caa a22 4500 475745981 CHVBK 20180406123513.0 cr unu---uuuuu 170329e20000401xx s 000 0 eng 10.1007/s002849910048 doi (NATIONALLICENCE)springer-10.1007/s002849910048 Purification and Characterization of an Extracellular Alkaline Serine Protease from Aspergillus terreus (IJIRA 6.2) [Elektronische Daten] [Syamal K. Chakrabarti, Nobuya Matsumura, Rajinder S. Ranu] Abstract.: 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. Springer-Verlag New York Inc., 2000 Chakrabarti Syamal K. Laboratory of Plant Molecular Biology and Biotechnology, Department of Bioagricultural Sciences & Pest Management, Colorado State University, Fort Collins, CO 80523, USA, US aut Matsumura Nobuya Laboratory of Plant Molecular Biology and Biotechnology, Department of Bioagricultural Sciences & Pest Management, Colorado State University, Fort Collins, CO 80523, USA, US aut Ranu Rajinder S. Laboratory of Plant Molecular Biology and Biotechnology, Department of Bioagricultural Sciences & Pest Management, Colorado State University, Fort Collins, CO 80523, USA, US aut https://doi.org/10.1007/s002849910048 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s002849910048 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Chakrabarti Syamal K. Laboratory of Plant Molecular Biology and Biotechnology, Department of Bioagricultural Sciences & Pest Management, Colorado State University, Fort Collins, CO 80523, USA, US aut NATIONALLICENCE 700 1- Matsumura Nobuya Laboratory of Plant Molecular Biology and Biotechnology, Department of Bioagricultural Sciences & Pest Management, Colorado State University, Fort Collins, CO 80523, USA, US aut NATIONALLICENCE 700 1- Ranu Rajinder S. Laboratory of Plant Molecular Biology and Biotechnology, Department of Bioagricultural Sciences & Pest Management, Colorado State University, Fort Collins, CO 80523, USA, US aut Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer