culture of genetically engineered bacteria containing a novel carbonyl reductase mutant encoding gene, or crude enzyme solution obtained from the crushing of wet bacterial cells, or pure enzyme extracted from the crushing of wet bacterial cells, as a catalyst, and using methyl 2-acetamido-3-(4-(methanesulfonyl)phenyl)-3-oxopropionate, to biocatalyze the preparation of (2... S ,3 R Methyl 2-acetamido-3-hydroxy-3-(4-(methylsulfonyl)phenyl)propionate is a key chiral intermediate in the synthesis of the drug florfenicol.

[0024] The application includes: using the wet cell culture obtained by fermentation of genetically engineered bacteria containing a novel carbonyl reductase mutant encoding gene, or the crude enzyme solution obtained by crushing the wet cell, or the pure enzyme extracted by crushing the wet cell, as a catalyst, and using ethyl 3-oxo-2-(3-thienyl)propionate (KEES) as a substrate, to biocatalyze the preparation of (… S Ethyl 3-hydroxy-3-(2-thienyl)propionate S )-HEES). S HEES is a drug. S It is a key chiral intermediate in the synthesis of duloxetine.

[0025] The application includes: using wet bacterial cells obtained by fermentation culture of genetically engineered bacteria containing a novel carbonyl reductase mutant encoding gene, or crude enzyme solution obtained from the crushing of wet bacterial cells, or pure enzyme extracted from the crushing of wet bacterial cells, as a catalyst, with ( S 6-chloro-5-hydroxy-3-hydroxyhexanoate tert-butyl ester (( S Using )-CHOH) as a substrate, (3) was prepared by biocatalysis. R 5 S 6-Chloro-3,5-dihydroxyhexanoate tert-butyl ester ((3) R 5 S )-CDHH). (3 R 5 S )-CDHH is a key chiral intermediate in the synthesis of the drug rosuvastatin.

[0026] The application includes: using wet bacterial cells obtained by fermentation culture of genetically engineered bacteria containing a novel carbonyl reductase mutant encoding gene, or crude enzyme solution obtained by crushing wet bacterial cells, or pure enzyme extracted by crushing wet bacterial cells as a catalyst, with 6-cyano-5 ( R Using tert-butyl 3-hydroxy-3-oxohexanoate as a substrate, (3) was prepared by biocatalysis. R 5 R 6-Cyano-3,5-dihydroxyhexanoate tert-butyl ester. This is a key chiral intermediate in the synthesis of the drug atorvastatin.

[0027] The application includes: using wet bacterial cells obtained by fermentation culture of genetically engineered bacteria containing a novel carbonyl reductase mutant encoding gene, or crude enzyme solution obtained from the crushing of wet bacterial cells, or pure enzyme extracted from the crushing of wet bacterial cells, as a catalyst, with ( S methyl 2-((tert-butoxycarbonyl)amino)-3-oxo-3-phenylpropionate (( S Using 2-aminoketone esters as substrates, biocatalysis was used to prepare (2) S ,3 R methyl 2-((tert-butoxycarbonyl)amino)-3-hydroxy-3-phenylpropionate ((2 S ,3 R )-aminohydroxy ester), (2 S ,3 R )-Aminohydroxy esters are key chiral intermediates in the synthesis of veberon.

[0028] Compared with the prior art, the present invention has the following technical effects:

[0029] This invention mutates the amino acid sequence shown in SEQ ID NO.2 to obtain carbonyl reductase mutants with varying degrees of enhanced activity compared to the wild type, especially the single-point mutants Mut-T75K, Mut-T75D, Mut-E216M and the combined mutant Mut-T75K-E216M. These mutants exhibit excellent relative enzyme activity, thermal stability, and substrate tolerance, and show high activity and stability against a variety of chiral alcohols. They also possess advantages such as high catalytic activity, high thermal stability, strong substrate tolerance, and ease of fermentation. Attached Figure Description

[0030] Figure 1 The image shows an SDS-PAGE image of carbonyl reductase and its mutants. Lane M represents the molecular weight of the protein, lane 1 represents the pure carbonyl reductase from the original strain WT, lane 2 represents the pure Mut-T75K enzyme, lane 3 represents the pure Mut-E216M enzyme, and lane 4 represents the pure Mut-T75K-E216M enzyme. Detailed Implementation

[0031] The present invention will be further described below with reference to embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only some, not all, of the embodiments of the present invention. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0032] In this embodiment of the invention, the amino acid sequence of the wild-type carbonyl reductase is shown in SEQ ID NO. 2, and its encoding gene is shown in SEQ ID NO. 1.

