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Alcohol dehydrogenase mutant and its application

A technology of alcohol dehydrogenase and mutants, applied in the field of enzyme and enzyme engineering, can solve the problems of high production cost, high diastereoisomer content, cumbersome post-treatment process, etc., and achieve improved enzyme activity and stereoselectivity Improved effect

Active Publication Date: 2018-10-30
ASYMCHEM LAB TIANJIN +5
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the application of industrial production, there are still some problems that need to be further solved, such as the high content of diastereoisomers produced after the catalytic reaction, the low catalytic activity of the enzyme, etc.
[0007] Therefore, there is still a need to improve the existing alcohol dehydrogenase to improve its catalytic activity, substrate specificity and / or stability, and then improve the existing technologies such as low stereoselectivity, cumbersome post-treatment process, production high cost

Method used

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  • Alcohol dehydrogenase mutant and its application
  • Alcohol dehydrogenase mutant and its application
  • Alcohol dehydrogenase mutant and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0057] Site-directed saturation mutation of the alcohol dehydrogenase (HTADH) gene (SEQ ID NO: 9) derived from Bacillus stearothermophilus LLD-R strain.

[0058] The amino acid sequence of alcohol dehydrogenase (HTADH) was simulated on the Swiss-model website to simulate the three-dimensional structure of the protein, and then the binding simulation between the substrate and the protein was carried out through Docking. Finally, through Pymol analysis, the selection may be related to the binding of the substrate and NAD. Amino acids associated with NAD proton transport were used as mutant amino acids.

[0059] According to the mutated amino acid and the base sequences on both sides (see the mutation site in Table 1 for the mutated amino acid), use Primmer 5.0 to design the corresponding mutation primers (Table 1). Using the pET22b(+) expression vector containing alcohol dehydrogenase gene (purchased from Novagen, product number 69744) as a template, the complete linear fragment...

Embodiment 2

[0062] Example 2: Cloning and expression of alcohol dehydrogenase mutants

[0063] In order to facilitate the expression and identification of alcohol dehydrogenase mutants, compatible restriction sites were designed at the 5' and 3' ends of the gene. NdeI and XhoI can be used to digest the target gene and pET-22b(+) (other expression plasmids that can express proteins in Escherichia coli can also be used) at the same time, respectively, and the target gene and the larger fragment of the plasmid after digestion Use T4 DNA ligase for ligation reaction, transform the ligated product into competent cells of Escherichia coli DH5α strain, then spread the transformed competent cells on LB culture plates containing 50 μg / ml ampicillin, and culture overnight at 37°C .

[0064] Pick a single colony grown on the above-mentioned petri dish and inoculate it in LB liquid medium containing 50 μg / ml ampicillin, culture it with shaking at 37°C overnight, collect the bacteria for plasmid extr...

Embodiment 3

[0065] Example 3: Screening of Alcohol Dehydrogenase Mutants

[0066] The mutant in Example 1 was inoculated in 500 ml of LB liquid medium containing 50 μg / ml ampicillin, cultured with shaking at 37°C until OD600=0.6, added IPTG to a final concentration of 0.2mM, and induced expression at 18°C . After 16 hours of induction, the cells were collected by centrifugation at 6000g for 10 minutes. The cells were disrupted by an ultrasonic breaker (JY92-2D, Ningbo Xinzhi Biotechnology Co., Ltd.), and the supernatant was collected by centrifugation at 10,000 g at 4°C for 20 min to obtain a crude enzyme solution of alcohol dehydrogenase for activity detection. Add 50.0mg of main raw materials (tetrahydrofuro[2,3-b]furan-3(2H)-one, 5.0mg NAD+, 50.0mg ammonium formate, 10mg coenzyme formate dehydrogenase and 1.5ml alcohol dehydrogenase to a 10ml reaction vial Enzyme crude enzyme solution, system pH = 6.0, and after incubating at 30±3°C for 17 hours, extract the reaction system with dich...

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Abstract

The invention discloses an alcohol dehydrogenase mutant and application thereof. The amino acid sequence of the alcohol dehydrogenase mutant is an amino acid sequence obtained by mutating an amino acid sequence as shown in SEQ ID NO:9; the mutated amino acid sequence has at least one mutation site selected from the following mutation sites: the 40th position, the 87th position, the 194th position and the 331st position; T on the 40th position is mutated into S, A or C; W on the 87th position is mutated into F, Y or H; V on the 194th position is mutated into I, L or E; R on the 331st position is mutated into A, K or M; or the amino acid sequence of the alcohol dehydrogenase mutant has the mutation sites in the mutated amino acid sequence, and the alcohol dehydrogenase mutant contains more than 90% of amino acid sequences which are homologous with the mutated amino acid sequence. The stereoselectivity and enzyme activity of the alcohol dehydrogenase mutant which is provided with the at least one mutation site or retains the mutation site and contains more than 90% of amino acid sequences which are homologous with the mutated amino acid sequence are greatly improved.

Description

technical field [0001] The invention relates to the fields of enzymes and enzyme engineering, in particular to an alcohol dehydrogenase mutant and its application. Background technique [0002] As a biocatalyst, enzymes can give full play to their high efficiency and high specificity in living organisms. However, in industrial applications, there are generally problems such as inability to adapt to industrial production conditions and low catalytic ability to unnatural substrates. Enzyme molecules must be modified by means of protein engineering methods to adapt to different application requirements. Protein engineering methods can be summarized into three types: rational design, irrational design and semi-rational design. [0003] Rational design refers to changing individual amino acids in protein molecules through site-directed mutation (Site-directed Mutagenesis) or other methods on the basis of understanding the spatial structure of proteins, thereby producing protein...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/04C12N15/53C12N15/70C12N1/21C12P17/18C12P7/62
CPCC12N9/0006C12P7/62C12P17/181
Inventor 洪浩詹姆斯·盖吉高峰刘立辉刘芳于文燕崔瑜霞唐芳荣张娜
Owner ASYMCHEM LAB TIANJIN
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