Ketoreductase mutants, methods, and uses
By constructing a ketone reductase mutant through site-directed mutagenesis, the problems of long synthetic routes and low yields of N-Boc-cis-3-hydroxy-L-proline methyl ester were solved, achieving highly selective and high-yield enzymatic production, thus improving production efficiency and environmental friendliness.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- NANJING NORMAL UNIVERSITY
- Filing Date
- 2026-04-20
- Publication Date
- 2026-06-09
AI Technical Summary
In the existing technology, the synthetic route of N-Boc-cis-3-hydroxy-L-proline methyl ester has many steps, low yield, and is difficult to prepare efficiently by pure chemical methods.
Using ketone reductase mutants, site-directed mutagenesis was performed to mutate isoleucine at position 91 to glycine (I91G) and alanine at position 139 to methionine (A139M) to construct ketone reductase mutants for catalyzing the formation of N-Boc-cis-3-hydroxy-L-proline methyl ester from 1-tert-butyl-2-methyl-3-oxopyrrolidine-1,2-dicarboxylic acid ester.
A highly selective and high-yield enzymatic production method for N-Boc-cis-3-hydroxy-L-proline methyl ester was achieved. The reaction conditions were mild, the production process was environmentally friendly, the conversion rate was as high as 96.5%, and the enantiomeric excess value was >99%.
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Figure CN122168557A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the fields of enzyme engineering and genetic engineering technology, and in particular to a ketone reductase mutant, method and application, specifically relating to its application in the production of N-BOC-cis-3-hydroxy-L-proline methyl ester. Background Technology
[0002] Hydroxyproline and its protected derivatives are important nitrogen-containing chiral building blocks, widely used in peptide drugs, antibiotics, and intermediates for the synthesis of chiral drugs. Introducing hydroxyl groups at different positions (C-3 or C-4) of the proline ring, followed by N-Boc protection and esterification, allows for precise control of reaction selectivity and improves stability and lipophilicity.
[0003] Currently, the most widely studied and mature research focuses on 4-hydroxy-L-proline and its N-Boc protected ester derivatives. For example, Chinese patent publication CN119350211A uses (2S,4R)-4-hydroxy-1,2-pyrrolinoline dicarboxylic acid-1-tert-butyl ester-2-methyl ester as a raw material to synthesize N-Boc-cis-4-hydroxy-L-proline methyl ester via a three-step chemical method. Chinese patent publication CN111057736B uses freeze-dried bacterial cells obtained from the fermentation culture of recombinant lipase engineered bacteria as a catalyst, racemic BOC-DL-proline methyl ester as a substrate, and pH 7.0 phosphate buffer solution as the reaction medium to resolve and prepare BOC-D-proline methyl ester, with an enantiomeric excess value >99% and a yield of 96.0% for BOC-D-proline methyl ester. Therefore, existing technologies have developed relatively mature chemical or enzymatic synthesis processes for the N-Boc protected ester of 4-hydroxy-L-proline. In stark contrast, there are extremely limited publicly reported synthesis reports for N-Boc-cis-3-hydroxy-L-proline methyl ester (hereinafter referred to as "target compound"). Cis-3-hydroxy-L-proline itself, as a natural amino acid derivative, has been confirmed to be part of the polypeptide antibiotic telomycin (Sheehan et al, Journal of the American Chemical Society, 1968, 90(2): 462–70) and cyclothialidine (Götschi et al, Helvetica Chimica Acta, 1996, 79, 2219-2234). To further utilize cis-3-hydroxy-L-proline as a molecular building block, it is necessary to synthesize an N-Boc protected ester of cis-3-hydroxy-L-proline to generate the target compound N-Boc-cis-3-hydroxy-L-proline methyl ester. However, existing methods generally rely on multi-step organic synthetic routes: for example, N-Boc-cis-3-hydroxy-L-proline methyl ester is synthesized from commercially available trans-3-hydroxy-L-proline through four chemical reactions in 64% yield. This synthetic route suffers from low overall yield due to the stereoconfigurational barrier of the hydroxyl group at position 3 (Gómez-Vidal and Silverman, Organic Letters, 2001, 3(16): 2481–4). Therefore, under current technological conditions, the main problems with the target compound are purely chemical synthetic routes, numerous steps, and low yields. Summary of the Invention
[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a ketone reductase mutant, its method, and its application.
[0005] The technical solution adopted by this invention to solve its technical problem is: A ketone reductase mutant, the mutant comprising the following point mutations: isoleucine at position 91 of the amino acid sequence shown in SEQ ID No. 1 is mutated to glycine (I91G) and alanine at position 139 is mutated to methionine (A139M).
