Carbonyl reductase mutants, combinatorial enzymes and their use in the synthesis of s-configured boswellic acids

Abstract

The application discloses a carbonyl reductase mutant, a combined enzyme and application of the combined enzyme in synthesis of S-configuration boselike, and belongs to the technical field of biological catalysis and pharmacy. The amino acid sequence of the wild-type carbonyl reductase mutant provided by the application is shown as SEQ ID NO:1, and the mutant is obtained by mutation of the wild-type carbonyl reductase, and the mutation mode is that F at the 147th position is mutated into L, V at the 163rd position is mutated into Y, L at the 176th position is mutated into E, and P at the 200th position is mutated into R. By using the mutant, a catalytic system is formed by combining the mutant with formic acid dehydrogenase (FDH), and optical pure S-configuration boselike can be efficiently and highly selectively prepared under high substrate concentration of 250-320 g / L, separation and purification problems caused by generation of a large amount of soluble by-products in catalysis of other combined enzymes are avoided, a post-treatment process is obviously simplified, and production cost and environmental burden are reduced.

Description

Technical Field

[0001] This invention relates to the field of biocatalysis pharmaceutical technology, and in particular to a carbonyl reductase mutant, a combinatorial enzyme, and its application in the synthesis of S-configuration Bosine. Background Technology

[0002] The information disclosed in the background section of this invention is intended only to enhance the understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Pro-Xylane is a bioactive β-C-glycoside compound widely used in anti-aging skincare products, capable of stimulating the synthesis of glycosaminoglycans in the skin's extracellular matrix. Studies have shown that its S-configuration isomer exhibits higher bioactivity than the R-configuration; therefore, developing efficient and selective methods for synthesizing the S-configuration of pro-Xylane has significant application value.

[0004] Currently, the synthesis of Bosein mainly involves two technical routes: chemical synthesis and biocatalysis. Chemical synthesis methods often involve the use of reagents such as boric acid, which makes it difficult to completely separate residual boric acid from the product. This not only increases purification costs but also poses potential safety hazards, thus restricting its large-scale production.

[0005] Biocatalysis, particularly the asymmetric reduction using carbonyl reductases, has become a promising synthetic approach due to its inherent high stereoselectivity and environmental friendliness. However, current biocatalytic technologies still face several bottlenecks. On the one hand, the catalytic activity and conversion efficiency of naturally occurring or reported carbonyl reductases for the substrate β-pyruvate are often insufficient, making it difficult to meet the industrial demands for complete conversion of high-concentration substrates. On the other hand, the coenzyme NADP is required during the catalytic process. + The continuous regeneration of nicotinamide adenine dinucleotide phosphate (NAPDP) is problematic, as commonly used regeneration systems (such as glucose dehydrogenase systems) produce a large number of acidic byproducts. These byproducts are similar in properties to the target product and are difficult to separate, which not only increases the difficulty and cost of post-processing but also affects the purity of the final product.

[0006] Therefore, developing a carbonyl reductase with high catalytic activity, high S-configuration selectivity, and the ability to avoid generating difficult-to-separate byproducts, along with a matching coenzyme regeneration system, is crucial for achieving efficient, green, and low-cost industrial production of S-configuration Bosein. Summary of the Invention

[0007] In view of this, the present invention provides a carbonyl reductase mutant, a combination enzyme, and its application in the synthesis of S-configuration Bosein. The carbonyl reductase mutant provided by the present invention is derived from [a specific enzyme] using error-prone PCR technology. Levilactobacillus brevisThe wild-type carbonyl reductase (EVBA) gene was randomly mutated and screened for activity and stereoselectivity. Using this mutant, it was combined with formate dehydrogenase (FDH) to form a catalytic system that can efficiently and selectively prepare optically pure S-configuration Bosein at high substrate concentrations.

[0008] In a first aspect, the present invention provides a carbonyl reductase mutant, which is obtained by mutating a wild-type carbonyl reductase with the amino acid sequence shown in SEQ ID NO:1, wherein the mutation method is as follows:

[0009] The F at position 147 is mutated to L, the V at position 163 to Y, the L at position 176 to E, and the P at position 200 to R.

[0010] Preferably, the amino acid sequence of the carbonyl reductase mutant is shown in SEQ ID NO: 3.

