Investigation into the chemical modification of α-amylase using octenyl succinic anhydride: enzyme characterisation and stability studies.

Abstract

The present study describes the chemical modification of α-amylase using succinic anhydride (SA), phthalic anhydride (PA) and a novel modifier viz. 2-octenyl succinic anhydride (2-OSA). SA-, PA- and 2-OSA-α-amylases displayed a 50%, 91% and 46% increase in stability at pH 9, respectively; as compared to unmodified α-amylase. PA-α-amylase showed a significant increase in Ea and ΔHa#, and a concomitant decrease in ΔSa#. The modified α-amylases exhibited improved thermostability as reflected by significant reductions in Kd and ΔSd#, and increments in t1/2, D-, Ed, ΔHd# and ΔGd# values. The modified α-amylases displayed variable stabilities in thepresence of different surfactants, inhibitors, metal ions and organic solvents. Interestingly, the chemical modification was found to confer resistance against inactivation by Hg2+ on α-amylase. The conformational changes in modified α-amylases were investigated using intrinsic tryptophan fluorescence, ANS (extrinsic) tryptophan fluorescence, and dynamic fluorescence quenching. Both intrinsic and extrinsic tryptophan fluorescence spectra showed increased fluorescence intensity for the modified α-amylases. Chemical modification was found to induce a certain degree of structural rigidity to α-amylase, as shown by dynamic fluorescence quenching. Analysis of the CD spectra by the K2d method using the DichroWeb online tool indicated evident changes in the α-helix, β-sheet and random coil fractions of the α-amylase secondary structure, following chemical modification using anhydrides. PA-α-amylase exhibited the highest productivity in terms of hydrolysis of starch at 60°C over a period of 5h indicating potential in varied biotechnological applications.