[0006] Furthermore, the amino acid sequence of the mutant is any one of the sequences shown in SEQ ID No. 2-4.
[0007] A gene encoding a ketone reductase mutant protein as described above.
[0008] Furthermore, the nucleotide sequence of the gene is any one of those shown in SEQ ID No. 5-7.
[0009] A cloning vector or expression vector containing the genes described above.
[0010] The method for preparing the ketone reductase mutant as described above includes the following steps: (1) Design point mutation primers, use plasmids containing wild-type ketone reductase gene (SEQ ID No. 5 ADH-WT) as templates, perform PCR reactions using point mutation primers, and obtain linearized plasmids of mutant genes after purification; (2) The linearized plasmid of the mutant gene is ligated to construct an expression vector, and the expression vector is transformed into the host bacteria for induction expression; (3) Collect the host bacteria expressing the ketone reductase mutant, and resuspend the bacterial cells to obtain a whole-cell catalytic solution containing the ketone reductase mutant.
[0011] Furthermore, the point mutation primer sequences in step (1) include:
[0012] In step (1), the reaction system for the PCR reaction is as follows (25 μL):
[0013] The method for producing N-Boc-cis-3-hydroxy-L-proline methyl ester using the ketone reductase mutant described above comprises the following steps: The method uses wet cells of recombinant engineered bacteria containing the gene encoding a ketone reductase mutant (SEQ ID No. 8 ADH-I91G / A139M) after induction expression as a catalyst, with 1-tert-butyl-2-methyl-3-oxopyrrolidine-1,2-dicarboxylic acid ester (CAS: 194924-96-4) as the substrate, and glucose, GDH, and NADP as the active ingredients. + or NAD + An electron circulation system is formed in a reaction system consisting of a potassium phosphate buffer aqueous solution with pH=6.5-7.5 (preferably 7), the shaking amplitude is 300-500 rpm (preferably 400 rpm), and the reaction is carried out overnight at 30°C for >18h or =18h. After the reaction is completed, N-BOC-cis-3-hydroxy-L-proline methyl ester is obtained.
[0014] Furthermore, the circulation system is a potassium phosphate buffer solution at pH=7 with 50 mg / ml 1sGDH (SEQ ID No. 9) and 2 mM NADP added. + / NAD + The lsGDH was obtained by mixing it with 200 mM glucose; wherein the amino acid sequence of the lsGDH is shown in SEQ ID No. 9. Alternatively, the potassium phosphate buffer solution system is an aqueous solution with a pH of 7.0 composed of 100 mM dimethyl hydrogen phosphate and potassium dihydrogen phosphate. Alternatively, the ketone reductase mutant in the catalyst may have an OD in the reaction system. 600 =10, the initial concentration of the substrate is 200mM.
[0015] Furthermore, the method for preparing the wet bacterial cells is as follows: Recombinant engineered bacteria containing the gene encoding the ketone reductase mutant were inoculated into liquid LB medium containing 100 ug / mL ampicillin sodium and cultured at 37°C for 8 h to obtain seed culture. The seed culture was then inoculated into sterilized TB medium containing 100 ug / mL ampicillin at a volume concentration of 2% and cultured at 37°C for about 8-12 h. When the bacterial OD600 reached 0.6, isopropyl thio-β-D-galactopyranoside (IPTG) was added to the medium at a final concentration of 0.1-1.0 mM (preferably 0.5 mM). After inducing expression at 18°C for 16-24 h, the bacteria were centrifuged at 4°C and 4000 rpm for 10-20 min, and the wet bacterial resuspended using the reaction buffer. The construction method of the recombinant engineered bacteria containing the gene encoding a ketone reductase mutant is as follows: The ketone reductase mutant group was integrated into the pET22b(+) vector plasmid, and the plasmid was transformed into BL21(DE3) competent cells. After incubating the strain with SOC for one hour, the strain was spread onto solid LB medium containing ampicillin sodium at a final concentration of 100 ug / mL and cultured at 37°C for 16 hours to obtain recombinant engineered bacteria containing the ketone reductase mutant encoding gene.
[0016] The advantages and positive effects of this invention are as follows: 1. The ketone reductase mutant obtained in this invention can produce N-BOC-cis-3-hydroxy-L-proline methyl ester with high selectivity and high yield, at OD 600 =10, 300mM glucose, 2mM NADP + / NAD + Under the conditions of 50 mg / ml ls GDH substrate concentration of 200 mM, the yield was 96.5% after 18 h of reaction, with de>99%.