[0011] In a second aspect, the present invention provides a nucleic acid molecule that encodes a carbonyl reductase mutant as described in the first aspect.

[0012] Preferably, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO: 4.

[0013] Thirdly, the present invention provides an expression vector comprising the nucleic acid molecule as described in the second aspect.

[0014] Fourthly, the present invention provides a recombinant strain that expresses the carbonyl reductase mutant as described in the first aspect, or carries the nucleic acid molecule as described in the second aspect, or contains the expression vector as described in the third aspect.

[0015] Fifthly, the present invention provides a combinatorial enzyme comprising a carbonyl reductase mutant and formate dehydrogenase as described in the first aspect.

[0016] Preferably, the formate dehydrogenase is derived from... Thiobacillus sp. KNK65MA, whose amino acid sequence is shown in SEQ ID NO: 5.

[0017] In a sixth aspect, the present invention provides the application of the carbonyl reductase mutant described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the recombinant strain described in the fourth aspect, or the combination enzyme described in the fifth aspect in the synthesis of S-configuration Bosonicine.

[0018] In a seventh aspect, the present invention provides a method for synthesizing S-configuration bosonicine, comprising the following steps:

[0019] The carbonyl reductase mutant described in the first aspect, the recombinant strain described in the fourth aspect, or the combination enzyme described in the fifth aspect are contacted with the reaction substrate β-acetone xyloside, and a catalytic reaction is carried out in the presence of a coenzyme cycle system to obtain S-configuration Bosein.

[0020] Preferably, the pH of the catalytic reaction system is 6.5-7.5; the concentration of the reaction substrate is 250-320 g / L; the reaction time is 12-24 hours; and the reaction temperature is 28-32℃.

[0021] Compared with the prior art, the present invention has achieved the following beneficial effects:

[0022] (1) This invention provides a carbonyl reductase mutant with mutations at four specific sites (F147L, V163Y, L176E, P200R). Compared with its wild type, this mutant exhibits significantly improved catalytic efficiency for the substrate β-pyruvate, enabling complete conversion of high-concentration substrates. Simultaneously, this mutant demonstrates strict enantioselectivity, with the S-configuration of Bosein in the catalytic product reaching 100%, ensuring the optical purity of the product and solving the common enantiomeric separation problem in chemical synthesis.

[0023] (2) This invention further provides a combined enzyme comprising the carbonyl reductase mutant and formate dehydrogenase. In traditional carbonyl reductase and glucose dehydrogenase combined systems, separation and purification are difficult due to the generation of large amounts of soluble sodium gluconate and residual glucose. However, the combined enzyme system of this invention enables highly efficient in-situ regeneration of the coenzyme, and the byproducts generated during the regeneration process are non-toxic gases that are easily and automatically removed from the reaction system. This feature fundamentally avoids the separation and purification difficulties of traditional combined enzymes, significantly simplifies the post-processing steps, and reduces production costs and environmental burden.

[0024] (3) The preparation method disclosed in this invention, under optimized reaction conditions, can achieve complete conversion of β-acetone xyloside within 16 hours at a high substrate concentration of 300 g / L. This process route combines a highly active and selective biocatalyst with a green and simplified reaction-separation system, which significantly improves production intensity and efficiency, and provides a practical technical solution for the large-scale, low-cost biomanufacturing of S-configuration Bosein. Attached Figure Description

[0025] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments and descriptions of the invention are used to explain the invention and do not constitute an undue limitation thereof. Obviously, those skilled in the art can obtain other drawings based on these drawings without any inventive effort.

[0026] Figure 1 This is a liquid chromatogram of the EVBA-T1 enzyme-catalyzed reaction solution in Example 4 of the present invention;

[0027] Figure 2 This is a liquid chromatogram of the EVBA-T2 enzyme-catalyzed reaction solution in Example 4 of the present invention;

[0028] Figure 3 This is a liquid chromatogram of the EVBA-T3 enzyme-catalyzed reaction solution in Example 4 of the present invention;

[0029] Figure 4 This is a liquid chromatogram of the EVBA-T4 enzyme-catalyzed reaction solution in Example 4 of the present invention;

[0030] Figure 5 This is a liquid chromatogram of the product obtained by post-treatment of the EVBA-T2 enzyme-catalyzed reaction solution in Example 4 of the present invention. Detailed Implementation

[0031] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0032] Unless otherwise stated, the terms used in this invention have the following meanings:

[0033] The term "vector" refers to a nucleic acid delivery system that allows the insertion of a target nucleotide sequence and the introduction of the carried genetic material into a host cell for replication and / or expression. When a vector contains a regulatory element that enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be transformed into host cells.