[0033] Example 1 Construction of carbonyl reductase mutant

[0034] (1) The wild-type carbonyl reductase encoding gene (nucleotide sequence as shown in SEQ ID NO. 1) was ligated to the expression vector pET28b to construct a heterologous expression recombinant plasmid pET28b(+)-WT containing the carbonyl reductase encoding gene. The expression recombinant plasmid was then transformed into the host bacteria. E. coli Recombinant genetically engineered bacteria containing a recombinant plasmid expressing carbonyl reductase were obtained from BL21(DE3) competent cells. E. coli BL21(DE3) / pET28b(+)-WT, i.e., engineered carbonyl reductase bacteria.

[0035] The engineered carbonyl reductase strain *E. coli* BL21(DE3) / pET28b(+)-WT was thawed on ice, and the bacterial culture was streaked onto kanamycin-resistant LB agar plates and incubated at 37 °C for 12 h. A single colony was picked and inoculated into a 10 mL LB tube with a final kanamycin concentration of 50 mg / L, and cultured at 37 °C with shaking at 180 rpm for 7 h. 2 mL of the bacterial culture from the tube was then inoculated into a 100 mL LB shake flask with a final kanamycin concentration of 50 mg / L, and cultured at 37 °C with shaking at 180 rpm for 2 h. 100 µL of 0.1 M IPTG was added, and the culture was incubated at 28 °C with shaking at 180 rpm for 12 h. The cells were collected by centrifugation at 4 °C and 8000 rpm for 15 min and stored at -20 °C for later use.

[0036] (2) Using the prepared vector heterologous expression recombinant plasmid pET28b(+)-WT as a template, the site-directed mutagenesis primers shown in Table 1 were used to introduce mutations by PCR. In Table 1, the underlined primer sequences indicate the mutation sites. Primers T75-R and T75K-F were used in PCR to mutate threonine at position 75 of the wild-type enzyme to lysine; primers T75-R and T75L-F were used in PCR to mutate threonine at position 75 of the wild-type enzyme to leucine; primers T75-R and T75D-F were used in PCR to mutate threonine at position 75 of the wild-type enzyme to aspartic acid; primers E216-R and E216M-F were used in PCR to mutate glutamate at position 216 of the wild-type enzyme to methionine; primers E216-R and E216S-F were used in PCR to mutate glutamate at position 216 of the wild-type enzyme to serine; and primers E216-R and E216K-F were used in PCR to mutate glutamate at position 216 of the wild-type enzyme to lysine.

[0037] Table 1

[0038]

[0039] The PCR reaction procedure for introducing mutations via PCR is as follows: 98 ℃ for 10 min, 98 ℃ for 30 s, 56 ℃ for 30 s, 72 ℃ for 3 min 40 s, repeated for 31 cycles; followed by a 5 min extension at 72 ℃.

[0040] The PCR products were inactivated by treating with DpnI at 37 °C for 3 h and then transformed into E. coli BL21(DE3) recipient bacteria. The products were plated on LB agar plates containing a final concentration of 50 mg / L kanamycin and cultured at 37 °C for 12 h. Single colonies were randomly selected for sequencing analysis to confirm the results, and the various carbonyl reductase mutants and their wet cells were obtained.

[0041] The mutant enzyme with the 75th threonine mutated to lysine is designated Mut-T75K; the mutant enzyme with the 75th threonine mutated to leucine is designated Mut-T75L; the mutant enzyme with the 75th threonine mutated to aspartic acid is designated Mut-T75D; the mutant enzyme with the 216th glutamate mutated to methionine is designated Mut-E216M; and the mutant enzyme with the 216th glutamate mutated to serine is designated Mut-E216. S The mutant enzyme in which the glutamic acid at position 216 of the wild-type enzyme is mutated to lysine is denoted as Mut-E216K.

