Production, purification and characterization of α-amylase from Trichoderma harzianum grown on mandarin peel

Abstract

The production, purification and characterization of α-amylase from Trichoderma harzianum grown on mandarin
peel were investigated. The effect of incubation time and mandarin peel concentration on the production of αamylase 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 Km and Vmax 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.