PRODUCTION AND CHARACTERIZATION OF α-AMYLASE FROM INDIGENOUSLY ISOLATED BACTERIAL STRAINS TREATING ORGANIC WASTE IN ANAEROBIC DIGESTER

Hydrolytic enzymes specifically amylases are among the most important biocatalysts for anaerobic digestion of organic waste. The current research is aimed at isolation, partial characterization and purification of alpha-Amylase by microorganisms from anaerobic digester carrying anaerobic co-digestion of cow manure (CM) with fruit-vegetable waste (FVW), and agricultural residues (AR) in continuous stirred tank reactor (CSTR). Six bacterial strains were isolated from anaerobic digester operating at mesophilic condition. With large hydrolytic zone producing two strains (RAS-1 and RAS-4) were selected for further study among the six isolates by plate assay. Selected isolates were identified as Bacillus subtilis (RAS-1) and Clostridium perfringens (RAS-4) by 16s rRNA gene sequencing. Temperature, pH, effect of inhibitor and metal ions on the stability of purified alpha-Amylase were studied. The enzyme was relatively stable between pH 7 to pH 9 and at temperatures ranging from 37 degrees C to 45 degrees C. The alpha-Amylase enzyme was purified by ammonium sulphate precipitation and column chromatography. The alpha-Amylase molecular weight estimation was confirmed by SDS-PAGE with a band of 52 kDa for B. subtilis (RAS-1) and 73 kDa of C. perfringens (RAS-4). The results suggested the cost effective production of commercially important enzyme alpha-Amylases from organic waste during biogas production.