Purification and properties of fungal alkaline ????-amylase from Aspergillus oryzae (Ah1b.) Cohn.

摘要

The fungal strain Aspergillus oryzae (Ah1b.) Cohn. was isolated from nearby potato grown soil sample. The isolated strain was cultivated for á- Amylase production in Czapeck dox broth at room temperature for 7 days. For á-Amylase production, it was determined that the best carbon source was soluble starch and the best nitrogen source was peptone. The optimum pH and temperature for enzyme activity was found to be 8.5 and 45°C respectively. The enzyme was stable for 2 hours at pH 8.5 and 45°C. á-Amylase was purified by Acetone Precipitation and Lectin agarose affinity chromatography. The purified amylase was a monomer showed a molecular mass of 44± 1 kDa as estimated by SDS-PAGE with 4.18 fold purification with 14.19% yield. The enzyme was found to have good activity in the presence of Cu2+and Fe2+ions. The group specific reagents PMSF, TLCK, NEM, IAA and EDTA substantially decrease the activity of the enzyme, indicating the presence of Serine and Cysteine at the active site of the enzyme. The enzyme was found to be Ca2+ dependent. The Km and Vmaxvalues of the enzyme for soluble starch hydrolysis was found to be 11.11mg/ml and 2.0μmol/min/ml respectively and it was an endoacting enzyme which was confirmed by its product analysis by thin layer chromatography.