Recombinant nitrilase, coding gene, mutant, engineering bacteria and application thereof

A technology of genetically engineered bacteria and nitrilase, applied in the field of nitrilase, can solve problems such as high cost, environmental pollution, and harsh reaction conditions, and achieve good regioselectivity and catalytic activity

Active Publication Date: 2014-12-17
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The purpose of the invention is to solve the traditional chemical hydrolysis of 1-cyanocyclohexyl acetonitrile in the process of 1-cyanocyclohexyl acetic acid, the reaction conditions are harsh, a large amount of organic solvent is needed in the reaction, the cost is higher, and the yield is lower. The more serious problem of environmental pollution

Method used

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  • Recombinant nitrilase, coding gene, mutant, engineering bacteria and application thereof
  • Recombinant nitrilase, coding gene, mutant, engineering bacteria and application thereof
  • Recombinant nitrilase, coding gene, mutant, engineering bacteria and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Acidovorax facilis (Acidovorax facilis) ZJB09122 strain cells were preserved in liquid nitrogen, and genomic DNA was extracted with a genome extraction kit (FastDNA SPIN Kit soil). Using the genomic DNA as a template, the upstream primer 1AcN1(F)5 PCR amplification was carried out under the action of '-ATGGTTTCGTATAACAGCAAG-3' and downstream primer 2AcN1(R) 5'-CTACTTTGCTGGGACCGG-3'.

[0053] The amount of each component in the PCR reaction system (50 μL): 10 μL of 10*Pfu DNA Polymerase Buffer (Takara), 1 μL of 2.5 mM dNTP mixture (2.5 mM each of dATP, dCTP, dGTP and dTTP), cloning primer 1 and primer at a concentration of 25 μM 2 1 μL each, 1 μL of genomic DNA, 1 μL of 5 U / μL Pfu DNA Polymerase (Takara), 40 μL of nucleic acid-free water.

[0054] Using Biorad’s PCR instrument, the PCR reaction conditions are: pre-denaturation at 95°C for 5 minutes, then enter the temperature cycle at 94°C for 50 seconds, 55°C for 1.5 minutes, and 72°C for 2 minutes, a total of 35 cycles...

Embodiment 2

[0057] Design expression primer (upstream primer 3AcN1 (F) 5'-AAT according to embodiment 1 analysis result GGATCC ATGGTTTCGTATAACAGCAAG-3', downstream primer 4 AcN1(R)5'-AGG GTC GAC CTACTTTGCTGGGACCGG-3'), and Nco I and Xho I restriction enzyme sites were introduced into primer 3 and primer 4, respectively. Under the triggering of primer 3 and primer 4, high-fidelity Pyrrobest DNA polymerase was used to amplify to obtain a 1116bp nitrilase gene fragment (the nucleotide sequence is shown in SEQ ID NO: 2), and after sequencing, use The amplified fragment was double-digested with Nco I and Xho I restriction enzymes, and the fragment was ligated with the expression vector pET28b treated with the same restriction endonuclease using T4 ligase to construct the expression vector pET28b(+) -AcN1. Transform the constructed expression vector pET28b(+)-AcN1 into Escherichia coli BL21(DE3), spread it on a solid medium plate containing kanamycin (Kan) LB with a final concentration of 5...

Embodiment 3

[0061] The recombinant Escherichia coli BL21(DE3) / pET28b(+)-AcN1 verified in Example 2 containing the expression vector pET28b(+)-AcN1 was subjected to site-directed mutation F168V (the F at position 168 was mutated to V, and the conventional gene Engineering operations add a histidine tag to the C-terminus of the protein to obtain a mutant of the nitrilase), thereby obtaining a gene after site-directed mutation (the nucleotide sequence is shown in SEQ ID NO: 4, and the mutant gene encodes The nitrilase is abbreviated as AcN2, and its amino acid sequence is shown in SEQ ID NO: 3).

[0062] According to site-directed mutagenesis, using SEQ ID NO: 4 as a template, design upstream primer 5 F168V (F) 5'-GAGCACGTTCAGCCGCTGTCCAAAT-3' and downstream primer 6 F168V (R) 5'-CGGCTGAACGTGCTCCCAGCAGTTC-3', with SEQ ID NO: 4 Perform PCR amplification for the template (PCR reaction parameters: 94°C for 4min; 98°C for 10s, 55°C for 15s, 72°C for 6min, repeat 30 cycles; 72°C for 10min.). PCR ...

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Abstract

The invention discloses a recombinant nitrilase, a coding gene, a mutant and engineering bacteria from Acidovorax facilis ZJB09122, and the application thereof to preparation of 1-cyano cyclohexyl acetate. The invention provides a nitrilase and its mutant for hydrolysis of 1-cyano cyclohexyl acetonitrile, the enzyme and its mutant have good regional selectivity and catalytic activity in catalysis of the above reaction, and the production process is environmentally friendly. The invention solves the problems in a traditional chemical hydrolysis of 1-cyano cyclohexyl acetonitrile into 1-cyano cyclohexyl acetate, such as harsh reaction conditions, a large amount of organic solvent required in the reaction, high cost, low yield and serious environmental pollution.

Description

(1) Technical field [0001] The present invention relates to a nitrilase, in particular to a recombinant nitrilase, a coding gene, a mutant, an engineering bacterium and its application, and the key to preparing gabapentin from 1-cyanocyclohexylacetonitrile by recombinant Escherichia coli expressing nitrilase Process for intermediate 1-cyanocyclohexylacetic acid. (2) Background technology [0002] The chemical name of gabapentin is 1-aminomethyl-1-cyclohexaneacetic acid. Developed by the Warner-Lambert Company of the United States, it was first launched in the UK in May 1993. It was approved by the FDA in 1994 and launched in the United States. Later, it was used in the treatment of epilepsy in many countries around the world. In 1996, Warner-Lambert Company began to expand Gabapentin indication research, in 2002, FDA approved it for the treatment of neuropathic pain drugs. In addition to being used alone to treat general epilepsy, gabapentin is also used as a superimposed ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/78C12P13/00C12N15/55C12N1/21C12N15/70
CPCC12N9/78C12P13/002C12Y305/05001
Inventor 郑裕国柳志强张新红薛亚平徐喆贾东旭沈寅初
Owner ZHEJIANG UNIV OF TECH
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