MECHANISM OF SOLVENT-INDUCED THERMAL STABILIZATION OF ALPHA-AMYLASE FROM BACILLUS-AMYLOLIGUEFACIENS

The transition temperature of irreversible thermal inactivation of a-a mylase from Bacillus amyloliquefaciens was estimated to be 60 degrees C. At this temperature, the enzyme inactivation followed first-order k inetics, having a half-life (t1/2) of 12 min with a rate constant (k) of 0.06 min(-1). Conformational change was a prerequisite for this the rmal inactivation. This is governed by stepwise temperature-dependent phenomena. Among the solvent stabilizers tested, the enzyme was therma lly stable in presence of DMSO and PEG 300 and the stabilizing efficie ncy of these cosolvents was concentration-dependent. The enzyme was pa rtially stabilized by 5.0 M DMSO and 1.9 M PEG 300 up to 78 degrees C. However, above 78 degrees C the enzyme was inactivated in these cosol vents also. The mechanism of stabilization has been explained by prefe rential hydration of the enzyme in these structure stabilizing solvent s by exclusion from the protein surface and interface by measurement o f partial specific volume in these cosolvents. The data suggest a high value of preferential interaction parameter, (delta g(3)/delta g(2))T ,mu(1), mu(3) being -0.606 g/g in 40% DMSO and a low value of -0.025 g /g in 5% glycerol. The preferential interaction parameters in sucrose and glycerol suggests that (delta g(3)/delta g(2))T,mu(1), mu(3) is hi ghest of -0.420 g/g in 10% glycerol than any other cosolvent. (C) Munk sgaard 1995.