A cysteine-dependent serine protease associated with the dormant spores of Bacillus cereus: purification of the protein and cloning of the corresponding gene.
Subtilisin-like serine protease, which is associated with the dormant spores of Bacillus cereus, was solubilized by washing the spores with 2 M KCl and purified to homogeneity by carbobenzoxy-D-phenylalanine-liganded affinity column chromatography and hydrophobic interaction column chromatography. Enzyme activity was completely inhibited by reagents for sulfhydryl groups such as HgCl2 as well as by conventional subtilisin inhibitors, suggesting the enzyme to be cysteine-dependent. The enzyme retained activity in 5 M urea at 4 degrees C for at least 2 months, and the specific activity was 50 times that of subtilisin BPN when measured for a common chromogenic substrate, carbobenzoxy-glycyl-glycyl-L-leucine p-nitroanilide. The gene encoding this protease was cloned in Escherichia coli, and its nucleotide sequence was analyzed. The deduced amino acid sequence suggested that the protease is produced as a precursor comprising three portions; a signal sequence (28 amino acid residues), a prosequence (80 amino acid residues) and a mature enzyme (289 amino acid residues). The mature region of the enzyme had high similarity with a thermitase from Thermoactinomyces vulgaris (72% identity) and a thermostable alkaline protease from Thermoactinomyces sp. E79 (66% identity), which have the N-terminal sequence showing scarcely noticeable similarity with corresponding stretches of subtilisins and mercuric ion-sensitive free cysteine in the equivalent position of the primary structure.
Moriyama
R
R
Department of Applied Biological Sciences, School of Agricultural Sciences, Nagoya University, Aichi, Japan. moriyama@agr.nagoya-u.ac.jp
Sugimoto
K
K
Zhang
H
H
Inoue
T
T
Makino
S
S
D000595:Amino Acid Sequence; D001409:Bacillus cereus / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000494:pharmacology; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D013171:Spores, Bacterial / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000737:chemistry
D011480:Protease Inhibitors; D013439:Sulfhydryl Reagents; D012697:Serine Endopeptidases; D003545:Cysteine