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A carbonyl reductase mutant with improved thermostability

A mutant and reductase technology, which is applied in the fields of genetic engineering and enzyme engineering, can solve the problems of poor thermal stability of carbonyl reductase ChKRED12, and achieve the effects of flexibility, improved thermal stability, and good industrial application prospects

Active Publication Date: 2022-05-31
CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The technical problem to be solved in the present invention is: the problem of poor thermal stability of carbonyl reductase ChKRED12

Method used

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  • A carbonyl reductase mutant with improved thermostability
  • A carbonyl reductase mutant with improved thermostability
  • A carbonyl reductase mutant with improved thermostability

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 14

[0036] The results of the rational design method for predicting potential thermotolerance sites are shown in Figure 1 of the description. single point mutant S79P,

[0041] V162I-F: 5' - GGCGCGCTGATTGGGGCGACC - 3'

Embodiment 2

[0050] The crude enzyme activity assay reaction conditions is shown in Table 1, and the coenzyme cycle is carried out with the glucose dehydrogenase GDH. The reaction system was at 40

[0053]

[0056] This round of mutation obtained 4 mutants S79P, L128M, V162I and G163A. Transduction of substrates by wild-type and mutants

[0058]

Embodiment 3

L128M-R: 5'-GTTACGCAGCATTTTCTGGATAAA G-3'

[0065] V162I-F: 5' - GGCGCGCTGATTGGGGCGACC - 3'

V162I-R: 5'-GGTCGCCCCAATCAGCGCGCC-3'

[0067] PCR conditions and operations are the same as in Example 1 to obtain a new mutant M722.

3.2 Construction of mutant M723

The site-directed mutagenesis method mutated the leucine at position 128 of mutant S79P to methionine (DNA sequence from TTA

to ATG), the glycine at position 163 was mutated to alanine (the DNA sequence was changed from GGG to GCA), and the mutant M723 was constructed.

The primers used are as follows:

L128M-F: 5'-CTTTATCCAGAAAATGCTGCGTAA C-3'

L128M-R: 5'-GTTACGCAGCATTTTCTGGATAAA G-3'

G163A-F: 5'-GGCGCGCTGGTGGCAGCGACCAAAGC-3'

G163A-R: 5' - GCTTTGGTCGCTGCCACCAGCGCGCC - 3'

[0074] PCR conditions and operations are the same as in Example 1 to obtain a new mutant M723.

3.3 Construction of mutant M7234

The site-directed mutagenesis method mutated the leucine at position 128 of mutant S79P to methionine (DNA sequ...

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Abstract

The invention belongs to the technical field of genetic engineering and enzyme engineering, in particular to a heat-resistant mutant of carbonyl reductase ChKRED12. Carbonyl reductase ChKRED12 can efficiently catalyze the preparation of duloxetine chiral alcohol and other intermediates, but its thermal stability is poor. Molecular evolution of the enzyme was carried out using a rationally designed scheme, and the heat resistance of the obtained mutant was greatly improved, which was more conducive to industrial application.

Description

A carbonyl reductase mutant with improved thermostability technical field The invention belongs to the technical field of genetic engineering and enzyme engineering, and concrete content relates to the carbonyl reductase that heat resistance improves mutant. Background technique [0002] Carbonyl reductase (Carbonyl reductase, EC 1.1.1.x) is an oxidoreductase A member of the (Oxidoreductases) family, refers to the ability to catalyze the reduction of carbonyl to the corresponding alcohol or its reverse reaction A class of oxidoreductases, the essence of which is the transfer of hydrogen or electrons between the substrate and the hydrogen donor. Chiral alcohols are heavy The essential organic synthesis building blocks have high application value in medicine, agriculture, food and chemical industries, and carbonyl reductase It can catalyze the asymmetric reduction of carbonyl groups to the corresponding chiral alcohols. With the development of industrial biotechnolo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/04C12N15/70
Inventor 吴中柳李孜一刘艳
Owner CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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