The use of enzymes in organic solvents has been proved to be a very interesting technology in organic synthesis, food-related transformation and analysis. There are numerous advantages using enzymes in non aqueous media since it is possible to increase their thermal stability, facilitate transformation of substrates unstable or poorly soluble in water, synthesis esters or amide bounds by hydrolases, prevent side reactions due to water. Nevertheless, a drawback of this technique is due to the low catalytic activity of enzymes in organic solvents, which can be of several orders of magnitude lower than that displayed in aqueous buffer. The reasons of this phenomenon have been ascribed to different causes such as restricted protein flexibility, low stabilization of the enzyme-substrate intermediate, partial enzyme denaturation because of the lyophilization, which becomes irreversible in anhydrous solvents, non-optimal pH before the lyophilisation process and diffusional limitations [1,2].

To shed light on this problem we studied the activity of different physical forms of Candida antarctica lipase B (CALB) such as crude CALB, purified CALB, purified CALB colyophilised with PEG (CALB+PEG) or oleic acid (CALB+OA) and a commercial form immobilised on a macroporous acrylic support (Novozym 435). The different forms were employed in several organic solvents (toluene, carbon tetrachloride and 1,4-dioxane) at various water activities (a_w) to catalyse the model reaction of vinyl acetate with 1-octanol, determining both transesterification and hydrolytic rates.

The study revealed that, for equal amounts of lipase protein, CALB+PEG had an higher activity than that of other forms. In fact in toluene (a_w<0.1) it was 260-fold higher than that of crude CALB and 1.8-fold higher than that of the very efficient commercial form Novozym 435. Purified CALB had at a_w=0.8 a total activity which was about 8-fold higher than that at a_w<0.1. These results suggest that the "activating" effect of PEG and oleic acid (CALB + OA showed an activity similar to that of Novozym 435) could be ascribed to a lyoprotectant effect of these additives, which could prevent the formation of intermolecular interactions during the dehydration process that might be responsible of reversible, in water but no in organic solvents, protein denaturation [3]. Moreover, since CALB+PEG had a transesterification activity in toluene which was 51% of the hydrolytic activity displayed by CALB in aqueous buffer with vinyl acetate as substrate, we proved that under suitable conditions, i.e., activated ester and "good" lipase form, lipase activity in organic solvents is comparable to that observed in water.

[1] Koskinen, A.M.P., Klibanov, A.M., Blackie Academic & Professional, 1996.

[2] Klibanov, A., Trends Biotechnol., 15, 97-101, 1997.

[3] Vecchio, G., Zambianchi, F., Zacchetti, P., Secundo, F., Carrea, G. Biotechnol. Bioeng., 64, 545-551, 1999.

[4] Secundo, F., Spadaro, S., Carrea, G., Overbeeke, P.L.A. Biotechnol. Bioeng., 62, 554-561, 1999.