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Aldehyde-ketone reductase mutant and its application

A reductase and mutant technology, applied to aldehyde-ketone reductase mutants and their application fields, can solve the problems of low asymmetric reduction activity and low substrate concentration, etc.

Active Publication Date: 2021-04-06
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 stereoselective method for the problems of low asymmetric reduction activity of 6-cyano-(5R)-hydroxy-3-carbonylhexanoic acid tert-butyl ester and low substrate concentration of existing aldehyde and ketone reductases. Sexual aldehyde and ketone reductase mutants and using the aldehyde and ketone reductase mutant gene recombinant bacteria and their crude enzyme solution as biocatalysts for the chiralization of 6-cyano-(3R,5R)-dihydroxyhexanoic acid tert-butyl ester Biosynthesis, the activity of the catalyst increased by 1.31 times, and the substrate concentration increased by 60%

Method used

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  • Aldehyde-ketone reductase mutant and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Example 1: Construction and screening of aldehyde and ketone reductase mutant library

[0026] The aldehyde and ketone reductase gene (shown in the amino acid sequence of SEQ ID No.2, and the nucleotide sequence shown in SEQ ID No.1) was constructed as an expression vector pET-28b(+)-klakr, transformed into Escherichia coli, and obtained the starting strain E .coliBL21(DE3) / pET28b-pklakr.

[0027] The preparation of the aldehyde and ketone reductase mutant library was achieved through 4 rounds of site-directed saturation mutagenesis. The primers were designed as shown in Table 1. In the first round, the vector pET-28b(+)-klakr was used as a template, and I125F and I125R in Table 1 were used as primers , through saturation mutation PCR, the 125th isoleucine in the amino acid sequence of aldehyde and ketone reductase shown in SEQ ID No.2 was mutated into the remaining 19 kinds of amino acids, and transformed, plated, and the aldehyde and ketone reductase mutation was obta...

Embodiment 2

[0031] Example 2: Induced expression of aldehyde and ketone reductase parent, mutant and glucose dehydrogenase

[0032] Glucose dehydrogenase gene esgdh (nucleotide sequence shown in SEQ ID No.3, amino acid sequence shown in SEQ ID No.4) was cloned from Exiguobacterium sibiricum, and connected to pET-28b(+) by double enzyme digestion On the vector, the recombinant plasmid was introduced into Escherichia coli E.coli BL21(DE3) to obtain the recombinant glucose dehydrogenase strain E.coliBL21(DE3) / pET28b-esgdh.

[0033] The starting strain E.coli BL21(DE3) / pET28b-pklakr and the aldehyde and ketone reductase mutant strain of Example 1 and the recombinant glucose dehydrogenase strain E.coli BL21(DE3) / pET28b-esgdh were respectively inoculated to a final concentration of 50 μg / In LB liquid medium containing 50 μg / mL kanamycin, cultured at 37°C for 9 hours, inoculated into fresh LB liquid medium containing 50 μg / mL kanamycin at a volume fraction of 2% (v / v), 37 Cultivate at 180 rpm ...

Embodiment 3

[0034] Example 3: Mutant library screening

[0035] Mix the wet cells of the mutant strain induced and expressed in Example 2 and the wet cells of glucose dehydrogenase at a mass ratio of 3:1, and add the amount of 50 g / L of the total amount of cells into pH 7.5, 100 mM phosphate buffer for resuspension , ultrasonic crushing on ice-water mixture for 10 minutes, ultrasonic crushing conditions: power of 400W, crushing for 1 second, pausing for 1 second, to obtain the crude enzyme solution of the mutant strain. Under the same conditions, replace the wet cells of the mutant strain with the starting strain E.coli BL21(DE3) / pET28b-pklakr to prepare the crude enzyme solution of the starting strain.

[0036] The crude enzyme solution of the mutant strain or the crude enzyme solution of the original strain was used as a catalyst, tert-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate was used as a substrate, glucose was used as an auxiliary substrate, and no exogenous sources were added. NADP...

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Abstract

The present invention discloses a mutant of aldehyde and ketone reductase and its preparation of 6-cyano-(3R,5R)-diphenoxylate by asymmetric reduction of 6-cyano-(5R)-hydroxy-3-carbonylhexanoic acid tert-butyl ester. For the application in tert-butyl hydroxycaproate, the aldehyde and ketone reductase mutant is obtained by performing single or multiple mutations at positions 125, 30, 212 and 63 of amino acids shown in SEQ ID No.2. The specific enzyme activity of the aldehyde and ketone reductase mutant pKlAKR-I125V-S30P-Q212R-I63W prepared by the present invention is 1.31 times higher than that of the parent aldehyde and ketone reductase, and the largest substrate is 6-cyano-(5R)-hydroxyl-3 The feed rate of tert-butyl carbonylhexanoate reaches 80g / L, and the aldehyde and ketone reductase mutant has more industrial application prospects.

Description

technical field [0001] The present invention relates to the construction of aldehyde and ketone reductase K1AKR 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] Cardiovascular and cerebrovascular diseases have become one of the main diseases that endanger human health. The incidence of cardiovascular and cerebrovascular diseases is closely related to the cholesterol level in human blood. Atorvastatin calcium can competitively inhibit the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, a key rate-limiting enzyme in the synthesis of cholesterol in the liver, reduce the synthesis of cholesterol, and lower blood hypotension. Density lipoprotein cholesterol (LDL-C) and triglyceride concentration are the only single lipid-lowering drugs with a cumulative sales of more than 10...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/04C12P13/00
CPCC12N9/0006C12P13/002C12Y101/01
Inventor 王亚军沈炜喻寒柳志强郑裕国
Owner ZHEJIANG UNIV OF TECH
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