Background:
Amylases are the most widely used biocatalysts in starch saccharification
and detergent industries. However, commercially available amylases have few limitations viz. limited
activity at low or high pH and Ca2+ dependency.
Objective:
The quest for exploiting amylase for diverse applications to improve the industrial processes
in terms of efficiency and feasibility led us to investigate the kinetics of amylase in the presence
of metal ions as a function of pH.
Methods:
The crude extract from soil fungal isolate cultures is subjected to salt precipitation, dialysis
and DEAE cellulose chromatography followed by amylase extraction and is incubated with divalent
metal ions (i.e., Ca2+, Fe2+, Cu2+, and Hg2+); Michaelis-Menton constant (Km), and maximum reaction
velocity (Vmax) are calculated by plotting the activity data obtained in the absence and presence of
ions, as a function of substrate concentration in Lineweaver-Burk Plot.
Results:
Kinetic studies reveal that amylase is inhibited un-competitively at 5mM Cu2+ at pH 4.5 and
7.5, but non-competitively at pH 9.5. Non-competitive inhibition of amylase catalyzed starch hydrolysis
is observed with 5mM Hg2+ at pH 9.5, which changes to mixed inhibition at pH 4.5 and 7.5. At
pH 4.5, Ca2+ induces K- and V-type activation of amylase catalyzed starch hydrolysis; however, the
enzyme has V-type activation at 7mM Ca2+ under alkaline conditions. Also, K- and V-type of activation
of amylase is observed in the presence of 7mM Fe2+ at pH 4.5 and 9.5.
Conclusion:
These findings suggest that divalent ions modulation of amylase is pH dependent. Furthermore,
a time-saving and cost-effective solution is proposed to overcome the challenges of the existing
methodology of starch hydrolysis in starch and detergent industries.
Effect of Divalent Metal Ions on Nucleotide Inhibition of Pig Heart Citrate Synthase
