CN110387359A - Carbonyl reduction enzyme mutant and its application - Google Patents

Carbonyl reduction enzyme mutant and its application Download PDF

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CN110387359A
CN110387359A CN201810343484.8A CN201810343484A CN110387359A CN 110387359 A CN110387359 A CN 110387359A CN 201810343484 A CN201810343484 A CN 201810343484A CN 110387359 A CN110387359 A CN 110387359A
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CN110387359B (en
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陶荣盛
朱傅赟
沈青
郑云
潘震华
沈正权
孙梁栋
刘萍
王亚夫
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Huzhou Yisheng Biotechnology Co., Ltd
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Huzhou Yi Hui Biotechnology Co Ltd
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    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01184—Carbonyl reductase (NADPH) (1.1.1.184)

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Abstract

The invention discloses a kind of carbonyl reduction enzyme mutant constructed by point mutation method, amino acid sequence is selected from SEQ ID NO:2-4.Compared to the wild type carbonyl reductase of 0705 bacterium source of Candida magnoliae ifo, the enzyme activity of carbonyl reduction enzyme mutant of the invention significantly improves, it is 6- cyano-(3R that 6- cyano-(5R)-hydroxyl -3- carbonyl hecanoic acid t-butyl ester asymmetric reduction, which can be efficiently catalyzed, 5R)-dihydroxy hecanoic acid t-butyl ester, and can be catalyzed the chloro- 5- hydroxyl -3- carbonyl hecanoic acid t-butyl ester asymmetric reduction of (S) -6- is the chloro- (3R of 6-, 5S)-dihydroxy hecanoic acid t-butyl ester produces statin compound intermediate.

Description

Carbonyl reduction enzyme mutant and its application
Technical field
The invention belongs to gene engineering technology fields, specifically, being related to the carbonyl reductase constructed by point mutation method Mutant and its application for producing statin compound intermediate.
Background technique
Atorvastatin and Rosuvastatin are that clinically most common Statins regulating plasma lipid medicine, category HMG-CoA are restored Enzyme inhibitor is domestic and international cardiovascular and cerebrovascular for treating hypercholesterolemia, combined hyperlipidemia familial, coronary heart disease and headstroke The great basic pharmaceutical of disease prevention and cure.For example active constituent was once more than for drug " Lipitor " annual sales amount of Atorvastatin 10000000000 dollars, be best-selling drug in history.
As the important chipal compounds intermediate of statins, Atorvastatin intermediate 6- cyano-shown in formula A7 (4R, 6R) -6- cyanogen methyl -2,2- two shown in (3R, 5R)-dihydroxy hecanoic acid t-butyl ester (No. CAS: 125971-93-9), formula A8 Methyl-1, shown in 3- dioxolanes-tert-butyl acetate (No. CAS is 125971-94-0, atorvastatin calcium side chain ATS-8), formula D3 Chloro- (3R, the 5S)-dihydroxy hecanoic acid t-butyl ester of Rosuvastatin intermediate 6- (No. CAS: 154026-93-4) generalling use Synthetic method preparation is learned, chemical method carbonyl substrates method of asymmetrically reducing is mainly used.Specifically using boron hydride as reducing agent, The carbonyl asymmetric reduction of latent chiral substrates is catalyzed using chiral catalysts such as chiral oxazaborolidine, transient metal complexes.It should The control of technology stereocpecificity is difficult, and the diastereomeric induction of reaction process is insufficient, and product optical purity is low；And reaction need to be in deep cooling Under the conditions of plus hydrogen, equipment requirement it is high；Chiral catalyst used is expensive, high production cost；Boron hydride is inflammable and explosive, safety Hidden danger is big, and the boride waste processing for reacting generation is difficult, does not meet the theory of Green Chemistry.Since chemical synthesis has ring The shortcomings that border pollution is big, high production cost, just gradually starts to be replaced with catalyzed by biological enzyme combination chemical method at present.
However, the catalytic activity of currently used enzyme such as carbonyl reductase and glucose dehydrogenase is generally lower, cause Enzyme dosage is very big in catalysis reaction, influences extract yield, and production cost is still higher.
Therefore, there is an urgent need in the art to develop a kind of enzyme with high-efficiency catalytic activity, to reduce production cost, and subtract Of low pollution.
Summary of the invention
The higher carbonyl reductase of enzymatic activity in order to obtain, the present invention is using technique for gene engineering come to candida magnoliae The wild type carbonyl reductase (SEQ ID NO:1) in 0705 source Candida magnoliae ifo is transformed and screens, The carbonyl reduction enzyme mutant for constructing enzymatic activity high, to realize the industrialization of Production by Enzymes statins drug midbody.
Therefore, the first purpose of this invention is to provide a kind of carbonyl reductase of high enzymatic activity, amino acid sequence Selected from SEQ ID NO:2-4:
Wherein SEQ ID NO:2 is that SEQ ID NO:1 the 95th phenylalanine F replaces with the mutant of isoleucine I, Its amino acid sequence are as follows:
MSTPLNALVTGASRGIGAATAIKLAENGYSVTLAARNVAKLNEVKEKLPVVKDGQKHHIWELDLASVEAASSFKGAP LPASDYDLFVSNAGIAQITPTADQTDKDFLNILTVNLSSPIALTKALLKGVSERSNEKPFHIIFLSSAAALHGVPQT AVYSASKAGLDGFVRSLAREVGPKGIHVNVIHPGWTKTDMTDGIDDPNDTPIKGWIQPEAIADAVVFLAKSKNITGT NIVVDNGLLA(SEQ ID NO:2)；
Wherein SEQ ID NO:3 is that SEQ ID NO:1 the 95th phenylalanine F replaces with isoleucine I, the 154th Threonine T replace with the mutant of alanine A, amino acid sequence are as follows:
MSTPLNALVTGASRGIGAATAIKLAENGYSVTLAARNVAKLNEVKEKLPVVKDGQKHHIWELDLASVEAASSFKGAP LPASDYDLFVSNAGIAQITPTADQTDKDFLNILTVNLSSPIALTKALLKGVSERSNEKPFHIIFLSSAAALHGVPQA AVYSASKAGLDGFVRSLAREVGPKGIHVNVIHPGWTKTDMTDGIDDPNDTPIKGWIQPEAIADAVVFLAKSKNITGT NIVVDNGLLA(SEQ ID NO:3)；
SEQ ID NO:4 is that SEQ ID NO:3 the 129th serine S replaces with the third ammonia of arginine R, the 145th again Sour A replaces with the mutant of valine V, amino acid sequence are as follows:
MSTPLNALVTGASRGIGAATAIKLAENGYSVTLAARNVAKLNEVKEKLPVVKDGQKHHIWELDLASVEAASSFKGAP LPASDYDLFVSNAGIAQITPTADQTDKDFLNILTVNLSSPIALTKALLKGVRERSNEKPFHIIFLSSVAALHGVPQA AVYSASKAGLDGFVRSLAREVGPKGIHVNVIHPGWTKTDMTDGIDDPNDTPIKGWIQPEAIADAVVFLAKSKNITGT NIVVDNGLLA(SEQ ID NO:4)；
The wherein amino acid sequence of SEQ ID NO:1 are as follows:
MSTPLNALVTGASRGIGAATAIKLAENGYSVTLAARNVAKLNEVKEKLPVVKDGQKHHIWELDLASVEAASSFKGAP LPASDYDLFVSNAGIAQFTPTADQTDKDFLNILTVNLSSPIALTKALLKGVSERSNEKPFHIIFLSSAAALHGVPQT AVYSASKAGLDGFVRSLAREVGPKGIHVNVIHPGWTKTDMTDGIDDPNDTPIKGWIQPEAIADAVVFLAKSKNITGT NIVVDNGLLA(SEQ ID NO:1)。
It is preferred that the amino acid sequence of above-mentioned carbonyl reductase is SEQ ID NO:4.