[0042] (3) Using plasmid DNA containing the Mut-T75K gene as a template, mutations were introduced by PCR. The following combination of mutation primers were used (the underlined positions are the mutation sites):

[0043]

[0044] The PCR reaction procedure for introducing the mutation via PCR is the same as step (2), yielding the recombinant carbonyl reductase mutant Mut-T75K-E216M and its wet cells. The nucleotide sequence of the mutant Mut-T75K-E216M is shown in SEQ ID NO. 3, and the amino acid sequence is shown in SEQ ID NO. 4.

[0045] (4) Using plasmid DNA containing the Mut-T75K gene as a template, mutations were introduced by PCR. The following are the combination of mutation primers (the underlined positions are the mutation sites):

[0046]

[0047] The PCR reaction procedure for introducing mutations by PCR is the same as step (2), and the recombinant carbonyl reductase combined mutant Mut-T75K-E216K and its wet cells are obtained.

[0048] like Figure 1 The image shown is an SDS-PAGE image of the above-mentioned wild-type carbonyl reductase and its mutants. Figure 1 In the diagram, lane M represents the molecular weight of the protein, lane 1 represents the carbonyl reductase from the original strain WT, lane 2 represents the Mut-T75K pure enzyme, lane 3 represents the Mut-E216M pure enzyme, and lane 4 represents the Mut-T75K-E216M pure enzyme.

[0049] Example 2 Screening of highly thermally stable carbonyl reductase mutants

[0050] The carbonyl reductase mutant obtained in Example 1 was analyzed by measuring its residual enzyme activity at 35 °C. oAfter incubation at C for 24 hours, 50 o After incubation at C for 1 hour, the remaining enzyme activity was measured to determine the optimal mutant. The steps are as follows:

[0051] (1) Preparation of crude enzyme solution: The strain containing the carbonyl reductase mutant encoding gene obtained in Example 1 was inoculated into LB liquid medium containing a final concentration of 50 mg / L kanamycin resistance and cultured at 37 ℃ and 180 Rpm for 7 h. Then, it was inoculated into fresh LB liquid medium containing a final concentration of 50 mg / L kanamycin resistance at a volume concentration of 2% and cultured at 37 ℃ and 180 Rpm for 2 h. 100 µL of 0.1 M IPTG was added and induced cultured at 28 ℃ and 180 Rpm for 12 h. After centrifugation at 4 ℃ and 8000 Rpm for 15 min, the supernatant was discarded and the wet bacterial cells were collected. 125 mg of the wet bacterial cells of the mutant was weighed and resuspended in 10 mL of sodium phosphate buffer and cultured at 4 ℃. o Sonication was performed under ice bath conditions until the bacterial solution became clear (220 W, 1 second, 2 seconds). Each crude enzyme solution was then incubated at 35°C. o Incubate at C for 24 hours, then at 50°C o After incubation at C for 1 hour.

[0052] (2) 500 µL reaction system: 150 µL NADP + 50 µL glucose, 50 µL of 35 o Crude enzyme solution incubated at C for 24 h, 100 µL substrate ( S The α-aminoketone ester stock solution was brought to a final volume of 500 µL with sodium phosphate buffer, shaken at 1100 rpm, and incubated at 35°C. o The reaction was carried out in a shaking reactor of type C, and samples were taken immediately after 10 minutes of reaction. The mother liquor substrate ( S The concentration of α-amino ketone ester was 50 g / L (solvent: dimethyl sulfoxide), the concentration of glucose as a cosubstrate in the mother liquor was 180 g / L (solvent: sodium phosphate buffer), and the concentration of NADP coenzyme in the mother liquor was... + The concentration was 1 g / L (solvent was sodium phosphate buffer). The concentration was determined by HPLC (2...). S ,3 R The yield of α-aminohydroxy esters was determined and the relative enzyme activity was calculated. The initial enzyme activity of each mutant was set as 100%, and the remaining enzyme activity after incubation for a certain period of time is shown in Table 2.