[0034] The term "recombinant strain" refers to a strain that has been introduced with exogenous nucleic acid molecules (such as the gene encoding the mutant of this invention) or expression vectors through genetic engineering, thereby altering its genetic background and enabling it to express proteins that were not originally present or to overexpress specific proteins.

[0035] This invention provides a carbonyl reductase mutant, which is obtained by mutating the wild-type carbonyl reductase with the amino acid sequence shown in SEQ ID NO: 1. The mutation method is as follows:

[0036] The phenylalanine (F) at position 147 was mutated to leucine (L), the valine (V) at position 163 was mutated to tyrosine (Y), the leucine (L) at position 176 was mutated to glutamic acid (E), and the proline (P) at position 200 was mutated to arginine (R).

[0037] The combined mutations at the aforementioned sites significantly altered the enzyme's catalytic performance, thereby simultaneously enhancing its catalytic activity and stereoselectivity towards the substrate β-pyruvate. Compared to the wild type, this carbonyl reductase mutant exhibited significantly improved catalytic efficiency, and in the asymmetric reduction reaction of β-pyruvate, the enantiomeric excess (ee) of the product S-configuration Bosein was greater than 99.9%, meaning its optical purity was close to 100%.

[0038] In this invention, the amino acid sequence (SEQ ID NO: 1) of the wild-type carbonyl reductase is shown below:

[0039] MSNRLDGKVAIVTGGTLGIGLAIATKFVEEGAKVMITGRHSDVGEKAAKSVGTPDQIQFFQHDSSDEDGWTKLFDATEKAFGPVSTLVNNAGIAVNKSVEETTTAEWRKLLAVNLDGVFFGTRLGI QRMKNKGLGASIINMSSIEGFVGDPSLGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGYIKTPLVDDLPGAEEAMSQRTKTPMGHIGEPNDIAYICVYLASNESKFATGSEFVVDGGYTAQ.

[0040] The codon-optimized nucleotide sequence of the wild-type carbonyl reductase (SEQ ID NO: 2) is shown below:

[0041] ATGTCCAACCGTCTGGATGGTAAAGTAGCCATCGTGACTGGTGGTACTCTGGGTATCGGCCTGGCTATCGCGACCAAATTCGTGGAAGAAGGTGCAAAGGTGATGATCACCGGCCGTCATAGCGACGTAGGTGAGAAAGCGGCGAAATCCGTAGGTACGCCGGATCAGATCCAGTTCTTCCAGCACGACAGCTCTGACGAAGATGGTTGGACCAAACTGTTCGACGCTACTGAGAAAGCGTTTGGTCCGGTGTCTACCTTGGTTAACAACGCGGGTATCGCGGTTAACAAATCCGTGGAAGAAACCACCACCGCAGAATGGCGTAAACTGCTGGCAGTTAACCTGGACGGTGTCTTCTTCGGCACCCGTCTGGGCATCCAGCGTATGAAGAACAAAGGTCTGGGTGCGTCCATCATCAACATGTCCAGCATCGAAGGTTTCGTAGGTGATCCAAGCCTGGGTGCTTACAACGCGTCTAAAGGTGCTGTTCGTATCATGAGCAAATCTGCTGCACTGGACTGTGCGCTGAAAGATTACGATGTGCGTGTGAACACCGTTCATCCGGGTTACATCAAGACTCCGTTGGTTGACGACCTGCCGGGCGCAGAAGAAGCTATGTCTCAGCGTACCAAAACTCCGATGGGCCATATCGGTGAACCGAACGACATCGCGTACATCTGCGTTTACCTGGCATCCAACGAATCCAAATTCGCTACTGGTAGCGAGTTCGTTGTTGACGGTGGCTATACCGCTCAGTAA。

[0042] In the present invention, the amino acid sequence of the carbonyl reductase mutant is as shown in SEQ ID NO: 3.

