title: Characterisation of an acid trehalase of Saccharomyces cerevisiae present in trehalase-sucrase aggregate.
abstract: An acid trehalase-sucrase aggregate was purified (by 780-fold) from Saccharomyces cerevisiae, following conventional protein purification techniques, to an apparent yield of 18.5%. The aggregate was electrophoretically 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 Km value of 5.0 mM but contained 3-times more sucrase activity. Only sucrose and trehalose were hydrolysed by this aggregate and both activities were inhibited by acetate or phosphate. Temperature and pH optima for trehalose hydrolysis appeared to be 40-45 degrees C and 5.0, respectively. The purified aggregate appeared to be disaggregating spontaneously resulting in inactivation 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 interaction chromatography resulted in inactivation. Rechromatography (HPGPLC) of the purified aggregate also gave disaggregation as well as inactivation of both enzymes. Disaggregated acid trehalase and sucrase contained 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.
authors.affiliation: Applied Biochemistry Department, Indian Institute of Chemical Biology, Calcutta, India.
authors.forename: N
authors.initials: N
authors.lastname: Biswas
authors.forename: A K
authors.initials: AK
authors.lastname: Ghosh
mesh_terms: D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013393:Sucrase / Q000302:isolation & purification / Q000378:metabolism*; D013696:Temperature; D014198:Trehalase / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D014198:Trehalase; D013393:Sucrase