[0017] 2. The ketone reductase mutant of the present invention is modified by site-directed mutagenesis based on the ketone reductase amino acid sequence shown in SEQ ID NO. 1, thereby altering its amino acid sequence and changing its protein structure and function, resulting in a ketone reductase mutant with the aforementioned mutation. Compared with the prior art, the present invention has the following significant advantages: (1) It is produced by enzymatic method, which is simple and the reaction conditions are mild. (2) Environmental protection in the production process (3) It has high conversion rate and high selectivity.
[0018] 3. The ketone reductase mutant provided by this invention is obtained by replacing at least one amino acid residue at points I91G and A139M in the amino acid sequence shown in SEQ ID No. 1, resulting in a mutant with high activity and high selectivity. This enzyme can efficiently convert 1-tert-butyl-2-methyl-3-oxopyrrolidine-1,2-dicarboxylic acid ester (CAS: 194924-96-4) to N-Boc-cis-3-hydroxy-L-proline methyl ester in a conventional aqueous phase. This characteristic improves the selectivity and yield of N-BOC-cis-3-hydroxy-L-proline production.
[0019] 4. This invention provides a ketone reductase mutant that can hydroxylate the 3-position of 1-tert-butyl-2-methyl-3-oxopyrrolidine-1,2-dicarboxylic acid ester with high selectivity and high yield to generate N-BOC-cis-3-hydroxy-L-proline methyl ester. Attached Figure Description
[0020] Figure 1This is a diagram of the reaction catalyzed by ketone reductase in the present invention to convert 1-tert-butyl-2-methyl-3-oxopyrrolidine-1,2-dicarboxylic acid ester into N-Boc-cis-3-hydroxy-L-proline methyl ester. Figure 2 This is a gas chromatogram of the catalytic sample of the mutant ADH-I91G / A139M in this invention; Figure 3 This is a diagram illustrating the effect of point mutation in this invention. Detailed Implementation
[0021] The present invention will be further described below with reference to the embodiments. The following embodiments are descriptive and not limiting, and should not be used to limit the scope of protection of the present invention.
[0022] The various experimental operations involved in the specific embodiments are all conventional techniques in the field. For parts not specifically annotated in this document, those skilled in the art can refer to various commonly used reference books, scientific and technological documents or related instructions and manuals prior to the filing date of this invention to carry out the operations.
[0023] The present invention provides a ketone reductase mutant, which is obtained by multiple mutations at positions 91 and 139 of the amino acid sequence shown in SEQ ID.1.
[0024] Furthermore, the mutant ketone reductase mutant is formed by mutating isoleucine at position 91 of the amino acid sequence shown in SEQ ID No. 1 to glycine (I91G) and alanine at position 139 to methionine (A139M).
[0025] This invention also relates to a recombinant vector encoding the ketone reductase mutant gene and recombinant genetically engineered bacteria prepared by transformation of the recombinant vector. The recombinant vector of this invention is not limited, as long as it can maintain its replication or autonomous replication in various host cells of prokaryotic and / or eukaryotic cells. The vector can be any conventional vector in the art, such as various plasmids, bacteriophages, or viral vectors, preferably using the pET22b(+) plasmid as the expression vector and *Escherichia coli* as the expression host (*E. coli* C43 cells or *E. coli* BL21).
[0026] This invention also provides an application method for the production of N-BOC-cis-3-hydroxy-L-proline methyl ester catalyzed by the ketone reductase mutant, wherein the method comprises: using wet cells of recombinant engineered bacteria containing the ketone reductase mutant encoding gene after induction expression as a catalyst, using 1-tert-butyl-2-methyl-3-oxopyrrolidine-1,2-dicarboxylic acid ester as a substrate, and using glucose dehydrogenase 1sGDH to catalyze the production of N-BOC-cis-3-hydroxy-L-proline methyl ester with glucose as a substrate. + / NAD +The reaction was carried out overnight (18 h) at 30°C in a potassium phosphate buffer solution with a pH of 6.5-7.5 (preferably 7) at 300-500 rpm (preferably 400 rpm) and 30°C. After the reaction, a reaction solution containing N-BOC-cis-3-hydroxy-L-proline methyl ester was obtained. Figure 1 As shown.
[0027] The circulation system consisted of 50 mg / ml 1sGDH (SEQ ID No. 9) and 2 mM NADP. + / NAD + It consists of 200mM glucose.
