Abstract

The production, purification and characterization of α-amylase from Trichodermaharzianum grown on mandarin peel were investigated. The effect of incubation time and mandarin peel concentration on the production of α-amylase by T. harzianum was studied. α-Amylase A3 was purified from T. harzianum to electrophoretic homogeneity by using DEAE-Sepharose and Sephacryl S-200 columns. The enzyme had molecular weight of 70 kDa using gel filtration and SDS-PAGE. The affinity of the substrates toward A3 was in the order of amylopectin > glycogen > starch > β-cyclodextrin > dextrin > α-cyclodextrin. These findings tend to suggest that the enzyme has high affinity toward high-molecular mass substrates. The K_m and V_max values of the enzyme for hydrolyzing potato soluble starch and glycogen were 6.53, 4.5 mg/ml and 2 and 2.2 μmol reducing sugar/ml, respectively. The maximum activity of enzyme against soluble starch was determined at pH 4.5 and 40°C. α-Amylase A3 was stable up to 40°C for 30 min of incubation and retained 70 and 50% of its activity at 50 and 60°C, respectively. While all the examined metal cations were effective in inhibiting the enzyme, Ca²⁺ considerably enhanced the activity. The metal chelators, EDTA, sodium citrate and sodium oxalate had inhibitory effects on A3. The rate of breakdown of starch was higher than the rate of formation of reduced sugar indicating A3 is endo-acting enzyme. These properties of A3 with its remarkable activity meet the prerequisites needed for liquefaction and saccharification of starch industry.

Key words: Trichoderma harzianum, mandarin, a-amylase, purification, characterization.