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Alcohol dehydrogenase mutant with improved activity and stereoselectivity, recombinant vector of alcohol dehydrogenase mutant, genetically engineered bacterium containing recombinant vector, and application of alcohol dehydrogenase mutant and genetically engineered bacterium

A stereoselective, alcohol dehydrogenase technology, applied in the field of protein engineering, can solve the problems of limited application of alcohol dehydrogenase, stereoselectivity and low activity substrate inhibition and product inhibition, etc., to achieve activity and stereoselectivity The effect of improving property, high stereoselectivity, and improving stereoselectivity

Active Publication Date: 2021-03-19
EAST CHINA UNIV OF SCI & TECH
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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 provide a kind of activity and the alcohol dehydrogenase mutant that stereoselectivity improves and its recombinant carrier, genetic engineering bacterium and application, thus solve the present problem Alcohol dehydrogenase in the technology prepares (S)-1-(2,6-dichloro-3-fluorophenyl)ethanol from ketones such as 2,6-dichloro-3-fluoroacetophenone and other ketones, etc. The stereoselectivity and activity of alcohol dehydrogenase are not high, and it is easy to produce substrate inhibition and product inhibition, etc., which leads to the problem that the industrial application of alcohol dehydrogenase is limited.

Method used

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  • Alcohol dehydrogenase mutant with improved activity and stereoselectivity, recombinant vector of alcohol dehydrogenase mutant, genetically engineered bacterium containing recombinant vector, and application of alcohol dehydrogenase mutant and genetically engineered bacterium
  • Alcohol dehydrogenase mutant with improved activity and stereoselectivity, recombinant vector of alcohol dehydrogenase mutant, genetically engineered bacterium containing recombinant vector, and application of alcohol dehydrogenase mutant and genetically engineered bacterium
  • Alcohol dehydrogenase mutant with improved activity and stereoselectivity, recombinant vector of alcohol dehydrogenase mutant, genetically engineered bacterium containing recombinant vector, and application of alcohol dehydrogenase mutant and genetically engineered bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Enzymatic activity detection and analysis stage biological reduction reaction

[0031] The corresponding lead-toteenate 2,6-dichloro-3-fluorophenylideteophenone of the target product (S) -1- (2,6-dichloro-3-fluorophenyl) ethanol is a substrate. The relative activity of the alcohol dehydrogenase CHADH90 to 2,6-dichloro-3-fluorophenylideophenone is determined by enzyme activity. The enzyme coupling circulatory system is then constructed for biological reduction. Then, the product EE value was detected to determine the stereo selectivity of the alcohol dehydrogenase.

[0032] The adjusted step of the coenzyme dependency of the alcohol dehydrogenase is: First, a crude enzyme solution of 5 μl of the alcohol dehydrogenase CHADH90, 182 μl of phosphate buffer (50 mM pH 7.0) is added to the pores of the enzyme label. 8 μlNADphPH and NADH (0.2 mm) were added to each of the adjuminations. Two min was incubated at 25 ° C. The enzyme board was placed in the enzyme label and se...

Embodiment 2

[0034] Example 2 Substrate Die and Molecular Motion Simulation

[0035] In order to obtain the spatial structure of the alcohol dehydrogenase CHADH90, the enzyme is homologous. The 3D model of the alcohol dehydrogenase CHADH90 is used with SwissModel. The 3D model of the alcohol dehydrogenase CHADH90 is from the short-chain alcohol dehydrogenase from Ralstonia Sp. DSM 6428 (PDB number: 4BMS) It is obtained for the template and the sequence similarity of both is 45%. The coenzyme NADH was laminated into the ChaDH90 model to form a CHADH90 / NADH complex. The energy minimized 2,6-dichloro-3-fluorophenylidetone was minimized to the access CHADH90 / NADH complex. According to the catalytic mechanism of the alcohol dehydrogenase, the docking results were screened, and the optimal docking posture was then selected as the initial structure as an initial structure for the initial structure. Molecular kinetic simulation.

[0036] The docking process is performed using Autodock Vina softwar...

Embodiment 3

[0038] Example 3 Construction and screening of mutants

[0039] The wild-type Chadh90 is first cultured and the plasmid of the bacteria is taken as a template for PCR. The primer is designed online with QuickChange (httore / primerdesign program.jsp). The specific method is: First, the base sequence of the alcohol dehydrogenase CHADH90 is pasted to the browser's text box; Second, click "Upload Translated", and the base sequence is automatically translated into an amino acid sequence; Third, there is a rectangular frame with a tickled in front of each amino acid after the translation, select the site you want to mutation; fourth, in the "Changeamino Acid (s) TO" location Select the target amino acid you want to suddenly Finally, click "Design Primers" system to automatically generate primers and include information such as primer length, annealing temperature.

[0040] The DNA polymerase used is a TAQ enzyme having high fidelity properties, which can achieve perfect amplification in ...

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Abstract

The invention discloses an alcohol dehydrogenase mutant with improved activity and stereoselectivity, a recombinant vector of alcohol dehydrogenase mutant, a genetically engineered bacterium containing the recombinant vector, and an application of the alcohol dehydrogenase mutant and the genetically engineered bacterium. The alcohol dehydrogenase mutant is mutated by taking wild type alcohol dehydrogenase as shown in SEQ ID NO.1 as a template. The 188th site of the wild type alcohol dehydrogenase plays a key role in improving stereoselectivity, and the 93rd site plays a key role in activity. According to the mutant N188L / Q93L of the alcohol dehydrogenase provided by the invention, (S)-1-(2, 6-dichloro-3-fluorophenyl) ethanol can be prepared by catalyzing 2, 6-dichloro-3-fluoroacetophenonewith high activity and high stereoselectivity, the activity and the stereoselectivity of various ketones are improved, and the mutant N188L / Q93L is more suitable for industrial production and has a wide application prospect.

Description

Technical field [0001] The present invention relates to the field of protein engineering, and more particularly to an alcohol dehydrogenase mutant and a recombinant vector, a genetic engineering, and a recombinant carrier, a genetic engineering, and an application thereof. Background technique [0002] The chiral molecule is a pair of enantiomers that are mirroring relationships. Their physicochemical properties are similar, but the optically interstitial is different, which can be divided into three types of racemic, R-type (right-rotating) and S-type (left-rotational). Chiral drugs are such a chiral molecule, often only one of the configurations have a pharmacodyne and another configuration has no drug effects or even side effects. Therefore, the optical purity (EE value) of the chiral drug is more than 98%. Chiral drugs occupy an important position in the list of heavy drugs. [0003] As an important block for synthetic chiral drugs, chromatography can be obtained by chemical ...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12N15/53C12P7/22
CPCC12N9/0006C12P7/22C12Y101/01
Inventor 王华磊魏东芝杨泽宇
Owner EAST CHINA UNIV OF SCI & TECH
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