Abstract
The application of lipases as catalysts in chemical reactions has been deterred by the high cost of isolation and purification of enzymes, the instability of their structure when they are isolated from their natural environment, contamination of products with residual protein, their sensitivity to process conditions, etc. These problems could be overcome using immobilized lipases. Immobilization is achieved by fixing enzymes to or within solid supports and as a result a heterogeneous system is obtained. The present paper reports on the immobilization of Candida rugosa lipase in hydrogels based on N-isopropylacrylamide and itaconic acid. Immobilization of lipase is carried out by two different methods. In the first method, lipase is added to the reaction mixture before polymerization and crosslinking (in situ polymerization), while in the second method the synthetized hydrogels are immersed in lipase solution and left to rich the equilibrium swelling. The specific activities of the immobilized lipase were determined in both cases and compared. The amount of the immobilized lipase is higher if the immobilization is carried out by immersing hydrogel in lipase solution. It was observed that in both cases lipase activity increases with an increase of the itaconic acid content up to 10 wt% and thereafter decreases. From the measurements of shear storage moduli (G') it was concluded that the increase of the itaconic acid content decreases the mechanical properties of the hydrogels. SEM analysis confirmed the highly porous structure of hydrogels. It was found that greater pores were achieved when the enzyme was immobilized by in situ polymerization. When the enzyme was immobilized by in situ polymerization the itaconic acid content had not great effect on the mass of the immobilized enzyme, except for the 100/0 sample. On the contrary, for the samples where the enzyme was immobilized by swelling, the increase of the itaconic acid content increases the mass of the immobilized enzyme. Concerning the activity of the immobilized lipase, the swelling degree and mechanical properties of the investigated hydrogels, the best results were performed by the 95/5 hydrogel sample.
Highlights
Imobilizacija je izvedena na dva načina, i to mešanjem enzima sa komponentama reakcione smeše pre izvođenja reakcije polimerizacije, pri čemu se enzim direktno imobiliše u toku formiranja hidrogela i bubrenjem prethodno sintetisanih hidrogelova u rastvoru lipaze na 5 i 25 °C
Nakon toga na uzorak je raspršena legura zlato/platina (15/85) pod vakuumom koristeći JEOL JEE-SS vakuum evaporator
Poređenjem vrednosti modula za pojedine uzorke, zapaža se da sa porastom sadržaja itakonske kiseline opada modul sačuvane energije, odnosno hidrogelovi imaju slabija mehanička svojstva
Summary
UTICAJ SASTAVA POLI(N-IZOPROPILAKRILAMIDA-KO-ITAKONSKA KISELINA) HIDROGELA NA AKTIVNOST IMOBILISANE LIPAZE IZ Candida rugosa. U radu je ispitana imobilizacija lipaze iz Candida rugosa u pH- i temperaturno-osetljive hidrogelove N-izopropilakrilamida i itakonske kiseline različitog sastava i pod različitim uslovima. Imobilizacija je izvedena na dva načina, i to mešanjem enzima sa komponentama reakcione smeše pre izvođenja reakcije polimerizacije, pri čemu se enzim direktno imobiliše u toku formiranja hidrogela (in situ polimerizacija) i bubrenjem prethodno sintetisanih hidrogelova u rastvoru lipaze na 5 i 25 °C. Ispitan je uticaj sastava hidrogelova, postupka i uslova imobilizacije na aktivnost imobilisanog enzima i kinetiku otpuštanja enzima iz hidrogela. Kako su za primenu imobilisanih enzima u reaktoru sa pakovanim slojem veoma važna mehanička svojstva nosača, u radu je ispitan uticaj sastava i različitih uslova sinteze na morfologiju i mehanička svojstva hidrogelova. Poroznost hidrogelova dozvoljava smeštanje enzima unutar gela, ali i njegovo potencijalno otpuštanje brzinom koja zavisi od veličine pora i koeficijenta difuzije enzima kroz mrežu gela [8]
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