[0028] The potassium phosphate buffer solution system is an aqueous solution with a pH of 7.0 composed of 100 mM dimethyl hydrogen phosphate and potassium dihydrogen phosphate.
[0029] Furthermore, the ketone reductase mutant in the catalyst has an OD value in the reaction system. 600 =10, the initial concentration of the substrate is 200mM.
[0030] Further, the wet bacterial cells are prepared as follows: recombinant engineered bacteria containing the gene encoding a ketone reductase mutant are inoculated into liquid LB medium containing 100 ug / mL ampicillin sodium and cultured at 37°C for 8 h to obtain a seed culture; the seed culture is inoculated into sterilized TB medium containing 100 ug / mL ampicillin at a volume concentration of 2% and cultured at 37°C for about 8-12 h. When the bacterial OD600 is 0.6, isopropyl thio-β-D-galactoside (IPTG) at a final concentration of 0.1-1.0 mM (preferably 0.5 mM) is added to the medium. After inducing expression at 18°C for 16-24 h, the cells are centrifuged at 4°C and 4000 rpm for 10-20 min, and the wet bacterial cells are resuspended using the reaction buffer. The one-pot catalytic reaction described in this invention can be carried out in whole-cell form, or it can be catalyzed using crude enzyme solution from cell disruption or completely disrupted pure enzyme. Furthermore, ketone reductase can be prepared into immobilized enzymes or immobilized cell-based enzymes using specific immobilization techniques.
[0031] Specifically, the relevant preparation and testing methods are as follows: Example 1: A ketoreductase mutant (hereinafter referred to as ADH-I91G) with the sequence shown in SEQ ID No. 2, wherein the isoleucine at position 91 of the original ketoreductase sequence (hereinafter referred to as ADH-WT) shown in SEQ ID No. 1 is mutated to glycine (I91G); The method for preparing mutants is as follows: 1. Construction of recombinant plasmids plasmid pET-22b(+), E. coli BL21(DE3) were all derived commercially. The wild-type ketoreductase gene of SEQ ID No. 1 was synthesized by Genewiz (Suzhou) Co., Ltd. The site-directed mutagenesis sequence of this enzyme was constructed by the applicant using PCR. Primers used to introduce the mutation site are shown in Table 1. The template for the point mutation PCR was the pET-22b(+) plasmid containing the wild-type ketoreductase gene sequence.
[0032] Table 1 Primers for mutant gene fragments and linearized plasmid primers
[0033] Table 2 PCR reaction system
[0034] Table 3 PCR reaction conditions
[0035] Linear mutant plasmids were obtained by digesting the template PCR product with DpnI at 37°C for 4 hours.
[0036] 2. Construction of recombinant strains and preparation of crude enzyme solution Transform the linear mutant plasmid into E. coli DH5α competent cells were plated on the surface of LB agar containing 100 μg / mL ampicillin and incubated at 37°C for 12 h. All single colonies growing on the plate were scraped into test tubes containing 5 mL of LB liquid agar containing 100 μg / mL ampicillin and incubated at 37°C with shaking at 200 rpm for 12 h. Plasmids were extracted using a plasmid extraction kit, and recombinant plasmids were stored at -20°C.
[0037] Transform the recombinant plasmid into E. coliBL21(DE3) host bacteria were plated on LB solid medium containing 100 μg / mL ampicillin and incubated at 37°C for 8 h. Individual *E. coli* colonies were inoculated into 3 mL of LB liquid medium containing 100 μg / mL ampicillin and incubated overnight at 37°C as a seed culture. The seed culture was then inoculated at a 5% inoculation rate into 250 mL Erlenmeyer flasks containing 50 mL of TB medium and incubated at 37°C and 200 rpm. TB liquid medium consisted of 12 g / L yeast extract, 12 g / L tryptone, 4 mL / L glycerol, 12.5 g / L dipotassium hydrogen phosphate, and 2.3 g / L potassium dihydrogen phosphate, with water as the solvent. After 8 h of incubation, IPTG was added to a final concentration of 0.5 mM, and the incubation temperature was set to 18°C for an additional 16-24 h. Centrifuge the fermentation broth, collect the cells, and resuspend them in 100mM phosphate buffer at pH 7.0 to obtain the ADH-I91G whole-cell catalyst (also known as whole-cell catalyst wet cells; the same applies below).
