Purification of alkaline proteases from a Bacillus strain and their possible interrelationship.
Alkalophilic Bacillus sp. KSM-K16 produced three alkaline proteases, as detected by polyacrylamide gel electrophoresis (PAGE). The major protease, designated M protease, was recently purified to homogeneity and its properties were characterized. In the present study, two minor proteases, designated H protease and N protease, were purified to homogeneity from cultures of this organism. H protease had a molecular mass of 28 kDa, as estimated by sodium dodecyl sulfate/PAGE (SDS-PAGE) and its maximum activity against casein was observed at pH 11.0 and at 55 degrees C. N protease consisted of two polypeptide chains with molecular masses of 12.5 kDa and 14.5 kDa, as estimated by SDS-PAGE, although it migrated as a single protein band during non-denaturing PAGE. Its maximum activity was observed at pH 11.0 and at 60 degrees C. The amino-terminal sequences of H protease and of the 14.5-kDa polypeptide of N protease were identical to that of M protease. The electrophoretic relationship between the three enzymes was examined after they had been stored at different pH values and at 5 degrees C. M protease was converted to H protease more rapidly at pH 11 than at pH 8 or below, and H protease was converted to M protease at pH 8 or below but not at pH 11. N protease appeared to be the autolytic product of the M and H proteases.
Kobayashi
T
T
Tochigi Research Laboratories of Kao Corporation, Japan.
Hakamada
Y
Y
Hitomi
J
J
Koike
K
K
Ito
S
S
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D013379:Substrate Specificity; D013696:Temperature
D010450:Endopeptidases