CHARACTERIZATION OF AN ACID TREHALASE OF SACCHAROMYCES-CEREVISIAE PRESENT IN TREHALASE-SUCRASE AGGREGATE

An acid trehalase-sucrase aggregate was purified (by 780-fold) from Sa ccharomyces cerevisiae, following conventional protein purification te chniques, to an apparent yield of 18.5%. The aggregate was electrophor etically homogeneous but contained 175, 90, 68, 60, 40 molar mass (kDa ) bands on SDS-electrophoresis. The purified aggregate had a specific activity (acid trehalase) of 22 U/mg; a K-m value of 5.0 mM but contai ned 3-times more sucrase activity. Only sucrose and trehalose were hyd rolysed by this aggregate and both activities were inhibited by acetat e or phosphate. Temperature and pH optima for trehalose hydrolysis app eared to be 40-45 degrees C and 5.0, respectively. The purified aggreg ate appeared to be disaggregating spontaneously resulting in inactivat ion of both enzymes, which was enhanced either at pH 3.5 or at pH 7.0. Separation of acid trehalase from the aggregate by hydrophobic intera ction chromatography resulted in inactivation. Rechromatography (HPGPL C) of the purified aggregate also gave disaggregation as well as inact ivation of both enzymes. Disaggregated acid trehalase and sucrase cont ained 20-fold and 13-fold lower specific activities, respectively, and appeared to be unstable. Based on these observations we suggest that acid trehalase is stabilised by aggregation with sucrase.