An isolated thermophilic Bacillus sp. produced two extracellular lipases. The expression of the two lipases was governed by the age of the culture. Hydrophobic interaction chromatography separated the lipolytic activity into two peaks. The second lipase got eluted with 80% ethylene glycol. The apparent molecular mass of Lip2 was approximately 60,000Da from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 62,000 +/- 3000 Da from gel filtration. The enzyme showed optimal activity at 60-65 degrees C and retained 100% activity after incubation at 60 degrees C and pH 8.0 for 1 h. The enzyme showed maximum activity at pH 8.0 and was very stable at pH 7.0-8.5 with optimum stability at pH 8.0. At this pH and 60 degrees C temperature, the half-life of enzyme was 170 h. It exhibited 50% of its original activity after 45 min incubation at 70 degrees C. It was demonstrated that SDS, DTT (100 mM each) and eserine (5 mM) did not affect enzyme activity while activity was inhibited in the presence of PMSF, DEPC and EDTA (100 mM each). Enzyme activity was stimulated in the presence of benzene or hexane (each 30%, v/v). With p-nitrophenyl laurate as a substrate, the enzyme exhibited a K-m and V-max of 0.19 mM and 0.032 mu M/min/ml. The enzyme showed preference for long chain triacylglycerol and hydrolyzed triolein at all positions. The enzyme had very high hydrophobic amino acid content (60.2%) with about 50% of total amino acids as alanine and 21% as glycine. Western blot analysis confirmed the cross-reactivity of antiserum against Lip I with Lip2. Dot blot analysis suggested that antibodies are specific for these isoforms as it did not react with a purified lipase from a mesophilic Bacillus sp. (c) 2006 Elsevier Inc. All rights reserved.