ELSEVIER Biochimica et Biophysica Acta 1290 (1996) 95-100 BB Biochi~ic~a et Biophysica A~ta Characterisation of an acid trehalase of Saccharomyces cerevisiae present in trehalase-sucrase aggregate Nilima Biswas, Anti Kumar Ghosh * Applied Biochemistry Department, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Calcutta 700 032, India Received 17 August 1995; revised 7 November 1995: accepted 10 January 1996 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 K m 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°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. Keywords: Acid trehalase; Sucrase; Enzyme aggregation: Characterization; (S. cerecisiae) 1. Introduction Trehalose (a-D-glucopyranosyl a-D-glucopyranoside) is an important disaccharide which is widely distributed in bacteria, yeast, fungi and insects [1]. Accumulation of trehalose in fungi is associated in general with periods of reduced growth rate, i.e., in derepressed stationary phase cells or during sporulation or nutrient starvation or temper- ature stress, etc. [2]. Consequently, trehalose appears to be primarily a storage carbohydrate during periods of non- proliferation. More recent studies have yielded convincing evidence of trehalose as a cell protectant containing a stabilising protein function against a variety of stress treat- ments [3,4]. The only enzyme known to be responsible for trehalose hydrolysis is trehalase (a, a-trehalose-1-D-gluco- hydrolase, EC 3.2.1.28) [2]. Studies on this enzyme from various laboratories revealed that two different trehalases can be discerned: a non-regulatory enzyme or acid treha- lase (AT) and a regulatory enzyme or neutral trehalase (NT) [2,5,6]. AT has an acid pH optimum, lower K m (0.5 to 3.0 mM) and is not inhibited by EDTA. On the other * Corresponding author. Fax: +91 33 4730284. 0304-4165/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PII S0304-4 165(96)00005-0 hand, NT with neutral pH optimum, with higher K m (around 4.8 mM or more), is inhibited by EDTA and is reported to be activated through cAMP-dependent protein phosphorylation [5,7]. The biological function of these two trehalases are also different [6]. AT is a glycoprotein and exists in multiple forms [8-11]. Since it is not known whether any regulatory mechanism exists for activity or not, interest to purify AT arose so that proper studies on AT may be undertaken. The present paper describes stabilisation of AT of Saccha- romyces cerevisiae by aggregation with sucrase (S). Com- plete purification of AT resulting in containing no S is extremely difficult. 2. Materials and methods 2.1. Organism and culture condition The diploid strain used throughout the entire work was derived from Saccharomyces cerevisiae strains, 8534-10A (MAT~, leu2, ura3, his4)and 6460-8D MATa, met3). Both strains were kindly provided by Dr. (Mrs.) P. Sinha, Bose Institute, Calcutta, India.