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A kind of highly stereoselective phenylalanine deaminase mutant and its application

A technology of phenylalanine deaminase and stereoselectivity, applied in the biological field, can solve the problems of reduced L-aromatic alanine generation ability, difficult separation, low optical purity, etc. Effects of reduced capacity and high thermal stability

Active Publication Date: 2020-11-06
XIHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But the stereoselectivity of phenylalanine deaminase is not high, and the product is L, the mixture of D-type aromatic alanine, and in industrial production, the product of by-product L-type is difficult to separate, causes product D-aryl alanine The optical purity of amino acid is not high, so it is necessary to improve the activity of phenylalanine deaminase and reduce the production capacity of L-aromatic alanine
[0004] In recent years, genetic engineering technology has been widely used to modify the stereoselectivity of enzymes, and achieved very good results, but the application of this technology to successfully improve the stereoselectivity of phenylalanine deaminase has not been reported in the literature

Method used

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  • A kind of highly stereoselective phenylalanine deaminase mutant and its application
  • A kind of highly stereoselective phenylalanine deaminase mutant and its application
  • A kind of highly stereoselective phenylalanine deaminase mutant and its application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] (1) With the gene of phenylalanine deaminase derived from prokaryotic organism Anabaena variabilis as a template, design 2 pairs of oligonucleotide primers, and use the unmutated recombinant plasmid pET-28-pal as a template, by PCR Methods Amplified to obtain mutant plasmid pET-28-pal / Gln311Glu / Glu448Thr.

[0022] (2) The sequences of 2 pairs of oligonucleotide primers are as follows:

[0023]

[0024] (3) The amplification reaction system of PCR is:

[0025]

[0026] (4) PCR amplification conditions were: pre-denaturation at 94°C for 1 min, denaturation at 94°C for 1 min, annealing at 56°C for 30 s, extension at 72°C for 7 min, 25 cycles.

[0027] (5) The PCR amplification product was purified and recovered using a DNA purification kit.

Embodiment 2

[0029] (1) The PCR product purified by the DNA purification kit was digested with Dpn I restriction endonuclease, and digested at 37° C. for 1 h. The digestion reaction system is as follows:

[0030]

[0031] (2) The digested product was purified and recovered using a DNA purification kit.

Embodiment 3

[0033] (1) Take the PCR product digested and purified by Dpn I restriction endonuclease, heat shock at 42°C for 60s, transform into Escherichia coli E. / L) on a solid LB plate and cultured at 37°C for 8h. Pick a single colony, insert it into LB liquid medium containing 50mg / L Kan for culture, extract the plasmid, carry out enzyme digestion and PCR verification. The plasmids of positive clones were selected and sent to Shanghai Sangon for sequencing. The correctly sequenced plasmid was heat-shocked at 42°C for 60s and then transformed into E. coli E.coli BL21 competent cells, cultured on LB plates containing Kan (10mg / L) resistance at 37°C for 8h, and the positive transformants were selected, namely phenylpropanoid Amino acid deaminase mutant producing bacteria.

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Abstract

The invention discloses a high-stereoselectivity phenylalanine deaminase mutant and application thereof. The mutant is produced by site-specific mutagenesis of an amino acid sequence of phenylalanine deaminase in a procaryotic organism Anabaena variabilis, namely the 311 glutamine is mutated into glutamic acid, the 448 glutamic acid is mutated into threonine, the mutant is subjected to catalyzed synthesis at specific selectivity to obtain D-aromatic alanine, and the protein amino acid sequence of the mutant is shown as SEQ ID No.1. The mutant disclosed by the invention has excellent performances such as high activity, high thermal stability and the like, and a foundation is laid for large-scale low-cost industrial application in synthesis of chiral D-aromatic alanine.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a highly stereoselective phenylalanine deaminase mutant and application thereof. Background technique [0002] Chiral D-aromatic alanine is widely used in the fields of medicine, pesticide and food industry. For example, D-phenylalanine is used to produce the drug "nateglinide" for the treatment of type 2 diabetes, β-lactam antibiotics and new antibacterial drugs. Precursors of cancer drugs. The methods for producing D-aromatic alanine include methods such as fermentation, chemical synthesis, and enzyme catalysis. Due to the complex metabolic process of D-aromatic alanine, the yield of engineering bacteria fermentation is too low to meet the requirements of industrial production. Therefore, the most important method for industrial production of D-aromatic alanine is chemical synthesis, but due to the synthetic Stereoselectivity is low, and the synthesized product is a r...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/88C12N15/60C12P13/22
CPCC12N9/88C12P13/222C12Y403/01005
Inventor 刘义孙伟峰丁文武黄玉坤刘平曾朝懿杨羚羚车振明周哲敏朱龙宝
Owner XIHUA UNIV
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