Sucrose fatty acid esters are obtained industrially by transesterification of sucrose with methyl or ethyl esters using alkaline catalysts. The enzymatic approach is attracting keen attention as a new manufacturing method for future application, but as yet is not used commercially. Although many reports deal with the enzymatic synthesis of sucrose esters, the intrinsic stability of the biocatalysts employed has seldom been analyzed. This is a crucial parameter when considering the substitution of chemical methods for the more selective enzyme-catalyzed processes.

Enzymatic synthesis of sucrose fatty acid esters is limited by the fact that most biological catalysts are inactivated by the polar solvents (dimethylsulfoxide, dimethylformamide, dimethylacetamide) where sucrose is soluble. In this context, we developed a methodology for the acylation of sucrose with vinyl fatty acid esters using mixtures of two miscible solvents [1,2]. More specifically, a low amount of a solvent where sucrose is notably soluble (dimethylsulfoxide) was added to a tertiary alcohol (2-methyl-2-butanol) in order to reduce the inactivation of the biocatalyst. The lipase from Humicola lanuginosa was the best of the biocatalysts assayed, acylating selectively the primary hydroxyl 6-OH.

In this work we have studied the stability under the conditions described above of the lipase from H. lanuginosa immobilised by different methods (deposition on diatomaceous earth, adsorption on polymeric carriers). To evaluate the residual activity, the reaction mixture was filtered, the biocatalyst thoroughly washed with 2-methyl-2-butanol, and dried. The residual activity was measured with a discontinuous spectrophotometric assay following the hydrolysis of p-nitrophenyl butyrate. In some cases, the inactivation curves were validated studying the hydrolytic activity with tributyrin.

The effect of different reaction parameters on lipase stability was analysed. In particular, temperature and solvent composition (i.e. percentage of dimethylsulfoxide in the mixture) were extensively studied.

[1] Ferrer, M., Cruces, M.A., Bernabé, M., Ballesteros, A., Plou, F.J. Biotechnol. Bioeng., 65, 10-16, 1999.

[2] F.J. Plou, F.J., Cruces, M.A., Pastor, E., Ferrer, M. Bernabé, M., Ballesteros, A. Biotechnol. Lett., 21, 635-639, 1999.