Second object of the present invention is to provide the gene for encoding above-mentioned carbonyl reduction enzyme mutant.
In a preferred embodiment, the gene for encoding above-mentioned carbonyl reduction enzyme mutant SEQ ID NO:2 is SEQ ID NO:5:
atgtctacgccgctgaatgctctggtgacgggtgcttctcgtggtattggtgctgcgaccgcgatcaaactggccga aaacggttacagcgtgaccctggcggcccgtaacgtcgcaaaactgaatgaagtgaaagaaaaactgccggtggtta aagatggccagaaacatcacatttgggaactggacctggcctctgtcgaagctgctagctcttttaaaggcgcaccg ctgccggcttcagattatgacctgtttgtttcgaacgcaggtatcgcacagatcaccccgacggcggatcaaaccga taaagacttcctgaacattctgacggtgaatctgagttccccgatcgcgctgaccaaagccctgctgaaaggcgtta gtgaacgctccaatgaaaaaccgtttcatattatcttcctgtcatcggcagcagcactgcacggtgtgccgcagacg gcagtttacagcgcgtctaaagccggcctggatggttttgttcgttcactggctcgcgaagtcggcccgaaaggtat tcatgttaacgtcatccacccgggctggaccaaaacggatatgaccgacggtattgatgacccgaatgatacgccga ttaaaggttggattcagccggaagctatcgcggacgccgtcgtgttcctggcgaaatcaaaaaacatcacgggcacg aacattgtggtggataacggtctgctggcgtga(SEQ IDNO:5)。
In a preferred embodiment, the gene for encoding above-mentioned carbonyl reduction enzyme mutant SEQ ID NO:3 is SEQ ID NO:6:
atgtctacgccgctgaatgctctggtgacgggtgcttctcgtggtattggtgctgcgaccgcgatcaaactggccga aaacggttacagcgtgaccctggcggcccgtaacgtcgcaaaactgaatgaagtgaaagaaaaactgccggtggtta aagatggccagaaacatcacatttgggaactggacctggcctctgtcgaagctgctagctcttttaaaggcgcaccg ctgccggcttcagattatgacctgtttgtttcgaacgcaggtatcgcacagatcaccccgacggcggatcaaaccga taaagacttcctgaacattctgacggtgaatctgagttccccgatcgcgctgaccaaagccctgctgaaaggcgtta gtgaacgctccaatgaaaaaccgtttcatattatcttcctgtcatcggcagcagcactgcacggtgtgccgcaggcg gcagtttacagcgcgtctaaagccggcctggatggttttgttcgttcactggctcgcgaagtcggcccgaaaggtat tcatgttaacgtcatccacccgggctggaccaaaacggatatgaccgacggtattgatgacccgaatgatacgccga ttaaaggttggattcagccggaagctatcgcggacgccgtcgtgttcctggcgaaatcaaaaaacatcacgggcacg aacattgtggtggataacggtctgctggcgtga(SEQ IDNO:6)。
In a preferred embodiment, the gene for encoding above-mentioned carbonyl reduction enzyme mutant SEQ ID NO:4 is SEQ ID NO:7:
atgtctacgccgctgaatgctctggtgacgggtgcttctcgtggtattggtgctgcgaccgcgatcaaactggccga aaacggttacagcgtgaccctggcggcccgtaacgtcgcaaaactgaatgaagtgaaagaaaaactgccggtggtta aagatggccagaaacatcacatttgggaactggacctggcctctgtcgaagctgctagctcttttaaaggcgcaccg ctgccggcttcagattatgacctgtttgtttcgaacgcaggtatcgcacagatcaccccgacggcggatcaaaccga taaagacttcctgaacattctgacggtgaatctgagttccccgatcgcgctgaccaaagccctgctgaaaggcgtta gggaacgctccaatgaaaaaccgtttcatattatcttcctgtcatcggtagcagcactgcacggtgtgccgcaggcg gcagtttacagcgcgtctaaagccggcctggatggttttgttcgttcactggctcgcgaagtcggcccgaaaggtat tcatgttaacgtcatccacccgggctggaccaaaacggatatgaccgacggtattgatgacccgaatgatacgccga ttaaaggttggattcagccggaagctatcgcggacgccgtcgtgttcctggcgaaatcaaaaaacatcacgggcacg aacattgtggtggataacggtctgctggcgtga(SEQ IDNO:7)。
Third object of the present invention is to provide the plasmid comprising said gene.
Fourth object of the present invention is to provide the microorganism for having converted above-mentioned plasmid.The microorganism can be selected from withered Careless bacillus, Lactobacillus brevis, candida magnoliae, Pichia pastoris, saccharomyces cerevisiae, Escherichia coli.
Preferably, the microorganism is e. coli bl21 (DE3).
Of the invention the 5th is designed to provide above-mentioned carbonyl reduction enzyme mutant or it is expressed microorganism and is producing him Purposes in the class pharmaceutical intermediate of spit of fland.The intermediate includes but is not limited to 6- cyano-(3R, 5R)-dihydroxy hecanoic acid t-butyl ester (No. CAS: 125971-93-9), (4R, 6R) -6- cyanogen methyl -2,2- dimethyl -1,3- dioxolanes-tert-butyl acetate (No. CAS For 125971-94-0, atorvastatin calcium side chain ATS-8), chloro- (3R, the 5S)-dihydroxy hecanoic acid t-butyl ester of 6- (No. CAS: 154026-93-4)。
In production 6- cyano-(3R, 5R)-dihydroxy hecanoic acid t-butyl ester (No. CAS: 125971-93-9), shown in formula A6 (R)-t-butyl 6-cyano-5-hydroxyl-3-oxo-hexanoate (No. CAS: 125988-01-4) be substrate raw material, with above-mentioned carbonyl It restores enzyme mutant or it expresses microorganism as the catalyst to catalysis reaction.
In production chloro- (3R, the 5S)-dihydroxy hecanoic acid t-butyl ester of 6-, with the chloro- 5- hydroxyl -3- of (S) -6- shown in formula D2 Oxo hecanoic acid t-butyl ester (No. CAS: 154026-92-3) is substrate raw material, with above-mentioned carbonyl reduction enzyme mutant or its expression Microorganism is as the catalyst to catalysis reaction.
In one embodiment, above-mentioned reaction is in glucose dehydrogenase, glucose and NADP+In the presence of carry out.
The amino acid sequence of above-mentioned glucose dehydrogenase is preferably SEQ ID NO:8:
MYPDLKGKVVAITGAASGLGKAMAIRFGKEQAKVVINYYSNKQDPNEVKEEVIKAGGEAVVVQGDVTKEEDVKNIVQ TAIKEFGTLDIMINNAGLENPVPSHEMPLKDWDKVIGTNLTGAFLGSREAIKYFVENDIKGNVINMSSVHEVIPWPL FVHYAASKGGIKLMTRTLALEYAPKGIRVNNIGPGAINTPINAEKFADPKQKADVESMIPMGYIGEPEEIAAVAAWL ASKEASYVTGITLFADGGMTLYPSFQAGRG(SEQ ID NO:8)。
Preferably, 6- cyano-(3R, 5R)-dihydroxy hecanoic acid t-butyl ester can further by chemical reaction, such as with 2, 2- dimethoxy propane and methanesulfonic acid reaction, synthesize (4R, 6R) -6- cyanogen methyl -2,2- dimethyl -1,3-dioxolane-acetic acid The tert-butyl ester (No. CAS is 125971-94-0).
