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Phenylalanine dehydrogenase as well as preparation method and application thereof

A technology of phenylalanine dehydrogenase and its use, which is applied in the field of extreme amino acid dehydrogenase and its preparation, and the ability to catalyze unnatural amino acids, and can solve the problems of less attention to stress resistance

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

AI Technical Summary

Problems solved by technology

At present, the research content of oxidoreductase mainly focuses on the catalytic properties such as separation and identification of enzymes, substrate specificity and stereoselectivity, while little attention has been paid to its stress resistance properties with industrial application properties.

Method used

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  • Phenylalanine dehydrogenase as well as preparation method and application thereof
  • Phenylalanine dehydrogenase as well as preparation method and application thereof
  • Phenylalanine dehydrogenase as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1: the preparation of phenylalanine dehydrogenase

[0036] 1) Preparation of a recombinant expression strain expressing the phenylalanine dehydrogenase: according to the nucleotide sequence shown in SEQ ID NO.2, design the upstream primer shown in SEQ ID NO.3 and the sequence shown in SEQ ID NO. The downstream primer shown in NO.4, and construct the phenylalanine dehydrogenase gene sequence with NdeI and Xhol restriction sites at the 5' end and 3' end respectively by PCR method, double enzyme digestion and connection with the vector pET28a , to obtain the pET28a-PheDH plasmid; transform the pET28a-PheDH plasmid into E.coliBL21(DE3), and obtain the recombinant expression strain E.coli BL21(DE3) that can express the phenylalanine dehydrogenase with the His-tag tag / pET28a.

[0037] 2) Cultivation of the recombinant expression strain E.coli BL21(DE3) / pET28a: Inoculate the recombinant expression strain E.coli BL21(DE3) / pET28a into 200 mL of LB medium at an inocu...

Embodiment 2

[0041] Embodiment 2: The influence of 20% organic solvent on phenylalanine dehydrogenase

[0042] 1) Preparation and cultivation of the recombinant expression strain: the same as Step 1)-2) of Example 1;

[0043] 2) Preparation of crude enzyme solution and pure enzyme: same as step 3) to 4) of Example 1;

[0044] 3) Determination of the influence of organic solvents on enzyme activity: the substrates 2-oxo-4-phenylbutyric acid ethyl ester and NADH were dissolved in the glycine-sodium hydroxide buffer solution of pH 9, the concentration of the substrate was 10mM, phenylpropanol The concentration of amino acid dehydrogenase is 20U / L. Under parallel conditions, add different organic solvents (ethanol, acetonitrile, methanol, ethylene glycol, isopropanol, etc.) Reaction, determination of enzyme activity, so as to determine the impact of different organic solvents on phenylalanine dehydrogenase, to add an equal amount of 0.05mol / L NH 4 Cl-NH 3 ·H 2 O (pH 8) was used as a contr...

Embodiment 3

[0046] Embodiment 3: The influence of 30% organic solvent on phenylalanine dehydrogenase

[0047] 1) Preparation and cultivation of the recombinant expression strain: the same as Step 1)-2) of Example 1;

[0048] 2) Preparation of crude enzyme solution and pure enzyme: same as step 3) to 4) of Example 1;

[0049] 3) Determination of the influence of organic solvents on enzyme activity: the substrates 2-oxo-4-phenylbutyric acid ethyl ester and NADH were dissolved in the glycine-sodium hydroxide buffer solution of pH 10, the concentration of the substrate was 100mM, phenylpropanol Amino acid dehydrogenase concentration is 50U / L. Under parallel conditions, add different organic solvents (ethanol, acetonitrile, methanol, ethylene glycol, isopropanol, etc.) reaction, to determine the enzyme activity, so as to determine the influence of different organic solvents on phenylalanine dehydrogenase, to add an equal amount of 0.15mol / L NH 4 Cl-NH 3 ·H 2 O (pH 10.5) was used as a cont...

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Abstract

The invention discloses phenylalanine dehydrogenase as well as a preparation method and application thereof, and relates to strain culture, enzyme expression, separation and purification, detection ofionic liquid tolerance and organic solvent system tolerance of enzymes and the like. According to the invention, high-stability phenylalanine dehydrogenase which is resistant to an organic solvent and an ionic liquid and is required by industrial biological catalysis application is obtained. The phenylalanine dehydrogenase has the characteristics of good stress resistance and high stability in anon-aqueous phase system, can effectively prolong the half-life period and improve the catalytic efficiency in industrial production, achieves the purpose of reducing the production cost, and has important industrial application value.

Description

technical field [0001] The invention belongs to the technical field of novel biocatalysts, and specifically relates to extreme amino acid dehydrogenases and their preparation methods and applications, to the anti-stress properties of oxidoreductases such as resistance to organic solvents and ionic liquids, and to catalyzing non-natural enzymes in non-aqueous phase systems. The ability of amino acids belongs to the field of new biocatalyst mining. Background technique [0002] Oxidoreductases account for the largest proportion of the six major enzymes, and are widely used in the production of fine chemicals and pharmaceuticals, medical diagnostic kits, coenzyme regeneration systems, biosensors, and the biodegradation of pollutants. Among them, NADH or NADPH-dependent oxidoreductase plays an important role in chiral synthesis, such as catalyzing the reduction of aldehydes, ketones, carbonyls and alkene carbon-carbon double bonds, so that many latent chiral substances can be co...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/06C12N15/53C12N15/70C12R1/19
CPCC12N9/0018C12Y104/0102C12N15/70
Inventor 王世珍刘凯泷王世燕
Owner XIAMEN UNIV
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