Abstract
A new heterofunctional support, octyl-glyoxyl agarose, is proposed in this study. The supports were prepared by simple periodate oxidation of the commercial octyl-agarose, introducing 25 mu mol of glyoxyl groups per wet gram of support. This support was assayed with three different lipases (those from Candida antarctica (form B), Thermomyces lanuginosus (TLL) or Rhizomucor miehei) and the artificial phospholipase Lecitase Ultra. Used at pH 7, the new support maintained as first immobilization step the lipase interfacial activation. Thus, it was possible to have the purification and immobilization of the enzyme in one step. Moreover, stabilization of the open form of the lipase was achieved. The covalent enzyme/support bonds cannot be obtained if the immobilized enzyme was not incubated at alkaline pH value. This incubation at pH 10 of the previously immobilized enzymes produced a smaller decrease in enzyme activity when compared to the direct immobilization of the enzymes on glyoxyl-agarose at pH 10, because the immobilization via interfacial activation promoted a stabilization of the lipases. Except in the case of TLL (covalent attachment involved 70% of the enzyme molecules), covalent immobilization yield was over 80%. The non-covalent attached enzyme molecules were discarded by washings with detergent solutions and the new biocatalysts were compared to the octyl-agarose immobilized enzymes. While the stability in thermal and organic solvents inactivations was increased for Lecitase Ultra, CALB and RML, TLL improved its stability in organic media but its thermal stability decreased after covalent attachment of the interfacially activated enzyme. This stabilization resulted in octyl-glyoxyllipase preparations which presented higher activity in the presence of organic solvents. Finally, while octyl-agarose released enzyme molecules after incubation at high temperatures or in the presence of organic solvents and detergents, the covalently immobilized enzyme remained attached to the support even after boiling the enzyme in SDS, eliminating the risks of product contamination.