Purification and Characterization of the Highly Thermostable Proteases from Bacillus stearothermophilus TLS33
| Autor: | Boonyaras Sookkheo, Supachok Sinchaikul, Suree Phutrakul, Shui-Tein Chen |
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| Rok vydání: | 2000 |
| Předmět: |
Proteases
Molecular Sequence Data Lysine Size-exclusion chromatography Coenzymes Chromatography Affinity Substrate Specificity Geobacillus stearothermophilus Affinity chromatography Sequence Analysis Protein Endopeptidases Enzyme Stability Protease Inhibitors Zymography Amino Acid Sequence Polyacrylamide gel electrophoresis chemistry.chemical_classification Chemistry Temperature Subtilisin Hydrogen-Ion Concentration Chromatography Ion Exchange Molecular Weight Enzyme Biochemistry Metals Chromatography Gel Electrophoresis Polyacrylamide Gel Holoenzymes Biotechnology |
| Zdroj: | Protein Expression and Purification. 20:142-151 |
| ISSN: | 1046-5928 |
| DOI: | 10.1006/prep.2000.1282 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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