Wild enzyme SEQ ID NO:1 is compared, the enzymatic activity of carbonyl reduction enzyme mutant SEQ ID NO:2-4 of the invention is big Amplitude improves, and the solid for all having height for Atorvastatin intermediate substrate and Rosuvastatin intermediate substrate is special Property, great industrial prospect.
Detailed description of the invention
Fig. 1 is the TLC contact plate tracking photo after CRHZ and 3 mutant catalytic cpd A6 reaction 4h；
Fig. 2 is the TLC contact plate tracking photo after CRHZ and 3 mutant catalytic cpd D2 reaction 4h.
Specific embodiment
The present invention is described in further details below in conjunction with specific embodiment.It should be understood that following embodiment is only used for The bright present invention is not for limiting the scope of the invention.
Additive amount, content and the concentration of many kinds of substance is referred to herein, wherein the percentage composition, except special instruction Outside, all refer to mass percentage.
In the present invention, term " carbonyl reduction enzyme mutant ", " mutant carbonyl reductase ", " mutation carbonyl reductase " " mutant enzyme " indicates identical meaning, all refers to the mutant of carbonyl reductase.It for simplicity, herein can be by " carbonyl Reduction enzyme mutant " is referred to as " carbonyl reductase ", as long as not obscuring with wild type carbonyl reductase SEQ ID NO:1.
In the present invention, term " wild (type) carbonyl reductase ", " wild carbonyl reductase " indicate identical meaning, all Refer to the carbonyl reductase or CRHZ (SEQ ID NO:1) of wild type.
For the sake of description simplicity, " carbonyl reductase " is referred to as " CRHZ " sometimes herein, they indicate identical Meaning may be used interchangeably.Sometimes " glucose dehydrogenase " is referred to as " GDH ", they indicate identical meaning, can be interchanged It uses.
The amino acid sequence of carbonyl reductase and glucose dehydrogenase of the invention is clear, therefore those skilled in the art are very It is easy to get its encoding gene, the expression cassette comprising these genes and plasmid and the transformant comprising the plasmid.
These genes, expression cassette, plasmid, transformant can be constructed by genetic engineering well-known to those skilled in the art Mode obtains.
When as being that biocatalyst is used to produce Atorvastatin and Rosuvastatin intermediate, carbonyl of the invention is also The form of enzyme or the form of thallus can be presented in protoenzyme and glucose dehydrogenase.The form of the enzyme includes resolvase, fixation Change enzyme, including the enzyme etc. that purifying enzyme, thick enzyme, fermentation liquid, carrier are fixed；The form of the thallus includes survival thallus and dead bacterium Body.
Carbonyl reductase and glucose dehydrogenase of the invention isolates and purifies including immobilised enzymes technology of preparing is also ability Known to field technique personnel.
Embodiment
Material and method
Full genome synthesis, primer synthesis and sequencing in embodiment is all complete by Nanjing Genscript Biotechnology Co., Ltd. At.
Molecular biology experiment in embodiment includes the main ginsengs such as plasmid construction, digestion, competent cell preparation, conversion According to " Molecular Cloning:A Laboratory guide " (third edition), J. Pehanorm Brooker, D.W. Russell (beauty) is write, and Huang Peitang etc. is translated, science Publishing house, Beijing, 2002) it carries out.Specific experiment condition can be determined by simple experiment when necessary.
The reaction condition or kit specification that PCR amplification experiment is provided according to plasmid or DNA profiling supplier carry out.It must It can be adjusted by simple experiment when wanting.
LB culture medium: 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride, pH7.2,121 DEG C of high temperature and pressure Sterilize 20min；
TB culture medium: 24g/L yeast extract, 12g/L tryptone, 16.43g/L K2HPO4.3H2O、2.31g/L KH2PO4, 5g/L glycerol, pH7.0-7.5,121 DEG C of autoclave sterilization 20min；
Slant medium: 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride, 20g/L agar powder mix It is dispensed into eggplant bottle by 30-40mL liquid amount afterwards, setting is placed in 121 DEG C of autoclave sterilization 20min, 100 μ are added after cooling G/mL kanamycin sulfate puts into inclined-plane, to be condensed at solid.
Fermented and cultured
Seed activation: taking seed glycerol stocks pipe, takes 100 μ L Seed Deposit liquid, eggplant bottle is uniformly applied to oese Inclined-plane is subsequently placed in 37 DEG C of incubator overnight incubations (18h)；
Seed culture: it takes 100mL sterile water to import eggplant bottle and is made into bacteria suspension, take 50 μ l of bacteria suspension access equipped with 50mL The 250mL shaking flask of TB culture medium, 30 DEG C, 220rpm cultivates 16h；
Fermentation: the access of first order seed culture solution is equipped with to the 5L shaking flask of 1L TB culture medium, 37 DEG C, 220rpm cultivates 4-6h Afterwards, 0.3mM IPTG is added and is cooled to 28 DEG C, 220rpm Fiber differentiation 12h.
Microorganism collection: collecting fermentation liquid, is placed in centrifuge 4000rpm centrifugation 30min, collects bacterium mud after abandoning supernatant, place It is collected in -20 DEG C of freezings.
The preparation of enzyme solution: weighing GDH, CRHZ and its each 5g of mutant bacterium mud respectively, and 20mL deionized water is then respectively added After resuspension, clasmatosis is carried out by ultrasonic wave, broken liquid takes supernatant, as enzyme solution after 12000rpm is centrifuged 10min, It is placed in ice bath stand-by.
The building of embodiment 1 wild type carbonyl reductase (CRHZ) gene recombined escherichia coli
According to 0705 source candida magnoliae Candida magnoliae ifo provided patent EP1152054A1 The amino acid sequence of carbonyl reductase CRHZ carries out the codon optimization suitable for Bacillus coli expression, and full genome synthesizes the gene Sequence designs restriction enzyme site NdeI and EcoRI at both ends, and is subcloned into corresponding positions on carrier pET24a (purchased from Novagen) Point, to obtain recombinant plasmid pET24a-CRHZ.The recombinant plasmid pET24a-CRHZ built Calcium Chloride Method is converted big Enterobacteria expressive host BL21 (DE3), obtain expression wild type carbonyl reductase recombination bacillus coli BL21 (DE3)/ pET24a-CRHZ。
The gene order of codon optimization is (SEQ ID NO:9):
atgtctacgccgctgaatgctctggtgacgggtgcttctcgtggtattggtgctgcgaccgcgatcaaactggccga aaacggttacagcgtgaccctggcggcccgtaacgtcgcaaaactgaatgaagtgaaagaaaaactgccggtggtta aagatggccagaaacatcacatttgggaactggacctggcctctgtcgaagcagctagctcttttaaaggcgcaccg ctgccggcttcagattatgacctgtttgtttcgaacgcaggtatcgctcagttcaccccgacggcggatcaaaccga taaagacttcctgaacattctgacggtgaatctgagttccccgatcgcgctgaccaaagccctgctgaaaggcgtta gtgaacgctccaatgaaaaaccgtttcatattatcttcctgtcatcggcagcagcactgcacggtgtgccgcagacg gcagtttacagcgcgtctaaagccggcctggatggttttgttcgttcactggctcgcgaagtcggcccgaaaggtat tcatgttaacgtcatccacccgggctggaccaaaacggatatgaccgacggtattgatgacccgaatgatacgccga ttaaaggttggattcagccggaagctatcgcggacgccgtcgtgttcctggcgaaatcaaaaaacatcacgggcacg aacattgtggtggataacggtctgctggcgtga(SEQ ID NO:9)。
Determined amino acid sequence is SEQ ID NO:1.
The building of 2 glucose dehydrogenase GDH of embodiment expression strain
Bacillus subtilis Bacillus subtilis subsp.subtilis str.168 is inoculated in the training of LB liquid It supports in base, 30 DEG C of 220rpm are cultivated 24 hours.The extraction reference gene group extracts kit specification of total DNA is (raw purchased from raw work Object engineering Shanghai limited liability company).
