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Carbonyl reductase mutant and application thereof

A reductase and mutant technology, applied in the fields of genetic engineering and enzyme engineering, can solve the problems of inability to meet huge demand, not easy to obtain, and the catalytic activity of enzymes cannot meet industrial production, and achieve the effect of enriching the biocatalysis toolbox

Active Publication Date: 2018-11-02
CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The carbonyl reductases studied in the laboratory usually have better stereoselectivity, but the catalytic activity of the enzymes often cannot meet the requirements of industrial production
On the other hand, most of the catalytic processes of carbonyl reductases screened from nature follow the Prelog rule, and only one configuration of chiral alcohols can be obtained
Another configuration that requires the catalytic process to follow the anti-Prelog is often not easy to obtain, so it cannot meet the huge demand for this type of chiral alcohol in the fields of medicine, fine chemicals and agricultural chemicals

Method used

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  • Carbonyl reductase mutant and application thereof
  • Carbonyl reductase mutant and application thereof
  • Carbonyl reductase mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Construction of a single-point saturation mutation library

[0030] use The site-directed mutagenesis kit performs single-point saturation mutations on the carbonyl reductase ChKRED12 gene (see SEQ ID NO.1).

[0031] The primers used are:

[0032] M191-SM:T7:5′-CCGGGTTTTATCAAAACGGAT NNK ACCCAGGAGTTT-3′

[0033] T7 ter : 5′-AAACTCCTGGGTM NNA TCCGTTTTGATAAAACCCGG-3′

[0034] Q151-SM:T7:5′-ACTGCGGGT NNK ACCAATTATAGCGCAGCGA-3′

[0035] T7 ter : 5′-TCGCTGCGCTATAATTGGT MNN ACCCGCAGT-3′

[0036] The reaction conditions were: pre-denaturation at 98°C for 3 minutes, denaturation at 98°C for 10 seconds, annealing at 55°C for 45 seconds, and extension at 72°C for 2 minutes, a total of 25 cycles. After electrophoresis, the gene fragments were recovered with a gel recovery kit. After the recovered fragment was digested with DpnI, it was transformed into Escherichia coli DH10B by electroporation to obtain a cloned mutant library.

Embodiment 2

[0037] Example 2 Screening of Carbonyl Reductase ChKRED12 Mutant Library

[0038]The mutant library clones in Example 1 were collected and the plasmids were extracted, transformed into E. coli expression strain BL21-DE3, spread on LB plates containing kanamycin (50 μg / mL), and cultured for 12 hours. Single clones were picked and placed in a 96-well plate, each well contained 200 μL TB medium (containing 50 μg / mL kanamycin, 0.5 mM IPTG), 37° C., 180 rpm, and cultured with shaking for 18 hours. The 96-well plate replicator replicated each single clone on an LB solid medium plate, cultured at 37°C for 12 hours, and stored in a refrigerator at 4°C. Centrifuge the 96-well plate after bacterial induction and expression at 4°C and 4000 rpm for 10 min, discard the supernatant, and add 200 μL of lysis buffer to each well to resuspend the cells (configuration of lysis buffer: 0.1 M, pH 8.0 potassium phosphate Buffer, 10mg / mL lysozyme, 1μg / mL DNase I, 10mM MgCl 2 ). Place the 96-well ...

Embodiment 3

[0039] The mensuration of embodiment 3 crude enzyme liquid enzyme activity

[0040] 3.1 Preparation of crude enzyme solution

[0041] Pick a single clone into LB (containing kanamycin 50 μg / mL) medium, culture overnight at 37 ° C, transfer to TB (containing kanamycin 50 μg / mL) medium with 1% inoculum size, 37 Cultivate at ℃ for 3h, add 0.5mM IPTG to induce, and continue to culture at 37°C for 18h. The bacteria liquid was centrifuged to collect the bacteria, the cell homogenizer was crushed, and the supernatant was centrifuged to obtain the crude enzyme liquid.

[0042] 3.2 Determination of crude enzyme activity

[0043] Crude enzyme activity assay reaction conditions: 3mg / mL crude enzyme solution (total protein concentration), 1mM NADP + , 0.1 M, pH 8.0 potassium phosphate buffer, 0.1 mL dimethyl sulfoxide, 10 mM substrate ethyl 2-methoxy-3-carbonylphenylpropionate (1a), 5% (w / v) Glucose, 2mg / mL glucose dehydrogenase. React at 40°C and 150rpm for 60min. After the reactio...

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Abstract

Carbonyl reductase ChKRED12 is subjected to molecular improvement through a semi-rational design method, so that a mutant with significant changes in enzymatic properties is obtained. The stereo-selectivity of the mutant M191S is improved, and the activity is improved by 4 times; the stereo-selectivity of each of combined mutants Q151F / M191L and Q151Y / M191L is reversed, and the activity is improved by about 3 times. The mutant with a reversed configuration can be catalyzed to obtain a chiral product with a parent-catalyzed reverse configuration, so that a bio-catalysis toolbox is enriched.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and enzyme engineering, and specifically relates to a carbonyl reductase mutant with improved heat resistance and its application. Background technique [0002] Carbonyl reductase (EC 1.1.1.184), a member of the oxidoreductase family, depends on the coenzyme NADH or NADPH, and can specifically catalyze the conversion between ketones (aldehydes) and alcohols. Chiral alcohols are important intermediates in the synthesis of chiral drugs. Carbonyl reductase can efficiently catalyze the reduction of latent chiral ketones, and is one of the important methods for preparing chiral alcohols. Carbonyl reductase is widely used in pharmaceutical industry, fine chemical industry and information industry. [0003] The catalytic efficiency and stereoselectivity of an enzyme are important indicators to measure its application value and the key to determine whether the biocatalytic process can be appl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C12P7/62
CPCC12N9/0006C12P7/62C12Y101/01184
Inventor 吴中柳李超刘艳
Owner CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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