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Short-chain dehydrogenase mutant and applications thereof,

A short-chain dehydrogenase and mutant technology, applied in the field of bioengineering, can solve the problems of asymmetric reduction of carvone, single stereoselectivity, and low stereoselectivity

Active Publication Date: 2020-04-21
SHENYANG PHARMA UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] 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 vector and recombinant expression transformant, recombinant mutant enzyme catalyzes the asymmetric reduction of 4R / S-carvone, and its application in the preparation of stereodiverse carveol

Method used

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  • Short-chain dehydrogenase mutant and applications thereof,

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Example 1 Construction of LfSDR1-M1 mutant gene by site-directed mutagenesis

[0030] Using the plasmid pET22b-LfSDR1 containing the short-chain dehydrogenase LfSDR1 gene as a template, the mutant gene was obtained by overlapping extension PCR.

[0031] First, the upstream and downstream primers of the LfSDR1 gene are as follows:

[0032] LfSDR1-NdeI-F: GGAATTCCATATGGGACAGTTTG

[0033] LfSDR1-XhoI-R: CCGCTCGAGTTGTGCCGTGTAGC

[0034] Then design mutation site primers as follows:

[0035] Mutant LfSDR1-M1

[0036] LfSDR1-V186W-F: TACCCTGGGTGGATTGCCACG

[0037] LfSDR1-V186W-R: CGTGGCAATCCACCCAGGGTA

[0038] The first step PCR reaction system is as follows:

[0039] PCR-a

[0040]

[0041] PCR-b

[0042]

[0043]Amplification program: 94°C: 10Min, (94°C: 30s, 45°C: 30s, 72°C: 30s) 30 cycles, 72°C, 10min. Two sets of PCR were used to obtain PCR fragment a and PCR fragment b respectively, and the two fragments were recovered with a common DNA product gel recove...

Embodiment 2

[0048] Example 2 Construction of LfSDR1-M2 mutant gene by site-directed mutagenesis

[0049] Using the plasmid pET22b-LfSDR1 containing the short-chain dehydrogenase LfSDR1 gene as a template, the mutant gene was obtained by overlapping extension PCR.

[0050] The upstream and downstream primers of the LfSDR1 gene are the same as in Example 1. LfSDR1 V186A / G92F / E141L / D146V / K206L

[0051] The primers for the mutation sites were designed as follows:

[0052] LfSDR1-G92F-F: GCCGGAATTTTTACTCCGCTG

[0053] LfSDR1-G92F-R: CAGCGGAGTAAAAATTCCGGC

[0054] LfSDR1-E141L-F: AGTTCGATCCTGGGGATGATC

[0055] LfSDR1-E141L-R: GATCATCCCCAGGATCGAACT

[0056] LfSDR1-D146V-F: ATGATCGGTGTGCCAACCGTT

[0057] LfSDR1-D146V-R: AACGGTTGGCACACCGATCAT

[0058] LfSDR1-V186A-F: TACCCTGGGGCAATTGCCACG

[0059] LfSDR1-V186A-R: CGTGGCAATTGCCCCAGGGTA

[0060] LfSDR1-K206L-F: TACATCGACCTGCACCCAATG

[0061] LfSDR1-K206L-R: CATTGGGTGCAGGTCGATGTA

[0062] Construction of LfSDR1-M2 mutants:

[0063] The fi...

Embodiment 3

[0082] Embodiment 3 construction mutant gene recombination plasmid

[0083] 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:

[0084]

[0085] 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 °C reaction for 2-6h) to obtain recombinant plasmids pET22b-LfSDR1-M1 and pET22b-LfSDR1-M2.

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Abstract

The invention belongs to the technical field of biology, and relates to short-chain dehydrogenase, a mutant and applications thereof, particularly to a stereoselective complementary short-chain dehydrogenase mutant, wherein the mutant is obtained by mutation of wild type short chain dehydrogenase (LfSDR1) excavated from Lactobacillus fermentum. The invention specifically relates to a short-chain dehydrogenase mutant and a preparation method thereof, and applications of the short-chain dehydrogenase mutant as a catalyst in catalysis of asymmetric reduction of 4R / S-carvone to prepare stereodiverse carveol. Compared with the existing chemical method, the method of the invention has the advantages of easy operation, mild reaction condition, environmentally friendly and the like.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a short-chain dehydrogenase LfSDR1 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 The use of mutants as catalysts to catalyze the asymmetric reduction of 4R / S-carvone to obtain carveol with stereodiversity. Background technique [0002] Carveol is a natural, unsaturated, monocyclic monocyclic alcohol that exists in spearmint oil in the (-)-cis form. Carveol is a colorless liquid, insoluble in water, soluble in organic solvents such as ethanol, and has a taste similar to spearmint or coriander, so carveol is used as a spice in cosmetics or food industries. In the field of medicine, carveol has been proved to be able to chemoprevent breast cancer (Carcinogenesis,1992,13(7),1261-1624.), and its derivatives (1R,2R,6S)-3-met...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C12N15/53C12N15/70C12N1/21C12P7/02
CPCC12N9/0006C12N15/70C12P7/02C12Y101/01184
Inventor 游松秦斌郭继阳张飞霆刘贵高秦凤玉张文鹤祝天慧唐军闫平泽张瑞李衡宇于召惠
Owner SHENYANG PHARMA UNIVERSITY
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