According to the reported source bacillus subtilis Bacillus subtilis subsp.subtilis str.168 Glucose dehydrogenase GDH gene order (NCBI accession number: AL009126.3), design primer is as follows:
Forward primer GDH-F:5 '-CGGGATCCATGTATCCGGATTTAAAAG-3 ' (BamHI),
Reverse primer GDH-R:5 '-CCCAAGCTTTTAACCGCGGCCTGCCTGG-3 ' (HindIII).
PCR reaction system includes: each 50pmol of GDH-F and GDH-R, total DNA 100ng, 1X KOD plus buffer, 0.2mM dNTP, 25mM MgSO4, KOD plus 2U, moisturizing to 50 μ L of total system.
PCR amplification condition are as follows: 95 DEG C of 5min, 94 DEG C of 45s, 55 DEG C of 45s, 68 DEG C of 1min repeat 30 and recycle, and 68 DEG C 10min。
PCR after reaction, is analyzed with agarose gel electrophoresis, detects the specific band of an about 800bp, For required band.Pcr amplification product is recycled using a small amount of plastic recovery kits, with BamHI and HindIII in 37 DEG C of double digestion 3- 6 hours, cross column purification recycling.The expression vector pET24a that recovery product and same digestion are handled is with T4DNA ligase in 16 DEG C Overnight, Transformed E .coli DH5 α competent cell, picking transformant carries out sequence verification for connection, obtains recombinant plasmid.
Rite-directed mutagenesis is done to the 252nd of GDH amino acid sequence and the 170th two sites, by the 170th glutamic acid (E) arginine (R) is sported, the 252nd glutamine (Q) is sported into leucine (L).According to amino acid to be mutated and Mutational site E170R, Q252L design primer is dashed forward using MEGA WHOP method (Arnold and Georgiou 2003) Become.Design primer is as follows:
Forward primer GDHE170R-F:AAGCTGATGACACGAACATTAGCGTT,
Reverse primer GDHQ252L-R:AATGAAGGATATAGTGTCATACCGC.
Sequence containing mutational site Q252L/E170R is gone out to primer amplification with this.PCR reaction system includes:
GDHE170R-F and GDHQ252L-R each 50pmol, plasmid template pET24a-GDH 50ng, 1X KOD plus Buffer, 0.2mM dNTP, 25mM MgSO4, KOD plus 2U, moisturizing to 50 μ L of total system.
PCR amplification condition are as follows: 95 DEG C of 5min, 94 DEG C of 45s, 55 DEG C of 45s, 68 DEG C of 30s repeat 30 and recycle, and 68 DEG C 10min.PCR after reaction, is analyzed with agarose gel electrophoresis, is detected the specific band of an about 250bp, is Required band.A small amount of plastic recovery kits recycle pcr amplification product.Using this PCR product as big primer, it is with pET24a-GDH Template does full plasmid linear amplification using high-fidelity DNA polymerase KOD plus, and PCR reaction system includes: big primer segments 50-100pmol, plasmid template pET24a-GDH 50ng, 1X KOD plus buffer, 0.2mM dNTP, 25mM MgSO4, KOD plus 2U, moisturizing to 50 μ L of total system.
PCR amplification condition are as follows: 95 DEG C of 5min, 94 DEG C of 45s, 55 DEG C of 45s, 68 DEG C of 6min repeat 25 and recycle, and 68 DEG C 10min.After the completion of amplification, DpnI is added in system and removes plasmid template in 37 DEG C of digestion, then directly turns digestion product Change E.coli BL21 (DE3) competent cell.Picked clones carry out sequence verification, and correct strain is sequenced and is named as BL21 (DE3)/pET24a-GDH。
Gene order is as follows:
atgtatccggatttaaaaggaaaagtcgtcgctattacaggagctgcttcagggctcggaaaggcgatggccattcg cttcggcaaggagcaggcaaaagtggttatcaactattatagtaataaacaagatccgaacgaggtaaaagaagagg tcatcaaggcgggcggtgaagctgttgtcgtccaaggagatgtcacgaaagaggaagatgtaaaaaatatcgtgcaa acggcaattaaggagttcggcacactcgatattatgattaataatgccggtcttgaaaatcctgtgccatctcacga aatgccgctcaaggattgggataaagtcatcggcacgaacttaacgggtgcctttttaggaagccgtgaagcgatta aatatttcgtagaaaacgatatcaagggaaatgtcattaacatgtccagtgtgcacgaagtgattccttggccgtta tttgtccactatgcggcaagtaaaggcgggataaagctgatgacacgaacattagcgttggaatacgcgccgaaggg cattcgcgtcaataatattgggccaggtgcgatcaacacgccaatcaatgctgaaaaattcgctgaccctaaacaga aagctgatgtagaaagcatgattccaatgggatatatcggcgaaccggaggagatcgccgcagtagcagcctggctt gcttcgaaggaagccagctacgtcacaggcatcacgttattcgcggacggcggtatgacactatatccttcattcca ggcaggccgcggttaa(SEQ ID NO:10)。
Determined amino acid sequence is SEQ ID NO:8.
The building of embodiment 3 fallibility PCR and random mutation library
Using the encoding gene of CRHZ as template, random mutant library is constructed using fallibility PCR and large primer PCR technology.If It is as follows to count primer:
Forward primer CRHZerr-F:5 '-GTTTAACTTTAAGAAGGAGATATAC；
Reverse primer CRHZerr-R:5 '-CTTGTCGACGGAGCTCGAAT-3 '.
100 μ L fallibility PCR reaction systems include: 50ng plasmid template, each 0.2 μM of pair of primers CRHZerr-F and CRHZerr-R, 1X Taq buffer, 0.2mM dGTP, 0.2mM dATP, 1mM dCTP, 1mM dTTP, 7mM MgCl2, (0.2mM,0.3mM,0.4mM)MnCl2, 1U Taq.
PCR reaction condition are as follows: 95 DEG C, 5min；94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1min, 40 circulations；72℃7min.Glue 1kb random mutation segment is recycled as big primer, is MegaPrimer PCR:94 DEG C with KOD FX neo archaeal dna polymerase, 2min, 68 DEG C of 10min；98 DEG C of 10s, 55 DEG C of 30s, 68 DEG C of 3min, 25 circulations；68℃10min.
DpnI is added in PCR product and removes plasmid template, electrotransformation e. coli after purification and recovery in 37 DEG C of digestion 37 DEG C of recovery 1h of 1mLLB culture medium are added in E.coli BL21 (DE3), are coated with 37 DEG C of overnight incubations of Kan plate, are more than 104The random mutant library of a clone.
4 high flux screening CRHZ mutant of embodiment
Transformant in mutant library is inoculated into the 96 hole depth well culture plates containing 200 μ L LB culture mediums with toothpick, is contained 100 μ g/mL Kan (kanamycins), 37 DEG C, 220rpm culture 6h or more；Add 200 μ L LB+100 μ g/mL, Kan+ in every hole 0.6mM IPTG culture medium, 28 DEG C, 220rpm overnight incubation.
The 96 orifice plates oscillation of overnight induction beats thallus, takes 15 μ L bacterium solutions to move to another piece of 96 orifice plates, is put into -70 DEG C of jelly 1- 2 hours, 37 DEG C of thawing 20min.The dilution of 100 μ L water is added in every hole, and oscillation inhales the enzyme solution transfer after 15 μ L dilution in every hole after mixing Onto another piece of 96 orifice plates, 185 hole μ L/ of chromogenic reaction liquid (60g/L glucose, tri- ethyl alcohol of 1.8g/L EDTA, 8.4g/L is added Amine, 6 μ L compound A6,9 μ L isopropanols, 46 μ L methyl reds, 5 μ L 4000U/mL GDH enzyme solutions, 0.05g/L NADP+, pH 7.0), 37 DEG C of 180rpm reaction 1.5h observe color change, and the muton that color is reddened detects progress from corresponding motherboard Secondary screening and enzyme activity determination.
