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Engineered transaminase polypeptide and application thereof

A transaminase and engineering technology, applied in the field of engineering transaminase polypeptides, can solve the problem of limited cost of natural raw materials and high

Active Publication Date: 2019-07-02
ENZYMASTER NINGBO BIO ENG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the direct extraction of biologically active chiral compounds from natural products is the most basic method, but the natural raw materials are limited and the cost is high

Method used

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  • Engineered transaminase polypeptide and application thereof
  • Engineered transaminase polypeptide and application thereof
  • Engineered transaminase polypeptide and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0295] Embodiment 1: the construction of gene cloning and expression vector

[0296] The amino acid sequence of the wild-type transaminase derived from Aspergillus fumigatus can be retrieved from NCBI, and then its corresponding nucleic acid is synthesized by common techniques in the art and cloned into the expression vector pACYC-Duet-1. Transform the recombinant expression plasmid into competent cells of E.coil BL21(DE3), the transformation condition is 42°C, heat shock for 90 seconds, the transformation solution is spread on the LB plate containing chloramphenicol, and cultured upside down at 37°C overnight, that is Recombinant transformants were obtained.

Embodiment 2

[0297] Embodiment 2: Shake flask expression of transaminase polypeptide

[0298] The recombinant E.coli BL21 (DE3) transformant obtained in Example 1 was inoculated into 50 mL of chloramphenicol-containing LB medium (peptone 10 g / L, yeast extract powder 5 g / L, sodium chloride 10 g / L, pH 7.0±0.2, 25°C) in a 250mL Erlenmeyer flask, placed at 30°C, shaking at 250rpm and culturing overnight. When the OD600 of the culture medium reaches 2, insert 250mL TB medium (tryptone 12g / L, yeast extract powder 24g / L, disodium hydrogen phosphate 9.4g / L , dipotassium hydrogen phosphate 2.2g / L, pH value 7.2±0.2, 30°C) into a 1000mL Erlenmeyer flask with a final concentration of 6g / L lactose as an inducer, placed at 30°C, 250rpm shaker shaking culture. After induction at 30°C for 20 h, the culture medium was centrifuged to collect the cells, washed twice with PBS buffer (pH 7.4), and the supernatant was centrifuged to obtain wet cells, which were placed in a -20°C refrigerator until use. If en...

Embodiment 3

[0299] Embodiment 3: Construction of transaminase mutant library

[0300] All the reagents used here are commercial reagents, preferably Quikchange kit (supplier: Agilent). The sequence design of the mutant primers was carried out according to the instructions of the kit. The construction of a saturation mutation library at a single residue position is now described as an example. The PCR system is: 5xBuffer 10uL, 10mM dNTP 1uL, plasmid DNA template 1uL (50ng / uL), upstream and downstream primers 0.75uL (10uM), high-fidelity enzyme 0.5uL, ddH2O 36uL. The codon of the PCR primer at the mutation position is NNK.

[0301] PCR amplification steps are: (1) 98°C, pre-denaturation for 3min; (2) 98°C, denaturation for 10s; (3) 72°C annealing and extension for 3min; steps (2)-(3) were repeated 25 times; (5) Continue to extend at 72°C for 10min, and cool to 4°C. Add 2uLDpnI to the PCR product, and digest overnight at 37°C to eliminate the plasmid template. The digested PCR product w...

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Abstract

The invention provides an amino acid sequence, a preparation method and a reaction process of engineered transaminase for asymmetric synthesis of chiral amine compounds under industrial-related conditions. The invention also provides a polynucleotide sequence encoding the engineered transaminase polypeptide, recombinant engineering bacteria capable of expressing the engineered transaminase polypeptide, and a method for producing the chiral amine compounds by using the engineered transaminase polypeptide. Compared with other preparation methods, the engineering transaminase polypeptide providedby the invention has better activity and stability, overcomes the inhibition of the product on enzymes, has higher unit activity, and has good industrial application prospect.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to an engineered aminotransferase polypeptide and its application. Background technique [0002] Chiral amine compounds are a very important class of compounds, which are widely used in fine chemistry, pharmaceuticals, health care, agriculture and material industries. Especially in the pharmaceutical industry, chiral amines are often used as intermediates for the preparation of various drugs, such as cephalosporins, neurological drugs, cardiovascular drugs, antihypertensive drugs, anti-infective drugs and vaccines, etc. Chiral amines are used as intermediates, and the main component of sitagliptin, an anti-diabetic oral drug Januvia, is R-type amines. The traditional chemical preparation of chiral amine compounds usually involves reactions under extreme conditions (high temperature, high pressure, etc.), and at the same time, in order to obtain products with high...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12P13/00C12P17/00C12P17/16
CPCC12N9/1096C12Y206/01C12P13/001C12P17/00C12P17/165C12P13/00C12N11/00C12N15/70
Inventor 陈海滨黄勇开王娟娟蔡宝琴尚传洋马克·博科拉
Owner ENZYMASTER NINGBO BIO ENG CO LTD
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