Trehalose (αDglucopyranosylαDglucopyra noside), a nonreducing disaccharide, is present in var ious organisms including bacteria, fungi, plants, and mammals [1]. Trehalose is found in spores, in resting cells, and in cells subjected to different forms of stress. This universality is related to the unique ability of this sugar to maintain the native state of membranes in con ditions of high ionic strength, dehydration, elevated temperature [15]. Many organisms can also use tre halose as an exogenous source of carbon. Certain grampositive and gramnegative bacteria including Escherichia coli can grow using trehalose as the only source of carbon at both low and high osmolarity, when the sugar is synthesized and accumulated inside a cell. It seems to be paradoxical: trehalose is cleaved as a source of carbon and simultaneously it is synthesized as an osmoprotector. For realization of these two pathways bacteria have developed two different systems of trehalose metabolism, one for high and the other one for low osmo larity. The scheme of trehalose metabolism has been stud ied in detail for E. coli [6]. Trehalose diffuses to the periplasm through LamB pores. Under conditions of low osmolarity, trehalose is transported through periplasm membrane and simultaneously phosphorylated by EIICB Tre (TreB) enzyme of the phosphotransferase sys tem (PTS) using EIIA of glucose PTS (EIIA Glc ) as phos phoryl donor. In cytoplasm, trehalose6phosphate is hydrolyzed to glucose and glucose6phosphate by the enzyme trehalose6phosphate hydrolase (TreC). Free glucose is phosphorylated by glucokinase, and glucose6 phosphate is subjected to glycolysis. At high osmolarity both TreB and TreC are repressed. Trehalose is hydrolyzed in periplasm to two glucose molecules by periplasmic trehalase (TreA). The latter is activated in turn at high osmolarity. Glucose is transported to the cytoplasm through the phosphotransferase system of glu cose. On the other hand, trehalose is synthesized at high osmolarity by the enzymes trehalose6phosphate syn thase (OtsA) and trehalose6phosphate phosphatase (OtsB) using glucose6phosphate and UDPglucose as the substrates. The objective of this study was cloning of the gene encoding the enzyme trehalose6phosphate hydrolase from the thermophilic bacterium Bacillus sp. GP16, and study of the principal enzymatic properties of this pro tein. Here we determined the Michaelis–Menten con stant as functions of pH, temperature, and ionic strength. It should be noted that this enzyme is poorly studied bio chemically, and the kinetic data in the literature are con troversial. The expression and purification of the enzyme from a thermophilic source was carried out for the first time in this work. Biochemistry (Moscow), Vol. 68, No. 9, 2003, pp. 10121019. Translated from Biokhimiya, Vol. 68, No. 9, 2003, pp. 12381246. Original Russian Text Copyright © 2003 by Karelov, Krasikov, Surjik, Firsov. 00062979/03/68091012$25.00 ©2003 MAIK “Nauka / Interperiodica” * To whom correspondence should be addressed. Expression, Isolation, Purification, and Biochemical Properties of Trehalose6phosphate Hydrolase from Thermoresistant Strain Bacillus sp. GP16 D. V. Karelov, V. V. Krasikov, M. A. Surjik, and L. M. Firsov* Konstantinov St. Petersburg Institute of Nuclear Physics, Russian Academy of Sciences, Gatchina 188350, Leningrad Region, Russia; Email: lfirsov@mail.wplus.net Received July 24, 2002 Revision received October 29, 2002 Abstract—Here we describe cloning, expression, and purification of the enzyme trehalose6phosphate hydrolase from ther moresistant strain Bacillus sp. GP16. Principal biochemical properties of the enzyme at different pH and temperature values were determined. Entropy and enthalpy of activation of the enzyme for substrates trehalose6phosphate and pnitrophenyl glucoside were calculated, and the dependence of the kinetic parameters from ionic strength was established. Key words: trehalose6phosphate hydrolase, cloning of a gene, gene expression, catalysis, temperature dependence, deter mination of Michaelis constants