Staphylococcal lipases stereoselectively hydrolyse the sn-2 position of monomolecular films of diglyceride analogs. Application to sn-2 hydrolysis of triolein.

Using the monomolecular film technique, a kinetic study on the stereoselectivity of nine staphylococcal lipase forms was carried out with three pairs of enantiomers from diglyceride analogs (didecanoyl-deoxyamino-O-methyl glycerol, DDG) containing a single hydrolysable decanoyl ester group and two lipase-resistant groups. Our results show that the kinetic profiles of the wild type, the recombinant untagged and the recombinant tagged forms of staphylococcal lipases are significantly different. As with most of the lipases investigated so far, these staphylococcal lipases showed higher catalytic rates with primary esters than with secondary esters. However, it is noteworthy that all these staphylococcal lipases were found to significantly hydrolyse the secondary ester group of diglyceride analogs, with a strong preference for the R configuration. This stereopreference, which was predicted on the basis of Kazlauskas' rule, was comparable to that of Candida rugosa and Pseudomonas glumae lipases. As was to be expected, all the staphylococcal lipases tested efficiently hydrolysed triolein at the sn-2 position. This hydrolytic activity was quantified by performing thin-layer chromatography to analyse the hydrolytic products of triolein. From the qualitative point of view, the sn-2 preferences observed with triolein and diglyceride analogs bearing a secondary ester function were in good agreement. Diglyceride analogs might therefore provide useful initial screening tools for use in future searches for strictly sn-2 specific lipases.

Snail hepatopancreatic lipase: a new member of invertebrates lipases' group.

Higher animal's lipases are well characterized; however, much less is known about lipases from mollusks. A lipolytic activity was located in the land snail (Eobania vermiculata) digestive glands (hepatopancreas), from which a snail digestive lipase (SnDL) was purified. Pure SnDL has a molecular mass of 60 kDa; it does not present the interfacial activation phenomenon. It was found to be more active on short-chain triacylglycerols than on long-chain triacylglycerols. The NH2-terminal sequence of the SnDL shows 66% of identity with the 17 NH2-terminal amino acids of a putative lipase from sea urchin (Strongylocentrotus purpuratus). No sequence identity was found with known lipases. Interestingly, neither colipase nor bile salts were detected in the snail hepatopancreas. This suggests that colipase evolved in vertebrates simultaneously with the appearance of an exocrine pancreas and a true liver which produces bile salts. Altogether, these results suggest that SnDL is a member of a new group of digestive lipases belonging to invertebrates.

Enzymatic synthesis of eugenol benzoate by immobilized Staphylococcus aureus lipase: optimization using response surface methodology and determination of antioxidant activity.

The ability of a non-commercial immobilized Staphylococcus aureus lipase to catalyze the esterification of eugenol with benzoic acid was checked and the antioxidant power of the ester formed was evaluated. Response surface methodology based on four variables (the reaction temperature, the amount of lipase, the benzoic acid/eugenol molar ratio and the volume of solvent) was used to optimize the experimental conditions of eugenol benzoate synthesis. The maximum conversion yield (75%) was obtained using 240 IU of immobilized lipase, a benzoic acid/eugenol molar ratio of 1.22 dissolved in 4.6 ml chloroform at 41 degrees Celsius. The antioxidant activities of eugenol and its ester were evaluated. Compared to BHT, used as a model synthetic antioxidant, the eugenol benzoate showed a higher antioxidative activity. The IC(50) value for 1,1-diphenyl-2-picrylhydrazyl was found to be 18.2 microg/ml versus 20.2 microg/ml for eugenol and eugenol benzoate.