Purification and characterization of an extracellular alkaline serine protease from Aspergillus terreus (IJIRA 6.2)

An extracellular alkaline serine protease has been purified from Aspergillu s terreus (IJIRA 6.2). The purification procedure involved chromatography o n DEAE-Sephadex A25, phosphocellulose, hydroxyapatite, casein-Sepharose, ge l filtration on Sephacryl-S-300 and by glycerol density gradient centrifuga tion. The enzyme was further purified to apparent homogeneity through a com bination of electrophoresis in polyacrylamide gel containing 0.1% sodium do decyl sulfate (SDS) with or without protease substrate (gelatin) and subseq uent regeneration of its activity in situ by removal of SDS. The active enz yme was visualized in a zymogram or on the basis of protease activity exhib ited on an X-ray film. The protein in the unstained segment of the gel was electroeluted. The eluted protein with protease activity exhibited a molecu lar 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 pu rified protease was found to be 0.055 mM. Antipain, phenylmethane sulfonyl fluoride, and chymostatin served as non-competitive inhibitors. Substrate s pecificity was determined by using a synthetic chromogenic peptide containi ng N-P-Tosyl-Gly-Pro-Arg-p-nitroanilide. Results showed that the protease c leaved the peptide on the -COOH end of arginine residue.