Thermostable alanine dehydrogenase of Bacillus sp. DSM730: gene cloning, purification, and characterization.
We have cloned the thermostable alanine dehydrogenase (EC 1.4.1.1) gene from a thermophile, Bacillus sp. DSM730, into Escherichia coli C600 with a vector plasmid, pBR322. The enzyme was overproduced by the transformed cells, and purified to homogeneity with a yield of 69% by heat treatment and another step. The enzyme has a molecular weight of about 250,000 and consists of 6 subunits identical in molecular weight (43,000). It is not inactivated by heat treatment at 75 degrees C for 60 min, or incubation in the pH range of 5.5-10.5 at 55 degrees C for 10 min. The enzyme ctalyzes the oxidative deamination of L-serine in addition to L-alanine. The oxo analogue of serine is as reactive as pyruvate. Thus, the enzyme differs markedly from alanine dehydrogenases so far studied.
Nagata
S
S
Department of Agricultural Chemistry, Faculty of Agriculture, Kochi University, Japan.
Misono
H
H
Nagasaki
S
S
Esaki
N
N
Tanaka
H
H
Soda
K
K
D050788:Alanine Dehydrogenase; D000594:Amino Acid Oxidoreductases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000596:Amino Acids / Q000378:metabolism; D001407:Bacillus / Q000201:enzymology / Q000235:genetics*; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D005798:Genes, Bacterial; D007700:Kinetics; D008970:Molecular Weight; D013379:Substrate Specificity; D013696:Temperature
D000596:Amino Acids; D000594:Amino Acid Oxidoreductases; D050788:Alanine Dehydrogenase