|
|
Effects of detergents on activity, thermostability and aggregation of immobilized lipases
Bančáková, Anna ; Voběrková, Stanislava (oponent) ; Hermanová, Soňa (vedoucí práce)
The diploma thesis deals with the issue of the effect of tweens on enzymatic activity of model hydrolase both free and immobilized on carbon-based carrier. In theoretical part, structural features, mechanism of action, and specialty applications of microbial lipases are reviewed along with detergent chemistry, with emphasize on tween family of detergents belonging into non-ionic surfactant group. In experimental part, effect of tweens on soluble as well as immobilized hydrolase was examined. Immobilization of commercial preparation of lipase was performed by non-covalent adsorption on graphene oxide as a carrier treated with different tweens (tween 20, 60, 80). The activity was determined spectrophotometrically by p-nitrophenyl laurate assay. Enhancement of soluble Rhizopus arrhizus lipase activity (activity coupling of 104 %) was observed at tween 20 concentration of 10 mmol•dm-3, which is highly above critical micelle concentration of this detergent. On the base of screening study, immobilization protocol comprised the incubation of soluble enzyme at concentration of 0.1 mg•ml-1 in phosphate buffer (pH 7.2) with tween 20 (10.8 mmol•dm-3) and the carrier for one hour. Both soluble and immobilized lipase exhibited maximum activity at approx. 35 °C. Optimal pH of immobilized lipase shifted to 8 compared to soluble form for which pH optimum at 9 was determined. Thermal stability profile follows almost same trend for both soluble and immobilized enzyme samples. The interactions between carrier and enzyme are suggested to be mainly non–covalent (adsorption, electrostatic interactions). No protein leaching was observed under studied conditions, and significant improvement of storage stability of immobilized lipase was achieved (activity retention of 41 % after 110 days) in comparison with soluble lipase (activity retention of 16 % after 42 days).
|
|
|
Biocatalysts based on lipases, their immobilization and characterization
Bančáková, Anna ; Voběrková, Stanislava (oponent) ; Hermanová, Soňa (vedoucí práce)
The thesis deals with the issue of immobilized enzymes. In the theoretical part, principal and novel methods of immobilization and types of carriers are reviewed together with practical applications of immobilized enzymes. Particularly, recent utilization of immobilized enzymes in a variety of industries such as food industry, medicine, chemical analysis, bioremediation, and biodiesel production is summarized. Different methodologies including adsorption, entrapment, covalent binding and cross-linking are discussed from the viewpoint of their dis/advantages resulting from the extent and the character of binding and the maintenance of enzyme activity. Among new carriers, the research work done on the utilization of graphene as a novel carrier for enzyme immobilization is particularly reported. In the experimental part, immobilization of commercial preparation of lipase (RA) isolated from microscopic fungi Rhizopus arrhizus was performed by adsorption on polyethylene terephthalate as a carrier. The basic parameters as lipolytic activity, temperature optimum, pH optimum and thermal stability of both soluble as well as immobilized enzyme were determined spectrophotometrically using p-nitrophenyl-laurate (pNPL) as a substrate. Both the soluble and immobilized lipase showed maximum activity at pH 7.2 and a decrease in activity was observed above pH 8 or below pH 6.5. The dependence of activity on pH of reaction medium was more pronounced in the case of immobilized form of enzyme. The soluble and immobilized lipases exhibited maximum activity at a temperature of approximately 30 °C. A drop in activity values was observed when the temperature was increased up to 50 °C and above this temperature the stability of the soluble lipase sharply decreased. On the contrary, thermal stability of immobilized RA lipase was significantly improved. Immobilized form of enzyme possesses activity 3.7 % in comparison to the soluble form.
|
|
|
Effects of detergents on activity, thermostability and aggregation of immobilized lipases
Bančáková, Anna ; Voběrková, Stanislava (oponent) ; Hermanová, Soňa (vedoucí práce)
The diploma thesis deals with the issue of the effect of tweens on enzymatic activity of model hydrolase both free and immobilized on carbon-based carrier. In theoretical part, structural features, mechanism of action, and specialty applications of microbial lipases are reviewed along with detergent chemistry, with emphasize on tween family of detergents belonging into non-ionic surfactant group. In experimental part, effect of tweens on soluble as well as immobilized hydrolase was examined. Immobilization of commercial preparation of lipase was performed by non-covalent adsorption on graphene oxide as a carrier treated with different tweens (tween 20, 60, 80). The activity was determined spectrophotometrically by p-nitrophenyl laurate assay. Enhancement of soluble Rhizopus arrhizus lipase activity (activity coupling of 104 %) was observed at tween 20 concentration of 10 mmol•dm-3, which is highly above critical micelle concentration of this detergent. On the base of screening study, immobilization protocol comprised the incubation of soluble enzyme at concentration of 0.1 mg•ml-1 in phosphate buffer (pH 7.2) with tween 20 (10.8 mmol•dm-3) and the carrier for one hour. Both soluble and immobilized lipase exhibited maximum activity at approx. 35 °C. Optimal pH of immobilized lipase shifted to 8 compared to soluble form for which pH optimum at 9 was determined. Thermal stability profile follows almost same trend for both soluble and immobilized enzyme samples. The interactions between carrier and enzyme are suggested to be mainly non–covalent (adsorption, electrostatic interactions). No protein leaching was observed under studied conditions, and significant improvement of storage stability of immobilized lipase was achieved (activity retention of 41 % after 110 days) in comparison with soluble lipase (activity retention of 16 % after 42 days).
|
|
|
Biocatalysts based on lipases, their immobilization and characterization
Bančáková, Anna ; Voběrková, Stanislava (oponent) ; Hermanová, Soňa (vedoucí práce)
The thesis deals with the issue of immobilized enzymes. In the theoretical part, principal and novel methods of immobilization and types of carriers are reviewed together with practical applications of immobilized enzymes. Particularly, recent utilization of immobilized enzymes in a variety of industries such as food industry, medicine, chemical analysis, bioremediation, and biodiesel production is summarized. Different methodologies including adsorption, entrapment, covalent binding and cross-linking are discussed from the viewpoint of their dis/advantages resulting from the extent and the character of binding and the maintenance of enzyme activity. Among new carriers, the research work done on the utilization of graphene as a novel carrier for enzyme immobilization is particularly reported. In the experimental part, immobilization of commercial preparation of lipase (RA) isolated from microscopic fungi Rhizopus arrhizus was performed by adsorption on polyethylene terephthalate as a carrier. The basic parameters as lipolytic activity, temperature optimum, pH optimum and thermal stability of both soluble as well as immobilized enzyme were determined spectrophotometrically using p-nitrophenyl-laurate (pNPL) as a substrate. Both the soluble and immobilized lipase showed maximum activity at pH 7.2 and a decrease in activity was observed above pH 8 or below pH 6.5. The dependence of activity on pH of reaction medium was more pronounced in the case of immobilized form of enzyme. The soluble and immobilized lipases exhibited maximum activity at a temperature of approximately 30 °C. A drop in activity values was observed when the temperature was increased up to 50 °C and above this temperature the stability of the soluble lipase sharply decreased. On the contrary, thermal stability of immobilized RA lipase was significantly improved. Immobilized form of enzyme possesses activity 3.7 % in comparison to the soluble form.
|
host ::
RSS.