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A kind of L-amino acid deaminase mutant with improved heat resistance and preparation method thereof

An amino acid and deaminase technology, applied in the biological field, can solve the problems of poor thermal stability, short enzyme life cycle, low enzyme specific activity, etc., and achieve the effects of reducing workload, prolonging service life and improving structural stability.

Active Publication Date: 2021-08-27
浙江正硕生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] The technical problem solved by the present invention is to overcome the defects of the existing L-amino acid deaminases, such as low enzyme specific activity, poor thermal stability, and short enzyme service life, and provide a kind of L-amino acid deaminase with improved heat resistance enzyme mutant

Method used

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  • A kind of L-amino acid deaminase mutant with improved heat resistance and preparation method thereof
  • A kind of L-amino acid deaminase mutant with improved heat resistance and preparation method thereof
  • A kind of L-amino acid deaminase mutant with improved heat resistance and preparation method thereof

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Embodiment 1

[0021] Below in conjunction with specific embodiment, further illustrate the present invention.

[0022] The medium formula involved in the embodiment is as follows:

[0023] LB liquid medium: 0.5% yeast extract, 1% peptone, 1% NaCl, pH7.0. Sterilize at 121°C for 20min. When making plates, add 2% agar and sterilize at 121°C for 20min.

[0024] When kanamycin antibiotics were screened, the final concentration was 50mg / L.

[0025] The unit in the above medium is % (W / V).

[0026] Example 1:

[0027] 1. Construction of 120 amino acid site mutants of L-amino acid deaminase

[0028] Total gene synthesis from Proteus mirabilis ( Proteus mirabilis ) L-amino acid deaminase (L-aminoacid deaminase, LAAD) gene (genbank accession number EU669819.1), Nde I and Hind Ⅲ Connected into pET24a, constructed plasmid LAAD, transformed into E. coli BL21(DE3), the recombinant strain PMLAAD was constructed.

[0029] The plasmid LAAD of the recombinant strain PMLAAD was extracted, and the ...

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Abstract

The invention belongs to the field of biotechnology, and relates to a method for improving the thermal stability of L-amino acid deaminase, which is beneficial to Proteus mirabilis ( Proteus mirabilis ) L-amino acid deaminase enzyme mutant obtained by performing site-directed mutation, wherein the mutation site is Trp at position 120. Based on the protein spatial structure information, the present invention analyzes the spatial environment around the special amino acid site inside the structure and the interaction between amino acids including hydrogen bonds, ionic bonds, van der Waals, etc., and combines molecular biology techniques to analyze specific amino acid site modification, by rationally designing mutation sites, using site-directed mutation method to obtain L-amino acid deaminase mutants, which can increase the probability of obtaining positive mutant enzyme mutants, and also greatly reduce the work of mutant screening quantity. The invention improves the structural stability of the enzyme, and further helps to prolong the service life of the enzyme.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to a method for improving the thermal stability of L-amino acid deaminase. Background technique [0002] L-amino acid deaminase, which exists in fungi, actinomycetes, and bacteria, is a type of FAD-dependent flavoenzyme, which directly oxidizes L-amino acids to produce corresponding ketoacids and ammonia under aerobic conditions. It is widely used in daily chemicals, pharmaceutical synthesis, food processing and other fields. Using L-amino acid deaminase to catalyze the synthesis of corresponding ketoacids can make the oxidation process efficiently synthesized under relatively mild conditions, avoiding high temperature, high pressure, and strong acid catalysis, and at the same time, it can be used without or with less inducer To achieve efficient expression. The whole process is efficient and the product quality is stable. More importantly, the ketoacid products produced by the enzymati...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/06C12N15/70
CPCC12N9/0022C12N15/70C12Y104/03002
Inventor 吴黎诚郭小雷章权
Owner 浙江正硕生物科技有限公司
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