Commercially available proteases and lipases were screened for their ability to acylate regioselectively sucrose with divinyladipate either in pyridine or dimethylformamide (DMF). The protease (EC 184.108.40.206) from Bacillus subtilis (Proleather FG-F) exhibited the highest conversion (100% in 24 h of reaction in DMF) yielding sucrose 2-O-vinyladipate as main product. The enzyme preference for a secondary hydroxyl group is a distinct feature of this biocatalyst compared to others described in the literature. Two sets of chemically distinct silica supports were used for Proleather immobilization presenting terminal amino (S(APTES)) or hydroxyl groups (S(TESPM)(-)(pHEMA)). The percentage of immobilized enzyme was smaller in S(APTES) (7-17%) than in S(TESPM)(-)(pHEMA) (52-56%), yet Proleather immobilized into S(APTES) supports presented higher total and specific hydrolytic activity. The highest total and specific activities were obtained with S(TESPM)(-)(pHEMA) and S(APTES), respectively. Silicas with large pore (bimodal distribution of pores, 130/1200 A, denoted as S(1000)) presented higher specific activities relative to those with smaller pore sizes. Furthermore, the synthetic specific activity of S(1000)S(APTES) immobilized protease was ca. 10-fold higher than that of the free enzyme. In addition to sucrose, the immobilized protease was used to acylate methyl alpha-D-glucopyranoside, trehalose, and maltose in nearly anhydrous DMF. Finally, immobilized Proleather was reasonably stable, retaining ca. 55% activity after six reaction cycles.