title: Characterization of solvent stable extracellular protease from Bacillus koreensis (BK-P21A).
abstract: A total of 18 protease producing bacterial strains were isolated from detergent effluent in South Korea using skim milk agar medium. A strain (BK-P21A) was selected and identified as Bacillus koreensis based on morphological, biochemical and molecular characterizations (16S rRNA gene sequence analysis). Optimized culture conditions for the production of protease were pH 8.5, 30 °C, sucrose (2%) and yeast extract (0.2%) during 36 h of incubation. Furthermore, the protease was partially purified by ammonium sulphate precipitation (80%) and again by Superdex 200 10/300 GL and Superdex 75 10/300 GL column chromatography, which resulted in 5.0 fold purification and a yield of 23%. The molecular mass of the protease was estimated to be 48 kDa by SDS-PAGE. The purified enzyme was further characterized and found to be most active at pH 9.0 and 60 °C. The activity of the purified protease was enhanced by CaCl₂ and CoCl₂, but inhibited by PMSF, which indicated it was a serine type protease. Moreover, the protease was moderately stable in surfactants and 81% stable in H₂O₂. Finally, the enzyme was more active and stable (94-126.5%) in various hydrophilic organic solvents. Considering the stability of protease towards the alkaline pH, high temperature and organic solvents (50%), the enzyme from B. koreensis can be used as an alternative biocatalyst for several industrial applications mainly for peptide synthesis in nonaqueous solvents.
authors.affiliation: Department of Biological Engineering, Inha University, Incheon, Republic of Korea. anbu25@yahoo.com
authors.forename: Periasamy
authors.initials: P
authors.lastname: Anbu
mesh_terms: D001407:Bacillus / Q000187:drug effects / Q000201:enzymology* / Q000302:isolation & purification; D002244:Carbon / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability / Q000187:drug effects; D005110:Extracellular Space / Q000187:drug effects / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D006868:Hydrolysis / Q000187:drug effects; D007477:Ions; D008670:Metals / Q000494:pharmacology; D009584:Nitrogen / Q000494:pharmacology; D016877:Oxidants / Q000494:pharmacology; D010447:Peptide Hydrolases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D012997:Solvents / Q000737:chemistry* / Q000494:pharmacology; D013501:Surface-Active Agents / Q000494:pharmacology; D013696:Temperature
chemical_list: D007477:Ions; D008670:Metals; D016877:Oxidants; D011480:Protease Inhibitors; D012997:Solvents; D013501:Surface-Active Agents; D002244:Carbon; D010447:Peptide Hydrolases; D009584:Nitrogen