Purification and characterization of kumamolysin, a novel thermostable pepstatin-insensitive carboxyl proteinase from Bacillus novosp. MN-32.
We have found a novel type of thermostable, pepstatin-insensitive carboxyl proteinase in the culture filtrate of Bacillus novosp. MN-32. The carboxyl proteinase, which was named kumamolysin, was purified about 8,300-fold by column chromatography including DEAE-Sepharose CL-6B, Sephadex G-100, and TSKgel DEAE-5PW. The purified kumamolysin gave a single band corresponding to 41 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular mass of kumamolysin was estimated to be 40 kDa by gel filtration. The isoelectric point of kumamolysin was estimated to be pH 3.5 by isoelectric focusing. Kumamolysin has maximum proteolytic activity at 70 degrees C and at pH 3.0. Kumamolysin specifically hydrolyzed the Leu15-Tyr16 peptide bond in oxidized insulin B-chain (Km = 9.0 x 10(-5) M, Kcat = 71 s-1; at pH 3.0, 30 degrees C), and additional cleavage at Phe25-Tyr26 was detected at a considerably lower rate. Kumamolysin is insensitive to the known carboxyl proteinase inhibitors pepstatin, diazoacetyl-DL-norleucine methyl ester, and 1,2-epoxy-3-(p-nitrophenoxy)propane. Kumamolysin has no similarity to the thermostable acid protease thermopsin from Sulfolobus acidocaldarius (Lin, X.-L., and Tang, J. (1990) J. Biol. Chem. 265, 1490-1495). Thus, the substrate specificity, the inhibitor sensitivity, the molecular mass, and the thermostability all suggest that kumamolysin is a novel type of carboxyl proteinase.
Murao
S
S
Department of Applied Microbial Technology, Kumamoto Institute of Technology, Japan.
Ohkuni
K
K
Nagao
M
M
Hirayama
K
K
Fukuhara
K
K
Oda
K
K
Oyama
H
H
Shin
T
T
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D016282:Aspartic Acid Endopeptidases; D001407:Bacillus / Q000201:enzymology*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism*; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010434:Pepsin A / Q000037:antagonists & inhibitors; D010436:Pepstatins / Q000494:pharmacology*; D011480:Protease Inhibitors / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D016339:Spectrometry, Mass, Fast Atom Bombardment; D013379:Substrate Specificity
D000596:Amino Acids; D007328:Insulin; D046911:Macromolecular Substances; D010436:Pepstatins; D011480:Protease Inhibitors; C014195:Streptomyces pepsin inhibitor; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C078894:kumamolysin; D010434:Pepsin A; C031375:pepstatin