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Immobilization of lipases via sol -gel procedures and application of the immobilized lipases in oleochemical reactions
Immobilization is one of the most used methods for enzymes to obtain stability. In this dissertation, lipases were chosen as the subject for immobilization research due to their potential applications in the oleochemical industry and biodiesel production. Sol-gel based procedures were explored for the immobilization of lipases. The lipase-containing sol-gel materials were prepared by polycondensation of hydrolyzed tetramethoxysilane and methyltrimethoxysilane, n-butyltrimethoxysilane, or iso-butyltrimethoxysilane. The most active immobilized lipase PS from P. cepacia so prepared retained full activity and dramatically increased its stability as measured in the hydrolysis and transesterification of soybean oil. Studies revealed that the retained activity of the immobilized lipase strongly depended on the type of silane precursors used in the sol-gel procedures. ^ In the immobilization of lipase AY from C. cepacia study, certain modifications were incorporated into the conventional immobilization procedure, which included the use of D-glucose as additive and the application of vacuum during the drying and aging stages. The modifications were incorporated into the immobilization procedure to prevent the interaction between the enzyme and methanol, which is a co-product of the sol-gel procedure and a strong inhibitor to lipase AY from C. rugosa. These modifications to the catalyst preparation protocol resulted in full retention of the activity of the immobilized lipase AY from C. rugosa as measured in the hydrolysis of soybean oil. ^ To correlate the activity of the immobilized lipases to their physical structures, the sol-gel materials were characterized for the particle size, specific surface area, degree of crosslinking, and the distribution of the enzyme in the solid matrix. The lipase/sol-gel materials were characterized by nitrogen adsorption to determine their specific surface area. Solid state NMR was used to reveal the degree of cross-linking of the sol-gel material. Scanning electron microscopy and atomic force microscopy were used to examine the morphology of the biocatalyst. Transmission electron microscopy and confocal microscopy were used to investigate the distribution of the enzyme within the sol-gel material. The characterization studies showed that the most active lipase-containing sol-gel material in the hydrolysis of triglyceride was a non-porous and amorphous material with random distribution of the enzyme throughout the sol-gel material. The activity of the immobilized lipases was not correlated to the cross-linking degree or the specific surface area of the sol-gel material. The enhanced activity of the immobilized enzyme was attributed to the massive external surface area of the lipase/sol-gel particles, the favorable hydrophobic microenvironment provided by the sol-gel matrix, and interactions between lipase and its hydrophobic support. ^
Gao, Xiaojian, "Immobilization of lipases via sol -gel procedures and application of the immobilized lipases in oleochemical reactions" (2004). ETD collection for University of Nebraska - Lincoln. AAI3148981.