[0038] Example 2: A ketoreductase mutant I91G / A139M (hereinafter referred to as ADH-I91G / A139M) with the sequence shown in SEQ ID No. 3. This mutant is formed by mutating isoleucine at position 91 of the original ketoreductase enzyme sequence shown in SEQ ID No. 1 to glycine (I91G) and alanine at position 139 to methionine (A139M).
[0039] The preparation method of mutant I91G / A139M is as follows: The plasmid obtained in Example 1 was used as the mutation template, and everything else was the same as in Example 1, except that the primers for the mutated gene fragment were as follows: Table 5 Primers for Mutant Gene Fragments
[0040] After the PCR product was verified by gel electrophoresis, the template PCR product was digested with DpnI at 37°C for 4 hours to obtain a linear mutant plasmid. The gene sequence of the mutant enzyme is shown in SEQ ID No. 7.
[0041] Example 3: Determination of the whole-cell catalytic yield of the ketone reductase mutant enzyme ADH-I91G / A139M prepared in Example 2. The whole-cell catalyst wet cells containing the gene encoding the ketone reductase mutant I91G / A139M obtained in Example 2 were used as catalysts in the catalyst OD. 600=10, substrate 1-tert-butyl-2-methyl-3-oxopyrrolidine-1,2-dicarboxylic acid ester (CAS: 194924-96-4) at a concentration of 200 mM, using 50 mg / mL glucose dehydrogenase 1sGDH (SEQ ID No. 9) as substrate to catalyze the reaction of 2 mM NADP with 300 mM glucose. + / NAD + In the constructed electronic reaction system, the reaction was carried out at 30°C for 18 hours. After the reaction, a reaction solution containing N-BOC-cis-3-hydroxy-L-proline methyl ester was obtained. After the reaction, 500 μL of the reaction solution was extracted with 500 µL of ethyl acetate, centrifuged at 1,2000 rpm for 1 min, and 300 µL of the supernatant was collected for gas chromatography analysis.
[0042] The electronic reaction system is prepared by adding 50 mg / ml 1s GDH and 2 mM NADP to a potassium phosphate buffer solution at pH 7.0. + / NAD + It is obtained by mixing with 200mM glucose; wherein the amino acid sequence of the lsGDH is shown in SEQ ID No. 9; The potassium phosphate buffer solution is an aqueous solution with a pH of 7.0 composed of 100 mM dimethyl hydrogen phosphate and potassium dihydrogen phosphate.
[0043] Gas chromatography conditions: Agilent Cyclosil-B column, gas program: 60℃ for 2 min, ramp-up to 240℃ at 45℃ / min, hold for 2 min. Retention time of N-BOC-cis-3-L-hydroxyproline methyl ester: 5.176 min.
[0044] The results are as follows Figure 2 As shown. From Figure 2 As can be seen, most of the substrate was converted into the product, specifically 96.56%.
[0045] like Figure 3 As shown, compared to the wild-type ADH-WT (144.2 mM, de=96%), the yield and selectivity of the first mutant ADH-I91G increased to 172.7 mM, de=98.4%, and the yield and selectivity of the second mutant ADH-I91G / A139M increased to 193.4 mM, de>99%. Compared to the existing chemical method for producing the chiral compound N-BOC-cis-3-L-hydroxyproline methyl ester with a yield of 64%, the enzymatically prepared second mutant ADH-I91G / A139M exhibits both high chiral selectivity (de>99%) and high yield (96.56%).
