Purification and Properties of a-Amylase from Trichoderma reesei

ABSTRACT

Two alpha-amylases AIV and AV from Trichoderma reesei, cultured on citrus peel, were purified with specific activity 2082 and 611 units/mg protein, by chromatography on DEAE-Sepharose and Sephacryl S-200 columns, respectively. The purified enzymes gave an apparent single protein band on SDS-polyacrylamide gel electrophoresis [SDS-PAGE]. The molecular mass of purified enzymes AIV and AV as estimated by SDS-PAGE and by gel filtration on Sephacryl S-200 to be 24 and 30 kDa, respectively, indicated that two enzymes are monomers. The same pH and temperature optima were detected at 5.5 and 40°C and are stable up to 40°C for AIV and AV. The K[m] for hydrolysis of soluble starch were 1.3 and 2.38 mg starch/ml for AIV and AV, respectively. AIV and AV display highest specificity towards starch followed by amylose, amylopectin, glycogen and beta-cyclodextrin. While Ba[+2] and Ca[+2] showed an activation effect for AIV and AV, Fe[+3] and Hg[+2] had a complete inhibition effect. At 20 Mm concentration, oxalic acid inhibited the two enzymes, and EDTA and citric acid had moderate inhibition effects for AIV and AV