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A method for improving the enzyme activity of l-amino acid deaminase heterologous expression

A technology of heterologous expression and deaminase is applied in the field of improving the enzyme activity of heterologous expression of L-amino acid deaminase, which can solve the problems of low enzyme activity expression, affecting industrial application, and low bacterial volume, etc., so as to improve the enzyme activity. Live, high industrial application value, improved quantitative effect

Active Publication Date: 2021-07-23
浙江正硕生物科技有限公司
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Problems solved by technology

When the active L-amino acid deaminase is expressed in Escherichia coli, the L-amino acid deaminase will deaminate the amino acid in the cell and produce hydrogen peroxide, and the hydrogen peroxide is toxic to the cell, causing the cell to prematurely automatize. Soluble, so that the expression of enzyme activity per unit cell is small, and the amount of cell per unit fermentation volume is small, which seriously affects the industrial application

Method used

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  • A method for improving the enzyme activity of l-amino acid deaminase heterologous expression
  • A method for improving the enzyme activity of l-amino acid deaminase heterologous expression
  • A method for improving the enzyme activity of l-amino acid deaminase heterologous expression

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Embodiment 1

[0033] A method for improving the enzyme activity of heterologous expression of L-amino acid deaminase, the specific operation steps are as follows:

[0034] 1. Construction of recombinant bacteria

[0035] Total gene synthesis from Proteus mirabilis ( Proteus mirabilis ) L-amino acid deaminase (L-amino acid deaminase, LAAD) gene (genbank accession number EU669819.1) LAAD, and connected into the pET28a vector ( NdeI / Hind ), construct the plasmid laad, transform it into E.Coli BL21 (DE3), and construct the recombinant strain PMLAAD;

[0036] Whole gene synthesis is derived from psychrophilic bacteria ( Psychrobacter piscatorii T-3) catalase (Catalase) gene (genbank accession number EU543218) PKTA, and connected into pET28a vector ( NcoI / Xhol ), construct the plasmid pkta, transform it into E.Coli BL21 (DE3), and construct the recombinant strain PKTA;

[0037] Plasmid pkta, amplified by PCR, adding a DNA fragment (aagctttctagaaataattttgtttaactttaagaaggagatatacc) u...

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Abstract

The invention belongs to the field of genetic engineering, and relates to a method for improving the enzyme activity of heterologous expression of L-amino acid deaminase, which uses L-amino acid deaminase (L-amino acid deaminase, LAAD) gene and catalase (Catalase) Gene expression in tandem. The present invention adopts L-amino acid deaminase to be expressed in series with catalase, which increases the enzyme activity per unit cell and increases the amount of cell per unit fermentation volume, thereby having high industrial application value.

Description

technical field [0001] The invention belongs to the field of genetic engineering and relates to a method for improving the enzyme activity of heterologous expression of L-amino acid deaminase. Background technique [0002] L-Amino Acid Deaminase (LAAD, L-Amino Acid Deaminase), also known as L-Amino Acid Oxidase (LAAO, L-Amino Acid Oxidase, EC 1.4.3.2), is a class of flavoenzyme Natural and unnatural L-amino acids have broad-spectrum α-deamination activity. With the participation of oxygen, LAAD oxidizes L-amino acid to de-α-amino, generates keto acid and ammonia, and produces hydrogen peroxide as a by-product. L-amino acid deaminase can be used to oxidize the amino group of L-amino acid to prepare corresponding α-keto acid or to oxidize L-amino acid in racemic amino acid to prepare D-amino acid. Both α-keto acid and D-amino acid are important pharmaceutical intermediates. [0003] Catalase is mainly derived from microorganisms and animal livers (such as bovine liver), and...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/06C12N9/08C12N15/70C12N1/21C12R1/19
CPCC12N9/0022C12N9/0065C12N15/70C12Y104/03002C12Y111/01006
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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