[0046] The relevant sequences are as follows: Amino acid sequence of the wild-type ADH MYRLLNKTAVITGGNSGIGLATAKRFVAEGAYVFIVGRRRKELEQAAAEIGRNVTAVKADVTKLEDLDRLYAIVREQRGSIDVLFANSGAIEQKTLEEITPEHYDRTFDVNVRGLIFTVQKALPLLRDGGSVILTSSVAGVLGLQAHDTYSAAKAAVRSLARTWTTELKGRSIRVNAVSPGAIDTPIIENQVSTQEEADELRAKFAAATPLGRVGRPEELAAAVLFLASDDSSYVAGIELFVDGGLTQV Amino acid sequence of the ADH-I91G mutant MYRLLNKTAVITGGNSGIGLATAKRFVAEGAYVFIVGRRRKELEQAAAEIGRNVTAVKADVTKLEDLDRLYAIVREQRGSIDVLFANSGAGEQKTLEEITPEHYDRTFDVNVRGLIFTVQKALPLLRDGGSVILTSSVMGVLGLQAHDTYSAAKAAVRSLARTWTTELKGRSIRVNAVSPGAIDTPIIENQVSTQEEADELRAKFAAATPLGRVGRPEELAAAVLFLASDDSSYVAGIELFVDGGLTQV Amino acid sequence of the ADH-A139M mutant MYRLLNKTAVITGGNSGIGLATAKRFVAEGAYVFIVGRRRKELEQAAAEIGRNVTAVKADVTKLEDLDRLYAIVREQRGSIDVLFANSGAIEQKTLEEITPEHYDRTFDVNVRGLIFTVQKALPLLRDGGSVILTSSVMGVLGLQAHDTYSAAKAAVRSLARTWTTELKGRSIRVNAVSPGAIDTPIIENQVSTQEEADELRAKFAAATPLGRVGRPEELAAAVLFLASDDSSYVAGIELFVDGGLTQV Amino acid sequence of the ADH-I91G / A139M mutant MYRLLNKTAVITGGNSGIGLATAKRFVAEGAYVFIVGRRRKELEQAAAEIGRNVTAVKADVTKLEDLDRLYAIVREQRGSIDVLFANSGAGEQKTLEEITPEHYDRTFDVNVRGLIFTVQKALPLLRDGGSVILTSSVMGVLGLQAHDTYSAAKAAVRSLARTWTTELKGRSIRVNAVSPGAIDTPIIENQVSTQEEADELRAKFAAATPLGRVGRPEELAAAVLFLASDDSSYVAGIELFVDGGLTQV SEQ ID No.5 Nucleotide sequence of the original enzyme ADH-WT atgTACCGTCTGCTGAACAAAACCGCGGTTATCACCGGTGGTAACAGCGGTATCGGTCTGGCAACCGCGAAACGTTTCGTTGCGGAAGGCGCGTACGTTTTCATCGTTGGCCGTCGTCGTAAAGAACTGGAACAGGCGGCGGCGGAAATCGGCCGTAACGTTACCGCAGTTAAAGCGGATGTTACCAAACTGGAAGATCTGGATCGTCTGTACGCGATCGTGCGTGAACAGCGTGGTAGCATCGATGTTCTGTTCGCGAACTCTGGCGCGATTGAACAGAAAACCCTGGAAGAAATCACCCCGGAACACTACGACCGTACCTTCGATGTTAACGTTCGTGGTCTGATTTTCACCGTTCAGAAAGCGCTGCCGCTGCTGCGTGATGGTGGTAGCGTTATCCTGACCAGCAGCGTTGCGGGTGTTCTGGGCCTGCAGGCGCACGATACCTACAGCGCGGCAAAAGCGGCGGTTCGTAGCCTGGCGCGTACCTGGACCACCGAACTGAAAGGTCGTAGCATCCGTGTTAACGCAGTGTCTCCGGGTGCGATCGATACCCCGATCATCGAAAACCAGGTTAGCACCCAGGAAGAAGCGGATGAACTGCGTGCGAAATTCGCGGCGGCGACCCCGCTGGGCCGTGTTGGTCGTCCGGAAGAACTGGCGGCGGCGGTTCTGTTCCTGGCGAGCGATGATTCTTCTTACGTTGCTGGTATCGAACTGTTCGTTGATGGCGGCCTGACCCAGGTT SEQ ID No.6 Nucleotide sequence of ADH-I91G mutant atgTACCGTCTGCTGAACAAAACCGCGGTTATCACCGGTGGTAACAGCGGTATCGGTCTGGCAACCGCGAAACGTTTCGTTGCGGAAGGCGCGTACGTTTTCATCGTTGGCCGTCGTCGTAAAGAACTGGAACAGGCGGCGGCGGAAATCGGCCGTAACGTTACCGCAGTTAAAGCGGATGTTACCAAACTGGAAGATCTGGATCGTCTGTACGCGATCGTGCGTGAACAGCGTGGTAGCATCGATGTTCTGTTCGCGAACTCTGGCGCG GGT