The preparation of methyl red: 0.1g methyl red is dissolved in 60% ethyl alcohol of 100mL.
Enzyme activity determination method: taking 250mL beaker, weighs 0.5g triethanolamine, and 24g glucose and 25g compound A6 are placed in In beaker, and the water of 25mL is added after mixing evenly and is warming up to 30 DEG C.With 20% H2SO4PH7.0 is adjusted, carbonyl is separately added into Base reductase enzyme solution (2.5mL) and GDH enzyme solution (1.39mL), are eventually adding NADP+(0.315mL) starts to react, reaction process The middle sodium carbonate liquor that 1M is added dropwise controls pH7.0, and the sodium carbonate liquor amount of solution being added dropwise with 1 hour characterizes enzyme activity.
By this screening technique, enzyme activity raising has been filtered out from the mutant library using CRHZ encoding gene as template Mutant CRHZ18；Random mutant library is rebuild by template of CRHZ18 encoding gene again, using same screening technique Filter out the mutant 154A6 that enzyme activity further increases；Repeat aforesaid operations step, continue on the basis of 154A6 with Machine mutation and directed screening finally obtain the mutant 246G12 that an enzyme activity greatly improves.
Carbonyl reductase mutant no Mutational site (compared with CRHZ) SEQ ID NO:
CRHZ18 F95I 2
154A6 F95I,T154A 3
246G12 F95I,T154A,S129R,A145V 4
5 wild type CRHZ of embodiment and 3 mutant compare the catalytic activity of compound A6
5.1, experimental method:
1) 250mL beaker is taken, 0.5g triethanolamine is weighed, 24g glucose and 25g compound A6 are placed in a beaker, and are added Enter the water of 25mL after mixing evenly and is warming up to 30 DEG C.
2) with 20% H2SO4PH7.0 is adjusted, carbonyl reductase enzyme solution (1.25mL) and GDH enzyme solution are separately added into (1.39mL), is eventually adding NADP+(0.315mL) starts to react, and controls pH7.0 with the sodium carbonate liquor of 1M in reaction process, And timesharing segment record dosage.It is tracked and is reacted with TLC.
3) carbonyl reductase that wherein 1# reaction is added is CRHZ；The carbonyl reductase that 2# reaction is added is CRHZ18；3# The carbonyl reductase that reaction is added is 154A6；The carbonyl reductase that 4# reaction is added is 246G12.
5.2, experimental data: due to 1 molecule substrate A6 of every conversion, 1 molecule glucose acid will be generated, 0.5 molecule is needed Sodium carbonate, so the operating speed of sodium carbonate liquor be represent catalysis reaction speed speed and enzyme activity height.
5.2.1, the dosage of sodium carbonate liquor compares:
The above results show to compare wild enzyme CRHZ, mutant 154A6 and mutant 246G12 when being catalyzed A6 reduction Enzymatic activity increase substantially, such as reaction 1h when, the enzymatic activity of mutant 154A6 is shown as more than 2 times of wild type, and is mutated The enzymatic activity of body 246G12 is shown as more than 7 times of wild type.When reacting 4h, the enzymatic activity of mutant 154A6 is shown as wild type More than 2 times, and the enzymatic activity of mutant 246G12 is shown as more than 4 times of wild type.
5.2.2, TLC contact plate:
Referring to Fig. 1, thin layer chromatography TLC testing result also demonstrates 246G12 and reacts most fast, and after reacting 4h, substrate is It is reacted to be over.
5.2.3 chiral determination
Since the testing requirements of product A7 are high, in production, all use is further blended into compound A-28 and carries out chirality again Detection.The specific method is as follows:
To above-mentioned 1#, 2# and 3# reaction, the reaction was continued to 16h, TLC detection all fully reactings, the feed liquid of end of reaction It handles respectively in accordance with the following steps:
Step 1: ethyl acetate 250mL and diatomite 25g being added in reacted feed liquid, is warming up to 50~60 DEG C, filtering After be layered, water layer is extracted twice with 150mL ethyl acetate, merge organic layer, be concentrated to get compound A7 crude product, be grease.
Grease, 2,2- dimethoxy propane 25g, toluene 50mL and methanesulfonic acid that step 1 obtains are added in flask 0.2g is warming up to 25 DEG C, insulation reaction 3 hours.After reaction, 100mL toluene is added, adds in saturated sodium bicarbonate aqueous solution With to pH7.5, dried organic layer is concentrated in branch vibration layer.75mL hexane is added, rising temperature for dissolving is subsequently cooled to 0 DEG C, crystallizes, mistake Filter, obtains compound A-28 crude product.It is recrystallized again with normal hexane (50mL) and ethyl alcohol (2.5mL) mixed solvent, chemical combination is dried to obtain in filtering Object A8.
The chiral analysis of compound A-28:
Detecting instrument: 1200 type high performance liquid chromatograph of Agilent
Liquid phase mobile phase: normal hexane: the mellow 98:2 of isopropyl
Wavelength X=215nm, flow velocity 0.6mL/min, solvent: mobile phase；
Chromatographic column: 5 μm of 250 × 4.6mm of Chiralcel OD-H；
Sample volume: 20 μ l, column temperature: 30 DEG C, runing time: 28min.
The retention time of compound A-28 is 19.8min or so；4R, 6S- isomers retention time 14.1min or so；4S, 6S- isomers retention time 15.5min or so；4S, 6R- isomers retention time 16.9min or so.
Sample analysis: CRHZ (1#), CRHZ18 (2#), 154A6 (3#) and 246G12 (4#) are weighed and is catalyzed and handles respectively Obtained A8 sample 200mg, is placed in the volumetric flask of 10ml, with solvent dissolution, constant volume, shakes up, as sample solution.Take sample 20 μ L of solution injects liquid chromatograph, is detected.
The result shows that the content of enantiomter 4S, 6S- isomers is equal in 4 samples under the conditions of liquid phase chiral analysis No more than 0.02%, other isomers are not detected.Show the wild type CRHZ of the invention constructed and 3 mutant for substrate A6 all has the stereospecificity of height.
6 wild type CRHZ of embodiment and 3 mutant compare the catalytic activity of compound D2
6.1, experimental method:
1) 250mL beaker is taken, 0.5g triethanolamine is weighed, 24g glucose and 25g substrate compounds D2 are placed in a beaker, And the water that 25mL is added is warming up to 30 DEG C after mixing evenly.
2) with 20% H2SO4PH7.0 is adjusted, carbonyl reductase enzyme solution (1.25mL) and GDH enzyme solution are separately added into (1.39mL), is eventually adding NADP+(0.315ml) starts to react, and controls pH7.0 with the sodium carbonate liquor of 1M in reaction process, And timesharing segment record caustic dosage.It is tracked and is reacted with TLC.
3) carbonyl reductase that wherein 1# reaction is added is CRHZ；The carbonyl reductase that 2# reaction is added is CRHZ18；3# The carbonyl reductase that reaction is added is 154A6；The carbonyl reductase that 4# reaction is added is 246G12.
6.2, experimental data: due to 1 molecule substrate D2 of every conversion, 1 molecule glucose acid will be generated, 0.5 molecule is needed Sodium carbonate, so the operating speed of sodium carbonate liquor be represent catalysis reaction speed speed and enzyme activity height.
