Carbonyl reductase mutant and application thereof in preparation of rosuvastatin chiral intermediate

A reductase and mutant technology, applied in the field of stereoselective carbonyl reductase mutants and their encoding genes, can solve the problem that the specific mechanism has not been accurately explained, and achieves good development and application value, low production cost, and simplification. Effects of chemical catalytic steps

Active Publication Date: 2022-04-29
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there are few carbonyl reductase enzyme sources available for industrial production, and the specific mechanism of carbonyl reductase catalysis has not been accurately elucidated. The use of protein engineering is a favorable tool to improve enzyme activity and stereoselectivity

Method used

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  • Carbonyl reductase mutant and application thereof in preparation of rosuvastatin chiral intermediate
  • Carbonyl reductase mutant and application thereof in preparation of rosuvastatin chiral intermediate
  • Carbonyl reductase mutant and application thereof in preparation of rosuvastatin chiral intermediate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Construction of recombinant carbonyl reductase mutant library

[0029] The carbonyl reductase coding gene SEQ ID NO.1 was connected with the expression vector pET28b to construct a heterologous expression recombinant plasmid containing the carbonyl reductase coding gene, and the expression recombinant plasmid was transformed into the host bacteria E. coli In BL21 (DE3), the recombinant genetically engineered bacteria containing the recombinant plasmid were obtained. With recombinant bacteria containing the expression vector pET28b-SCR ( E. coli BL21(DE3) / pET28b-SCR) was used as the starting strain, and the mutation library was constructed by semi-rational design method, and the carbonyl reductase substrate ( S )-CHOH catalytic activity.

[0030] Site-directed saturation mutation:

[0031] Val (V) 153:

[0032] Upstream primer 1: 5'-GGCGATCCGACT NNK GGCGCTTATAAC-3'

[0033] Downstream primer 3: 5'-GTTATAAGCGCC MNN AGTCGGATCGCC-3'

[0034] Gly(G) 23...

Embodiment 2

[0038] Example 2: Screening of recombinant carbonyl reductase mutants

[0039] Randomly pick a single colony from the obtained mutant library for sequencing analysis to obtain different mutant amino acids at different mutation points, and then perform enzyme activity assay analysis to establish the optimal mutation point.

[0040] Screening of positive clones:

[0041] Through the saturation mutation analysis of Val(V)153 and Gly(G)233 sites, a series of different amino acid mutants were obtained. The optimal mutant was determined by comparing the conversion rate of different mutants when catalyzing the substrate. The transformation reaction was carried out in a 10mL transformation bottle, the concentration of the substrate ((S)-CHOH was 100g / L, isopropanol was 4mL, the mutant wet bacteria was 15g / L, 30°C, 600rpm magnetic stirring for 20min, and the reaction solution was passed through HPLC analysis is used to determine the conversion rate of the reaction to determine the op...

Embodiment 3

[0043] Example 3: Preparation of recombinant carbonyl reductase mutant wet thallus

[0044] Inoculate the recombinant Escherichia coli (recombinant Escherichia coli BL21(DE3) / pET28b-mut-Val153Cys, recombinant Escherichia coli BL21(DE3) / pET28b-mut-Gly233Asn) obtained in Example 2 containing the recombinant carbonyl reductase mutant gene into LB liquid medium containing kanamycin resistance at a final concentration of 50 μg / mL, cultivated at 37°C for 8 hours at 200 rpm, and then inoculated with 2% ( v / v ) was inoculated into fresh LB liquid medium containing a final concentration of 50 μg / mL kanamycin resistance, and cultured at 37°C and 200 rpm until the cell OD 600 After reaching 0.6-0.8, add IPTG with a final concentration of 0.1mM, induce culture at 28°C for 12h, centrifuge at 8000 rpm for 15min at 4°C, discard the supernatant, collect the precipitate, and obtain the mutant gene containing the expression of recombinant carbonyl reductase Recombinant Escherichia coli wet ce...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to a carbonyl reductase mutant, a coding gene and application of the carbonyl reductase mutant, and the carbonyl reductase mutant is obtained by mutating the 153rd valine of an amino acid sequence shown in SEQ ID NO.2 into cysteine; or the amino acid sequence is obtained by mutating the 233rd glycine of the amino acid sequence as shown in SEQ ID NO.2 into asparagine. Compared with a wild enzyme, the mutant disclosed by the invention has the advantages that the catalytic activity is obviously improved during conversion of the reaction, the catalytic preparation efficiency of (3R, 5S)-CDHH is high, the reaction time is short, the enzyme expression quantity of recombinant engineering bacteria is high, fermentation preparation is easy, the unit enzyme activity of wet thalli is high, the mutant can be directly used for enzymatic catalytic preparation of rosuvastatin intermediates, and the application prospect is wide. The method has the outstanding advantages of low production cost and high efficiency, and has good development and application values; compared with a chemical method for preparing (3R, 5S)-CDHH, the method provided by the invention has the advantages that the stereoselectivity of the product is high, the tedious chemical catalysis step is simplified, the reaction condition is milder, the requirement on equipment is low, the reaction cost is reduced, and the method is environment-friendly.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a stereoselective carbonyl reductase mutant and its coding gene, and its biocatalytic preparation of a chiral intermediate of rosuvastatin (3 R ,5 S )-6-Chloro-3,5-dihydroxyhexanoic acid tert-butyl ester. Background technique [0002] Stereoselective carbonyl reductase (Carbonyl reductase, E.C.1.1.1.148) is a class of bidirectional reversible reactions that can catalyze alcohols and aldehydes / ketones, requiring coenzyme nicotinamide adenine dinucleotide (NAD(H)) and tobacco Amide adenine dinucleotide phosphate (NADP(H)) acts as a hydrogen transporter. Carbonyl reductase exists widely in bacteria, fungi, animals and plants, and there are many types and quantities. In recent years, stereoselective carbonyl reductases have been widely used in the asymmetric synthesis of chiral alcohols. [0003] Rosuvastatin calcium is a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) re...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C12N15/53C12N15/70C12N1/21C12P7/62C12R1/19
CPCC12N9/0006C12Y101/01148C12N15/70C12P7/62
Inventor 张晓健刘倩柳志强郑裕国
Owner ZHEJIANG UNIV OF TECH
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