title: Purification and characterization of the highly thermostable proteases from Bacillus stearothermophilus TLS33.
abstract: Three thermostable proteases, designated S, N, and B, are extracellular enzymes produced by Bacillus stearothermophilus strain TLS33. They were purified by lysine affinity chromatography, strong anion exchange Q HyperD chromatography, and Ultrogel AcA44 gel filtration. The molecular masses of the enzymes determined by SDS-PAGE and zymography were approximately 36, 53, and 71 kDa, respectively. Thermostable protease S bound strongly to the lysine affinity column and could be purified by this single step. The optimum pH values of proteases S, N, and B were shown to be 8.5, 7.5, and 7.0, respectively. The maximum activities for the enzymes were at 70, 85, and 90 degrees C, respectively. Proteases S, N, and B at pH 7.0 in the presence of 5 mM CaCl(2) retained half their activities after 30 min at 72, 78, and 90 degrees C, respectively. All three thermostable proteases were strongly inhibited by the metal chelators EDTA and 1,10-phenanthroline, and the proteolytic activities were restored by addition of ZnCl(2). They can thus be classified as Zn(2+) metalloproteases. The cleavage specificities of proteases S, N, and B on a 30-residue synthetic peptide from pro-BPN' subtilisin were Tyr-Ile, Phe-Lys, and Gly-Phe, respectively.
authors.affiliation: Department of Chemistry, Chiang Mai University, Chiang Mai, 50200, Thailand.
authors.forename: B
authors.initials: B
authors.lastname: Sookkheo
authors.forename: S
authors.initials: S
authors.lastname: Sinchaikul
authors.forename: S
authors.initials: S
authors.lastname: Phutrakul
authors.forename: S T
authors.initials: ST
authors.lastname: Chen
mesh_terms: D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003067:Coenzymes / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D004795:Enzyme Stability; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D020035:Holoenzymes / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008239:Lysine / Q000378:metabolism; D008670:Metals / Q000378:metabolism / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000494:pharmacology; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D003067:Coenzymes; D020035:Holoenzymes; D008670:Metals; D011480:Protease Inhibitors; D010450:Endopeptidases; D008239:Lysine