Effects of ribonucleic acid removal methods on proteolytic activity and protein solubility in Candida utilis

Abstract

AbstractCandida utilis was grown under controlled conditions and nucleic acids were removed from whole cells and homogenates by alkaline hydrolysis techniques, en‐zymatic methods, and washing with buffer. Homogenization released hydrolytic enzymes and proteolytic activity increased with incubation at elevated temperatures under acidic conditions. Slight proteolysis occurred in all incubated samples and this may contribute to protein insolubilization. Very little protein was lost during incu‐bation when compared to similar processes using bakers' yeast. This can be due to lower levels of protease activities in C. utilis. Alkaline hydrolysis methods resulted in hydrolysis of some proteins and irreversible insolubilization of the protein. These methods also destroyed any residual enzymatic activities. Heat denaturation studies suggest that protein insolubilization occurs at neutral pH when heat treatments equivalent to or greater than 85° C for 15 min are used. SDS‐PAGE methods were used to characterize and monitor changes in protein. Eighteen proteins and/or sub‐units were present at levels of 1% or greater. Results may help to explain changesin functional properties of sample preparations which accompany RNA removal.