L-alanine dehydrogenase mutant zymoprotein and preparation method thereof

An alanine dehydrogenase, mutant technology, applied in the fields of botanical equipment and methods, microorganism-based methods, biochemical equipment and methods, etc., can solve the problems of low enzyme content, low activity, limited use, etc. The effect of improving enzyme activity

Inactive Publication Date: 2013-05-01
HEBEI NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the low content and low activity of these enzymes in microorganisms, their use has been limited.

Method used

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  • L-alanine dehydrogenase mutant zymoprotein and preparation method thereof
  • L-alanine dehydrogenase mutant zymoprotein and preparation method thereof
  • L-alanine dehydrogenase mutant zymoprotein and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 L-alanine dehydrogenase gene ald acquisition and expression vector construction

[0039] (1) ald gene acquisition

[0040] Primers were designed according to the known L-alanine dehydrogenase gene (NCBI number: YP_003426902) of Bacillus pseudofirmus OF4 (Bacillus pseudofirmus), and B. pseudofirmus genomic DNA was used as a template for PCR amplification. As a result, a specific fragment was obtained ( figure 1 ). ald amplification primers and PCR conditions were as follows:

[0041] Sense: 5′-G CATATG ATTATCGGTATTCCA-3′ (the underline is the restriction site);

[0042] Anti-sense: 5′-C CTCGAG TGCTTGAACAGGTGTTTTC -3′ (the underline is the restriction site);

[0043] Pre-denaturation at 94°C for 4min; denaturation at 94°C for 45sec, annealing at 55°C for 1min, extension at 72°C for 2min, cycle 25 times; full extension at 72°C for 10min. The amplified PCR fragment was recovered by gel, ligated with the carrier pMD18-T, and the ligated product was transfo...

Embodiment 2

[0048] Example 2 Selection of mutation sites

[0049] Using ClustalX software, the amino acid sequence of Bacillus pseudostrongis L-alanine dehydrogenase was compared with the sequence of L-alanine dehydrogenase (PDB ID: 2VOE) from Mycobacterium tuberculosis, In ESPript (http: / / espript.ibcp.fr / ESPript / ESPript / index.php; Gouet P, Courcelle E, Stuart DI, Metoz F. ESPript: multiple sequence alignments in PostScript. Bioinformatics. 1999, 15, 305-308 ) to predict the secondary structure of the protein, and determine that the site of site-directed mutation is lysine K at position 73, and the mutation direction is that lysine K at position 73 is replaced by alanine A.

Embodiment 3

[0050] Example 3 Construction of mutant plasmid pET22b-K73A

[0051] According to the gene sequence of L-alanine dehydrogenase of Bacillus pseudotenus (NCBI number: YP_003426902) and the selected mutation site 73K, the following two site-directed mutagenesis primers were designed:

[0052] Sense-K73A: 5′-ATGGTGATG GCA GTTAAAGA-3′ (the underline is the mutated base);

[0053] Anti-sense-K73A: 5′-TCTTTAAC TGC CATCACCAT-3′ (the underline is the mutated base);

[0054] Referring to the Quick Change Site-Directed Mutagenesis technique (Quick Change Site-Directed Mutagenesis), PCR was carried out using the recombinant expression vector pET22b-Ald as a template. The PCR reaction conditions were: pre-denaturation at 94°C for 4 minutes; denaturation at 94°C for 35 seconds, annealing at 55°C for 1 minute, and extension at 72°C for 7 minutes , cycled 16 times; fully extended at 72°C for 10min.

[0055] PCR product ( figure 2 ) was digested with DpnⅠ, transformed into Escherichia ...

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Abstract

The present invention discloses L-alanine dehydrogenase mutant zymoprotein and a preparation method thereof. According to a homologous sequence secondary structure comparison result, site-specific mutagenesis of 73th lysine near an L-alanine dehydrogenase activity center of Bacilluspseudofirmus into alanine through PCR is performed to construct a mutant expression vector, and prokaryotic expression and Ni-NTA affinity chromatography purification are performed to obtain a mutant enzyme K73A, wherein specific activity of the obtained mutant enzyme K73A is 202% of specific activity the wild type, and other enzymatic properties are not changed. In addition, a turnover number Kcat on a substrate L-alanine by the mutant zymoprotein is 376.7 min<-1> and is 2.0 times the turnover number Kcat of the wild type, a turnover number Kcat on beta-NAD<+> by the mutant zymoprotein is 290.1 min<-1> and is 2.1 times the turnover number Kcat of the wild type, and the mutant zymoprotein can be applicable for production processes of production of pyruvic acid, L/D-alanine, and the like through improvement biology methods.

Description

technical field [0001] The invention relates to a mutant enzyme protein of L-alanine dehydrogenase, belonging to the technical fields of genetic engineering and enzyme engineering. Background technique [0002] L-alanine dehydrogenase (L-alanine dehydrogenase, Ald, EC 1.4.1.1) is an amino acid dehydrogenase with nicotinamide adenine (NAD) as a coenzyme. L-alanine dehydrogenase reversibly catalyzes the oxidative deamination of L-alanine to pyruvate, ammonia and NADH. The response looks like this: [0003] L-alanine + NAD + + H 2 O <-> NH 4 + + pyruvate + NADH + H + [0004] L-alanine dehydrogenase is an important enzyme involved in the regulation of amino acid and sugar metabolism. The product of the catalytic reaction, pyruvate or L-alanine, is widely used in medicine, pesticide, food and other fields, and has a good development prospect. If this enzyme works together with alanine racemase, pyruvate, ammonia, etc. can be finally generated into D-alanine. The...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/06C12N15/53C12N15/70C12R1/19C12R1/07
Inventor 鞠建松徐书景赵宝华何广正李兵杰
Owner HEBEI NORMAL UNIV
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