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Alcohol dehydrogenase mutant and application thereof

A technology of alcohol dehydrogenase and mutants, applied in the field of enzyme engineering and microbial engineering, can solve the problems of low S/C, low conversion efficiency, easy inactivation of alcohol dehydrogenase, etc., and achieve high thermal stability and conversion High efficiency and high catalytic efficiency

Active Publication Date: 2020-04-10
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Existing enzymes that can be used for the asymmetric reduction of latent chiral bisaryl ketones to produce chiral bisaryl alcohols are mainly alcohol dehydrogenases (ADH, EC 1.1.1.1), but most of the alcohol dehydrogenases Enzymes have low thermostability, T 50 15 The value is less than 50°C (see references for details: "Min Li, et al., Applied and Environmental Microbiology., 2017, 83, 12, e00603-17", "Xumin Gong, et al, ACS Catalysis, 2019, 9, 1, 147-153" ), which makes alcohol dehydrogenases easily inactivated at industrial production temperatures, and most of the alcohol dehydrogenases asymmetrically reduce latent chiral bis-aryl ketones to produce chiral bis-aryl alcohols with low conversion efficiency and space-time production The rate is low, and the S / C is not high (see references for details: "Xumin Gong, et al, ACS Catalysis, 2019, 9, 1, 147-153", "Jieyu Zhou, et al, J.Am.Chem.Soc.2018, 140, 12645-12654"), which greatly hindered the industrialization of the production of chiral bisaryl alcohols by enzymatic asymmetric reduction

Method used

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  • Alcohol dehydrogenase mutant and application thereof
  • Alcohol dehydrogenase mutant and application thereof
  • Alcohol dehydrogenase mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Example 1: Preparation, expression and purification of alcohol dehydrogenase mutants

[0072] Chemically synthesize the gene (the nucleotide sequence of the gene as shown in SEQ ID NO.2) of the alcohol dehydrogenase encoding amino acid sequence as shown in SEQ ID NO.1; The gene obtained and pET-28a (+) plasmid pass through Ligation was performed after double enzyme digestion (NdeI and XhoI), and the ligation product was transformed into E. coli BL21(DE3). The transformed product was spread on LB solid medium, cultured at 37°C for 8-10h, and picked on LB solid medium. Take 5 transformants, insert them into LB liquid medium for culture, and extract the plasmid after culturing at 37°C for 10 hours. The extracted plasmid will be verified by enzyme digestion and sequencing. If the verification is correct, the gene encoding wild-type alcohol dehydrogenase will be obtained. The recombinant plasmid pET28a-KpADH and the recombinant strain E.coli BL21 / pET28a-KpADH containing the ...

Embodiment 2

[0104] Example 2: Temperature Stability of Alcohol Dehydrogenase Mutants

[0105] Warm the pure enzyme solutions of wild-type alcohol dehydrogenase, alcohol dehydrogenase mutants M1-M64 and alcohol dehydrogenase mutant S5 obtained in Example 1 in a water bath at a temperature of 40-60°C for 15 minutes and 15 minutes respectively. Afterwards, the enzyme activity of wild-type alcohol dehydrogenase, alcohol dehydrogenase mutants M1-M64 and alcohol dehydrogenase mutant S5 was measured, and the activity before warming at different temperatures was 100%, and the remaining activity after warming was comparable to it. The relative activity was calculated to determine the T of wild-type alcohol dehydrogenase, alcohol dehydrogenase mutants M1-M64 and alcohol dehydrogenase mutant S5 50 15 value (T 50 15 The value is the temperature corresponding to the activity of alcohol dehydrogenase after warm bath 15min for half of the activity before the warm bath, and the test results are shown ...

Embodiment 3

[0117] Example 3: Kinetic parameters of alcohol dehydrogenase mutants and asymmetric reduction of latent chiral bisaryl ketone (4-chlorophenyl)-(pyridin-2-yl)-methanone to generate chiral bisaryl alcohol Stereoselectivity of (4-chlorophenyl)-(pyridin-2-yl)-methanol

[0118] The wild-type alcohol dehydrogenase obtained in Example 1 and the alcohol dehydrogenase mutants M64, M64 and S5 were selected, and the latent chiral bisaryl ketone (4-chlorophenyl)-(pyridine -2-yl)-methanone is a substrate, and the initial hydrolysis activity of the wild-type alcohol dehydrogenase obtained in Example 1 and the alcohol dehydrogenase mutants M64, M64 and S5 is determined by using the non-linear function in Graph PadPrism 7.0 software The regression method is used to fit the data to obtain the K of the Michaelis-Menten equation respectively. m and V max value, and then calculate K cat and K cat / K m value, the calculation results are shown in Table 3;

[0119] Among them, K cat The form...

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Abstract

The invention, which belongs to the technical field of enzyme engineering and microbial engineering, discloses an alcohol dehydrogenase mutant and application thereof. The alcohol dehydrogenase mutantdisclosed by the invention is high in thermal stability, and high in catalytic efficiency and conversion efficiency (namely space-time yield) of asymmetric reduction of prochiral diarylketone to produce chiral diarylalcohol. Therefore, the alcohol dehydrogenase mutant disclosed by the invention has the extremely broad application prospects in production of chiral diarylalcohol such as (S)-(4-chlorphenyl)-(pyridine-2-yl)-methanol, (R)-(4-chlorphenyl)-(pyridine-2-yl)-methanol and the like.

Description

technical field [0001] The invention relates to an alcohol dehydrogenase mutant and its application, belonging to the technical fields of enzyme engineering and microbe engineering. Background technique [0002] Chiral bisaryl alcohols are an important class of chiral compounds that can be used to synthesize various drugs such as betahistine and rotoxamine. Therefore, chiral bisaryl alcohols are widely used in the field of medicine. [0003] At present, the method for producing chiral bisaryl alcohols is mainly a chemical asymmetric synthesis method, which uses latent chiral bisaryl ketones as raw materials and trans-RuCl 2 [(R)-xylbinap][(R)-daipen], (S)-[Ru(BINAP)Cl 2 ] 2 (NE 3 ), (S,S)-6-CHOONa, etc. as a catalyst or chiral BINAL-H, etc., as a chiral reducing agent, under certain conditions (high pressure) for an asymmetric reduction reaction to obtain chiral bis-aryl alcohols ( For details, see references "C.Y.Chen, et al., Org. Lett., 2003, 5, 5039-5042", "Zhao Zhiq...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C12N15/53C12N15/70C12N1/21C12P7/18C12P17/12C12R1/19
CPCC12N9/0006C12N15/70C12P7/18C12P17/12C12Y101/01001C12P7/22C12Y101/01006C12Y101/01027C12Y101/01047C12Y120/01001C12Y102/02001C12P41/002C12N9/0008C12N9/0093
Inventor 倪晔戴威许国超周婕妤韩瑞枝董晋军
Owner JIANGNAN UNIV
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