Development of immobilized horse radish (Armoracia rusticana) peroxidase for polymerization reactions and removal of phenols from waste waters

U prvom delu ove teze je makroporozni kopolimer glicidil metakrilata i etilen glikol dimetakrilata, poly(GMA-co-EGDMA) sa dijametrom čestica od 150 do 500 μm i različitim karakteristikama površine i različitom srednjom veličinom dijametra pora od 44 do 200 nm sintetisan, modifikovan 1,2-diaminoetanom i testiran kao nosač za imobilizaciju peroksidaze iz rena (HRP) koristeći dve kovalentne metode, glutaraldehidnu i perjodatnu. Najveća specifična aktivnost od oko 35 U/g suve mase nosača je postignuta na poly(GMA-co-EGDMA) kopolimeru sa srednjom veličinom dijametra pora od 200 i 120 nm koristeći perjodatni metod. Proučavanjem kinetike deaktivacije na 65 oC i u 80% (v/v) dioksanu je otkriveno da perjodatna metoda takođe dovodi do značajne stabilizacije biokatalizatora, i da stabilizacioni faktor zavisi snažno od površinskih karakteristika kopolimera. HRP imobilizovana na kopolimeru sa srednjom veličinom dijametra pora od 120 nm perjodatnom metodom je pokazala najveću specifičnu aktivnost ali takođe i dobru stabilnost, tako da je u daljem radu dodatno karakterisana. Imobilizacija je dovela do stabilizacije enzima u širem opsegu pH, dok je Mihaelisova konstanta (Km) imobilizovane HRP bila 10,8 mM što je otprilike 5,6 puta više nego za slobodni enzim. Posle 6 upotreba u bač reaktoru za oksidaciju pirogalola imobilizovana peroksidaza je zadržala 45% polazne aktivnosti. U drugom delu ove teze ista serija makroporoznih kopolimera glicidil metakrilata i etilen glikol dimetakrilata, poly(GMA-co-EGDMA) sa veličinom pora od 200 nm (ZS9) pa do neporoznog (ZS6) je testirana kao nosač za imobilizaciju peroksidaze iz rena, ali ovaj put sa dijametrom čestica od 2 do 2,5 μm. HRP je imobilizovana na 4 načina, preko epoksidne grupe na kopolimeru, i nakon aminovanja kopolimera glutaraldehidnom, perjodatnom i karbodiimidnom metodom. Najveća aktivnost imobilizovane HRP od 14,5 U/g je dobijena glutaraldehidnom metodom, a odmah zatim najveća aktivnost od 13,7 U/g karbodiimidnom metodom na poly(GMA-co-EGDMA) označenom kao ZS9 čija je srednja veličina pora bila oko 200 nm. Prinos imobilizacije je bio veći u odnosu na seriju kopolimera sa većom veličinom čestica i iznosio je 33,5% za glutaraldehidnu metodu i 30,3 % za karbodiimidnu u odnosu na oko 5% prinosa dobijenog za seriju kopolimera sa većom veličinom čestica i...In the first part of this thesis macroporous copolymer of glycidyl methacrylate and ethylene glycol dimethacrylate, poly(GMA-co-EGDMA) with various surface characteristics and mean pore size diameters ranging from 44 to 200 nm was synthesized, modified with 1,2-diaminoethane and tested as a carrier for immobilization of horseradish peroxidase (HRP) by two covalent methods, glutaraldehyde and periodate. The highest specific activity of around 35 U/g of dry weight of carrier was achieved on poly(GMA-co-EGDMA) copolymers with mean pore diameter of 200 and 120 nm by the periodate method. Study of deactivation kinetics at 65oC and in 80% (v/v) dioxane revealed that the periodate immobilization also produced an appreciable stabilization of the biocatalyst, while stabilization factor depended strongly on the surface characteristics of the copolymers. HRP immobilized on copolymer with mean pore diameter of 120 nm by periodate method showing the highest specific activity but also good stability was further characterized. It appeared that the immobilization resulted in stabilization of enzyme over a broader pH range while the Michaelis constant value (Km) of the immobilized HRP was 10.8 mM, approximately 5.6 times higher than that of the free enzyme. After 6 cycles of repeated use in batch reactor for pyrogallol oxidation, the immobilized HRP retained 45% of its original activity. In the second part of this thesis the same series of macroporous copolymer of glycidyl methacrylate and ethylene glycol dimethacrylate, poly(GMA-co-EGDMA) with pore diameter from 200 nm (ZS9) to the nonporous (ZS6) was tested as carier for immobilization of HRP, but this time with particle diameter between 2 and 2.5 μm. HRP was immobilized in 4 ways, via epoxide group on copolymer and after amination of copolymer using glutaraldehyde, periodate and carbodiimide method. The highest activity of immobilized HRP of 14.5 U/g was obtained using glutaraldehyde method and immediately after 13.75 U/g using carbodiimide method on poly(GMA-co-EGDMA) named ZS9 with average pore size of 200 nm. Yield of immobilization was..