Solid/gas biocatalysis: an appropriate tool to study the influence of organic components on kinetics of lipase catalyzed alcoholysis

Abstract : The influence of the addition of an extra component in a gaseous reaction medium, on the kinetics of alcoholysis of methyl propionate and n-propanol catalyzed by immobilized lipase B from Candida antarctica was studied in a continuous solid/gas reactor. In this reactor, the solid phase is composed of a packed enzymatic sample which is percolated by gaseous nitrogen, simultaneously carrying gaseous substrates and additional components to the enzyme while removing reaction products. The system permits to set thermodynamic activity of all gaseous components (substrates or not) independently at the desired values. This allows in particular to study the influence of an extra added component at a constant thermodynamic activity value, contrary to classical solid/liquid system, which involves large variations of thermodynamic activity of added solvent, when performing full kinetic studies. Alcohol inhibition constant (KI) and methyl propionate and propanol dissociation constants (KMP and KP) have been determined in the solid/gas reactor in presence of 2-methyl-2-butanol, and compared with values previously obtained in absence of added component and in presence of water. Complementary experiments were carried out in the presence of an apolar compound (hexane) and led to the conclusion that the effect of added organic component on lipase catalyzed alcoholysis is related to their competitive inhibitory character towards first substrate methyl propionate. The comparison of data obtained in liquid or with gaseous 2-methyl-2-butanol shows that lower KMP and KI are found in gaseous medium, which would correspond on the one hand to a lower acylation rate k2, and on the other hand to a higher binding rate k1 between substrate and free enzyme in gaseous medium.
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Fabien Létisse, Sylvain Lamare, Marie-Dominique Legoy, Marianne Graber. Solid/gas biocatalysis: an appropriate tool to study the influence of organic components on kinetics of lipase catalyzed alcoholysis. Biochimica et Biophysica Acta Proteins and Proteomics, Elsevier, 2003, 1652, pp.27-34. ⟨hal-00329687⟩

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