Penicillin acylase (PAC) is an important industrial enzyme for the production
of many ?-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
was 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.
Effect of internal diffusional restrictions on kinetic parameters of hydrolysis of penicillin G and synthesis of cephalexin with immobilized penicillin acylase