Abstract

Guanidine hydrochloride (GdnHCl) denaturation of native and Ca-depleted Bacillus licheniformis α-amylase (BLA) was investigated both in the absence and presence of 2 mM calcium chloride (CaCl₂) using circular dichroism, fluorescence spectroscopy and biological activity. In both states (Ca-depleted and native form), the protein was denatured to a considerable extent in the absence of 2 mM CaCl₂with concomitant loss of biological activity upon increasing GdnHCl concentration. On the other hand, this effect was significantly reduced when 2 mM CaCl₂ was included in the incubation mixture as revealed by a higher relative mean residue ellipticity, higher relative fluorescence intensity, smaller change in emission maximum and lesser reduction in biological activity. Interestingly, using these probes, 2 mM CaCl₂ seemed to offer the same degree of stability to Ca-depleted BLA as that observed with native BLA in the absence of 2 mM CaCl₂. All these results suggest calcium-induced stabilization of BLA against GdnHCl denaturation.

Key words: α-Amylase, Bacillus licheniformis, calcium, denaturation, guanidine hydrochloride, circular dichroism, intrinsic fluorescence.

Abbreviation

Abbreviations: BLA, Bacillus licheniformis α-amylase; GdnHCl, guanidine hydrochloride; CaCl₂, calcium chloride; MRE, mean residue ellipticity; EGTA,ethylene glycol-bis (2-aminoethyl-ether)-N, N, N’, N’-tetraacetic acid; CD, circular dichroism.