Transaminase mutant and application thereof

A technology of mutants and transaminases, which is applied in the field of transaminase mutants and its applications, can solve the problems of high enzyme quantity and low activity

Active Publication Date: 2019-12-20
ASYMCHEM LAB TIANJIN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] But its activity is low, and the amount of enzyme added during the reaction is more

Method used

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  • Transaminase mutant and application thereof
  • Transaminase mutant and application thereof
  • Transaminase mutant and application thereof

Examples

Experimental program
Comparison scheme
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Embodiment approach

[0043] According to a typical embodiment of the present invention, a recombinant plasmid is provided. The recombinant plasmid contains any one of the above DNA molecules. The DNA molecules in the above recombinant plasmids are placed in appropriate positions of the recombinant plasmids, so that the above DNA molecules can be replicated, transcribed or expressed correctly and smoothly.

[0044] Although the qualifier used in the present invention is "contains" when limiting the above-mentioned DNA molecule, it does not mean that other sequences irrelevant to its function can be arbitrarily added to both ends of the DNA sequence. Those skilled in the art know that in order to meet the requirements of recombination operations, it is necessary to add suitable restriction endonuclease cutting sites at both ends of the DNA sequence, or additionally add start codons, stop codons, etc., therefore, if using A closed-ended statement will not truly cover these situations.

[0045]The t...

Embodiment 1

[0060]

[0061] 10 mg of Substrate 1 / Substrate 2 / Substrate 3 were dissolved in 30 μL of DMSO to prepare substrate solutions. Add transaminase 10mg, 66.6μL isopropylamine hydrochloride solution (6M), 0.1mg PLP, 0.1M Tris-Cl buffer (pH9.0) solution to the total volume of 500μL, and finally add the prepared substrate to the reaction system Solution, adjust pH to 9.0, 200rpm, 45°C constant temperature reaction for 16h. Add 2 times the volume of acetonitrile to the reaction system, mix well, let stand for 10 minutes, centrifuge at 12000 rpm for 10 minutes, take the supernatant, dilute it 10 times and send it to liquid phase to measure the conversion rate. ee value detection method: add 2 times the volume of acetonitrile to the reaction system, mix well, let stand for 10 minutes, centrifuge at 12000rpm for 10 minutes, take the supernatant, add anhydrous MgSO 4 Remove water, centrifuge at 12000rpm for 10min, take the supernatant, and use N 2 Blow dry, add 1mL methanol, dissolve ...

Embodiment 2

[0068]

[0069] 10 mg of Substrate 4 / Substrate 5 were dissolved in 30 μL DMSO respectively to prepare substrate solutions. Add transaminase 10mg, 66.6μL isopropylamine hydrochloride solution (6M), 0.1mg PLP, 0.1M Tris-Cl buffer (pH 9.0) solution to the total volume of 500μL, and finally add the prepared substrate solution to the reaction system , adjust the pH to 9.0, 200rpm, and react at a constant temperature of 45°C for 16h. Add 2 times the volume of acetonitrile into the reaction system, mix well, let stand for 10 minutes, centrifuge at 12000 rpm for 10 minutes, take the supernatant, dilute it 10 times, and send it to the liquid phase to measure the conversion rate. ee value detection method: add 2 times the volume of acetonitrile to the reaction system, mix well, let stand for 10 minutes, centrifuge at 12000rpm for 10 minutes, take the supernatant, add anhydrous MgSO 4 Remove water, centrifuge at 12000rpm for 10min, take the supernatant, and use N 2 Blow dry, add 1mL...

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Abstract

The invention discloses a transaminase mutant and application thereof. An amino acid sequence of the transaminase mutant is an amino acid sequence obtained by mutation of an amino acid sequence shownin SEQ ID NO:1, and mutation at least comprises one of the following mutation site combinations: T7C+S47C, Q78C+A330C, V137C+G313C, A217C+Y252C and L295C+C328C; or the amino acid sequence of the transaminase mutant is an amino acid sequence which has a mutation site in a mutant amino acid sequence and has 80% homology or above with the mutant amino acid sequence. The transaminase mutant realizes change of protein structure and functions, reduces enzyme dose, increased the ee value of the product and reduces after treatment difficulty, so that the transaminase mutant can be suitable for industrial production.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a transaminase mutant and its application. Background technique [0002] Chiral amine compounds are key intermediates in the synthesis of many chiral drugs. Many important neurological drugs, cardiovascular drugs, antihypertensive drugs, anti-infective drugs, etc. are synthesized using chiral amines as intermediates. At present, the synthesis of chiral amines is mainly achieved by chemical methods, such as asymmetric reduction of Schiff bases (Chiral amine synthesis: methods, developments and applications [M]. West Sussex, United Kingdom: John Wiley & Sons. 2010), but there are reaction conditions in the reaction Harshness, the use of toxic transition metal catalysts, and low product stereoselectivity are disadvantages. [0003] Transaminases can catalyze the transfer of amino groups from amino donors to prochiral acceptor ketones, resulting in chiral amines and by-product ketones....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12P13/00
CPCC12N9/1096C12P13/001C12Y206/01
Inventor 洪浩詹姆斯·盖吉肖毅马玉磊张娜焦学成牟慧艳程逸冰曹姗
Owner ASYMCHEM LAB TIANJIN
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