Mechanisms of aggregation of alpha- and beta-amylases in aqueous dispersions

Abstract

A lot of attention has been paid to elucidate the mechanisms of enzyme inactivation and aggregation so that stabilization strategies can be specifically applied to prevent thermal and/or physical denaturation of enzymes in industrial processes. In the present study, we have investigated the mechanisms of aggregate formation of two model enzymes, α-amylase from Bacillus subtilis and soybean β-amylase, under different experimental conditions. Aggregation can be observed among α-amylase particles after 1 week of aging either through physical contacts or through the intermolecular disulfide bond formed by the surface Cys residues from two enzyme monomer. In contrast, β-amylase does not form any aggregation probably due to the electrostatic repulsion and steric hindrance. Both surfactant sodium n-dodecyl sulfate and reducing agent beta-mercaptoethanol (BME) dramatically reduce the aggregate size, especially for α-amylase, by destroying the intermolecular disulfide linkages. Sonication appears to improve the formation of aggregates only for α-amylase. The average aggregate sizes are usually the largest at the isoelectric points of these two enzymes. A protein having more free Cys residues on its surface does not guarantee disulfide bond-induced aggregation.