Carbonyl reductase, gene and mutant and application thereof to asymmetrical reduced carbonyl compound

A technology of carbonyl reductase and carbonyl compounds, applied in the field of bioengineering, can solve the problems of low product concentration, low optical purity, low enzyme catalytic activity and the like

Active Publication Date: 2012-08-01
EAST CHINA UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Therefore, the technical problem to be solved by this invention is, prepare (R)-o-chloromandelic acid methyl ester, (R)-2-hydroxyl-4-phenylbutyric acid ethyl ester and ( The reaction of R)-4-chloro-3-hydroxybutyric acid ethyl ester requires different sources of enzymes, low enzyme catalytic activity, low product concentration, low optical purity, and the need to add additional expensive coenzymes. A carbonyl reductase with high catalytic activity, strong enantioselectivity, and good substrate tolerance, the gene of the carbonyl reductase, a recombinant expression vector containing the gene, a recombinant expression transformant and an efficient preparation method thereof, and the carbonyl reductase Use of reductases in catalyzing asymmetric reduction of carbonyl substrates

Method used

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  • Carbonyl reductase, gene and mutant and application thereof to asymmetrical reduced carbonyl compound
  • Carbonyl reductase, gene and mutant and application thereof to asymmetrical reduced carbonyl compound
  • Carbonyl reductase, gene and mutant and application thereof to asymmetrical reduced carbonyl compound

Examples

Experimental program
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Effect test

Embodiment 1

[0084] Example 1 Cloning of carbonyl reductase gene

[0085] According to the gene sequence (NCBI accession number: CAG58832) predicted to be Candida glabrata carbonyl reductase included in Genbank, the PCR primers were designed as follows:

[0086] CgKR1f: 5'- CATATG GCTTCTGATAACAGCAAC-3';

[0087] CgKR1r: 5'- GGATCC TTAATTAGAGTTCTTCTCGGC-3'.

[0088] Among them, the underlined part of the upstream primer is the Nde Ⅰ restriction site, and the underlined part of the downstream primer is the BamH Ⅰ restriction site.

[0089] The genomic DNA of Candida glabrata CGMCC 2.234 was used as a template for PCR amplification. PCR system: 10 μl of 2×Taq PCR MasterMix, 1 μl of upstream primer and downstream primer (0.3 μmol / L), 1 μl of DNA template (0.1 μg) and ddH 2 O 7 μl. PCR amplification steps are: (1) 95°C, pre-denaturation for 3min; (2) 94°C, denaturation for 1min; (3) 55°C for 30s; (4) 72°C extension for 1min; steps (2) to (4) repeated 30 times; (5) Continue extending at...

Embodiment 2

[0090] Example 2 Site-directed mutation of CgKR1 gene

[0091] Base mutation was performed on the full-length gene sequence of carbonyl reductase (SEQ ID No.1) of Candida glabrata CGMCC 2.234 obtained in Example 1.

[0092] Design PCR primers as follows:

[0093] Mutation V85I:

[0094] Upstream primer: 5'-CAAGGTTATCTTACACACCGCCTCTCC-3'

[0095] Downstream primer: 5'-CGGTGTGTAAGATAACCTTGATATCCTTGCCATG-3'

[0096] Mutation F92L:

[0097] Upstream primer: 5'-CCGCCTTCCACTGCACTTCAAACACCACTGACATT-3'

[0098] Downstream primer: 5'-GTGCAGTGGAGAGGCGGTGTGTAAG-3'

[0099] Mutation F94V:

[0100] Upstream primer: 5'-CACACCGCCTCTCCATTCCACGTTAACACCACTGA

[0101] CATTGAA-3'

[0102] Downstream primer: 5'-TGGAGAGGCGGTGTG-3'

[0103] Mutation I99Y:

[0104] Upstream primer: 5'-CCACTGACTATGAAAAGGATCTATTGATCCC-3'

[0105] Downstream primer: 5'-GATCCTTTTCATAGTCAGTGGTGTTGAAGTGG-3'

[0106] Mutation G174A:

[0107] Upstream primer: 5'-CCAATCAGAGCTTACTGTGGTTCAAAGAAGTTTG-3'

[0108] Do...

Embodiment 3

[0123] Example 3 Construction of recombinant expression vector (plasmid) and preparation of recombinant expression transformant

[0124] Digest the carbonyl reductase gene DNA fragment obtained in Example 1 or 2 at 37°C with restriction endonucleases NdeI and BamHI for 12 hours, purify by agarose gel electrophoresis, and recover using an agarose gel DNA recovery kit target fragment. Under the action of T4 DNA ligase, the target fragment was ligated with the plasmid pET28a that was also digested with NdeI and BamHI at 4°C overnight to obtain the recombinant expression plasmid pET28a-CgKR1 and its mutants. The plasmid construction map is as follows: Figure 5 shown.

[0125] Transform the above-mentioned recombinant expression plasmid into Escherichia coli (E.coli) DH5α competent cells, the transformation conditions are 45°C, heat shock for 90 seconds, and positive recombinants are screened on the resistance plate containing kanamycin , pick a single clone, colony PCR verifica...

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Abstract

The invention discloses a novel carbonyl reductase, a gene, a mutant thereof, a recombinant expression vector containing the gene and the mutant, a recombinant expression transformant, a recombinase preparation method, and applications of the carbonyl reductase and recombinase to preparation of active chiral alcohols with a chiral carbonyl compound before asymmetrical reduction. The carbonyl reductase is derived from candida glabrata, is applied to preparation of a plurality of optically-active chiral alcohols such as (R)-chloromandelic acid methyl ester, (R)-2-hydroxy-4-phenyl ethyl butyrate, (R)-4-chlorin-3-phenyl ethyl butyrate and the like. Compared with other preparation methods, a product prepared through the method has high concentration, does not require additionally or slightly adding any expensive coenzyme, has high optical purity, and has the advantages of mild reaction conditions, easiness and convenience for operating, easiness for amplifying and the like, and has a good industrial application prospect in the production of clopidogrel, L-carnitine and perindopril antihypertensive medicinal intermediates.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and specifically relates to a carbonyl reductase and its gene and mutant, a recombinant expression vector and a recombinant expression transformant containing the gene, its recombinase and a preparation method of the recombinase, and the carbonyl reduction The use of the enzyme or its recombinant enzyme as a catalyst in the asymmetric reduction of chiral carbonyl compounds to prepare optically active chiral alcohols. Background technique [0002] Chiral alcohols are important intermediates for many popular drugs and chiral chemicals. For example, (R)-o-chloromandelic acid methyl ester (molecular formula is o-Cl-C 6 h 4 CH(OH)COOCH 3 , molecular weight 200.62, CAS number: 32345-59-8) is an important chiral intermediate for the synthesis of the platelet aggregation inhibitor clopidogrel. Clopidogrel is an adenosine diphosphate (ADP) receptor blocker, which can bind to the ADP receptor on...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C12N15/53C12N15/63C12N1/21C12P7/62C12P7/22C12R1/72
Inventor 马宏敏李春秀潘江郑高伟郁惠蕾许建和
Owner EAST CHINA UNIV OF SCI & TECH
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