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Recombinant aldo-keto reductase mutant and application thereof

A reductase and mutant technology, which is used in recombinant aldo-ketone reductase mutants and application fields, and can solve the problems of low substrate feeding amount and low asymmetric reduction activity.

Active Publication Date: 2019-07-12
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to provide a three-dimensional ketone reductase for the problems of low asymmetric reduction activity of 6-cyano-(5R)-hydroxyl-3-carbonylhexanoic acid tert-butyl ester and low substrate dosage. Selective aldehyde and ketone reductase mutants and the gene recombinant bacteria using the aldehyde and ketone reductase mutants and their crude enzyme solution as biocatalysts for 6-cyano-(3R,5R)-dihydroxyhexanoic acid tert-butyl ester Sexual biosynthesis, catalyst activity increased by 1.15 times, substrate dosage increased to 200g / L

Method used

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  • Recombinant aldo-keto reductase mutant and application thereof
  • Recombinant aldo-keto reductase mutant and application thereof
  • Recombinant aldo-keto reductase mutant and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0022] Example 1: Construction and screening of aldehyde and ketone reductase mutant library

[0023] The preparation of the aldehyde and ketone reductase mutant library was achieved by three rounds of site-directed saturation mutagenesis. The primers were designed as shown in Table 1. In the first round, E.coli BL21(DE3) / pET28a(+)-kmakr-W297H / Y296W was used for construction (see The vector pET28a(+)-kmakr-W297H / Y296W in the patent application 201710282633.X) was used as a template, Lys29-F and Lys29-R in Table 1 were used as primers, and the aldehyde and ketone shown in SEQ ID NO.2 were synthesized by saturation mutation PCR. The 29th lysine of the reductase KmAKR-W297H / Y296W amino acid sequence was mutated into the remaining 19 amino acids, and transformed, smeared on a plate, and obtained the aldehyde and ketone reductase mutant KmAKR-W297H / Y296W / K29H (ie SEQ The 29th lysine of the amino acid shown in ID NO.2 is mutated to histidine) and KmAKR-W297H / Y296W / K29R (that is, the...

Embodiment 2

[0028] Embodiment 2: Induced expression of control group aldehyde and ketone reductase, mutant and glucose dehydrogenase

[0029] Preparation of glucose dehydrogenase wet cells: the E.coli BL21(DE3) / pET28b(+)-esgdh is inserted into pET28b(+) by the glucose dehydrogenase gene (GenBank NO.KM817194.1) from Exiguobacterium sibirium DSM 17290 A recombinant expression vector was constructed, and the expression vector was transformed into E. coli BL21(DE3) to obtain E coli BL21(DE3) / pET28b(+)-esgdh.

[0030] The starting strain E.coli BL21(DE3) / pET28a(+)-kmakr-W297H / Y296W in Example 1 and the aldehyde and ketone reductase mutant strain screened in Example 1 and E coli BL21(DE3) / pET28b(+)-esgdh They were inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin, cultured at 37°C for 10 h, and inoculated with a volume concentration of 1.5% (v / v) into fresh LB liquid medium containing a final concentration of 50 μg / mL kanamycin. Cultivate in LB liquid medi...

Embodiment 3

[0031] Example 3: Mutant library screening

[0032] Mix the wet cells of the mutant strain induced and expressed in Example 2 and the wet cells of glucose dehydrogenase at a dry weight ratio of 3.5:1 (w / w) to form a mixed cell, add pH 7.0, 100mM PBS buffer to resuspend, Obtain the mixed bacterial liquid of mutant strains. Under the same conditions, the control strain E.coli BL21(DE3) / pET28a(+)-kmakr-W297H / Y296W was used to replace the wet cells of the mutant strain to prepare a mixed bacterial solution.

[0033] The mixed bacterial solution of the mutant strain and the mixed bacterial solution of the control strain were used as catalysts, tert-butyl 6-cyano-(5R)-hydroxy-3-carbonylhexanoate was used as the substrate, glucose was used as the auxiliary substrate, and no external derived NADPH or NADP + , using the endogenous NADPH of the bacteria to establish a coenzyme circulation system. The reaction system is selected as 10mL, the amount of catalyst is 25g / L based on the to...

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Abstract

The invention discloses a recombinant aldo-keto reductase mutant and an application thereof in asymmetric reduction of 6-cyano-(5R)-hydroxy-3-tertiary butyl carbonyl hexanoate to prepare 6-cyano-(3R,5R)-tertiary butyl dihydroxycaproate. The recombinant aldo-keto reductase mutant is obtained by single or multiple mutations of 29th and 28th amino acids shown in SEQ ID NO.2. The specific enzyme activity of the prepared aldo-keto reductase mutant KmAKR-Y296W / W297H / K29H / Y28A is 1.15 times higher than that of a control group KmAKR-Y296W / W297, and the feeding amount of the maximum substrate 6-cyano-(5R)-hydroxy-3-tertiary butyl carbonyl hexanoate is up to 200 g / L, the conversion rate of the substrate is more than 99%, and the dep value of the product is remained at 99.5% or more. The concentration of the product is 549 mM and the space-time yield reaches 670.5 g / L d.

Description

technical field [0001] The present invention relates to the construction of aldehyde and ketone reductase KmAKR mutants, the development of aldehyde and ketone reductase recombinant bacteria and the chiral biosynthesis of atorvastatin side chain 6-cyano-(3R,5R)-tert-butyl dihydroxyhexanoate aspects of application. Background technique [0002] Atorvastatin calcium (7-[2-(4-fluorophenyl)-3-phenyl-4-(anilinoformyl)-5-(2-propyl)pyrrol-1-yl]-3, Calcium 5-dihydroxyheptanoate) belongs to the third generation of synthetic statins, trade name for calcium salt It has high lipid-lowering efficacy, safety and long-term clinical benefits, and can reduce the morbidity and mortality of cardiovascular and cerebrovascular diseases. So far, the cumulative sales of atorvastatin calcium have exceeded 100 billion US dollars, and it is the most successful single drug variety in the history of the pharmaceutical industry. [0003] tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate is the key chira...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12P13/00
CPCC12N9/0006C12P13/002
Inventor 王亚军喻寒邱帅程峰郑裕国
Owner ZHEJIANG UNIV OF TECH
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