6.2.1, caustic dosage compares:
The above results show to compare wild enzyme CRHZ, mutant 154A6 and mutant 246G12 when being catalyzed D2 reduction Enzymatic activity increase substantially, such as reaction 1h when, the enzymatic activity of mutant 154A6 is shown as more than 3 times of wild type, and is mutated The enzymatic activity of body 246G12 is shown as 8 times of wild type.When reacting 4h, the enzymatic activity of mutant 154A6 is shown as wild type Nearly 4 times, and the enzymatic activity of mutant 246G12 is shown as more than 4 times of wild type.
6.2.2, TLC contact plate:
Referring to fig. 2, thin layer chromatography TLC testing result also demonstrate 246G12 reaction it is most fast, react 4h after, substrate is It is reacted to be over.
6.2.3 liquid phase and chiral detection
Detecting instrument: 1200 type high performance liquid chromatograph of Agilent.
The detection method of product in the present invention:
HPLC chromatogram column: ZORBAX SB-C8,4.6 × 150mm, 5-Micron,
Mobile phase: 30% acetonitrile, flow velocity: 1.0mL/min,
Column temperature: 40 DEG C, Detection wavelength: 210nm.
Product D3 retention time: 7.1min or so；Substrate D2 retention time: 12.0min or so.
Liquid phase detection conversion 4h the results show that the substrate transformation rate with add sodium carbonate liquor amount it is corresponding, respectively 22.7%, 48.26%, 84.6% and 99.7%；
When detection is chiral, using chiral chromatographic column OD-H column (250 × 4.6mm, 5 μm), mobile phase: n-hexane: isopropanol =85:15, flow velocity 1mL/min, Detection wavelength 215nm.Compound D3 and the retention time of (3S, 5S)-isomers are respectively 5.1min and 4.9min.Testing result shows that the product ee value of CRHZ, CRHZ18,154A6 and 246G12 catalysis reduction all exists 99.9% or more, show that the wild type CRHZ that the present invention constructs and the solid that 3 mutant all have height for substrate D2 are special It is anisotropic.
In conclusion the present invention constructs carbonyl reduction enzyme mutant SEQ ID NO:2-4, wild type carbonyl reduction is compared The enzyme activity of enzyme, two mutant is significantly improved, and for substrate Atorvastatin intermediate 6- cyano-(3R, 5R)- Dihydroxy hecanoic acid t-butyl ester and chloro- (3R, the 5S)-dihydroxy hecanoic acid t-butyl ester of Rosuvastatin intermediate 6- all have the vertical of height Body specificity, has wide industrial prospect.
Sequence table
<110>Huzhou Yi Hui Biotechnology Co., Ltd
<120>carbonyl reduction enzyme mutant and its application
<130> SHPI810323
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 241
<212> PRT
<213> Candida magnoliae ifo 0705
<400> 1
Met Ser Thr Pro Leu Asn Ala Leu Val Thr Gly Ala Ser Arg Gly Ile
1 5 10 15
Gly Ala Ala Thr Ala Ile Lys Leu Ala Glu Asn Gly Tyr Ser Val Thr
20 25 30
Leu Ala Ala Arg Asn Val Ala Lys Leu Asn Glu Val Lys Glu Lys Leu
35 40 45
Pro Val Val Lys Asp Gly Gln Lys His His Ile Trp Glu Leu Asp Leu
50 55 60
Ala Ser Val Glu Ala Ala Ser Ser Phe Lys Gly Ala Pro Leu Pro Ala
65 70 75 80
Ser Asp Tyr Asp Leu Phe Val Ser Asn Ala Gly Ile Ala Gln Phe Thr
85 90 95
Pro Thr Ala Asp Gln Thr Asp Lys Asp Phe Leu Asn Ile Leu Thr Val
100 105 110
Asn Leu Ser Ser Pro Ile Ala Leu Thr Lys Ala Leu Leu Lys Gly Val
115 120 125
Ser Glu Arg Ser Asn Glu Lys Pro Phe His Ile Ile Phe Leu Ser Ser
130 135 140
Ala Ala Ala Leu His Gly Val Pro Gln Thr Ala Val Tyr Ser Ala Ser
145 150 155 160
Lys Ala Gly Leu Asp Gly Phe Val Arg Ser Leu Ala Arg Glu Val Gly
165 170 175
Pro Lys Gly Ile His Val Asn Val Ile His Pro Gly Trp Thr Lys Thr
180 185 190
Asp Met Thr Asp Gly Ile Asp Asp Pro Asn Asp Thr Pro Ile Lys Gly
195 200 205
Trp Ile Gln Pro Glu Ala Ile Ala Asp Ala Val Val Phe Leu Ala Lys
210 215 220
Ser Lys Asn Ile Thr Gly Thr Asn Ile Val Val Asp Asn Gly Leu Leu
225 230 235 240
Ala
<210> 2
<211> 241
<212> PRT
<213>artificial sequence ()
<400> 2
Met Ser Thr Pro Leu Asn Ala Leu Val Thr Gly Ala Ser Arg Gly Ile
1 5 10 15
Gly Ala Ala Thr Ala Ile Lys Leu Ala Glu Asn Gly Tyr Ser Val Thr
20 25 30
Leu Ala Ala Arg Asn Val Ala Lys Leu Asn Glu Val Lys Glu Lys Leu
35 40 45
Pro Val Val Lys Asp Gly Gln Lys His His Ile Trp Glu Leu Asp Leu
50 55 60
Ala Ser Val Glu Ala Ala Ser Ser Phe Lys Gly Ala Pro Leu Pro Ala
65 70 75 80
Ser Asp Tyr Asp Leu Phe Val Ser Asn Ala Gly Ile Ala Gln Ile Thr
85 90 95
Pro Thr Ala Asp Gln Thr Asp Lys Asp Phe Leu Asn Ile Leu Thr Val
100 105 110
Asn Leu Ser Ser Pro Ile Ala Leu Thr Lys Ala Leu Leu Lys Gly Val
115 120 125
Ser Glu Arg Ser Asn Glu Lys Pro Phe His Ile Ile Phe Leu Ser Ser
130 135 140
Ala Ala Ala Leu His Gly Val Pro Gln Thr Ala Val Tyr Ser Ala Ser
145 150 155 160
Lys Ala Gly Leu Asp Gly Phe Val Arg Ser Leu Ala Arg Glu Val Gly
165 170 175
Pro Lys Gly Ile His Val Asn Val Ile His Pro Gly Trp Thr Lys Thr
180 185 190
Asp Met Thr Asp Gly Ile Asp Asp Pro Asn Asp Thr Pro Ile Lys Gly
195 200 205
Trp Ile Gln Pro Glu Ala Ile Ala Asp Ala Val Val Phe Leu Ala Lys
210 215 220
Ser Lys Asn Ile Thr Gly Thr Asn Ile Val Val Asp Asn Gly Leu Leu
225 230 235 240
Ala
<210> 3
<211> 241
<212> PRT
<213>artificial sequence ()
<400> 3
Met Ser Thr Pro Leu Asn Ala Leu Val Thr Gly Ala Ser Arg Gly Ile
1 5 10 15
Gly Ala Ala Thr Ala Ile Lys Leu Ala Glu Asn Gly Tyr Ser Val Thr
20 25 30
