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
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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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