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Pantoea amidase, gene, vector, engineering bacterium and application thereof

A technology for encoding bacteramidase and bacteramidase, which is applied in genetic engineering, application, plant genetic improvement, etc., can solve the problems of large amount of catalyst and long reaction time, and achieve short reaction time, small amount of catalyst, and high substrate concentration. high effect

Active Publication Date: 2016-05-25
ZHEJIANG UNIV OF 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 an amidase gene, a recombinant vector containing the amidase gene, the genetically engineered bacteria transformed by the recombinant vector and the recombinant amidase expressed, and its ability to catalyze the hydrolysis of 1-cyanocyclohexylacetamide The application in the synthesis of 1-cyanocyclohexylacetic acid lays the foundation for the nitrile hydratase / amidase coupling catalyzed synthesis of key intermediates of gabapentin, and solves the problems of excessive catalyst consumption and excessive reaction time in the current selective hydrolysis route of nitrilase. long wait question

Method used

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  • Pantoea amidase, gene, vector, engineering bacterium and application thereof
  • Pantoea amidase, gene, vector, engineering bacterium and application thereof
  • Pantoea amidase, gene, vector, engineering bacterium and application thereof

Examples

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Embodiment 1

[0026] Embodiment 1: the acquisition of pantea amidase gene pa-ami

[0027] The amidase gene is synthesized by means of molecular biology. The specific screening method is to search for the amino acid sequence encoded by the gene encoded by NCBI according to the conserved sequence of amidase of the AS family and the NF family, and further screen for proteins that can match the catalytic triad . Finally, an amidase (GenBankaccessionNO.WP_008109374) derived from Pantoeasp.YR343 was established, the amino acid sequence of which is shown in SEQ ID NO:1. According to the preferred codon of Escherichia coli, the amidase gene was optimized, and the amidase gene pa-ami was synthesized by the conventional operation of genetic engineering, with a full length of 1445bp (its nucleotide sequence is as SEQ ID NO: 2 shown), the sequence encodes a complete open reading frame.

Embodiment 2

[0028] Embodiment 2: Construction of recombinant expression vector pET28a-Pa-Ami

[0029] The pa-ami fragment of the amidase gene synthesized in Example 1 was connected to the pUC57 vector by using the PMD-18T kit of TaKaRa Company to obtain the cloning recombinant plasmid pUC57-Pa-Ami. The recombinant plasmid was transformed into Escherichia coli JM109, a single colony was randomly picked and sequenced, and the gene whose nucleotide sequence was shown in SEQ ID NO:2 was obtained.

[0030] Cultivate E.coliJM109 cells containing the recombinant plasmid pUC57-Pa-Ami, use the plasmid extraction kit to extract the plasmid pUC57-Pa-Ami, perform double digestion with restriction endonuclease NcoI / HindⅢ (Fermentas), and recover after gel cutting The fragment was ligated overnight with the commercial vector pET-28a (Novagen) treated with the same restriction endonuclease using T4 ligase (Promega) to construct the recombinant expression plasmid pET28a-Pa-Ami.

Embodiment 3

[0031] Embodiment 3: Construction of genetically engineered bacteria E.coliBL21 / pET28a-Pa-Ami

[0032] The recombinant expression vector pET28a-Pa-Ami constructed in Example 2 was transformed into Escherichia coli BL21(DE3), spread on an LB plate containing a final concentration of 50 μg / mL kanamycin, and cultured overnight at 37°C. Clones were randomly selected for colony PCR identification, and positive clones were sequenced for verification. The results showed that the recombinant expression vector pET28a-Pa-Ami was successfully transformed into the expression host E.coliBL21(DE3), and the amidase gene was successfully cloned into the NcoI and HindⅢ sites of pET-28a, and the recombinant genetically engineered bacteria E.coliBL21( DE3) / pET28a-Pa-Ami.

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Abstract

The invention discloses a Pantoea amidase, a gene, a vector, an engineering bacterium and an application thereof in preparation of 1-cyancyclohexylacetic acid through biological catalysis of 1-cyancyclohexylacetamide. The amino acid sequence of the amidase is represented by SEQ ID NO.1. A synthesis technology of 1-cyancyclohexylacetic acid through biological catalysis by using the amidase is reported in the invention for the first time, and the technology has the substantial advantages of small catalyst dosage (2g / L), high substrate concentration (100g / L), short reaction time (20min), and high product conversion rate reaching 100%.

Description

(1) Technical field [0001] The present invention relates to a gene encoding amidase, an encoding enzyme protein, a recombinant vector containing the gene, a transformed recombinant genetically engineered bacterium and its key intermediate 1-cyano in the synthesis of gabapentin from 1-cyanocyclohexylacetamide Application of cyclohexylacetic acid. (2) Background technology [0002] Gabapentin, whose chemical name is 1-(aminomethyl)cyclohexylacetic acid (I), is an epilepsy drug developed by the Warner-Lambert Company of the United States, and has been used for the treatment of neuropathic pain. Compared with the current similar drugs, gabapentin has significant advantages in oral absorption, side effects, tolerance and therapeutic effect. Moreover, gabapentin is not easily metabolized in the body, does not induce the production of liver enzymes, and rarely interacts with other antiepileptic drugs. Hence, gabapentin occupies a significant position in the epilepsy treatment dru...

Claims

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Application Information

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IPC IPC(8): C12N9/78C12N15/55C12N15/70C12N1/21C12P13/00
CPCC12N9/80C12P13/002C12Y305/01004
Inventor 郑仁朝郑裕国吴哲明
Owner ZHEJIANG UNIV OF TECH
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