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Short-chain dehydrogenase and mutant thereof as well as preparation of gene and application of short-chain dehydrogenase

A technology of short-chain dehydrogenase and mutants, which can be used in applications, genetic engineering, plant gene improvement, etc., and can solve problems such as low stereoselectivity and single stereoselectivity

Active Publication Date: 2018-09-18
SHENYANG PHARMA UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to solve the problem of low stereoselectivity and single stereoselectivity of the existing Lactobacillus fermentum short-chain dehydrogenase (LfSDR1), and obtain the enzyme mutant, the nucleic acid encoding the mutant, and the nucleic acid containing the nucleic acid through site-directed mutagenesis. Recombinant expression vectors and recombinant expression transformants, recombinant mutant enzymes catalyze the asymmetric reduction of 1-phenylethanones, and the application in the preparation of optically pure chiral alcohols of corresponding opposite configurations (R and S configurations)

Method used

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  • Short-chain dehydrogenase and mutant thereof as well as preparation of gene and application of short-chain dehydrogenase
  • Short-chain dehydrogenase and mutant thereof as well as preparation of gene and application of short-chain dehydrogenase
  • Short-chain dehydrogenase and mutant thereof as well as preparation of gene and application of short-chain dehydrogenase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 Site-directed mutagenesis construction of a mutant gene with improved stereoselectivity based on the wild type

[0057] Using the plasmid pET22b-LfSDR1 containing the short-chain dehydrogenase LfSDR1 gene as a template, the mutant gene was obtained by overlapping extension PCR.

[0058] First, the upstream and downstream primers of the LfSDR1 gene are as follows:

[0059] LfSDR1-NdeI-F: GGAATTCCATATGGGACAGTTTG

[0060] LfSDR1-XhoI-R: CCGCTCGAGTTGTGCCGTGTAGC

[0061] Then design mutation site primers as follows:

[0062] Mutant LfSDR1-V186F

[0063] LfSDR1-V186F-F: TACCCTGGGTTTATTGCCACG

[0064] LfSDR1-V186F-R: CGTGGCAATAAACCCAGGGTA

[0065] Mutant LfSDR1-V186W

[0066] LfSDR1-V186W-F: TACCCTGGGTGGATTGCCACG

[0067] LfSDR1-V186W-R: CGTGGCAATCCACCCAGGGTA

[0068] Take the construction of mutant LfSDR1-V186F as an example:

[0069] The first step PCR reaction system is as follows:

[0070] PCR-a

[0071]

[0072] PCR-b

[0073]

[0074] Amplific...

Embodiment 2

[0081] Example 2 Site-directed mutagenesis construction of a mutant gene that is stereoselectively reversed on the basis of the wild type

[0082] Using the plasmid pET22b-LfSDR1 containing the short-chain dehydrogenase LfSDR1 gene as a template, the mutant gene was obtained by overlapping extension PCR.

[0083] The upstream and downstream primers of the LfSDR1 gene are the same as in Example 1.

[0084] The primers for the mutation sites were designed as follows:

[0085] LfSDR1-V186A-F: TACCCTGGGGCAATTGCCACG

[0086] LfSDR1-V186A-R: CGTGGCAATTGCCCCAGGGTA

[0087] LfSDR1-E141L-F: AGTTCGATCCTGGGGATGATC

[0088] LfSDR1-E141L-R: GATCATCCCCAGGATCGAACT

[0089] LfSDR1-G92F-F: GCCGGAATTTTTACTCCGCTG

[0090] LfSDR1-G92F-R: CAGCGGAGTAAAAATTCCGGC

[0091] LfSDR1-G92E-F: GCCGGAATTGAAACTCCGCTG

[0092] LfSDR1-G92E-R: CAGCGGAGTTTCAATTCCGGC

[0093] Take the construction of the LfSDR1-V186A-E141L-G92F mutant as an example:

[0094] The first step of PCR is as follows:

[0095] ...

Embodiment 3

[0111] Embodiment 3 construction mutant gene recombination plasmid

[0112]The mutant gene obtained by overlap extension PCR and the vector plasmid were subjected to double enzyme digestion, and the reaction system was as follows:

[0113]

[0114] After reacting at 37°C for 4-8 hours, use the ordinary DNA product gel recovery kit to recover the cleaved nucleic acid fragments; use T4 DNA ligase to ligate the double-digested mutant gene fragments with cohesive ends and the carrier plasmid fragments (16 ℃ reaction for 2-6h) to obtain recombinant plasmids pET22b-LfSDR1-V186F, pET22b-LfSDR1-V186W, pET22b-LfSDR1-V186A-E141L-G92F and pET22b-LfSDR1-V186A-E141L-G92E.

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Abstract

The invention belongs to the technical field of biology, and relates to wild type short-chain dehydrogenase LfSDR1 and a gene of mutant enzyme of the short-chain dehydrogenase, construction of a recombinant expression vector containing the gene, preparation of recombinant expression transformant, recombinase and a preparation method of the recombinase. Through mutation of the wild type short-chaindehydrogenase LfSDR1, the mutant enzyme having two stereoselectivities (R or S stereoselectivities) is obtained, and catalytic asymmetrical reduction on prochiral ketone is carried out by utilizing the mutant enzyme, such as asymmetrical reduction of catalytic 1-phenyl ethanone compounds. Compared with the wild type short-chain dehydrogenase, the mutant disclosed by the invention has the advantages that the stereoselectivities is improved or reversed, a substrate is catalyzed so as to obtain chiral alcohol having two types (R or S type), such as optical pure (R)- or (S)-2-chloro-1-phenylethanol; moreover, the mutant has a good application value in the field of preparation of chiral alcohol.

Description

technical field [0001] The present invention relates to the technical field of bioengineering, in particular to a short-chain dehydrogenase LfSDH1 mutant and its coding gene, a recombinant expression vector containing the short-chain dehydrogenase mutant gene and the construction of a transformant, a method for preparing a recombinant enzyme, and Using different mutants as catalysts to catalyze the asymmetric reduction of acetophenone compounds to obtain corresponding optically pure chiral phenylethanol products with different configurations (S and R configurations). Background technique [0002] Optically pure 1-phenylethanol compounds, especially α-halogen substituted 1-phenylethanol compounds are important chiral intermediates in the synthesis of pharmaceuticals and other fine chemicals. At present, a variety of methods for obtaining optically pure and chirally pure materials have been developed, including kinetic resolution and asymmetric synthesis. The theoretical yiel...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12N15/53C12P7/22
CPCC12N9/0006C12P7/22C12Y101/01184
Inventor 游松秦斌秦凤玉郭继阳刘亚林张飞霆张文鹤祝天慧
Owner SHENYANG PHARMA UNIVERSITY
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