GAACAGAAAACCCTGGAAGAAATCACCCCGGAACACTACGACCGTACCTTCGATGTTAACGTTCGTGGTCTGATTTTCACCGTTCAGAAAGCGCTGCCGCTGCTGCGTGATGGTGGTAGCGTTATCCTGACCAGCAGCGTTatgGGTGTTCTGGGCCTGCAGGCGCACGATACCTACAGCGCGGCAAAAGCGGCGGTTCGTAGCCTGGCGCGTACCTGGACCACCGAACTGAAAGGTCGTAGCATCCGTGTTAACGCAGTGTCTCCGGGTGCGATCGATACCCCGATCATCGAAAACCAGGTTAGCACCCAGGAAGAAGCGGATGAACTGCGTGCGAAATTCGCGGCGGCGACCCCGCTGGGCCGTGTTGGTCGTCCGGAAGAACTGGCGGCGGCGGTTCTGTTCCTGGCGAGCGATGATTCTTCTTACGTTGCTGGTATCGAACTGTTCGTTGATGGCGGCCTGACCCAGGTT Nucleotide sequence of ADH-A139M mutant, SEQ ID No.7 atgTACCGTCTGCTGAACAAAACCGCGGTTATCACCGGTGGTAACAGCGGTATCGGTCTGGCAACCGCGAAACGTTTCGTTGCGGAAGGCGCGTACGTTTTCATCGTTGGCCGTCGTCGTAAAGAACTGGAACAGGCGGCGGCGGAAATCGGCCGTAACGTTACCGCAGTTAAAGCGGATGTTACCAAACTGGAAGATCTGGATCGTCTGTACGCGATCGTGCGTGAACAGCGTGGTAGCATCGATGTTCTGTTCGCGAACTCTGGCGCGATTGAACAGAAAACCCTGGAAGAAATCACCCCGGAACACTACGACCGTACCTTCGATGTTAACGTTCGTGGTCTGATTTTCACCGTTCAGAAAGCGCTGCCGCTGCTGCGTGATGGTGGTAGCGTTATCCTGACCAGCAGCGTTatgGGTGTTCTGGGCCTGCAGGCGCACGATACCTACAGCGCGGCAAAAGCGGCGGTTCGTAGCCTGGCGCGTACCTGGACCACCGAACTGAAAGGTCGTAGCATCCGTGTTAACGCAGTGTCTCCGGGTGCGATCGATACCCCGATCATCGAAAACCAGGTTAGCACCCAGGAAGAAGCGGATGAACTGCGTGCGAAATTCGCGGCGGCGACCCCGCTGGGCCGTGTTGGTCGTCCGGAAGAACTGGCGGCGGCGGTTCTGTTCCTGGCGAGCGATGATTCTTCTTACGTTGCTGGTATCGAACTGTTCGTTGATGGCGGCCTGACCCAGGTT SEQ ID No.8 ADH-I91G / A139M nucleotide mutant atgTACCGTCTGCTGAACAAAACCGCGGTTATCACCGGTGGTAACAGCGGTATCGGTCTGGCAACCGCGAAACGTTTCGTTGCGGAAGGCGCGTACGTTTTCATCGTTGGCCGTCGTCGTAAAGAACTGGAACAGGCGGCGGCGGAAATCGGCCGTAACGTTACCGCAGTTAAAGCGGATGTTACCAAACTGGAAGATCTGGATCGTCTGTACGCGATCGTGCGTGAACAGCGTGGTAGCATCGATGTTCTGTTCGCGAACTCTGGCGCG GGT GAACAGAAAACCCTGGAAGAAATCACCCCGGAACACTACGACCGTACCTTCGATGTTAACGTTCGTGGTCTGATTTTCACCGTTCAGAAAGCGCTGCCGCTGCTGCGTGATGGTGGTAGCGTTATCCTGACCAGCAGCGTT atg GGTGTTCTGGGCCTGCAGGCGCACGATACCTACAGCGCGGCAAAAGCGGCGGTTCGTAGCCTGGCGCGTACCTGGACCACCGAACTGAAAGGTCGTAGCATCCGTGTTAACGCAGTGTCTCCGGGTGCGATCGATACCCCGATCATCGAAAACCAGGTTAGCACCCAGGAAGAAGCGGATGAACTGCGTGCGAAATTCGCGGCGGCGACCCCGCTGGGCCGTGTTGGTCGTCCGGAAGAACTGGCGGCGGCGGTTCTGTTCCTGGCGAGCGATGATTCTTCTTACGTTGCTGGTATCGAACTGTTCGTTGATGGCGGCCTGACCCAGGT SEQ ID No.9 lsGDH amino acid sequence MYSDLEGKVIVITGASTGLGKAMALRFGEEKAKVIVNFRSDENEANAVVEGVKKAGGDAIAVKGDVTVEEDVINLVQTAVNKFGTLDVMINNAGIENPVASHEMPLSDWNRVINTNLTGAFLGSREAIKYF VENDIKGSVINMSSVHEMIPWPLFVHYAASKGGIKLMTQTLALEYAPKGIRINNIGPGAINTPINAEKFADPAKRADVESMVPMGYIGKPEEIAAVAAWLASSQASYVTGITLFADGGMTLYPDFQAGRGLE Although embodiments of the invention have been disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, variations, and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, the scope of the invention is not limited to the contents disclosed in the embodiments.
