Purification and characterization of an extracellular alkaline serine protease from Aspergillus terreus (IJIRA 6.2).
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 degrees C. Purified protease was active between pH 5.5 and 9.5 and was found to be stable up to 60 degrees 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.
Chakrabarti
S K
SK
Laboratory of Plant Molecular Biology and Biotechnology, Department of Bioagricultural Sciences & Pest Management, Colorado State University, Fort Collins, CO 80523, USA.
Matsumura
N
N
Ranu
R S
RS
D001230:Aspergillus / Q000201:enzymology*; D002499:Centrifugation, Density Gradient; D002845:Chromatography / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005780:Gelatin; D006863:Hydrogen-Ion Concentration; D011480:Protease Inhibitors / Q000494:pharmacology; D012697:Serine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification*; D013379:Substrate Specificity; D013696:Temperature; D014962:X-Ray Film
D011480:Protease Inhibitors; D005780:Gelatin; D012697:Serine Endopeptidases; C026109:oryzin