[0043] SEQ ID NO: 3 is as follows:

[0044] MSNRLDGKVAIVTGGTLGIGLAIATKFVEEGAKVMITGRHSDVGEKAAKSVGTPDQIQFFQHDSSDEDGWTKLFDATEKAFGPVSTLVNNAGIAVNKSVEETTTAEWRKLLAVNLDGVFFGTRLGI QRMKNKGLGASIINMSSIEGLVGDPSLGAYNASKGAYRIMSKSAALDCAEKDYDVRVNTVHPGYIKTPLVDDLRGAEEAMSQRTKTPMGHIGEPNDIAYICVYLASNESKFATGSEFVVDGGYTAQ.

[0045] In a second aspect, the present invention provides a nucleic acid molecule that encodes a carbonyl reductase mutant as described in the first aspect.

[0046] In this invention, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO: 4.

[0047] SEQ ID NO: 4 is as follows:

[0048] .

[0049] Thirdly, the present invention provides an expression vector comprising the nucleic acid molecule as described in the second aspect.

[0050] In this invention, the expression vector can be any prokaryotic or eukaryotic expression vector commonly used in the art. In a preferred embodiment, a prokaryotic expression vector is used, such as the pET series (e.g., pET-28a, pET-30a), pUC series, or pRSFDuet series vectors. The nucleic acid molecule is typically operatively linked to a promoter (e.g., T7 lac promoter), ribosome binding site, and terminator on the vector to form a complete expression cassette. The vector typically also contains a selection marker gene (e.g., kanamycin resistance gene) and a replication origin.

[0051] Fourthly, the present invention provides a recombinant strain that expresses the carbonyl reductase mutant as described in the first aspect, or carries the nucleic acid molecule as described in the second aspect, or contains the expression vector as described in the third aspect.

[0052] The recombinant strain can be obtained by modifying various host cells suitable for protein expression. The host cells include, but are not limited to, prokaryotic microbial cells or eukaryotic fungal cells. In a preferred embodiment, the host cell is a prokaryotic cell, such as *Escherichia coli* (E. coli). Escherichia coli (including but not limited to BL21(DE3) strains).

[0053] The recombinant strain can be obtained by introducing the nucleic acid molecule as described in the second aspect or the expression vector as described in the third aspect into the host cells. The introduction method includes, but is not limited to, transformation and electroporation. The nucleic acid molecule is present on a self-replicating vector. The expression of the carbonyl reductase mutant can be achieved by culturing the recombinant strain.

[0054] Fifthly, the present invention provides a combinatorial enzyme comprising a carbonyl reductase mutant and formate dehydrogenase as described in the first aspect.

[0055] In this invention, the formate dehydrogenase (FDH) is used to catalyze the oxidation of formate, while simultaneously converting NADP... + It is reduced to NADPH, thereby achieving the cyclic regeneration of the coenzyme. Preferably, the formate dehydrogenase is derived from... Thiobacillus sp. KNK65MA, whose amino acid sequence is shown in SEQ ID NO: 5. This enzyme exhibits high specific activity and stability. Of course, formate dehydrogenases or formate dehydrogenase mutants from other sources with similar functions can also be used in this invention.

[0056] SEQ ID NO: 5 is as follows:

[0057] MAKILCVLYDDPVDGYPKTYARDDLPKIDHYPGGQTLPTPKAIDFTPGQLLGSVSGELGLRKYLEANGHTFVVTSDKDGPDSVFEKELVDADVVISQPFWPAYLTPERIAKAKNLKLALTAGIGSDHVDLQSAIDRGITVAEVTYCNSISVAEHVVMMILGLVRNYIPSHDWARKGGWNIADCVEHSYDLEGMTVGSVAAGRIGLAVLRRLAPFDVKLHYTDRHRLPEAVEKELGLVWHDTREDMYPHCDVVTLNVPLHPETEHMINDETLKLFKRGAYIVNTARGKLADRDAIVRAIESGQLAGYAGDVWFPQPAPKDHPWRTMKWEGMTPHISGTSLSAQARYAAGTREILECFFEGRPIRDEYLIVQGGALAGTGAHSYSKGNATGGSEEAAKFKKAG。

[0058] The nucleotide sequence of formate dehydrogenase (SEQ ID NO: 6) is as follows:

[0059]

[0060] In a sixth aspect, the present invention provides the application of the carbonyl reductase mutant described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the recombinant strain described in the fourth aspect, or the combination enzyme described in the fifth aspect in the synthesis of S-configuration Bosonicine.

