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New application of (R)-omega-transaminase mutant

A technology of mutants and transaminases, applied in the field of molecular biology, can solve the problems of low optical purity, environmental pollution, and low yield, and achieve the effects of strong stereoselectivity, simple preparation process, and easy separation and extraction

Inactive Publication Date: 2020-04-07
ZHEJIANG UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] The present invention provides a new application of (R)-ω-transaminase mutant, using the transaminase as a catalyst to biosynthesize (R)-2,3-dichlorophenethylamine or (R)-2,5-dichlorophenethylamine Phenylethylamine, the synthesis method of this application has strong stereoselectivity, simple preparation process and convenient extraction, and solves the problems of low optical purity, low yield and environmental pollution that are widely existing in the synthesis of chiral compounds

Method used

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Experimental program
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Effect test

Embodiment 1

[0027] 1. Experimental materials

[0028] (1) LB medium: 10g / L tryptone (purchased from Oxoid), 5g / L yeast powder (purchased from Oxoid), 10g / L sodium chloride (purchased from Sangon Bioengineering Co., Ltd. (Shanghai)), pH 7.0. LB solid medium: add 2% (mass ratio) agar powder to LB liquid medium.

[0029] (2) 2,3-dichloroacetophenone, 2,5-dichloroacetophenone and Marfey reagent were purchased from Aladdin Reagent Co., Ltd. Sodium chloride, glycerin, calcium chloride, imidazole, glacial acetic acid, disodium hydrogen phosphate, sodium dihydrogen phosphate, pyridoxal 5-phosphate, Coomassie brilliant blue protein concentration determination kit, Ni-NTA chromatography medium, isopropyl Base-β-D-thiogalactoside (IPTG), kanamycin sulfate, coenzyme pyridoxal-5-phosphate (PLP) DNA and protein markers were purchased from Sangon Bioengineering Co., Ltd. (Shanghai) .

[0030] (3) Bacterial strain: ω-transaminase gene (ω-TA gene) (SEQ ID NO.2) General Biosystems (Anhui) Co., Ltd. wa...

Embodiment 2

[0069] (R)-ω-transaminase mutants catalyze the transfer of amino groups from amino donors to 2,3-dichloroacetophenone or 2,5-dichloroacetophenone: the substrate solution was phosphate buffered saline (50mmol / L, pH 8.0), the 1mL reaction system includes 50mM sec-butylamine, 30mM aminoacceptor ketones (substrate ketones are 2,3-dichloroacetophenone or 2,5-dichloroacetophenone), 0.1 mM PLP, 0.1mg / mL (R)-ω-transaminase mutant, catalyzed reaction at 30°C for 30h, and the blank control was replaced by buffer solution (R)-ω-transaminase mutant.

[0070] Add 2% of the Marfey chiral derivatization reagent to the reacted mixed solution (the Marfey reagent reacts with the amino groups and other groups in the chiral molecules of the reaction product, and derivates to obtain optical isomers containing two or more chiral centers , through HNMR, HPLC and other analytical means, convenient and quick analysis of the optical purity of the derivatized prochiral compound), derivatized at 40°C for...

Embodiment 3

[0072] (R)-ω-transaminase mutants catalyze the transfer of amino groups from amino donors to 2,3-dichloroacetophenone or 2,5-dichloroacetophenone: the substrate solution was phosphate buffered saline (50mmol / L, pH 8.0), the 1mL reaction system includes 50mM sec-butylamine, 1mM aminoacceptor ketones (substrate ketones are 2,3-dichloroacetophenone or 2,5-dichloroacetophenone), 0.1 mM PLP, 0.1mg / mL (R)-ω-transaminase mutant, catalyzed reaction at 30°C for 30h, and the blank control was replaced by buffer solution (R)-ω-transaminase mutant.

[0073] The reacted mixture was added with 2% Marfey chiral derivatization reagent, and derivatized at 40° C. for 2 h. After the derivatization was completed, 2M HCl was added to terminate the derivatization. The product was extracted with ethyl acetate, dried by a nitrogen blower, reconstituted in acetonitrile, filtered through a filter membrane, and then filled into a sample bottle.

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Abstract

The invention discloses an application of a (R)-omega-transaminase mutant. The amino acid sequence of the (R)-omega-transaminase mutant is shown as SEQ ID NO.1. 2,3-dichloroacetophenone or 2,5-dichloroacetophenone is taken as a substrate. The (R)-omega-transaminase mutant is taken as a catalyst. Catalytic reaction is carried out in the presence of an amino donor, and (R)-2,3-dichlorophenylethylamine or (R)-2,5-dichlorophenylethylamine is synthesized. According to the method, (R)-omega-transaminase is used as a biocatalyst for catalytic generation of a substrate 2,3-dichloroacetophenone or 2,5-dichloroacetophenone, and the product can be used as a drug intermediate for synthesis of related drugs. The method is high in stereoselectivity, simple in preparation process and convenient to extract. The problems of low optical purity, low yield, environmental pollution and the like widely existing in current chiral compound synthesis are solved.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to the new application of (R)-ω-transaminase mutants, namely the synthesis of (R)-2,3-dichlorophenethylamine or (R)-2,5-dichloro Biocatalysis of phenethylamine. Background technique [0002] Due to the special three-dimensional structure of chiral compounds, their enantiomers often exhibit different physical and chemical properties and physiological activities. In recent years, they have played an important role in the fields of new material development, new drug design, and fine chemical synthesis. [0003] The PPARG gene is the gene encoding peroxisome proliferative activated receptor γ (peroxisome proliferative activated receptor, gamma), which belongs to the nuclear receptor peroxisome proliferative activated receptor subfamily. PPARG is expressed in different degrees in fat, liver, skeletal muscle, kidney, pancreas and other tissues. It heterodimerizes with retinal-l...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12P13/00
CPCC12N9/1096C12P13/001C12Y206/01
Inventor 黄俊刘春燕梅乐和吕常江王宏鹏于林凯胡升赵伟睿蒋成君
Owner ZHEJIANG UNIVERSITY OF SCIENCE AND TECHNOLOGY
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