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Carbonyl reductase, mutant and application of carbonyl reductase in preparation of diltiazem intermediate

A technology of carbonyl reductase and reductase, which is applied in the direction of oxidoreductase, application, and microbial-based methods, can solve the problems of low loading of enzymatic conversion substrates, low conversion rates, and long bacterial cell culture cycles, etc., to achieve Easy to scale up, less raw material consumption, and easy operation

Active Publication Date: 2021-07-27
EAST CHINA UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, the existing technology for the preparation of diltiazem synthetic intermediates by enzymatic reduction has problems such as long cell culture period, low loading capacity of enzymatic conversion substrate, time-consuming reaction, low conversion rate, etc., which greatly limits the asymmetric synthesis of enzymatic methods. application, it is necessary to discover new carbonyl reductases with superior performance to meet the industrial requirements of high substrate concentration, high catalytic reaction efficiency, high yield and simple operation

Method used

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  • Carbonyl reductase, mutant and application of carbonyl reductase in preparation of diltiazem intermediate
  • Carbonyl reductase, mutant and application of carbonyl reductase in preparation of diltiazem intermediate
  • Carbonyl reductase, mutant and application of carbonyl reductase in preparation of diltiazem intermediate

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Embodiment 1

[0079] Example 1 Gene Cloning of Carbonyl Reductase CpKR

[0080] The genome of Candida parapsilosis (Candidaparapsilosis) with deposit number CGMCC No. 2.4312 was obtained by phenol-chloroform extraction method. Candidaparapsilosis (Candidaparapsilosis) with the preservation number CGMCC No.2.4312 was inoculated into 200mL of YM medium (formulation: peptone 5g / L, yeast extract 3g / L, beef extract 3g / L, glucose 10g / L, NaCl 10g / L), cultured at 30°C for 48h, took 100mL of bacterial liquid, centrifuged at 8,000rpm for 10min, collected the bacterial cells, washed with 20 mL of normal saline, repeated twice, and then resuspended the cells with 20 mL of normal saline. Add 1mL lysozyme solution (50mg / mL) to the suspended bacteria liquid, keep the temperature in a water bath at 37°C for 1h; add 1.6mL sodium dodecylsulfonate solution (10%, w / v), 160μL proteinase K (20mg / mL) , keep warm in a water bath at 55°C until the suspension becomes clear. Add 1 / 3 volume of saturated NaCl solutio...

Embodiment 2

[0088] Example 2 Preparation of Carbonyl Reductase CpKR Recombinant Expression Plasmid and Recombinant Expression Transformant

[0089] The carbonyl reductase DNA fragment obtained above and the empty plasmid pET28a were double digested with restriction endonucleases EcoR I and Xho I respectively, and the respective digested fragments were subjected to electrophoresis separation to collect corresponding DNA fragments. The recovered DNA fragment was ligated with the plasmid pET28a fragment using T4 DNA ligase at a molar concentration ratio of 5:1, and ligated overnight at 16°C. All the ligation products were transformed into E.coli DH5α, spread on LB solid medium plate containing 50 μg / mL kanamycin, and cultured at 37°C for 12h. Use a sterile yellow pipette tip to select several single clones into corresponding LB test tubes containing 50 μg / mL kanamycin, incubate with shaking at 37°C for 12 hours, extract the corresponding plasmids and verify them by sequencing to obtain the r...

Embodiment 3

[0090] Embodiment 3 carbonyl reductase CpKR mutant construction

[0091]Construct the mutant library of carbonyl reductase CpKR by using the method of cosense analysis: use the CpKR protein sequence as a probe for sequence alignment, and preferably select different protein sequences with a homology of more than 30% from thermophilic sources. Through ClustalX2 sequence alignment and Espript synonymous analysis, a series of non-conserved residues were selected for single-point mutation, and the corresponding mutation primers were designed, and the plasmid pET28a-CpKR was used as a template, and the high-fidelity polymerase PrimeSTAR was used for PCR amplification. increase. The PCR reaction conditions are as follows: to a PCR reaction system with a total volume of 20 μL, add 0.5–20 ng of template, 10 μL of 2×PrimeSTAR (Premix), 0.4 μL of each pair of mutant primers (10 μM), and add sterilized distilled water to 20 μL. PCR reaction program: (1) Denaturation at 98°C for 10 second...

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Abstract

The invention relates to carbonyl reductase, a mutant and application of the carbonyl reductase in preparation of diltiazem intermediates, in particular to carbonyl reductase derived from Candida parapsilosis, the mutant with remarkably improved stability of the carbonyl reductase, coding nucleic acid of the carbonyl reductase, a recombinant expression vector and a recombinant expression transformant containing the nucleic acid sequence, and an application of the recombinant carbonyl reductase or the recombinant expression transformant as a catalyst for asymmetric reduction preparation of a chiral hydroxyl compound, especially in production of a cardiovascular drug diltiazem chiral intermediate (2R, 3S)-p-methoxyphenyl glycidyl acid methyl ester [(2R, 3S)-MPGM]. Compared with other methods (including a chemical method or a lipase hydrolysis resolution method and the like) for preparing (2R, 3S)-MPGM, the enzyme and the technical method thereof disclosed by the invention have the advantages that the theoretical yield is up to 100%, the reaction condition is mild, the enzyme is environment-friendly, the operation is simple and convenient, the amplification is easy and the like, and the enzyme and the technical method thereof have a good application prospect in the production of diltiazem and other drug intermediates.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a carbonyl reductase derived from Candida parapsilosis (Candidaparapsilosis) and a mutant whose stability is significantly improved, its encoding nucleic acid, a recombinant expression vector containing the nucleic acid sequence and Recombinant expression transformant, and the use of the recombinant carbonyl reductase or recombinant expression transformant as a catalyst to prepare chiral hydroxyl compounds through asymmetric reduction, especially in the production of cardiovascular drug diltiazem chiral intermediate (2R,3S)-MPGM. . Background technique [0002] The cardiovascular drug diltiazem (diltiazem) is a benzothiazepine calcium ion channel blocker developed in the 1970s, which belongs to non-dihydropyridine calcium ion antagonists and is used for the treatment of supraventricular arrhythmias and variant angina pectoris , senile hypertension and other di...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C12N15/53C12N15/70C12N1/21C12P7/62C07D303/48C07D301/26C12R1/19
CPCC12N9/0006C12N15/70C12P7/62C07D303/48C07D301/26C12Y101/01184
Inventor 潘江陈铖许建和陈琦
Owner EAST CHINA UNIV OF SCI & TECH
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