[0061] In a seventh aspect, the present invention provides a method for synthesizing S-configuration bosonicine, comprising the following steps:

[0062] The carbonyl reductase mutant described in the first aspect, the recombinant strain described in the fourth aspect, or the combination enzyme described in the fifth aspect are contacted with the reaction substrate β-acetone xyloside, and a catalytic reaction is carried out in the presence of a coenzyme cycle system to obtain S-configuration Bosein.

[0063] The coenzyme recycling system described above in this invention refers to a reaction system capable of achieving coenzyme recycling and regeneration, which includes a catalytic amount of coenzyme (NADP). + The enzyme may contain NADPH, a carbonyl reductase mutant as described in the first aspect, formate dehydrogenase, and formate as a substrate for regeneration. Preferably, the carbonyl reductase mutant and formate dehydrogenase are provided in the form of a combinatorial enzyme as described in the fifth aspect.

[0064] In this invention, the pH of the catalytic reaction system is 6.5-7.5, more preferably 6.8-7.2; the concentration of the reaction substrate is 250-320 g / L; the reaction time is 12-24 hours, more preferably 15-20 hours; and the reaction temperature is 28-32°C, more preferably 30°C.

[0065] The method described in this invention may specifically include the following steps:

[0066] (1) Providing the reaction system: In a suitable buffer solution, add the substrate β-pyruvate and a catalytic amount of coenzyme (NADP). + ), as described in the fifth aspect, combination enzymes, and formates (such as sodium formate or ammonium formate).

[0067] (2) Catalytic reaction: The reaction system is placed in a constant temperature shaker or reactor and the reaction is carried out under specific conditions.

[0068] (3) Termination of reaction and separation of product: After the reaction is completed, the reaction can be terminated by heating, adjusting pH or centrifuging to remove the catalyst. The product S-configuration Bosein can be obtained from the reaction solution by conventional separation and purification methods, including decolorization, electroosmosis, crystallization and other methods.

[0069] This invention employs a coenzyme cyclic regeneration scheme consisting of formate dehydrogenase, formate, and formic acid. In this scheme, formate and formic acid are oxidized to generate carbon dioxide gas, simultaneously completing coenzyme regeneration. The majority of the byproduct is gaseous carbon dioxide, which can escape directly. A small amount of residual formate can be easily removed by electrodialysis, thus significantly simplifying the product separation and purification steps and offering both environmental and economic advantages.

[0070] The S-configuration seoin prepared by this invention can be used as a key active ingredient in the preparation of anti-aging skin care products, cosmetics and other personal care products.

[0071] The technical solution of the present invention will be further described below with reference to specific embodiments. Experimental methods in this invention that do not specify specific conditions are generally performed according to conventional conditions in the art or according to the conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0072] The culture media used in the following examples are as follows:

[0073] LB liquid medium: 10.0 g / L tryptone, 5.0 g / L yeast extract, 10.0 g / L NaCl.

[0074] LB solid medium: 10.0 g / L tryptone, 5.0 g / L yeast extract, 10.0 g / L NaCl, 15.0 g / L agar.

[0075] TB liquid medium: 12.0 g / L tryptone, 24.0 g / L yeast extract, 12.54 g / L K2HPO4, 2.31 g / L KH2PO4, 4 mL / L glycerol.

[0076] The relevant solutions used in the embodiments of this invention are as follows:

[0077] 10% Formic Acid Solution: Add 10 g of formic acid to 90 g of water to prepare a 10% formic acid solution.

[0078] Example 1

[0079] This embodiment provides the construction of a carbonyl reductase mutant library.

[0080] This embodiment is derived from... Levilactobacillus brevis The wild-type carbonyl reductase (EVBA) gene was used as the starting sequence, with its amino acid sequence shown in SEQ ID NO: 1 and its nucleotide sequence shown in SEQ ID NO: 2. Random mutations were introduced into the coding gene using error-prone PCR to construct a mutant library.

