Purification and Characterization of an Extracellular Alkaline Serine Protease from Aspergillus terreus (IJIRA 6.2)
| Autor: | Syamal K. Chakrabarti, Rajinder S. Ranu, Nobuya Matsumura |
|---|---|
| Rok vydání: | 2000 |
| Předmět: |
medicine.medical_treatment
Size-exclusion chromatography Applied Microbiology and Biotechnology Microbiology Substrate Specificity chemistry.chemical_compound Centrifugation Density Gradient medicine Protease Inhibitors Aspergillus terreus Sodium dodecyl sulfate Polyacrylamide gel electrophoresis Serine protease Differential centrifugation Chromatography Protease biology X-Ray Film Serine Endopeptidases Temperature General Medicine Hydrogen-Ion Concentration biology.organism_classification Aspergillus chemistry Biochemistry biology.protein Gelatin Electrophoresis Polyacrylamide Gel Antipain |
| Zdroj: | Current Microbiology. 40:239-244 |
| ISSN: | 1432-0991 0343-8651 |
| DOI: | 10.1007/s002849910048 |
| Popis: | An extracellular alkaline serine protease has been purified from Aspergillus terreus (IJIRA 6.2). The purification procedure involved chromatography on DEAE-Sephadex A25, phosphocellulose, hydroxyapatite, casein-Sepharose, gel filtration on Sephacryl-S-300 and by glycerol density gradient centrifugation. The enzyme was further purified to apparent homogeneity through a combination of electrophoresis in polyacrylamide gel containing 0.1% sodium dodecyl sulfate (SDS) with or without protease substrate (gelatin) and subsequent regeneration of its activity in situ by removal of SDS. The active enzyme was visualized in a zymogram or on the basis of protease activity exhibited on an X-ray film. The protein in the unstained segment of the gel was electroeluted. The eluted protein with protease activity exhibited a molecular mass of 37,000-daltons on electrophoresis in SDS-polyacrylamide gel. A sedimentation coefficient of 3.2S was obtained by glycerol density gradient contrifugation. Maximum activity of protease was observed at pH 8.5 and at 37°C. Purified protease was active between pH 5.5 and 9.5 and was found to be stable up to 60°C. With Na-caseinate, the K m of the purified protease was found to be 0.055 mM. Antipain, phenylmethane sulfonyl fluoride, and chymostatin served as non-competitive inhibitors. Substrate specificity was determined by using a synthetic chromogenic peptide containing N-P-Tosyl-Gly-Pro-Arg-p-nitroanilide. Results showed that the protease cleaved the peptide on the -COOH end of arginine residue. |
| Databáze: | OpenAIRE |
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