Purification and characterization of α-amylase from Trichoderma pseudokoningii.

Abstract

Background: Previous studies have demonstrated that members of Trichoderma are able to generate appreciable amount of extracellular amylase and glucoamylase on soluble potato starch. In this study the α-amylase was purified and characterized from Trichoderma pseudokoningii grown on orange peel under solid state fermentation (SSF).

Results: Five α-amylases A1-A5 from Trichodrma pseudokoningii were separated on DEAE-Sepharose column. The homogeneity of α-amylase A4 was detected after chromatography on Sephacryl S-200. α-Amylase A4 had molecular weight of 30 kDa by Sephacryl S-200 and SDS-PAGE. The enzyme had a broad pH optimum ranged from 4.5 to 8.5. The optimum temperature of A4 was 50 °C with high retention of its activity from 30 to 80 °C. The thermal stability of A4 was detected up to 50 °C and the enzyme was highly stable till 80 °C after 1 h incubation. All substrate analogues tested had amylase activity toward A4 ranged from 12 to 100% of its initial activity. The Km and Vmax values of A4 were 4 mg starch/ml and 0.74 μmol reducing sugar, respectively. The most of metals tested caused moderate inhibitory effect, except of Ca²⁺ and Mg²⁺ enhanced the activity. Hg²⁺ and Cd^+ 2 strongly inhibited the activity of A4. EDTA as metal chelator caused strong inhibitory effect.

Conclusions: The properties of the purified α-amylase A4 from T. pseudokoningii meet the prerequisites needed for several applications.