Impact of Metal Ion Substitution on the Activity and Stability of Saccharifying Raw Starch Digesting Amylase from Aspergillus carbonarius

Abstract

Though raw starch digesting amylases can be utilized in numerous bioprocesses, poor activity and stability remain a limiting factor.  In this study, the effect of metal ion substitution on the activity and stability of the RSDA from Aspergillus carbonarius was investigated. The amylase was inactivated using the chelating agent ethylene di aminotetraacetic acid (EDTA) and reactivated using different metal ions. The effect of different metal ions on the reactivation of the amylase activity was investigated. Impact of the metal ions on the stability of the amylase was also studied. Kinetic constants of the native enzyme were compared to the metal reactivated holoenzyme. Most efficient was 5 mM concentration of Co2+ with 94.6% activity recovery. Others included 5 mM Zn2+ (77.7%) and 5 mM Ca2+ (68.7%). Incubating the Co2+ activated amylase in 10 mM Mn2+ further stimulated the activity of the amylase to 136.7%. Compared to the metal ions tested, Mn2+ had the most stabilizing effect on the amylase; the amylase exhibited 148.2% and 136.5% activity at 70ºC and 80ºC respectively in the presence of 5 mM Mn2+. Ca2+ inhibited the amylase activity and inhibition rate increased with increasing concentration of Ca2+ concentrations. Km of the reactivated amylase was 0.18 mg/ml.