Mutated arginine deiminase as well as preparation method and application thereof

A technology of arginine deiminase and arginine, which is applied in the field of mutant arginine deiminase and its preparation, can solve the problems of low fermentation unit and the like, and achieves simple ingredients and is conducive to separation and purification. Effect

Active Publication Date: 2015-04-29
石药集团中诺药业(石家庄)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is currently reported that most arginine deiminases in Escherichia coli have low fermentation units, and most of them exist in the form of inactive inclusion bodies. The early stage of enzymatic catalysis requires the denaturation and refolding of inclusion bodies, which seriously restricts the industrial production of direct enzymatic methods. Can be produced indirectly by fermentation

Method used

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  • Mutated arginine deiminase as well as preparation method and application thereof
  • Mutated arginine deiminase as well as preparation method and application thereof
  • Mutated arginine deiminase as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1 Construction of arginine deiminase (ADI) encoding gene, establishment of error-prone PCR system, establishment of ADI activity detection system, conditions for determining arginine and citrulline by HPLC

[0058] 1. Primer design

[0059] According to the full gene sequence of ADI and the characteristics of the expression vector pET-21a of Escherichia coli, the initial construction and the mutation primers before and after error-prone PCR (both are the same):

[0060] Primer ADI1F: 5'-CGCGGATCCATGAAAATGGAACAAGCATTG-3' (underlined bases are Bam HI recognition sites);

[0061] Primer ADI1R: 5'-ACGCGTCGACTTATTTTAGATTTTCTCTAAC-3' (bases underlined are Sal I recognition sites).

[0062] 2. PCR primers to amplify ADI target gene

[0063] ADI1F and ADI1R were used as primers, and the plasmid PUC 57-ADI (synthesized by Nanjing KingScript Biotechnology Co., Ltd.) was used as a template to amplify the ADI gene. The polymerase used in PCR, the corresponding amplificati...

Embodiment 2

[0099] Example 2 Construction and expression of the Bacillus subtilis recombinant plasmid of the gene encoding arginine deiminase (ADI)

[0100] 1. Primer design

[0101] Cloning primers were designed according to the obtained arginine deiminase gene sequence containing five mutation sites screened in Example 1 and the multiple cloning site of pWB980 (secretion type) Bacillus subtilis.

[0102] ADI2F: CGCGGATCCCATGAAAATGGAACAAGCATTG (underlined bases are BamHI recognition sites);

[0103] ADI2R: ACGCGTCGACTTATTTTAGATTTTCTCTAAC (the underlined base is the Sal I recognition site);

[0104] 2. PCR primers to amplify ADI target gene

[0105] ADI2F and ADI2R were used as primers, and the plasmid PUC 57-ADI (synthesized by Nanjing KingScript Biotechnology Co., Ltd.) was used as a template to amplify the ADI gene. The polymerase used in PCR, the corresponding amplification buffer, and dNTP solution were all provided by TaKaRa Company purchased.

[0106] The PCR reaction system is...

Embodiment 3

[0116] Example 3 Construction and expression of recombinant plasmid of Pichia pastoris encoding gene for arginine deiminase (ADI)

[0117] 1. Primer design

[0118] According to the obtained arginine deiminase (ADI) gene sequence containing five mutation sites screened in Example 1, and the multiple cloning sites of the Pichia pastoris vector were designed to connect intracellular and extracellular expression Vector primers.

[0119] ADI3F: CCG GAATTC ATGAAAATGGAACAAGCATTG (underlined bases are EcoRI recognition sites);

[0120] ADI3R: CCG GAATTC TTATTTTAGATTTTCTCTAAC (underlined bases are EcoRI recognition sites);

[0121] 2. Construction of Pichia pastoris recombinant expression vectors pAO815-ADI and pPic9k-ADI

[0122] ADI3F and ADI3R were used as primers, and the plasmid PUC 57-ADI (synthesized by Nanjing KingScript Biotechnology Co., Ltd.) was used as a template to amplify the ADI gene. The polymerase used in PCR, the corresponding amplification buffer, and dNTP ...

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Abstract

The invention discloses a mutated arginine deiminase as well as a preparation method and an application thereof. Amino acids in one or more sites of K137A, F198W, V230A, R257L and A260D of arginine deiminase mutate, and the mutated arginine deiminase is improved in activity significantly, can be used for catalyzing L-arginine to produce L-citrulline and can achieve the conversion rate of more than 95%. The mutated arginine deiminase is prepared by adopting an error-prone PCR (polymerase chain reaction) system.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a mutant arginine deiminase and its preparation method and application. Background technique [0002] L-citrulline (L-Citrulline), the chemical name is 2-amino-5-(carbamoylamino) n-pentanoic acid. L-citrulline is an α-amino acid that has the ability to synthesize peptide bonds but is not a normal constituent amino acid for proteins. It was named "citrulline" because it was first obtained from watermelon. Citrulline is an intermediate product of the urea cycle of amino acid metabolism in animals. It is produced from ornithine and carbamoyl phosphate in the urea cycle, or a by-product of arginine catalyzed by nitric oxide synthase (NOS). product. L-citrulline is involved in a series of metabolic processes, such as urea cycle, arginine-citrulline cycle, etc. Recent studies have shown that L-citrulline can be widely used in medicine, food, and health care pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/78C12N15/55C12N15/70C12N15/75C12N15/81C12N1/21C12N1/19C12P13/10C12R1/19C12R1/125C12R1/84
CPCC12N9/78C12Y305/03006
Inventor 任丽梅刘健袁国强郭军臣赵英杰徐永龙李鹤李晓静成志远
Owner 石药集团中诺药业(石家庄)有限公司
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