Hyperactivation of Rhizomucor miehei lipase by hydrophobic xerogels

Although a variety of approaches exist for the immobilization of enzymes, the "science" of enzyme immobilization is still in its infancy. In recent years, considerable interest has developed regarding the use of xerogels for enzyme immobilization. There are several advantages to xerogels for enzyme immobilization, including the opportunity to produce them in defined shapes or thin films and the ability to manipulate their physical characteristics (e.g., porosity, hydrophobicity, and optical properties). In this study we examined the effect of xerogel hydrophobicity on the activity of lipase (EC 3.2.2.3) from Rhizomucor miehei. The hydrophobicity of the xerogels was manipulated by generating xerogels with various molar ratios of propyltrimethoxysilane (PTMS) to tetramethoxysilane (TMOS), from 1:1 to 10:1. The belief was that, by increasing the proportion of propyl groups, the hydrophobicity of the resulting xerogel would be increased. Differences in the hydrophobicity of the resulting xerogels were confirmed using water-affinity studies. Two approaches were taken for water-affinity determinations by examining the ability of the xerogels to remove water from air (controlled humidity) and from water-saturated isopropyl ether. Xerogels with higher propyl content showed a reduced affinity for water. A crude lipase preparation from Rhizomucor miehei was then contacted with sized xerogel particulates and the effect of the xerogel on lipase activity was determined. The presence of the xerogel resulted in hyperactivation of the lipase. Analysis of the protein adsorption revealed changes in the profile of proteins adsorbed to the xerogel based on the hydrophobicity of the xerogel. Based on estimations of the specific activity of the hyperactivated lipase, a minimum hyperactivation of 207% was observed. Part of the hyperactivation may be attributable to xerogel-lipase interactions, but also to the adsorption of a component from the crude lipase preparation that may complex with the lipase and the xerogel producing a stabilizing effect. Further improvements in hyperactivation and selectivity of the xerogels is likely possible by working at lower PTMS:TMOS ratios than those investigated in this study.