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Amyloglucosidase from A. niger was covalently immobilized onto poly(GMA-co-EGDMA) by the glutaraldehyde and periodate method. The immobilization of amyloglucosidase after periodate oxidation gave a preparate with the highest specific activity reported so far on similar polymers. The obtained immobilized preparates show the same pH optimum, but a higher temperature optimum compared with the soluble enzyme. The kinetic parameters for the hydrolysis of soluble starch by free and both immobilized enzymes were determined. Amiloglukozidaza iz A.niger je imobilizovana na poly(GMA-co-EGDMA) glutaraldehidnom i perjodatnom metodom. Imobilizacija amiloglukozidaze nakon perjodatne oksidacije daje preparat sa najvećom do sada objavljenom specifičnom aktivnosti na sličnim polimerima. Dobijeni imobilizovani preparat ima isti pH optimum ali povećani termooptimum u poređenju sa rastvornim enzimom. Određeni su i kinetički parametri za hidrolizu rastvornog skroba imobilizovanim kao i rastvornim enzimom.

Penicillin acylase (PAC) is an important industrial enzyme for the production of many β-lactam antibiotics. It is capable of catalyzing the hydrolysis of penicillin G (Pen G) to generate phenylacetic acid (PAA) and 6-aminopenicillanic acid (6-APA). In this paper, in order to prevent enzyme inactivation, an attempt of coupling enzyme modification and immobilization is presented. Chemical modification was promoted to introduce carbohydrate moiety into the PAC molecule, capable of being covalently linked to an amino support. This seems to provide a possibility to couple the enzyme without risking a reaction at the active site which might cause a loss of activity. PAC molecules were modified by cross-linking with polyaldehyde derivatives of alginate in order to add them new and useful functions. Immobilization of alginate-PAC on Sepabeads EC-HA was used as a model system in order to demonstrate the potential of this strategy. Optimal conditions for covalent immobilization of alginate-PAC from Escherichia coli on support Sepabeads EC-HA were investigated. The immobilized enzyme was then characterized by evaluating the potential effects of immobilization on its thermal stability, temperature and pH profile in comparison with native non-modified PAC and modified non-immobilized PAC. The maximum amount of the alginate-PAC coupled on the dry support of 99 mg/g was satisfactory. Deactivation rate constants at 50°C for free PAC, alginate-PAC and alginate-PAC immobilized on Sepabeads EC-HA were 2.32, 50.65 and 1.68 h-1, respectively. Alginate-PAC and alginate-PAC immobilized on Sepabeads EC-HA had the same pH and temperature optimum as the native non-modified PAC. U ovom radu započeto je sistematsko ispitivanje imobilizacije enzima modifikovanih derivatima alginata. Penicilin-acilaza (PAC) modifikovana je polialdehidnim derivatom alginata i zatim imobilisana na Sepabeads EC-HA nosač. Ispitani su optimalni uslovi za kovalentnu imobilizaciju modifikovane PAC i imobilisani enzim je okarakterisan u pogledu efekata imobilizacije na njegovu termalnu stabilnost, pH i temperaturni profil. Dodatno, imobilisani enzim je po pitanju ovih parametara upoređen kako sa nativnom tako i sa modifikovanom (alginat-PAC) formom enzima. Konstante brzine dezaktivacije za PAC, alginat-PAC i modifikovan enzim imobilisan na Sepabeads EC-HA (alginat-PAC-Sepabeads EC-HA) iznosile su redom 2,03, 36,48 i 1,23 h-1 na 40°C, odnosno 2,32, 50,65 i 1,68 h-1 na 50°C. Pokazano je da alginat-PAC i alginat-PAC-Sepabeads EC-HA imaju isti pH i temperaturni optimum kao i nativna PAC.

An approach is presented for the stable covalent immobilization of Upase from Candida rugosa on Eupergit® with a high retention of hydrolytic activity. It comprises covalent bonding via lipase carbohydrate moiety previously modified by periodate oxidation, allowing a reduction in the involvement of the enzyme functional groups that are probably important in the catalytic mechanism. The hydrolytic activities of the lipase immobilized on Eupergif1 by two conventional methods (via oxirane group and via glutaralde-hyde) and with periodate method were compared. Results of lipase assays suggest that periodate method is superior for lipase immobilization on Eupergit® among methods applied in this study with respect to both, yield of immobilization and hydrolytic activity of the immobilized enzyme. U ovom radu je ispitana mogućnost primene metode za kovalentno vezivanje lipaze iz Candida rugosa za komercijalni polimerni nosač Eupergit® kojom se dobijaju stabilni i aktivni imobilisani enzimi. Vezivanje se odvija preko ugljenohidratne komponente enzima, koja je prethodno modifikovana oksidacijom pomoću perjodata, a ne preko proteinske komponente, koja je važna za katalitičku aktivnost enzima. Hidrolitička aktivnost na ovaj način imobilisane lipaze upoređena je sa aktivnostima lipaza koje su imobilisane pomoću dve konvencionalne metode (vezivanje preko epoksidnih grupa nosača i vezivanje za nosač modifikovan glutaraldehidom). Rezultati ovog istraživanja pokazuju da je perjodatna metoda pogodnija za imobilizaciju lipaze na Eupergit® sa oba ispitivana aspekta: prinosa imobilizacije i hidrolitičke aktivnosti imobilisanog enzima.