title: Extremely thermostable serine-type protease from Aquifex pyrophilus. Molecular cloning, expression, and characterization.
abstract: A gene encoding a serine-type protease has been cloned from Aquifex pyrophilus using a sequence tag containing the consensus sequence of proteases as a probe. Sequence analysis of the cloned gene reveals an open reading frame of 619 residues that has three canonical residues (Asp-140, His-184, and Ser-502) that form the catalytic site of serine-type proteases. The size of the mature form (43 kDa) and its localization in the cell wall fraction indicate that both the NH2- and COOH-terminal sequences of the protein are processed during maturation. When the cloned gene is expressed in Escherichia coli, it is weakly expressed as active and processed forms. The pH optimum of this protease is very broad, and its activity is completely inactivated by phenylmethylsulfonyl fluoride. The half-life of the protein is 6 h at 105 degreesC, suggesting that it is one of the most heat-stable proteases. The cysteine residues in the mature form may form disulfide bonds that are responsible for the strong stability of this protease, because the thermostability of the protein is significantly reduced in the presence of reducing reagent.
authors.affiliation: Structural Biology Center, Korea Institute of Science and Technology, Seoul, 136-791 Korea.
authors.forename: I G
authors.initials: IG
authors.lastname: Choi
authors.forename: W G
authors.initials: WG
authors.lastname: Bang
authors.forename: S H
authors.initials: SH
authors.lastname: Kim
authors.forename: Y G
authors.initials: YG
authors.lastname: Yu
mesh_terms: D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D016946:Gram-Negative Aerobic Rods and Cocci / Q000201:enzymology*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D017386:Sequence Homology, Amino Acid; D013381:Subtilisins / Q000235:genetics* / Q000378:metabolism
chemical_list: D004269:DNA, Bacterial; C116912:Aquifex pyrophilus serine-type protease; D013381:Subtilisins