Claims
1. A ketone reductase mutant, characterized in that: The mutant includes the following point mutations: isoleucine at position 91 of the amino acid sequence shown in SEQ ID No. 1 is mutated to glycine (I91G), and alanine at position 139 is mutated to methionine (A139M).
2. The ketone reductase mutant according to claim 1, characterized in that: The amino acid sequence of the mutant is any one of the sequences shown in SEQ ID No. 2-4.
3. A gene encoding the ketone reductase mutant protein of claim 1 or 2.
4. The gene according to claim 3, characterized in that: The nucleotide sequence of the gene is any one of those shown in SEQ ID No. 6-8.
5. A cloning vector or expression vector containing the gene as described in claim 3 or 4.
6. The method for preparing the ketone reductase mutant according to claim 1, characterized in that: Includes the following steps: (1) Design point mutation primers, use plasmids containing wild-type ketone reductase gene as templates, perform PCR reactions using point mutation primers, and obtain linearized plasmids of mutant genes after purification; (2) The linearized plasmid of the mutant gene is ligated to construct an expression vector, and the expression vector is transformed into the host bacteria for induction expression; (3) Collect the host bacteria expressing the ketone reductase mutant, and resuspend the bacterial cells to obtain a whole-cell catalytic solution containing the ketone reductase mutant.
7. The method for preparing the ketone reductase mutant according to claim 6, characterized in that: The point mutation primer sequences in step (1) include: In step (1), the reaction system for the PCR reaction is as follows (25 μL): 。 8. A method for producing N-Boc-cis-3-hydroxy-L-proline methyl ester using the ketoreductase mutant as described in claim 1 or 2, characterized in that: Includes the following steps: The method uses wet cells of recombinant engineered bacteria containing a ketone reductase mutant encoding gene, induced to express the gene, as a whole-cell catalyst. It employs 1-tert-butyl-2-methyl-3-oxopyrrolidine-1,2-dicarboxylic acid ester as a substrate, and glucose, GDH, and NADP as the active ingredients. + / NAD + An electron circulation system is formed in a reaction system consisting of a potassium phosphate buffer solution with pH=6.5-7.
5. The shaking amplitude is 300-500 rpm, and the reaction is carried out overnight at 30°C for >18h or =18h. After the reaction is completed, N-BOC-cis-3-hydroxy-L-proline methyl ester is obtained.
9. The method according to claim 8, characterized in that: The circulation system was prepared by adding 50 mg / ml 1s GDH and 2 mM NADP to a potassium phosphate buffer solution at pH 7.
0. + / NAD + It is obtained by mixing with 200mM glucose; wherein the amino acid sequence of the lsGDH is shown in SEQ ID No. 9; Alternatively, the potassium phosphate buffer solution is an aqueous solution with a pH of 7.0 composed of 100 mM dimethyl hydrogen phosphate and potassium dihydrogen phosphate. Alternatively, the ketone reductase mutant in the whole-cell catalyst has an OD in the reaction system. 600 =10, the initial concentration of the substrate is 200mM.
10. The method according to claim 8 or 9, characterized in that: The whole-cell catalyst is prepared as follows: Recombinant engineered bacteria containing the gene encoding ketone reductase mutant were inoculated into liquid LB medium containing ampicillin sodium at a final concentration of 100 ug / mL and cultured at 37°C for 8 h to obtain seed culture. The seed culture was inoculated at a volume concentration of 2% into sterilized TB medium containing a final concentration of 100 μg / mL ampicillin. The culture was incubated at 37°C for about 8-12 h. When the bacterial OD600 was 0.6, isopropyl thio-β-D-galactoside was added to the medium at a final concentration of 0.1-1.0 mM. Expression was induced at 18°C for 16-24 h. After centrifugation at 4°C and 4000 rpm for 10-20 min, the wet bacterial body was resuspended using the reaction buffer. The construction method of the recombinant engineered bacteria containing the gene encoding a ketone reductase mutant is as follows: The ketone reductase mutant gene was constructed into the pET22b(+) vector plasmid, which was then transformed into BL21(DE3) competent cells. After incubating the strain with SOC for one hour, the strain was spread onto solid LB medium containing ampicillin sodium at a final concentration of 100 ug / mL and cultured at 37°C for 16 hours to obtain recombinant engineered bacteria containing the ketone reductase mutant encoding gene.