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Method for improving conversion rate of chiral amine

A conversion rate and chiral amine technology, applied in the chemical field, can solve problems such as improving the conversion rate of chiral amines, and achieve the effect of increasing the conversion rate

Active Publication Date: 2020-07-28
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the asymmetric synthesis of chiral amines using transaminases, no one has reported that the conversion rate of chiral amines has been improved through the coupling system of formate acetyltransferase and lactate dehydrogenase

Method used

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  • Method for improving conversion rate of chiral amine
  • Method for improving conversion rate of chiral amine
  • Method for improving conversion rate of chiral amine

Examples

Experimental program
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Effect test

Embodiment 1

[0048] A method for improving the conversion rate of chiral amines, comprising the steps of:

[0049] (1) In vitro synthesis and amplification of genes:

[0050] Obtain the amino acid sequence of the (R)-selective type omega transaminase derived from Arthrobacter from the NCBI database, and the (R)-selective type omega transaminase is abbreviated as: ArR-ωTA; the amino acid sequence of the ArR-ωTA is as SEQ ID NO .1 shown;

[0051] The amino acid sequence of the ArR-ωTA is on the JCAT website, and the optimized ArR-ωTA nucleotide sequence is obtained by inputting: avoiding XbaI, NdeI, SpeI and HindIII restriction sites as conditions;

[0052] Before and after the optimized ArR-ωTA nucleotide sequence, XbaI restriction enzyme cutting site and HindIII restriction enzyme cutting site were respectively added to obtain the optimized ArR-ωTA nucleotide sequence containing restriction enzyme cutting site, The optimized ArR-ωTA nucleotide sequence containing restriction sites is shown...

Embodiment 2

[0066] A method for improving the conversion rate of chiral amines, comprising the steps of:

[0067] (1) with embodiment 1 step (1);

[0068] (2) replace tdcE in Example 1 with ybiw, and others are the same as Example 1 step (2);

[0069] (3) Introduce p2A4-ArR-ωTA; p2A4-ybiw; p2A4-ldh into Escherichia coli (E.coli MG1655) respectively to obtain recombinant bacteria 1, recombinant bacteria 2 and recombinant bacteria 6;

[0070] (4) Fermenting recombinant bacteria 1, recombinant bacteria 2 and recombinant bacteria 6 respectively to obtain recombinant bacteria 1 fermentation broth, recombinant bacteria 2 fermentation broth and recombinant bacteria 6 fermentation broth;

[0071] Fermentation process is with embodiment 1;

[0072] (5) The fermentation broth obtained in step (4) was centrifuged at 4°C and 6500rpm to collect the precipitate, and the precipitate was washed with 0.01M PBS (pH=7.5) to obtain the recombinant bacteria 1 fermentation broth precipitation, recombinant ba...

Embodiment 3

[0076] A method for improving the conversion rate of chiral amines, comprising the steps of:

[0077] (1) with embodiment 1 step (1);

[0078] (2) Replace tdcE in Example 1 with pflB, and others are the same as Example 1 step (2);

[0079] (3) Introduce p2A4-ArR-ωTA; p2A4-pflB; p2A4-ldh into Escherichia coli (E.coli MG1655) respectively to obtain recombinant bacteria 1, recombinant bacteria 3 and recombinant bacteria 6;

[0080] (4) Fermenting recombinant bacteria 1, recombinant bacteria 3 and recombinant bacteria 6 respectively to obtain recombinant bacteria 1 fermentation broth, recombinant bacteria 3 fermentation broth and recombinant bacteria 6 fermentation broth;

[0081] Fermentation process is with embodiment 1;

[0082] (5) The fermentation broth obtained in step (4) was centrifuged at 4°C and 6500rpm to collect the precipitate, and the precipitate was washed with 0.01M PBS (pH=7.5) to obtain the recombinant bacteria 1 fermentation broth precipitate, recombinant bact...

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Abstract

The invention discloses a method for improving conversion rate of chiral amine. The method comprises the following steps: synthesizing and amplifying an optimized ArR-omega TA nucleotide sequence containing enzyme cutting sites, constructing p2A4-ArR-omega TA, p2A4-FA and p2A4-ldh, wherein FA is a gene for expressing formic acid acetyl transferase, and correspondingly introducing escherichia colito obtain recombinant bacteria 1, FA and 6; fermenting to obtain fermentation liquor of the recombinant bacteria 1, FA and 6; centrifuging to obtain fermentation liquor precipitates of the recombinantbacteria 1, FA and 6; and correspondingly preparing crude FA enzyme liquid and crude ldh enzyme liquid; resuspending the fermentation liquor precipitate of the recombinant bacteria 1 by using PBS, adding PLP and NAD< +>, and reacting; and adding the crude FA enzyme liquid, the crude ldh enzyme liquid, formate dehydrogenase, D-alanine, a substrate ketone and CoA, adding a cosolvent, reacting, terminating, extracting and drying. According to the method disclosed by the invention, a byproduct pyruvic acid is removed, and the conversion rate of the chiral amine is improved. The formate dehydrogenase is added to recover a cofactor reduced coenzyme I.

Description

technical field [0001] The invention belongs to the field of chemistry, in particular to a method for improving the conversion rate of chiral amines. Background technique [0002] Enantiomerically pure amines have played a vital role in the chemical, pharmaceutical and agrochemical industries in recent years. Chiral amines are widely used in therapeutic drugs, such as neurological drugs, cardiovascular drugs, antihypertensive drugs, anti-infective drugs and vaccines. At present, chemical method, biological resolution method and biological asymmetric synthesis method are the main methods for preparing chiral amines. Asymmetric synthesis has become a research hotspot because of its low pollution to the environment and the theoretical yield can reach 100%. During the asymmetric synthesis of chiral amines using transaminase, the transamination reaction is reversible and the by-product pyruvate will hinder the reaction to produce amines. At present, the coupling system of lact...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N15/70C12N1/21C12P13/00
CPCC12N9/0036C12Y206/01C12N15/70C12P13/001
Inventor 宋浩赵妍淑张金华
Owner TIANJIN UNIV
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