Leu Ala Ala Arg Asn Val Ala Lys Leu Asn Glu Val Lys Glu Lys Leu
35 40 45
Pro Val Val Lys Asp Gly Gln Lys His His Ile Trp Glu Leu Asp Leu
50 55 60
Ala Ser Val Glu Ala Ala Ser Ser Phe Lys Gly Ala Pro Leu Pro Ala
65 70 75 80
Ser Asp Tyr Asp Leu Phe Val Ser Asn Ala Gly Ile Ala Gln Ile Thr
85 90 95
Pro Thr Ala Asp Gln Thr Asp Lys Asp Phe Leu Asn Ile Leu Thr Val
100 105 110
Asn Leu Ser Ser Pro Ile Ala Leu Thr Lys Ala Leu Leu Lys Gly Val
115 120 125
Ser Glu Arg Ser Asn Glu Lys Pro Phe His Ile Ile Phe Leu Ser Ser
130 135 140
Ala Ala Ala Leu His Gly Val Pro Gln Ala Ala Val Tyr Ser Ala Ser
145 150 155 160
Lys Ala Gly Leu Asp Gly Phe Val Arg Ser Leu Ala Arg Glu Val Gly
165 170 175
Pro Lys Gly Ile His Val Asn Val Ile His Pro Gly Trp Thr Lys Thr
180 185 190
Asp Met Thr Asp Gly Ile Asp Asp Pro Asn Asp Thr Pro Ile Lys Gly
195 200 205
Trp Ile Gln Pro Glu Ala Ile Ala Asp Ala Val Val Phe Leu Ala Lys
210 215 220
Ser Lys Asn Ile Thr Gly Thr Asn Ile Val Val Asp Asn Gly Leu Leu
225 230 235 240
Ala
<210> 4
<211> 241
<212> PRT
<213>artificial sequence ()
<400> 4
Met Ser Thr Pro Leu Asn Ala Leu Val Thr Gly Ala Ser Arg Gly Ile
1 5 10 15
Gly Ala Ala Thr Ala Ile Lys Leu Ala Glu Asn Gly Tyr Ser Val Thr
20 25 30
Leu Ala Ala Arg Asn Val Ala Lys Leu Asn Glu Val Lys Glu Lys Leu
35 40 45
Pro Val Val Lys Asp Gly Gln Lys His His Ile Trp Glu Leu Asp Leu
50 55 60
Ala Ser Val Glu Ala Ala Ser Ser Phe Lys Gly Ala Pro Leu Pro Ala
65 70 75 80
Ser Asp Tyr Asp Leu Phe Val Ser Asn Ala Gly Ile Ala Gln Ile Thr
85 90 95
Pro Thr Ala Asp Gln Thr Asp Lys Asp Phe Leu Asn Ile Leu Thr Val
100 105 110
Asn Leu Ser Ser Pro Ile Ala Leu Thr Lys Ala Leu Leu Lys Gly Val
115 120 125
Arg Glu Arg Ser Asn Glu Lys Pro Phe His Ile Ile Phe Leu Ser Ser
130 135 140
Val Ala Ala Leu His Gly Val Pro Gln Ala Ala Val Tyr Ser Ala Ser
145 150 155 160
Lys Ala Gly Leu Asp Gly Phe Val Arg Ser Leu Ala Arg Glu Val Gly
165 170 175
Pro Lys Gly Ile His Val Asn Val Ile His Pro Gly Trp Thr Lys Thr
180 185 190
Asp Met Thr Asp Gly Ile Asp Asp Pro Asn Asp Thr Pro Ile Lys Gly
195 200 205
Trp Ile Gln Pro Glu Ala Ile Ala Asp Ala Val Val Phe Leu Ala Lys
210 215 220
Ser Lys Asn Ile Thr Gly Thr Asn Ile Val Val Asp Asn Gly Leu Leu
225 230 235 240
Ala
<210> 5
<211> 726
<212> DNA
<213>artificial sequence ()
<400> 5
atgtctacgc cgctgaatgc tctggtgacg ggtgcttctc gtggtattgg tgctgcgacc 60
gcgatcaaac tggccgaaaa cggttacagc gtgaccctgg cggcccgtaa cgtcgcaaaa 120
ctgaatgaag tgaaagaaaa actgccggtg gttaaagatg gccagaaaca tcacatttgg 180
gaactggacc tggcctctgt cgaagctgct agctctttta aaggcgcacc gctgccggct 240
tcagattatg acctgtttgt ttcgaacgca ggtatcgcac agatcacccc gacggcggat 300
caaaccgata aagacttcct gaacattctg acggtgaatc tgagttcccc gatcgcgctg 360
accaaagccc tgctgaaagg cgttagtgaa cgctccaatg aaaaaccgtt tcatattatc 420
ttcctgtcat cggcagcagc actgcacggt gtgccgcaga cggcagttta cagcgcgtct 480
aaagccggcc tggatggttt tgttcgttca ctggctcgcg aagtcggccc gaaaggtatt 540
catgttaacg tcatccaccc gggctggacc aaaacggata tgaccgacgg tattgatgac 600
ccgaatgata cgccgattaa aggttggatt cagccggaag ctatcgcgga cgccgtcgtg 660
ttcctggcga aatcaaaaaa catcacgggc acgaacattg tggtggataa cggtctgctg 720
gcgtga 726
<210> 6
<211> 726
<212> DNA
<213>artificial sequence ()
<400> 6
atgtctacgc cgctgaatgc tctggtgacg ggtgcttctc gtggtattgg tgctgcgacc 60
gcgatcaaac tggccgaaaa cggttacagc gtgaccctgg cggcccgtaa cgtcgcaaaa 120
ctgaatgaag tgaaagaaaa actgccggtg gttaaagatg gccagaaaca tcacatttgg 180
gaactggacc tggcctctgt cgaagctgct agctctttta aaggcgcacc gctgccggct 240
tcagattatg acctgtttgt ttcgaacgca ggtatcgcac agatcacccc gacggcggat 300
caaaccgata aagacttcct gaacattctg acggtgaatc tgagttcccc gatcgcgctg 360
accaaagccc tgctgaaagg cgttagtgaa cgctccaatg aaaaaccgtt tcatattatc 420
ttcctgtcat cggcagcagc actgcacggt gtgccgcagg cggcagttta cagcgcgtct 480
aaagccggcc tggatggttt tgttcgttca ctggctcgcg aagtcggccc gaaaggtatt 540
catgttaacg tcatccaccc gggctggacc aaaacggata tgaccgacgg tattgatgac 600
ccgaatgata cgccgattaa aggttggatt cagccggaag ctatcgcgga cgccgtcgtg 660
ttcctggcga aatcaaaaaa catcacgggc acgaacattg tggtggataa cggtctgctg 720
gcgtga 726
<210> 7
<211> 726
<212> DNA
<213>artificial sequence ()
<400> 7
atgtctacgc cgctgaatgc tctggtgacg ggtgcttctc gtggtattgg tgctgcgacc 60
gcgatcaaac tggccgaaaa cggttacagc gtgaccctgg cggcccgtaa cgtcgcaaaa 120
ctgaatgaag tgaaagaaaa actgccggtg gttaaagatg gccagaaaca tcacatttgg 180
gaactggacc tggcctctgt cgaagctgct agctctttta aaggcgcacc gctgccggct 240
tcagattatg acctgtttgt ttcgaacgca ggtatcgcac agatcacccc gacggcggat 300
caaaccgata aagacttcct gaacattctg acggtgaatc tgagttcccc gatcgcgctg 360
accaaagccc tgctgaaagg cgttagggaa cgctccaatg aaaaaccgtt tcatattatc 420
ttcctgtcat cggtagcagc actgcacggt gtgccgcagg cggcagttta cagcgcgtct 480
aaagccggcc tggatggttt tgttcgttca ctggctcgcg aagtcggccc gaaaggtatt 540
catgttaacg tcatccaccc gggctggacc aaaacggata tgaccgacgg tattgatgac 600
ccgaatgata cgccgattaa aggttggatt cagccggaag ctatcgcgga cgccgtcgtg 660
ttcctggcga aatcaaaaaa catcacgggc acgaacattg tggtggataa cggtctgctg 720
gcgtga 726
<210> 8
<211> 261
<212> PRT
<213>artificial sequence ()
<400> 8
Met Tyr Pro Asp Leu Lys Gly Lys Val Val Ala Ile Thr Gly Ala Ala
1 5 10 15
Ser Gly Leu Gly Lys Ala Met Ala Ile Arg Phe Gly Lys Glu Gln Ala
20 25 30
Lys Val Val Ile Asn Tyr Tyr Ser Asn Lys Gln Asp Pro Asn Glu Val
35 40 45
Lys Glu Glu Val Ile Lys Ala Gly Gly Glu Ala Val Val Val Gln Gly