[0053] (3) 500 µL reaction system: 150 µL NADP + 50 µL glucose, 50 µL of 50 o Crude enzyme solution incubated at C for 1 h, 100 µL substrate ( SThe α-aminoketone ester stock solution was brought to a final volume of 500 µL with sodium phosphate buffer, shaken at 1100 rpm, and incubated at 50 °C. o The reaction was carried out in a shaking reactor of type C, and samples were taken immediately after 10 minutes of reaction. The mother liquor substrate ( S The concentration of α-amino ketone ester was 50 g / L (solvent: dimethyl sulfoxide), the concentration of glucose as a cosubstrate in the mother liquor was 180 g / L (solvent: sodium phosphate buffer), and the concentration of NADP coenzyme in the mother liquor was... + The concentration was 1 g / L (solvent was sodium phosphate buffer). The concentration was determined by HPLC (2...). S ,3 R The yield of α-aminohydroxy esters was determined and the relative enzyme activity was calculated. The initial enzyme activity of each mutant was set as 100%, and the remaining enzyme activity after incubation for a certain period of time is shown in Table 2.

[0054] Table 2 Comparison of Remaining Enzyme Activities

[0055] enzymes <![CDATA[35 o Remaining enzyme activity (%) after 24 hours of incubation at C <![CDATA[50 o Remaining enzyme activity (%) after 1 h of incubation at C wild type 36.89 1.75 Mut-T75K 89.21 68.95 Mut-T75L 75.86 57.56 Mut-T75D 77.32 58.79 Mut-E216M 55.65 19.81 Mut-E216 43.62 12.50 Mut-E216K 48.57 11.65 Mut-T75K-E216K 53.23 12.36 Mut-T75K-E216M 91.61 78.67

[0056] As shown in Table 2, the results of the residual enzyme activity determination indicate that Example 1 improved the stability of carbonyl reductase through site-directed mutagenesis. The optimal mutant for a single mutant was Mut-T75K, and the optimal mutant obtained after combining mutations at different mutation sites was Mut-T75K-E216M. In addition, the single mutants Mut-T75D and Mut-E216M also showed good thermal stability.

[0057] As shown in Table 2, the carbonyl reductase mutants Mut-T75K, Mut-T75D, and Mut-T75K-E216M not only showed improved catalytic activity, but also significantly enhanced thermal stability.

[0058] Example 3: Determination of relative enzyme activity of carbonyl reductase mutant

[0059] The relative enzyme activity of the carbonyl reductase mutant obtained in Example 1 was determined. The steps are as follows:

[0060] (1) Preparation of crude enzyme solution: The method is the same as in Example 2.

[0061] (2) 500 µL reaction system: 150 µL NADP + 50 µL glucose, 50 µL of 35 o Crude enzyme solution incubated at C for 24 h, 100 µL substrate ( S The α-aminoketone ester stock solution was brought to a final volume of 500 µL with sodium phosphate buffer, shaken at 1100 rpm, and incubated at 35°C. o The reaction was carried out in a shaking reactor of type C, and samples were taken immediately after 10 minutes of reaction. The mother liquor substrate (S The concentration of α-amino ketone ester was 50 g / L (solvent: dimethyl sulfoxide), the concentration of glucose as a cosubstrate in the mother liquor was 180 g / L (solvent: sodium phosphate buffer), and the concentration of NADP coenzyme in the mother liquor was... + The concentration was 1 g / L (solvent was sodium phosphate buffer). The concentration was determined by HPLC (2...). S ,3 R The yield of α-aminohydroxy esters and the relative enzyme activity were calculated, and the results are shown in Table 3.

[0062] Table 3 Comparison of relative enzyme activities

[0063] enzymes Relative enzyme activity (100%) wild type 100.00 Mut-T75K 111.55 Mut-T75L 102.31 Mut-T75D 101.79 Mut-E216M 105.65 Mut-E216 98.51 Mut-E216K 95.59 Mut-T75K-E216M 99.56 Mut-T75K-E216M 116.55

[0064] As shown in Table 3, the relative enzyme activity results indicate that Example 1 improved the stability of carbonyl reductase through site-directed mutagenesis, and the mutants Mut-T75K, Mut-E216M, and Mut-T75K-E216M exhibited superior relative enzyme activity.