[0081] Error-prone PCR amplification: Using plasmid pET30a(+)-EVBA containing the wild-type EVBA gene as a template, amplification was performed using the primers shown in Table 1. The PCR reaction system (50 μL) contained: 1 μL template DNA, 2 μL upstream primer EVBA-F, 2 μL downstream primer EVBA-R, 1 μL 10 mM dATP, 5 μL 10 mM dTTP, 1 μL 10 mM dGTP, 5 μL 10 mM dCTP, 14 μL 20 mM MgCl2, 2.5 μL 25 mM MnCl2, 1 μL Taq DNA polymerase, 5 μL 10× buffer, and ultrapure water to a final volume of 50 μL. The reaction program was: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 45 s, for 30 cycles; and a final extension at 72℃ for 5 min.

[0082] Table 1 Primers used in this embodiment

[0083]

[0084] Library construction: The error-prone PCR product was cloned into the pET30a(+) vector digested with Nde I and Hind III via homologous recombination. The ligation system (20 μL) consisted of: 1 μL linearized vector, 1 μL purified PCR product, 2 μL homologous recombinase, 4 μL buffer, and ultrapure water. The ligation product was then transformed into *E. coli*. E. coli BL21(DE3) competent cells were spread on LB solid medium containing kanamycin (50 μg / mL) and cultured overnight at 37°C to obtain a carbonyl reductase mutant library.

[0085] Example 2

[0086] This embodiment provides the induced expression and preliminary screening of carbonyl reductase mutants.

[0087] Small-scale induced expression: Single clones were randomly selected from the carbonyl reductase mutant library constructed in Example 1 and seeded into 96-well plates, each well containing 600 μL of LB liquid medium (containing 50 μg / mL kanamycin), and cultured at 37°C and 200 rpm for 16 h. 20 μL of the culture was then transferred to a new 96-well plate (each well containing 800 μL of TB liquid medium) and cultured until OD... 600 When the concentration was 0.8-1.0, isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 0.1 mg / mL, and expression was induced at 25℃ for 15 h. The bacterial cells were collected by centrifugation and obtained as crude enzyme solution by high pressure disruption.

[0088] Preliminary activity screening: A 200 μL screening system was established, comprising: 10 μL of the above crude enzyme solution, 250 g / L β-pyruvyl xyloside, and 1 mg / mL NADP. + The volume was replenished with phosphate buffer at pH 7.0. The reaction was carried out at 30℃ for 6 h, and enzyme activity was initially assessed by monitoring the rate of decrease in absorbance at 340 nm (the characteristic absorption peak of NADPH). If the enzyme activity was relatively high, NADPH would be consumed more quickly, resulting in a steeper slope of the decrease curve. Several mutants with enhanced activity were screened, among which EVBA-T1, EVBA-T2, EVBA-T3, and EVBA-T4 showed high catalytic activity in the initial screening and proceeded to the secondary screening.

[0089] Example 3

[0090] This embodiment provides the construction of recombinant formate dehydrogenase (FDH) bacteria.

[0091] To construct a complete coenzyme regeneration system, the source of... Thiobacillus The FDH gene of sp. KNK65MA (its amino acid sequence is shown in SEQ ID NO: 5, and its nucleotide sequence is shown in SEQ ID NO: 6) underwent codon optimization in *E. coli* and was synthesized by Sangon Biotech (Shanghai) Co., Ltd. The optimized FDH gene was cloned into the pET30a(+) vector between the Nde I and Hind III sites to construct the recombinant plasmid pET30a-FDH, which was then transformed into... E. coli Recombinant strains were obtained from BL21(DE3). E. coli BL21 / pET30a-FDH. After induction, the wet cells of this strain can be used as a coenzyme regeneration catalyst.

[0092] Example 4

[0093] This embodiment provides a highly efficient method for screening and identifying carbonyl reductase mutants.

[0094] This embodiment is used to verify the performance of the mutant in a whole-cell catalytic system, especially the efficiency of the combined system with formate dehydrogenase.

[0095] Catalyst preparation: The four mutant plasmids EVBA-T1, EVBA-T2, EVBA-T3, and EVBA-T4 obtained in Example 2 were transformed into catalysts respectively. E. coli In BL21(DE3), wet bacterial cells were collected by centrifugation after induction of expression and used as a catalyst for carbonyl reductase. Simultaneously, the enzyme constructed in Example 3 was cultured... E. coli The BL21 / pET30a-FDH strain was used to collect wet cells as a cocatalyst for formate dehydrogenase.

