STUDIES ON α- AMYLASE PRODUCTION BY MICROORGANISMS ASSOCIATED WITH FLOURS OF SELECTED CEREALS USING COMPOSITE GRAINS AS SUBSTRATE

Abstract

The process of complex starch conversion into simple unit of glucose is initiated by a class of enzymes called amylase. The increase in demand of local enzyme for industrial application has stimulated the exploration of the extracellular enzymes of several microorganisms and in solid state fermentation. This study aimed at isolating and screening microorganisms associated with fermented cereal flour of corn, rice, wheat and millet for amylase production, purifying and characterizing them. In this study, microorganisms were isolated from five days spontaneous fermentation of corn, rice, wheat and millet flour and identified by standard microbiological techniques. The isolated microorganisms were screened for amylase production on starchcontaining agar medium using plate assay condition. Qualitatively, selected amylase producing microorganisms from above were further screened under solid state fermentation of corn, rice, wheat, and millet at different ratios (100%, 1:1, 1:2:1,1:1:1:1) and assayed for by standard assay procedure. The selected amylase producing isolates were presumptively identified by standard conventional method and their identities were authenticated by 16SrRNA-region sequence analysis for Bacillus amyloliquefaciens and ITS-region for Aspergillus flavus which had the highest amylase production from the substrate corn, rice, wheat and millet (CRWM) at 1:1:1:1 and rice and millet (RM) at ratio 2:2 respectively. Process parameters (incubation time, substratemoisture concentration, temperature, pH, carbon and nitrogen sources) were optimized using onefactor at a time technique on the identified bacterium and fungus and their best substrate respectively. Maximum amylase production was obtained from B. amyloliquefaciens in WMC (ratio1:2:1) at 72hrs in 2:5 substrate-moisture concentration at 400C, pH 6.0, in 1% fructose and yeast extract supplementation as carbon and nitrogen source. Maximum amylase from A. flavus in RM (ratio 1:1) was obtained at 72hrs in 4:5 substrate-moisture concentration at 300C, pH 6.0, and with 1% sucrose and yeast extract supplementation as carbon and nitrogen source. A. flavus had the highest amylase production compared to B.amyloliquefaciens therefore its full purification and biochemical characterization was done. The purified amylase from A. flavus had optimum Temperature at 500C and was stable between 400C-600C for 90 minutes, optimum pH of 6.0 and was stable between 6-8 pH. The enzyme activity was stimulated by Ca2+, Zn2+, and Co2+ while it was inhibited in Mg2+, Hg2+, and EDTA. The apparent Michaelis-Menten constant (Km) and Maximum Velocity (Vmax) for the purified amylase were 2.133 μmol/min/ml and 1.71mg/ml with a molecular weight of 45 KDa. The biochemical properties of the purified α-amylase such as thermal tolerant, acidic stability, and high affinity for substrate from A. flavus makes it applicable in starch degrading industries.