[0065] Example 4: Application of carbonyl reductase mutant in the preparation of florfenicol intermediates

[0066] The single-point mutants Mut-T75K, Mut-E216M, and the combined mutant Mut-T75K-E216M, which exhibit superior thermal stability, were selected for the preparation of florfenicol intermediates. The specific steps are as follows:

[0067] The composition and catalytic conditions of the catalytic system are as follows:

[0068] In a 20 mL reaction system: add 100 mM potassium phosphate buffer solution (pH 7.0), DMSO (20% concentration), crude carbonyl reductase solution (20 g / L, prepared in the same way as in Example 2), glucose dehydrogenase (10 g / L), 200 mM glucose, and NADP. + 2 g / L, substrate methyl 2-acetamido-3-(4-(methanesulfonyl)phenyl)-3-oxopropionate 100 g / L, and bring to 20 mL with sodium phosphate buffer.

[0069] The reaction system was placed at 35°C. o The reaction was carried out in a C water bath at 600 rpm with a magnetic stirrer for 24 h. Samples were taken periodically during the reaction, with a sample volume of 200 μL. After separation and purification, the samples were diluted 100 times with pure acetonitrile, and the conversion rate was determined by HPLC analysis.

[0070] The results showed that after 24 h of catalysis, WT catalytic production (2 S ,3 RThe yields of methyl 2-acetamido-3-hydroxy-3-(4-(methanesulfonyl)phenyl)propionate reached 76.9%, with ee>99%; Mut-T75K reached 92.1%, with ee>99%; Mut-E216M reached 87.2%, with ee>99%; and Mut-T75K-E216M reached 99.2%, with ee>99%.

[0071] Example 5: Application of carbonyl reductase mutant in the preparation of duloxetine intermediates

[0072] The single-point mutants Mut-T75K, Mut-E216M, and the combined mutant Mut-T75K-E216M, which exhibit superior thermal stability, were selected for the preparation of duloxetine intermediates. The specific steps are as follows:

[0073] The composition and catalytic conditions of the catalytic system are as follows:

[0074] 20 mL reaction system: 20 g / L crude enzyme solution (prepared using the same method as in Example 2), 150 g / L substrate KEES, 100 mM glucose co-substrate (solvent: sodium phosphate buffer), glucose dehydrogenase (10 g / L), and coenzyme NADP. + The concentration is 3 g / L (solvent is sodium phosphate buffer). Bring the volume to 20 mL using sodium phosphate buffer.

[0075] The reaction system was placed at 35°C. o A water bath at C was used, and a magnetic stirrer at 600 rpm was used to react for 24 hours. Samples were taken at regular intervals, with a sample volume of 200 μL. After separation and purification, the samples were diluted 100 times with pure acetonitrile, and the conversion rate was determined by HPLC analysis.

[0076] The results showed that after 24 h of catalysis, WT catalytic production ( S The yield of )-HEES reached 84.9%, ee>99%; Mut-T75K reached 90.1%, ee>99%; Mut-E216M reached 89.8%, ee>99%; Mut-T75K-E216M reached 98.8%, ee>99%.

[0077] Example 6: Application of carbonyl reductase mutant in the preparation of rosuvastatin intermediates

[0078] The single-point mutants Mut-T75K, Mut-E216M, and the combined mutant Mut-T75K-E216M, which exhibit superior thermal stability, were selected for the preparation of rosuvastatin intermediates. The specific steps are as follows:

[0079] The composition and catalytic conditions of the catalytic system are as follows:

[0080] 20 mL reaction system: Weigh 20 g / L of crude enzyme solution (preparation method is the same as in Example 2), substrate ( S 150 g / L CHOH, 100 mM glucose (sodium phosphate buffer), glucose dehydrogenase (10 g / L), and NADP coenzyme. + The concentration is 3 g / L (solvent is sodium phosphate buffer). Bring the volume to 20 mL using sodium phosphate buffer.

[0081] The reaction system was placed at 35°C. o A water bath at C was used, and a magnetic stirrer at 600 rpm was used to react for 24 hours. Samples were taken at regular intervals, with a sample volume of 200 μL. After separation and purification, the samples were diluted 40 times with 30% acetonitrile, and the conversion rate was determined by HPLC analysis.