[0096] Whole-cell catalytic reaction: A 100 mL reaction system was established containing: 300 g / L β-pyruvate xyloside and 1 mg / mL NADP. + Add 100 g / L carbonyl reductase catalyst, 100 g / L formate dehydrogenase co-catalyst, and 40 g / L sodium formate, and bring the volume up to 100 mL with water.

[0097] Reaction and detection: The reaction system was carried out at 30℃ and 220 rpm. The pH of the reaction solution was dynamically controlled at 7.0 ± 0.5 by adding 10% formic acid solution dropwise. After 16 h of reaction, samples were taken, and the substrate conversion and product configuration were detected by high performance liquid chromatography (HPLC).

[0098] Screening results: The liquid chromatograms of the enzyme-catalyzed reaction solutions of EVBA-T1, EVBA-T2, EVBA-T3, and EVBA-T4 are shown below. Figure 1 , Figure 2 , Figure 3 and Figure 4 As shown, the substrate conversion rates were 97.496%, 100%, 80.953%, and 79.897%, respectively. It can be seen that the EVBA-T2 mutant exhibited the best performance, with a substrate conversion rate of 100%. The EVBA-T2 enzyme-catalyzed reaction solution was dehydrated, desalted, and recrystallized to obtain the product. The high-performance liquid chromatography chromatogram of the obtained product is shown below. Figure 5 As shown, the proportion of S-configuration Bosonic in the product is 100% (S:R = 100:0), indicating that an optically pure S-configuration Bosonic product was obtained.

[0099] Mutant sequence determination: The EVBA-T2 mutant gene was sequenced, and the results showed that its amino acid sequence was SEQ ID NO: 3 and its nucleotide sequence was SEQ ID NO: 4. Compared with wild-type EVBA (SEQ ID NO: 1), its amino acid sequence had four mutations: phenylalanine (F) at position 147 was mutated to leucine (L), valine (V) at position 163 was mutated to tyrosine (Y), leucine (L) at position 176 was mutated to glutamic acid (E), and proline (P) at position 200 was mutated to arginine (R).

[0100] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A carbonyl reductase mutant, characterized in that, The wild-type carbonyl reductase with amino acid sequence shown in SEQ ID NO: 1 is mutated to obtain the mutant, and the mutation mode is as follows: F at position 147 is mutated to L, V at position 163 is mutated to Y, L at position 176 is mutated to E, and P at position 200 is mutated to R.

2. A nucleic acid molecule, characterized in that, The mutant of the carbonyl reductase as claimed in claim 1.

3. An expression vector, characterized by, The nucleic acid molecule as claimed in claim 2.

4. A recombinant bacterial strain, characterized in that, The mutant of the carbonyl reductase as claimed in claim 1, or carrying the nucleic acid molecule as claimed in claim 2, or containing the expression vector as claimed in claim 3.

5. A combination enzyme characterized in that, The mutant of the carbonyl reductase as claimed in claim 1 and formate dehydrogenase.

6. The combination enzyme of claim 5, wherein, The formate dehydrogenase is derived from Thiobacillus sp. KNK65MA, the amino acid sequence of which is shown in SEQ ID NO:

5.

7. The mutant of the carbonyl reductase as claimed in claim 1, the nucleic acid molecule as claimed in claim 2, the expression vector as claimed in claim 3, the recombinant strain as claimed in claim 4, or the combined enzyme as claimed in claim 5 or 6 is used for synthesizing S-form Bose factor.

8. A method of synthesizing S-configuration somatotropin, characterized by, The method comprises the following steps: The mutant of the carbonyl reductase as claimed in claim 1, or the recombinant strain as claimed in claim 4, or the combined enzyme as claimed in claim 5 or 6 is contacted with the reaction substrate β-ketoxylose, and a catalytic reaction is carried out in the presence of a coenzyme cycle system, so as to obtain S-form Bose factor.

9. The method of synthesizing S-form somatotropin according to claim 8, wherein, The pH of the catalytic reaction system is 6.5-7.5; the concentration of the reaction substrate is 250-320 g / L; the reaction time is 12-24 hours; and the reaction temperature is 28-32℃.

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