Purification and characterization of a halotolerant intracellular protease from Bacillus subtilis strain FP-133.
A halotolerant strain FP-133, able to grow at concentrations of 0-12.5% (w/v) NaCl, was isolated from a fish paste and identified as Bacillus subtilis . B. subtilis strain FP-133 produced an intracellular protease which showed catalytic activity under saline conditions. The enzyme was purified to homogeneity 143-fold with a yield of 0.9%. The purified enzyme showed an optimum activity at a concentration of 5% (w/v) NaCl. After storage in 7.5% (w/v) NaCl at 4 degrees C for 24 h, the enzyme kept 100% of its activity. The molecular mass of the protease was determined to be 59 kDa by gel filtration; the protein consisted of four subunits each with a molecular mass of 14 kDa. The enzyme showed aminopeptidase activity. It acted on L-leucyl-p-nitroanilide, L-leucyl-beta-naphthylamide, and oligopeptides containing glycine, L-histidine, or L-leucine. The K(m ) and V (max) values for L-leucyl-p-nitroanilide were 18 microm and 2.2 mm/h mg, respectively. The enzyme was activated by Fe(2+), Fe(3+), and Ni(2+) in synergism with Mg(2+).
Setyorini
Endang
E
Division of Life Science, Graduate School of Science and Technology, Kobe University, Rokko, Kobe 657-8501, Japan.
Kim
Young-Ju
YJ
Takenaka
Shinji
S
Murakami
Shuichiro
S
Aoki
Kenji
K
D000595:Amino Acid Sequence; D000818:Animals; D001412:Bacillus subtilis / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D002413:Cations, Divalent; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005396:Fish Products / Q000382:microbiology; D005516:Food Microbiology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D012965:Sodium Chloride / Q000494:pharmacology; D013379:Substrate Specificity; D013696:Temperature
D001426:Bacterial Proteins; D002413:Cations, Divalent; D015853:Cysteine Proteinase Inhibitors; D008670:Metals; D009842:Oligopeptides; D012965:Sodium Chloride; D010447:Peptide Hydrolases