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Method for immobilizing L-amino acid deaminase

A deaminase and amino acid technology, applied in the field of immobilizing L-amino acid deaminase, can solve the problems of high cost and low activity of L-amino acid deaminase

Inactive Publication Date: 2018-12-18
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

[0005] The present invention overcomes the defects of low enzymatic activity and high cost of industrial application of L-amino acid deaminase derived from microorganisms, and provides a method for immobilizing L-amino acid deaminase

Method used

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  • Method for immobilizing L-amino acid deaminase
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  • Method for immobilizing L-amino acid deaminase

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Embodiment Construction

[0039] The present invention will be described in further detail below in conjunction with specific embodiments.

[0040] A method for immobilizing L-amino acid deaminase, comprising the steps of:

[0041] 1. Construction of recombinant bacteria

[0042] will be derived from Clostridium thermocellum ( Clostridium thermocellum ) fused to the cellulose binding domain (CBD) (genbank accession number AF283517.1) derived from Proteus mirabilis ( Proteus mirabilis ) C-terminal or N-terminal of L-amino acid deaminase (L-amino acid deaminase, LAAD) (genbank accession number EU669819.1).

[0043] The whole gene is synthesized into two gene fragments, the CBD gene fragment is connected to the 3' or 5' end of the LAAD gene fragment through a short DNA strand (GGAGGAGGAGGAGGAGGAGGAGGAGGA), and connected to the pET24a vector (using NdeI / Hind for double enzyme digestion) to construct a plasmid LAAD-CBD was transformed into E. Coli BL21 (DE3), and the recombinant strain PMLAAD-CBD was...

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Abstract

The invention belongs to the field of biotechnology, and relates to a method for immobilizing L-amino acid deaminase; a cellulose binding domain (CBD) gene fragment is connected a 3' or 5' end of an L-amino acid deaminase (LAAD) gene fragment by a short DNA strand; the L-amino acid deaminase is fused with a cellulose binding domain and combined with microcrystalline cellulose for immobilizing, because the microcrystalline cellulose is cheap and easy to obtain, the immobilization cost is low, and the specificity of the immobilizing process plays an enzyme purifying role, so that enzyme catalysis by-products are very few, and fixed L-AAD-CBD can be used multiple times due to low cost and has a large industrial application value.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to a method for immobilizing 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] L-amino acid deaminase is derived from various microorganisms, such as Proteus, ProÍidencia, Morganella In addition, L-amino acid deaminase in s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/12C12N15/70C12N1/21C12R1/19
CPCC12N9/0022C12N11/12C12N15/70C12Y104/03002
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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