Lipases at Solid Surfaces ­ An Adsorption and Activity Study

Abstract: The adsorption of lipases to solid surfaces was studied by means of in situ ellipsometry. In addition, the activities of the adsorbed lipases were measured in situ, by the hydrolysis of p-nitro phenyl acetate. Adsorption was made to methylated silica surfaces with varying degree of wettability as well as to clean silica surfaces. Lipases from Humicola lanuginosa (HLL) ­ the wild-type (WT) and a mutant with increased hydrophobicity in the active site region, and lipase B from Candica antarctica (CALB) were investigated. HLL is known to be activated in the presence of an interface. By the displacement of a 'lid', covering the active site in the closed form, the hydrophobic active site region is revealed. CALB does not have this lid but the active site is, however, situated in the middle of a large hydrophobic area. Studies were performed with respect to concentration, pH, ionic strength and temperature. For HLL-WT and -mutant the effects of surfactants, in mixture with the lipase or sequentially added, on the adsorption and activities of the adsorbed lipases were also investigated. The surfactants used were SDS, C12E5 and Na-dodecanoate. The adsorbed amounts for all lipases in the study decreased with the hydrophobicity of the surface, the decrease was most pronounced, however, for HLL-WT. The adsorbed amounts were significantly higher for HLL-mutant and CALB than for HLL-WT. Measurements of the activities of the adsorbed lipases show that the specific activity, especially for HLL-WT, increased with increasing wettability of the surface. It is indicated that the lipase adsorbs with its active site region directed towards the surface when adsorbed to a surface with high hydrophobicity. As the surface hydrophobicity decreases, conformational/orientational changes of the adsorbed lipase presumably occur. These changes probably lead to an increased exposure of the active site to the solution, i. e. access for the water soluble substrate, resulting in increased activity. From the results it is shown that HLL-WT is significantly interfacially activated. Further, the adsorbed amounts of lipases were low when adsorbed from mixtures with surfactants (conc > cmc). The desorption effects of the surfactants at low concentrations (< cmc) were significant when sequentially added, the non ionic surfactant being more efficient than the anionic ones. No significant effects on the lipase activities were found of SDS or C12E5, at the actual concentrations. The presence of Na-dodecanotae, however, increased the specific activities of the lipases.