Short-chain dehydrogenase and mutant thereof, and preparation and applications of gene

A short-chain dehydrogenase and mutant technology, which is applied in the fields of application, genetic engineering, plant genetic improvement, etc.

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

If the selectivity is not controlled, then 17 hydrogenation products (carveol, dihydrocarvone or dihydrocarveol) are produced, such as Bogel- The research group used palladium (Pd), rhodium (Rh) or ruthenium (Ru) as catalysts in supercritical carbon dioxide (Green Chem, 2011, 13, 2825), and the Santos research group used some fungi on human skin to biotransform 4R / S-fragrance Apione has obtained at least 4 hydrogenation mixtures (Biocatalysis and Biotransformation, 2017, 1-5), others utilize bacteria (Chimie, 2005, 8, 849–852), fungi (J. Mol. Catal. B: Enzym, 1998, 5, 149–154 ) and yeast (J.Mol.Catal.B: Enzym, 2012,77,98–104.) reported biotransformation of carvone can only obtain a mixture of saturated ketones / alcohols and enols, indicating that alkenes in whole cells Reductase and carbonyl reductase can compete to reduce the substrate, and the product carveol after carvone is reduced by carbonyl reductase cannot be accepted by alkene reductase due to its C=C double bond inertness, resulting in the by-product carveol produce

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Embodiment 1 Expression of stereoselective complementary enzyme

[0018] In order to obtain the mutant of OYE1, the wild-type OYE1 gene (SEQ ID NO.1) is used as a template, and the mutation primers containing the mutation point are used (select the bases of 15-20bp upstream and downstream of the mutation point, and the bases of the mutation point The codon replaced with the mutated amino acid was used as the PCR forward primer, and its reverse complementary sequence was used as the reverse primer), and the genes of the mutants OYE1-M1 and OYE1-M2 were obtained by PCR amplification.

[0019] In order to obtain mutants with increased stereoselectivity, the wild-type short-chain dehydrogenase LfSDR1 gene (SEQ ID No.1) was used as a template, and mutation primers containing mutation points were used (choose 15-20 bp bases upstream and downstream of the mutation point, The base of the mutation point was replaced with the codon of the mutated amino acid as the PCR forward pri...

Embodiment 2

[0022] Embodiment 2 (1R, 2S, 4R) - the synthesis of dihydrocarveol

[0023] In a 1ml reaction system, first add 800 μl of olefin reductase OYE1 prepared in Example 1 and 200 μl of glucose dehydrogenase GDH, then add NADP+ with a final concentration of 0.3 g / L and 5.4 g / L of glucose, and finally add The concentration was 5 mM 4R-carvone (co-solvent: petroleum ether, 20% v / v). The reaction was shaken at 28° C. and 200 rpm, and the reaction time was 12 hours. After the reaction is finished, use 1ml of ethyl acetate to extract twice. After the GC detects that the substrate has completely reacted, it is dried by rotary evaporation and used as the substrate of the second step reaction (5% ethanol solubilization, v / v). The second step reaction The system was the same as the first reaction except that the enzyme was short-chain dehydrogenase LfSDR1, and the reaction conditions were 37°C and 200rpm shaking for 12h. After the reaction was finished, it was also extracted twice with 1ml...

Embodiment 3

[0024] Embodiment 3 (1R, 2R, 4R) - the synthesis of dihydrocarveol

[0025]Except for the enzymes used in the two-step reaction, the synthesis method, reaction system and conditions of this configuration dihydrocarveol are the same as in Example 2. The enzymes used in the two-step reaction were: OYE1 and BmSDR5, the conversion rate was >99%, the single-step de value was 98.4%, and the total de value was 90.5%.

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PUM

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Abstract

The invention belongs to the technical field of biology, and relates to short-chain dehydrogenase and a mutant thereof, and preparation and applications of a gene, specifically to a wild type C=C double bond reductase OYE1 derived from Saccharomyces pastoris, and a mutant thereof, short-chain dehydrogenases LfSDR1, BmSDR5 and BsSDR8 respectively derived from Lactobacillus fermentum, Bacillus megaterium and Bacillus subtilis, mutants thereof, C=C double bond reductase and C=O double bond reductase with stereoselectivity complementation, genes of C=C double bond reductase and C=O double bond reductase, construction and preparation of a recombinant expression vector containing the gene, expression of recombinase, an enzyme catalytic two-step asymmetric reduction reaction of carvone, the conditions of the reaction, and a detection method of the product dihydrocarveol. Compared with the existing chemical method, the method of the invention has the advantages of being easy to operate, mild in reaction condition, environmentally friendly and the like.

Description

technical field [0001] The invention relates to the technical field of bioengineering, and relates to the preparation and application of a short-chain dehydrogenase and its mutants and genes, in particular to catalyzing 4R by using stereoselective complementary alkene reductase and short-chain dehydrogenase as biocatalysts Synthesis of stereodiverse dihydrocarveol by asymmetric reduction of / S-carvone. Background technique [0002] Dihydrocarveol is a colorless to light yellow viscous liquid with a spearmint aroma and a peppery pungent taste. Insoluble in water, soluble in acetone, ether and ethanol. Dihydrocarveol is a kind of spice, which can be used as an artificial spice for food. It can be used to prepare mint-flavored essence for food; it can be used to prepare spearmint essence for cosmetics; it can also be used as raw material for other spices. Dihydrocarveol has 3 chiral centers (C-1, C-2 and C-4), and 8 stereoisomers, which are currently on the market in the form...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C12N15/53C12N15/70C12N1/21C12P7/02C12R1/19
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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