title: Purification and characterization of serine proteinase from a halophilic bacterium, Filobacillus sp. RF2-5.
abstract: In order to find a unique proteinase, proteinase-producing bacteria were screened from fish sauce in Thailand. An isolated moderately halophilic bacterium was classified and named Filobacillus sp. RF2-5. The molecular weight of the purified enzyme was estimated to be 49 kDa. The enzyme showed the highest activity at 60 degrees C and pH 10-11 under 10% NaCl, and was highly stable in the presence of about 25% NaCl. The activity was strongly inhibited by phenylmethane sulfonyl fluoride (PMSF), chymostatin, and alpha-microbial alkaline proteinase inhibitor (MAPI). Proteinase activity was activated about 2-fold and 2.5-fold by the addition of 5% and 15-25% NaCl respectively using Suc-Ala-Ala-Phe-pNA as a substrate. The N-terminal 15 amino acid sequence of the purified enzyme showed about 67% identity to that of serine proteinase from Bacillus subtilis 168 and Bacillus subtilis (natto). The proteinase was found to prefer Phe, Met, and Thr at the P1 position, and Ile at the P2 position of peptide substrates, respectively. This is the first serine proteinase with a moderately thermophilic, NaCl-stable, and NaCl-activatable, and that has a unique substrate specificity at the P2 position of substrates from moderately halophilic bacteria, Filobacillus sp.
authors.affiliation: Department of Applied Biology, Faculty of Textile Science, Kyoto, Japan.
authors.forename: Kazumi
authors.initials: K
authors.lastname: Hiraga
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Nishikata
authors.forename: Sirilak
authors.initials: S
authors.lastname: Namwong
authors.forename: Somboon
authors.initials: S
authors.lastname: Tanasupawat
authors.forename: Katsumi
authors.initials: K
authors.lastname: Takada
authors.forename: Kohei
authors.initials: K
authors.lastname: Oda
mesh_terms: D001406:Bacillaceae / Q000201:enzymology*; D005396:Fish Products; D006863:Hydrogen-Ion Concentration; D010455:Peptides / Q000378:metabolism; D012697:Serine Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D010455:Peptides; D015842:Serine Proteinase Inhibitors; D012697:Serine Endopeptidases