An extracellular sucrase from the culture filtrate of filamentous basidiomycota Termitomyces clypeatus grown on high sucrose (5%. w/v) was purified by gel filtration chromatography, ion exchange chromatography and HPGPLC. The biochemical properties. molecular weight and conformation of sucrase produced were significantly different from the sucrase earlier purified from sucrose (1%, w/v) medium in the fungus. Purified sucrase was characterized as a low molecular weight protein of 13.5 kDa as approximated by SDS-PAGE and HPGPLC and exhibited predominantly random coil conformation in far-UV CD spectra. The enzyme was optimally active at 47 degrees C and pH 5.0. K-m and catalytic activity of the enzyme for sucrose were found to be 3.5 mM and 1.06 U/mg/mM, respectively. The enzyme was maximally active towards sucrose than to raffinose and sucrase activity was significantly inhibited by bivalent metal ions and reducing group agents. The results indicated that due to changes in aggregation pattern, molecular organization of purified sucrase, produced in high sucrose medium, was altered and was different from the previously reported enzyme. This is the first report of a sucrase of such low size showing activity. (C) 2009 Elsevier Ltd. All rights reserved.