A thermostable extracellular α-amylase from Aspergillus flavus S2-OY: Purification, characterisation and application in raw starch hydrolysis

Abstract

An amylolytic fungus was isolated locally from the soil of a cassava waste dumpsite. It was identified as Aspergillus flavus S2-OY using 18S rRNA gene sequencing with gene bank accession number MZ267053.1. α-Amylase was produced from the fungus through solid substrate fermentation of potato (Solanum tuberosum L.) peel, a cheap and renewable substrate. The fungal enzyme was purified 13.41-fold by ammonium sulphate and ion exchange chromatography (CM Sephadex C-50), with final yield of 13.41%. Apparent molecular weight of 55.0 kDa was obtained for the purified enzyme, using SDS-PAGE. Optima pH (6.0) and temperature (70 °C) of α-amylase activity were determined for the enzyme which also showed thermal stability up to 70 °C, with 80% residual activity after 120 min. The enzyme exhibited a wide range of pH stability (4.0 to 7.0). Kinetic parameters Km and Vmax values obtained with soluble starch were 158 µg/ml/min and 1.51 mg/ml, respectively. The enzyme was slightly inhibited by Mg2+. Mn2+, Zn2+, Na+, K+, Hg2+ and the chelating agent, EDTA, at 5.0 mM concentration. However, it was activated by Sn2+. The enzyme hydrolysed gelatinised and ungelatinized raw yam, corn, rice and millet starches strongly but cassava starch moderately. These results revealed that cost-effective α-amylase production from Aspergillus flavus S2-OY could be realised with the use of potato peel as a cheap substrate, under solid substrate fermentation condition. Also, the fungal α-amylase is a raw starch-hydrolysing, acid to neutral and thermostable enzyme with potential for broad biotechnological applications.