Immobilization of enzymes on a porous poly(glycidyl methacrylate)

V diplomski nalogi je prikazana študija imobilizacije encima na poliHIPE material, poli(glicidilmetakrilat-ko-etilenglikol dimetakrilat). Najprej smo pripravili poliHIPE materiale z različnimi deleži vodne faze (med 75 vol% in 90 vol%) in zamreževala (15 mol% in 30 mol%). Nato pa smo ustrezno očiščen in okarakteriziran material (FTIR spektroskopija, elementna analiza, vrstična elektronska mikroskopija in adsorpcijskaporozimetrija) uporabili kot nosilec za encim lipazo, katerega smo uporabili kot biokatalizator za hidrolizo 4-nitrofenil butirata. Za ta namen smo uporabili dve vrsti lipaze, lipazo Aspergilius niger in Candida cylindracea. Poli(glicidilmetakrilat-ko-etilenglikol dimetakrilatne) monolite smo uspešno sintetizirali s polimerizacijo emulzije z visokim deležem notranje faze tipa voda v olju. Oljna (organska, monomerna oz. zunanja) faza emulzije je vsebovala monomer glicidil metakrilat, zamreževalo etilenglikol dimateakrilat, iniciator azobisizobutironitril in surfaktant Pluronica L 121. Kot notranjo fazo smo uporabili 1,8% vodno raztopino kalcijevega klorida heksahidrata. Polimerne monolite smo okarakterizirali in funkcionalizirali. S karakterizacijo smo potrdili vključenost obeh monomerov v polimerne verige in njihovo porozno strukturo. Funkcionalizirali smo jih z nukleofilno substitucijo z encimom lipazo. Uspešnost reakcije smo preverili z elementno analizo in testno reakcijo, hidrolizo 4-nitrofenil butirata v p-nitrofenol in butirično kislino. Elementna analiza je pokazala vsebnost dušika v polimeru z vezanim (imobiliziranim) encimom, medtem ko dušika pri osnovnem polimeru elementni analizator ni zaznal. Da je bila imobilizacija encima uspešna smo potrdili tudi z reakcijo hidrolize in sicer z nastankom rumenega kompleksa p-nitrofenola, ki se pri katalizi s polimerom brez encima ni formiral.The thesis presents a study of enzyme immobilization on polyHIPE material, poly(glycidyl methacrylate-co-ethyleneglycol dimethacrylate). Firstly, we prepared polyHIPE materials with different proportions of the aqueous phase (between 75 vol% and 90 vol%) and crosslinker (15 mol% and 30 mol%). Secondly, the properly purified and characterized material (FTIR spectroscopy, elemental analysis, scanning electron microscopy and adsorption porosimetry) was used as a carrier for the enzyme lipase, which was used as a biocatalyst for the hydrolysis of 4-nitrophenyl butyrate. For this purpose, we used two types of lipase, the lipase of Aspergilius niger and Candida cylindracea. Poly(glycidyl methacrylate-co-ethyleneglycol dimethacrylate) monoliths were successfully synthesized by polymerization of high internal phase emulsion type water-in-oil. The oily (organic, monomeric or external) phase of the emulsion contained the monomer glycidyl methacrylate, the crosslinker ethyleneglycol dimethacrylate, the initiator azobisiso-butyronitrile and the surfactant Pluronica L 121. A 1.8% aqueous solution of calcium chloride hexahydrate was used as the internal phase. The polymer monoliths were characterized and functionalized. Characterization confirmed the inclusion of both monomers in the polymer chains and their porous structure. They were functionalized by nucleophilic substitution with the enzyme lipase. The success of the reaction was verified by elemental analysis and a test reaction, the hydrolysis of 4-nitrophenyl butyrate into p-nitrophenol and butyric acid. Elemental analysis showed nitrogen content in the polymer with bound (immobilized) enzyme, while nitrogen was not detected by the elemental analyzer in the basic polymer. The fact that the immobilization of the enzyme was successful was also confirmed by the hydrolysis reaction, namely by the formation of a yellow complex of p-nitrophenol, which was not formed during catalysis with a polymer without the enzyme