[0082] The results showed that after 24 h of catalysis, WT catalytic production (3 R 5 S The yield of )-CDHH reached 86.9%, ee>99%; Mut-T75K reached 95.8%, ee>99%; Mut-E216M reached 94.9%, ee>99%; Mut-T75K-E216M reached 99.8%, ee>99%.

[0083] Example 7: Application of carbonyl reductase mutant in the preparation of atorvastatin intermediates

[0084] The single-point mutants Mut-T75K, Mut-E216M, and the combined mutant Mut-T75K-E216M, which exhibit superior thermal stability, were selected for the preparation of atorvastatin intermediates. The specific steps are as follows:

[0085] The composition and catalytic conditions of the catalytic system are as follows:

[0086] 20 mL reaction system: 20 g / L crude enzyme solution (preparation method same as in Example 2), substrate 6-cyano-5 ( R 150 g / L tert-butyl 3-hydroxy-3-oxohexanoate, 100 mM glucose (sodium phosphate buffer), glucose dehydrogenase (10 g / L), and NADP coenzyme. + The concentration is 3 g / L (solvent is sodium phosphate buffer). Bring the volume to 20 mL using sodium phosphate buffer.

[0087] The reaction system was placed at 35°C. oA C-type water bath was used, and a magnetic stirrer was employed at 600 rpm. The reaction was carried out for 24 hours, with 200 μL samples taken periodically. After separation and purification, the samples were diluted 100-fold with acetonitrile, and the conversion rate was determined by HPLC. The results showed that after 24 hours of catalysis, WT catalyzed the production of (3... R 5 R The yields of tert-butyl 6-cyano-3,5-dihydroxyhexanoate reached 80.2%, with ee>99%; Mut-T75K reached 90.8%, with ee>99%; Mut-E216M reached 88.5%, with ee>99%; and Mut-T75K-E216M reached 99.8%, with ee>99%.

[0088] Example 8: Application of carbonyl reductase mutant in the preparation of viberon intermediate

[0089] The single-point mutants Mut-T75K, Mut-E216M, and the combined mutant Mut-T75K-E216M, which exhibit superior thermal stability, were selected for the preparation of viberon intermediates. The specific steps are as follows:

[0090] The composition and catalytic conditions of the catalytic system are as follows:

[0091] 20 mL reaction system: substrate ( S The α-aminoketone ester was dissolved in 10 mL of n-butyl acetate (final concentration 100 g / L), and 1 mL of 0.1 mM, pH 6.5 NaH₂PO₄-Na₂HPO₄ buffer solution was added. Crude enzyme solution (final concentration 20 g / L, prepared in the same way as in Example 2) was added, along with 2 mL of crude glucose dehydrogenase solution (final concentration 10 g / L), 100 mg of NADPH coenzyme, and 2.63 g of glucose cosubstrate. The solution was brought to a final volume of 20 mL using sodium phosphate buffer.

[0092] The reaction system was placed at 35°C. o A C water bath was used, and a magnetic stirrer was employed at 600 rpm. Samples were taken periodically at 200 µL. After separation and purification, the samples were diluted 100 times with pure acetonitrile, and the conversion rate was determined by HPLC analysis.

[0093] The results showed that after 24 h of catalysis, WT catalytic production (2 S ,3 R The yield of α-aminohydroxy ester reached 89.2%, ee>99%; Mut-T75K reached 94.2%, ee>99%; Mut-E216M reached 91.1%, ee>99%; Mut-T75K-E216M reached 99.7%, ee>99%.

[0094] Example 9: Effect of Feeding Process on the Preparation of Viberon Intermediate from Carbonyl Reductase Mutant

[0095] The single-point mutants Mut-T75K, Mut-E216M, and the combined mutant Mut-T75K-E216M, which exhibit superior thermal stability, were selected for use in the fed-batch process to prepare viberon intermediates. The specific steps are as follows:

[0096] (1) 20 mL reaction system: the substrate ( S Dissolve α-aminoketone ester in 10 mL of n-butyl acetate (final concentration 80 g / L), add 1 mL of 0.1 mM, pH 6.5 NaH₂PO₄-Na₂HPO₄ buffer solution, add crude enzyme solution (final concentration 20 g / L), add glucose dehydrogenase (final concentration 10 g / L), add 100 mg of coenzyme NADPH, and add 2.63 g of glucose as a cosubstrate. Bring the volume to 20 mL using sodium phosphate buffer.

