Purification and Characterization of the Highly Thermostable Proteases from Bacillus stearothermophilus TLS33

• Published in: Protein expression and purification 2000-11, Vol.20 (2), p.142-151
• Main Authors: Sookkheo, Boonyaras, Sinchaikul, Supachok, Phutrakul, Suree, Chen, Shui-Tein
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Chromatography, Affinity; Chromatography, Gel; Chromatography, Ion Exchange; Coenzymes - analysis; Coenzymes - chemistry; Coenzymes - metabolism; Electrophoresis, Polyacrylamide Gel; Endopeptidases - chemistry; Endopeptidases - isolation & purification; Endopeptidases - metabolism; Enzyme Stability; Geobacillus stearothermophilus - enzymology; Holoenzymes - analysis; Holoenzymes - chemistry; Holoenzymes - metabolism; Hydrogen-Ion Concentration; Lysine - metabolism; lysine affinity chromatography; metalloprotease; Metals - metabolism; Metals - pharmacology; Molecular Sequence Data; Molecular Weight; Protease Inhibitors - pharmacology; Sequence Analysis, Protein; Substrate Specificity; Temperature; thermostable protease; zymography
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1006/prep.2000.1282

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°C, respectively. Proteases S, N, and B at pH 7.0 in the presence of 5 mM CaCl2 retained half their activities after 30 min at 72, 78, and 90°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 ZnCl2. They can thus be classified as Zn2+ 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.