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Study on interactions between lipase and carbon-based support
Hamrlová, Romana ; Voběrková, Stanislava (oponent) ; Hermanová, Soňa (vedoucí práce)
The thesis deals with issue of immobilized lipase, which was studied concretely from the aspect of interactions between lipase and carbon-based carrier. Immobilization of lipase from Rhizopus arrhizus (RA) was performed by adsorption onto graphene oxide types of carrier (a1, a, b, c and d) and by covalent attachment onto poly(ethylene glycol) modified carrier a1, PEG-a1. Adsorbed enzyme was additionally cross-linked with glutaraldehyde, GA(RA-a1). The influence of hydrophobic character of the carrier surface was confirmed since enzyme immobilized onto carriers with more hydrophobic surface (less polar groups) achieved higher activity retention at raising enzyme concentration. All activity assays were done spectrophotometrically using p-nitrophenyl laurate (p-NPL) as a substrate. The basic biochemical and kinetics characteristics were determined for immobilized as well as soluble enzyme. The pH optimum of covalently attached and adsorbed enzyme was shifted to more acidic environment (pH 7-8), whereas for soluble enzyme the optimum was achieved at pH 9. The thermostability of immobilized samples was significantly improved in the case of GA(RA-a1), where the glutaraldehyde chemistry was additionally involved. The glutaraldehyde cross-linking of adsorbed enzyme lead to the enhancement of the thermostability, which may be the consequence of intermolecular covalent linkage. The storage stability evaluation showed great improvement of lipolytic activity retention during the condition of the samples at 4 °C in phosphate buffer. Soluble enzyme lost more than 84 % of its initial activity after 42 days, while the immobilized enzyme onto c carrier kept still 100 % of its initial activity. The best storage stability showed the c carrier while after 180 days still kept 87 % of its initial activity.
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Study on interactions between lipase and carbon-based support
Hamrlová, Romana ; Voběrková, Stanislava (oponent) ; Hermanová, Soňa (vedoucí práce)
The thesis deals with issue of immobilized lipase, which was studied concretely from the aspect of interactions between lipase and carbon-based carrier. Immobilization of lipase from Rhizopus arrhizus (RA) was performed by adsorption onto graphene oxide types of carrier (a1, a, b, c and d) and by covalent attachment onto poly(ethylene glycol) modified carrier a1, PEG-a1. Adsorbed enzyme was additionally cross-linked with glutaraldehyde, GA(RA-a1). The influence of hydrophobic character of the carrier surface was confirmed since enzyme immobilized onto carriers with more hydrophobic surface (less polar groups) achieved higher activity retention at raising enzyme concentration. All activity assays were done spectrophotometrically using p-nitrophenyl laurate (p-NPL) as a substrate. The basic biochemical and kinetics characteristics were determined for immobilized as well as soluble enzyme. The pH optimum of covalently attached and adsorbed enzyme was shifted to more acidic environment (pH 7-8), whereas for soluble enzyme the optimum was achieved at pH 9. The thermostability of immobilized samples was significantly improved in the case of GA(RA-a1), where the glutaraldehyde chemistry was additionally involved. The glutaraldehyde cross-linking of adsorbed enzyme lead to the enhancement of the thermostability, which may be the consequence of intermolecular covalent linkage. The storage stability evaluation showed great improvement of lipolytic activity retention during the condition of the samples at 4 °C in phosphate buffer. Soluble enzyme lost more than 84 % of its initial activity after 42 days, while the immobilized enzyme onto c carrier kept still 100 % of its initial activity. The best storage stability showed the c carrier while after 180 days still kept 87 % of its initial activity.
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