A thermostable hydantoinase of Bacillus stearothermophilus NS1122A: cloning, sequencing, and high expression of the enzyme gene, and some properties of the expressed enzyme.
A DNA fragment containing the gene for a thermostable hydantoinase was cloned from a thermophile, Bacillus stearothermophilus NS1122A in Escherichia coli. Nucleotide sequencing showed that the DNA fragment contains one open reading frame, which is predicted to encode a peptide of 471 amino acids, with a calculated molecular weight of 51,724. When the hydantoinase gene was under the control of both the lpp promoter and the lac promoter-operator, and its expression was induced by isopropyl-1-thio-beta-D-galactopyranoside, it was overexpressed in E. coli leading to the formation of an insoluble aggregate. The enzyme was purified to homogeneity from the insoluble aggregate. The molecular mass of the purified active enzyme was approximately 200 kDa by gel filtration. Although the monomer had no activity, the activity was restored by incubation with Mn2+ or Co2+ at pH 8.1. These findings suggested that the hydantoinase is a metalloenzyme and the oligomeric structure is required for activity. The oligomeric structure is suggested to contribute to thermostability.
Mukohara
Y
Y
Odawara Research Center, Nippon Soda Co., Ltd., Kanagawa, Japan.
Ishikawa
T
T
Watabe
K
K
Nakamura
H
H
D000581:Amidohydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D002614:Chelating Agents / Q000494:pharmacology; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D011487:Protein Conformation
D001426:Bacterial Proteins; D002614:Chelating Agents; D008670:Metals; D000581:Amidohydrolases; C020047:dihydropyrimidinase