The combined effects of two anti-aggregatory agents, alpha-cyclodextrin and Ca2+, on the refolding process of denatured alpha-amylase.

It is now a well-known fact that alpha-CD (alpha-cyclodextrin) and Ca(2+) are two important folding agents in the re-activation and refolding processes of many denatured enzyme solutions. Bacillus sp. alpha-amylase was extensively denatured in a 6 M guanidinium chloride solution overnight. Under suitable renaturation conditions, 20-30% more activity was recovered in the presence of various concentrations of alpha-CD (0-100 mM). Similarly, 25-30% more activity was recovered under the influence of different concentrations of Ca(2+) (0-100 mM). Regardless of these results, both alpha-CD and Ca(2+) at 100 mM concentrations were separately capable of preventing the extent of aggregate formation by 86 and 77% respectively. These observations clearly indicate that the effects of alpha-CD and/or Ca(2+) on the process of aggregation and refolding (activity recovery) are not synchronized: both agents mostly act as anti-aggregatory agents instead of acting as refolding agents.