A thermostable lipase was partially purified from the culture supernatant of a thermophilic Bacillus sp. The enzyme is optimally active at 60ºC and pH 8.0. The enzyme showed enhancement in activity in presence of benzene or hexane (30% v/v each). The activity (assayed by determining the release of pNP from pNP laurate) was stimulated up to 60% of these solvents in enzyme reaction mixture. The catalytic properties of this thermostable enzyme can be further improved via the use of different immobilization techniques and reaction conditions. Enzyme was immobilized on different solid supports and their enzyme activity and stability was compared. The enzyme was adsorbed on silica and HP-20 beads followed by cross-linking with gluteraldehyde on HP-20, which improved the thermostability of enzyme. The optimum pH (pH 8.5) was nearly same for aqueous and immobilized enzyme while optimum temperature was nearly 5ºC higher in case of immobilized enzyme. The immobilized/cross linked enzyme was more thermostable at 70 and 80ºC in comparison to aqueous and surface adsorbed lipase on silica and HP-20. The optimum temperature for esterification reactions was determined to be 60-65ºC. Half-life of immobilized lipase was nearly 2.5 x higher than the aqueous enzyme at 70ºC. Esterification of methanol and oleic acid to methyl oleate by immobilized enzyme was studied in detail.

A thermophilic Bacillus sp. was isolated that secreted an extracellular, thermostable lipolytic enzyme. The enzyme was purified to 58 folds with a specific activity of 9730 units/mg of protein and yield of 10% activity by ammonium sulphate precipitation, Phenyl Sepharose chromatography, gel-permeation followed by Q Sepharose chromatography. The relative molecular mass of the protein was determined to be 61 kDa by SDS-PAGE and approximately 60 kDa by gel permeation chromatography. The enzyme showed optimal activity at 60-65 ( composite function)C and retained 100% activity after incubation at 60 ( composite function)C and pH 8.0 for 1 h. The optimum pH was determined to be 8.5. It exhibited 50% of its original activity after 65 min incubation at 70 ( composite function)C and 23 min incubation at 80 ( composite function)C. Catalytic function of lipase was activated by Mg(++) (10 mM), while mercury (10 mM) inactivated the enzyme completely. No effect on enzyme activity was observed with trypsin and chymotrypsin treatment, while 50% inhibition was observed with thermolysin. It was demonstrated that PMSF, SDS, DTT, EDTA, DEPC, betaME (100 mM each) and eserine (10 mM) inhibited the activity of the lipolytic enzyme. With p-nitrophenyl laurate as a substrate, the enzyme exhibited a K ( m ) and V (max) of 0.5 mM and 0.139 microM/min/ml. The enzyme showed preference for short chain triacylglycerol and hydrolyzes triolein at all positions. In contrast to other thermostable Bacillus lipases, this enzyme has very low content of hydrophobic amino acids (22.58 %). Immunological studies showed that the active site and antigen-binding site of enzyme do not overlap.

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A thermostable lipase produced by a thermophilic Bacillus sp. J33 was purified to 175-fold with 15.6% recovery by ammonium sulphate and Phenyl Sepharose column chromatography. The enzyme is a monomeric protein having molecular weight of 45 kDa. It hydrolyzes triolein at all positions. The fatty acid specificity of lipase is broad with little preference for C12 and C4. The Km and Vmax for lipase with pNP-laurate as substrate was calculated to be 2.5 mM and 0.4 microM min(-1) ml(-1) respectively. The immobilized enzyme was stable for 12 h at 60 degrees C. Polyhydric alcohols such as ethylene glycol (2.5 M), sorbitol (2.5 M) and glycerol (2.5 M) were used as thermostabilizers. Lipase acquired a remarkable stability, since no deactivation occurred at 70 degrees C for 150 min in the presence of additives.