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Mutant enzyme of glutamate dehydrogenase and construction method thereof

A technology of glutamate dehydrogenase and mutants, applied in the field of mutant enzymes and their construction, can solve the problems of limited use and inability to catalyze other amino acids

Active Publication Date: 2013-06-19
ANHUI NORMAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the above enzymes can only catalyze glutamic acid, but not other amino acids, so their uses are limited.

Method used

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  • Mutant enzyme of glutamate dehydrogenase and construction method thereof
  • Mutant enzyme of glutamate dehydrogenase and construction method thereof
  • Mutant enzyme of glutamate dehydrogenase and construction method thereof

Examples

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Embodiment 1

[0022] 1. Acquisition of glutamate dehydrogenase gene gdh

[0023] Primers were designed based on the known glutamate dehydrogenase gene of Salmonella typhimurium atcc 14028 (Salmonella typhimurium), and S. typhimurium genomic DNA was used as a template for PCR amplification. As a result, only one specific fragment was obtained ( figure 1 ). The gdh amplification primers and PCR conditions were as follows:

[0024] Sense: 5′-GAACCACGTCATATGGATCAGACATGTTC-3′;

[0025] Anti-sense: 5′-ATCCCTCGAGAAAGCTATCTGGCCTGAC-3′;

[0026] Pre-denature at 95°C for 3 minutes; cycle 35 times at 94°C for 30s, 60°C for 30s, and 72°C for 1min and 30s; fully extend at 72°C for 10 minutes.

[0027] 2. Construction of recombinant vector pET28b-gdh

[0028] The amplified product and the vector pET28b were digested with NdeI and XhoI respectively, then ligated and transformed into E.coliDH5α. Positive clones were screened to obtain the expression plasmid pET28b-gdh. After double-enzyme digestion a...

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Abstract

The invention discloses mutant enzyme of glutamate dehydrogenase and a construction method thereof. The mutant enzyme has an amino acid sequence shown as SEQ ID NO: 1. The mutant gene is well expressed in a heterologous host, namely Escherichia coli; and expressed proteins are one kind of dehydrogenases which can catalyze various amino acids. The method mainly comprises the following steps of: (1) obtaining a glutamate dehydrogenase gene gdh and constructing a recombinant plasmid; (2) constructing a mutant strain; (3) constructing an expression strain; and (4) expressing and purifying. Compared with the prior art, the generated engineering bacteria not only can catalyze glutamic acid, but also can catalyze methionine and norleucine.

Description

technical field [0001] The present invention relates to the use of mutant enzymes and a construction method thereof, in particular to glutamate dehydrogenase mutant enzymes and a construction method thereof. Background technique [0002] Amino acid dehydrogenases (amino acid dehydrogenases, EC 1.4.1), the family of proteins using NAD + or NADP + It is a cofactor that catalyzes the oxidative deamination of amino acids to form the corresponding ketoacids. The response looks like this: [0003] [0004] This family includes Glutamate dehydrogenase (GDH), Leucine dehydrogenase, Valine dehydrogenase and Phenylalanine dehydrogenase . Because these amino acid dehydrogenases can catalyze specific reactions, they are often used in the synthesis of dietary and pharmaceutical amino acids, and can also be used in the clinical detection of amino acids and ammonia. Some members of this family share similar sequence structures but differ in substrate specificity, stability, and sal...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/06C12N15/53C12N15/10C12N15/70C12R1/42C12R1/19
Inventor 朱国萍潘蔚宋平曹正宇王鹏王晖王文才靳明明
Owner ANHUI NORMAL UNIV
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