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Use of oxidoreductase or recombinase thereof and recombinant oxidoreductase

A reductase and reaction technology, applied in the field of recombinant oxidoreductase, can solve the problems of expensive coenzyme and high catalytic activity carbonyl reductase, etc., and achieve the effect of high optical purity, excellent catalytic activity and easy preparation

Active Publication Date: 2013-11-06
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The technical problem to be solved by the present invention is to provide an oxidoreductase for the defects that the existing high catalytic activity and high selectivity carbonyl reductases are rare, and most of them require expensive coenzymes, which cannot meet the ideal needs of industrial applications. A new application of its recombinant enzyme in the field of asymmetric reduction of carbonyl compounds, and a recombination redox that can efficiently and specifically catalyze the asymmetric reduction of carbonyl compounds, especially ethyl 4-chloroacetoacetate, and realize its own coenzyme cycle enzyme

Method used

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  • Use of oxidoreductase or recombinase thereof and recombinant oxidoreductase
  • Use of oxidoreductase or recombinase thereof and recombinant oxidoreductase
  • Use of oxidoreductase or recombinase thereof and recombinant oxidoreductase

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Example 1 Construction of Escherichia coli (E.coli) BL21(DE3) / pET-28a-ScCR

[0040] 1. Amplification of ScCR gene

[0041] According to the base sequence shown in SEQ ID No.1, the primer sequence is designed as follows:

[0042] Upstream primer (ScCR-NdeI): GGAGATT CATATG AGCACCACCGGAACCACC;

[0043] Downstream primer (ScCR-HindIII): GTG AAGCTT TCAGACGGCGGTGTAGGCGC.

[0044] Using the genomic DNA of Streptomyces coelicolor A3(2) NRRL B-16638 as a template, PCR amplification was performed to obtain the ScCR gene. PCR system: 2×Taq PCRMasterMix 15μl, upstream primer and downstream primer 1μl (0.3μmol / L), DNA template 2μl (0.1μg) and ddH 2 O 11 μl. PCR amplification steps are: (1) 95°C, pre-denaturation for 3 minutes; (2) 94°C, denaturation for 1 minute; (3) 55°C annealing for 30s; (4) 72°C extension for 90s; steps (2) to (4) cycle 30 times; (5) 72°C, extended for 10min, cooled to 4°C for storage. The PCR product was purified by agarose gel electrophoresis, and t...

Embodiment 2

[0049] Example 2 Preparation of recombinant oxidoreductase ScCR

[0050]The Escherichia coli (E.coli) BL21 (DE3) / pET28a-ScCR that embodiment 1 gained is inoculated in the LB culture medium (comprising yeast extract 5g / L) containing kanamycin (final concentration 50mg / L, Peptone 10g / L and NaCl 10g / L), cultured with shaking at 37°C for 8h. Then by 5% (v / v) inoculum size, the seed liquid is inoculated in the 5L fermentor that 3L LB' medium (comprising yeast extract 10g / L, peptone 20g / L and NaCl 10g / L) is housed, 37 ℃ Grow to OD 600 At about 8 o'clock, IPTG with a final concentration of 0.5 mM was added and induced at 25°C for 12 hours.

[0051] The obtained fermentation culture liquid was centrifuged at 9000 rpm to remove the supernatant, and the wet cells were collected. Part of the wet cells were washed twice with physiological saline and freeze-dried to obtain freeze-dried cells for future use. Another part of wet cells was suspended in pH 6.5, 100mM phosphate-sodium phosp...

Embodiment 3

[0052] Example 3 Activity determination of recombinant oxidoreductase ScCR

[0053] Determination of the reduction reaction activity of the oxidoreductase ScCR: the reaction system is 1ml, including pH 6.5, 100mM sodium phosphate buffer, 0.1mM NADH, 2mM COBE, react at 30°C, and use a spectrophotometer to measure the decrease in absorbance at 340nm. Enzyme activity is defined as the amount of enzyme required to oxidize 1 μmol NADH per minute as one enzyme activity unit U. Protein content was determined by the Brandford method.

[0054] The above method was used to measure the activity of the recombinant oxidoreductase obtained in Example 2, and the result was that the reduction reaction activity of the recombinant oxidoreductase ScCR was 10.4 U / mg.

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Abstract

The invention discloses the use of an oxidoreductase having an amino acid sequence represented by SEQ ID No.2 in a sequence table or recombinase thereof as a carbonyl reductase catalyst in the asymmetric reduction of a prochiral carbonyl compound for preparing optically active chiral alcohol. The invention also discloses a recombinant oxidoreductase prepared by a method which is to culture a recombinant expression transformant having an oxidoreductase gene which has a base sequence represented by SEQ ID No.1 in a sequence table. The oxidoreductase the invention relates to is simple and convenient in preparation, and as a carbonyl reductase catalyst, the oxidoreductase is applicable to more prochiral carbonyl compounds. The oxidoreductase has high catalytic activity, can obtain optically active chiral alcohol with high conversion rate and high optical purity, realize the regeneration of own coenzyme NADH without requiring an extra coenzyme regeneration system. In whole reaction process, the pH control is not needed, the reaction conditions are mild, and the oxidoreductase has a bright industrial application and development prospect.

Description

technical field [0001] The invention relates to the application of an oxidoreductase or its recombinant enzyme as a carbonyl reductase catalyst in the asymmetric reduction of prochiral carbonyl compounds to prepare optically active chiral alcohols, and a recombinant oxidoreductase. Background technique [0002] Optically active chiral alcohols are important intermediates in the synthesis of pharmaceuticals and fine chemicals. At present, the preparation methods of optically active chiral alcohols mainly include chemically catalyzed carbonyl asymmetric reduction, biocatalyzed carbonyl asymmetric reduction and biocatalyzed chiral resolution of racemic alcohols. Among them, chemically catalyzed asymmetric reduction of carbonyl groups usually requires expensive metal catalysts such as rhodium and ruthenium. These catalysts are difficult to recover, pollute the environment, and have high reaction energy consumption. The advantage of the biocatalytic asymmetric reduction method ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P7/02C12N15/53C12N15/63C12N9/02C12R1/19
Inventor 许建和王丽娟潘江倪燕沈乃东邱勇隽邱立欢
Owner EAST CHINA UNIV OF SCI & TECH
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