Mikrobinių lipolizinių fermentų imobilizavimas ir imobilizuotų preparatų analizė /

Particular attention is currently being paid to the microbial enzyme class of lipases (E.C. 3.1.1.3), whose functional versatility and unique conformational structure allow the hydrolysis and synthesis of triacylglycerols. These properties are advantageous in a wide range of industrial applications, but the use of free-form enzymes is limited by the instability, decrease in activity under unfavorable conditions and difficulty in isolating the enzymes from the reaction mixture. Immobilization is a method of improving the kinetic and physicochemical properties of enzymes. It results in an insoluble carrier-enzyme system that enables the isolation and reuse of the preparations, ensuring environmental friendliness and cost-effectiveness. The aim of this work was the immobilization of six commercial lipases, namely Palatase 20000 L, Resinase A 2X, Lipozyme TL 100 L, Lipolase 100 L, type EX, Lecitase Ultra and Lipex 100 L, on a sugar industry pyrolysis product and octylsepharose carriers. Optimization of the conditions was first carried out by determining the stabilizing pH values by thermal shift assay and the optimum reaction temperature by spectrophotometric determination. The enzyme immobilization was based on adsorption. The lipolytic activity of the preparations and their supernatants and the concentration of the protein bound to the carrier were determined during the research study. Finally, three preparations were selected for further investigation of their physicochemical properties and the capability of synthesis reactions in organic media. The range of stabilizing buffer conditions for free enzymes was determined to be pH 6–9 and the optimum operating temperature 30–40 °C. Under these conditions, immobilization on the sugar industry pyrolysis product was not effective and the preparations lost most of their enzymatic activity, therefore no further studies were carried out. Optimization of the enzyme load did not result in a significant decrease in the lipolytic activity of the preparations in the case of octylsepharose, and therefore Palatase 20000 L, Resinase A 2X and Lecitase Ultra preparations immobilized for 24 h were selected with relative activities of 77,3 %, 107,0 % and 110,3 %, respectively. It was found that the optimum operating temperature for these preparations remained almost unchanged at 30 °C compared to the free enzymes, but that at this temperature Resinase A 2X and Lecitase Ultra showed better thermostability than the free enzymes after 4 h and 6 h incubation. The highest substrate specificity for free and immobilized enzymes was observed for the p-NPB substrate. For the p-NPC substrate, preparations showed a slightly higher specificity than the free form enzymes. Moreover, the hydrolysis of the p-NPD substrate by Palatase 20000 L was 14.4% more efficient than the free enzyme. For the remaining preparations, the lipolytic activity obtained remained either similar or lower than that of the free enzyme. The synthesis study of the aromatic ester 2-phenylethylbutanoate showed that after 24 h and 48 h, Resinase A 2X and Palatase 20000 L were more efficient than free enzymes in synthesizing the product, while Lecitase Ultra did not synthesize the product. This work attempted to obtain immobilized preparations of commercial lipases with better kinetic and physicochemical properties. Immobilization on sugar industry waste product did not succeed, but the selected preparations of Lecitase Ultra and Resinase A 2X immobilized on octylsepharose showed increased thermostability, while Palatase 20000 L and Resinase A 2X showed higher substrate specificity for either p-NPD or p-NPC substrates. In addition, these 2 immobilized preparations have potential applications in organic synthesis, whereas Lecitase Ultra requires further optimization of conditions.

Grantor dc:publisher

Institutional Repository of Vilnius University

Year dc:date

2024

Author dc:creator

• Gineikaitė, Augustė,

Statement dc:rights

• info:eu-repo/semantics/openAccess

Language dc:language

lit

Repository record dc:identifier

https://repository.vu.lt/VU:ELABAETD210638837&prefLang=en_US

OAI identifier oai:identifier

oai:vu.lt:elaba:210638837