Immobilization of alginate-PAC on sepabeads EC-HA support

Abstract

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.