50 55 60
Asp Val Thr Lys Glu Glu Asp Val Lys Asn Ile Val Gln Thr Ala Ile
65 70 75 80
Lys Glu Phe Gly Thr Leu Asp Ile Met Ile Asn Asn Ala Gly Leu Glu
85 90 95
Asn Pro Val Pro Ser His Glu Met Pro Leu Lys Asp Trp Asp Lys Val
100 105 110
Ile Gly Thr Asn Leu Thr Gly Ala Phe Leu Gly Ser Arg Glu Ala Ile
115 120 125
Lys Tyr Phe Val Glu Asn Asp Ile Lys Gly Asn Val Ile Asn Met Ser
130 135 140
Ser Val His Glu Val Ile Pro Trp Pro Leu Phe Val His Tyr Ala Ala
145 150 155 160
Ser Lys Gly Gly Ile Lys Leu Met Thr Arg Thr Leu Ala Leu Glu Tyr
165 170 175
Ala Pro Lys Gly Ile Arg Val Asn Asn Ile Gly Pro Gly Ala Ile Asn
180 185 190
Thr Pro Ile Asn Ala Glu Lys Phe Ala Asp Pro Lys Gln Lys Ala Asp
195 200 205
Val Glu Ser Met Ile Pro Met Gly Tyr Ile Gly Glu Pro Glu Glu Ile
210 215 220
Ala Ala Val Ala Ala Trp Leu Ala Ser Lys Glu Ala Ser Tyr Val Thr
225 230 235 240
Gly Ile Thr Leu Phe Ala Asp Gly Gly Met Thr Leu Tyr Pro Ser Phe
245 250 255
Gln Ala Gly Arg Gly
260
<210> 9
<211> 726
<212> DNA
<213> Candida magnoliae ifo 0705
<400> 9
atgtctacgc cgctgaatgc tctggtgacg ggtgcttctc gtggtattgg tgctgcgacc 60
gcgatcaaac tggccgaaaa cggttacagc gtgaccctgg cggcccgtaa cgtcgcaaaa 120
ctgaatgaag tgaaagaaaa actgccggtg gttaaagatg gccagaaaca tcacatttgg 180
gaactggacc tggcctctgt cgaagcagct agctctttta aaggcgcacc gctgccggct 240
tcagattatg acctgtttgt ttcgaacgca ggtatcgctc agttcacccc gacggcggat 300
caaaccgata aagacttcct gaacattctg acggtgaatc tgagttcccc gatcgcgctg 360
accaaagccc tgctgaaagg cgttagtgaa cgctccaatg aaaaaccgtt tcatattatc 420
ttcctgtcat cggcagcagc actgcacggt gtgccgcaga cggcagttta cagcgcgtct 480
aaagccggcc tggatggttt tgttcgttca ctggctcgcg aagtcggccc gaaaggtatt 540
catgttaacg tcatccaccc gggctggacc aaaacggata tgaccgacgg tattgatgac 600
ccgaatgata cgccgattaa aggttggatt cagccggaag ctatcgcgga cgccgtcgtg 660
ttcctggcga aatcaaaaaa catcacgggc acgaacattg tggtggataa cggtctgctg 720
gcgtga 726
<210> 10
<211> 786
<212> DNA
<213>artificial sequence ()
<400> 10
atgtatccgg atttaaaagg aaaagtcgtc gctattacag gagctgcttc agggctcgga 60
aaggcgatgg ccattcgctt cggcaaggag caggcaaaag tggttatcaa ctattatagt 120
aataaacaag atccgaacga ggtaaaagaa gaggtcatca aggcgggcgg tgaagctgtt 180
gtcgtccaag gagatgtcac gaaagaggaa gatgtaaaaa atatcgtgca aacggcaatt 240
aaggagttcg gcacactcga tattatgatt aataatgccg gtcttgaaaa tcctgtgcca 300
tctcacgaaa tgccgctcaa ggattgggat aaagtcatcg gcacgaactt aacgggtgcc 360
tttttaggaa gccgtgaagc gattaaatat ttcgtagaaa acgatatcaa gggaaatgtc 420
attaacatgt ccagtgtgca cgaagtgatt ccttggccgt tatttgtcca ctatgcggca 480
agtaaaggcg ggataaagct gatgacacga acattagcgt tggaatacgc gccgaagggc 540
attcgcgtca ataatattgg gccaggtgcg atcaacacgc caatcaatgc tgaaaaattc 600
gctgacccta aacagaaagc tgatgtagaa agcatgattc caatgggata tatcggcgaa 660
ccggaggaga tcgccgcagt agcagcctgg cttgcttcga aggaagccag ctacgtcaca 720
ggcatcacgt tattcgcgga cggcggtatg acactatatc cttcattcca ggcaggccgc 780
ggttaa 786

Claims (10)

1. a kind of carbonyl reductase, amino acid sequence are as follows:
SEQ ID NO:2；
SEQ ID NO:3 replaces with the mutant of A for SEQ ID NO:2 the 154th T；Or
SEQ ID NO:4, for SEQ ID NO:3 the 129th S replaces with R, the 145th A replaces with the mutant of V.
2. encoding the gene of carbonyl reductase as described in claim 1.
3. gene as described in claim 1, which is characterized in that the encoding gene of SEQ ID NO:2 is SEQ ID NO:5, SEQ The encoding gene that the encoding gene of ID NO:3 is SEQ ID NO:6, SEQ ID NO:4 is SEQ ID NO:7.
4. the plasmid comprising the gene as described in Claims 2 or 3.
5. having converted the microorganism of plasmid as claimed in claim 4.
6. microorganism as claimed in claim 5, which is characterized in that the microorganism be selected from bacillus subtilis, Lactobacillus brevis, Candida magnoliae, Pichia pastoris, saccharomyces cerevisiae, Escherichia coli.
7. microorganism as claimed in claim 6, which is characterized in that be the microorganism be e. coli bl21 (DE3).
8. carbonyl reductase as described in claim 1 or microorganism as claimed in claim 6 are in production statin compound Purposes in mesosome 6- cyano-(3R, 5R)-dihydroxy hecanoic acid t-butyl ester, chloro- (3R, the 5S)-dihydroxy hecanoic acid t-butyl ester of 6-.
9. purposes as claimed in claim 8, which is characterized in that reaction is in glucose dehydrogenase, glucose and NADP+Presence Lower progress.
10. purposes as claimed in claim 9, which is characterized in that the amino acid sequence of the glucose dehydrogenase is SEQ ID NO:8。
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CN111647591A (en) * 2020-06-24 2020-09-11 湖州颐盛生物科技有限公司 Method for preparing statin intermediate by using immobilized enzyme
CN111978286A (en) * 2020-08-21 2020-11-24 江苏万年长药业有限公司 Method for preparing ATS-8
CN113652407A (en) * 2021-07-09 2021-11-16 浙江工业大学 Carbonyl reductase mutant and application thereof in asymmetric synthesis of bi-chiral compound
CN114774491A (en) * 2022-05-05 2022-07-22 湖州颐盛生物科技有限公司 Process for preparing (2S,3R) -2- (phthalimidomethyl) -3-hydroxybutyrate

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Publication number Priority date Publication date Assignee Title
CN111647591A (en) * 2020-06-24 2020-09-11 湖州颐盛生物科技有限公司 Method for preparing statin intermediate by using immobilized enzyme
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