[0097] The reaction system was placed at 35°C. o C-type water bath, magnetic stirring device 600 R At 8 h, 1.4 g of substrate was added as a feedstock (total substrate concentration after feedstock addition was 150 g / L). Samples were taken at regular intervals, with a sample volume of 200 µL. After separation and purification, the samples were diluted 150 times with pure acetonitrile, and the conversion rate was determined by HPLC analysis.

[0098] The results showed that after 24 h of catalysis, WT catalytic production (2 S ,3 R The yields of )-aminohydroxy esters reached 89.5% (WT) and 99% (ee>99%); Mut-T75K reached 96.2% (ee>99%); Mut-E216M reached 92.1% (ee>99%); and Mut-T75K-E216M reached 99.8% (ee>99%).

[0099] (2) 500 mL reaction system: the substrate ( S )-Amino ketone ester dissolved in 10 mL of n-butyl acetate (( S Add the following: α-aminoketoester to a final concentration of 80 g / L; crude enzyme solution (final concentration 20 g / L); glucose dehydrogenase (final concentration 10 g / L); 3.0 g of NADPH coenzyme; and 35.5 g of glucose cosubstrate. Bring the solution to a final volume of 500 mL using sodium phosphate buffer.

[0100] The reaction system was placed in a 35°C water bath with the stirring device set to 600 Rpm. After 8 h, 35 g of substrate was added as feed (the total substrate concentration after feed was 150 g / L). Samples were taken at regular intervals, with a sample volume of 200 μL. After separation and purification, the sample was diluted 150 times with pure acetonitrile, and the conversion rate was determined by HPLC analysis.

[0101] The results showed that after 24 h of catalysis, WT catalytic production (2 S ,3 R The yield of α-aminohydroxy ester reached 91.7%, with ee>99%; Mut-T75K reached 97.5%, with ee>99%; Mut-E216M reached 95.8%, with ee>99%; and Mut-T75K-E216M reached 99.9%, with ee>99%. Compared with the non-feeding process, the feeding process further improved production efficiency.

[0102] Unless otherwise specified, the raw materials and equipment used in this invention are all commonly used in the field; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0103] 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, alterations, and equivalent transformations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A carbonyl reductase mutant, characterized by: It is obtained by performing any of the following mutations on the amino acid sequence shown in SEQ ID NO.2: The threonine at position 75 is mutated to lysine; Alternatively, threonine at position 75 may be mutated to lysine and glutamic acid at position 216 may be mutated to methionine.

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

3. An expression vector comprising the encoding gene of claim 2.

4. The expression vector of claim 3, wherein: The expression vector is a plasmid, bacteriophage, or viral vector.

5. A host cell comprising the encoding gene of claim 2.

6. The host cell of claim 5, wherein: The cells were Escherichia coli.

7. Use of the carbonyl reductase mutant according to claim 1 for the synthesis of chiral alcohols, characterized in that: The synthesized chiral alcohol is: Methyl (2S,3R)-2-acetamido-3-hydroxy-3-(4-(methanesulfonyl)phenyl)-3-oxopropionate was synthesized using methyl 2-acetamido-3-(4-(methanesulfonyl)phenyl)-3-oxopropionate as a substrate. (S)-3-hydroxy-3-(2-thienyl)propionate ethyl ester was synthesized using 3-oxo-2-(3-thienyl)propionate ethyl ester as a substrate. (3R,5S)-CDHH was synthesized using (S)-CHOH as a substrate. Using tert-butyl 6-cyano-5(R)-hydroxy-3-oxohexanoate as a substrate, tert-butyl (3R,5R)-6-cyano-3,5-dihydroxyhexanoate was synthesized. Alternatively, (2S,3R)-aminohydroxy esters can be synthesized using (S)-amino ketone esters as substrates.

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