Evaluation of affinity and pseudo-affinity adsorption processes for penicillin acylase purification.
Affinity ligand (6-Aminopenicillanic acid, Amoxycillin, Ampicillin, Benzylpenicillin and 4-Phenylbutylanzine) of penicillin acylase (EC 3.5.1.11) were attached to hydrophilic gels like Sepharose 4B-CNBr and Minileak 'medium'. Ampicillin and 4-Phenylbutylamine were the affinity ligands that presented the higher concentrations attached to both gels. Penicillin acylase adsorption on these affinity gels was mainly dependent on the activated group of the gel, the affinity ligand attached and the experimental conditions of enzyme adsorption. Under affinity conditions only the ligands Amoxycillin, Ampicillin and 4-Phenylbutylamine, immobilized on Minileak, adsorbed the enzyme from osmotic shock extracts at different pH values. These affinity ligand systems were characterized by low adsorption capacities of penicillin acylase activity (1.2-2.1 IU mL-1 gel) and specific activity (1.5-2.9 IU mg-1 prot). Under pseudo-affinity conditions all the ligands attached both activated to gels (Sepharose 4B-CNBr and Minileak) adsorbed the enzyme. The affinity gels were characterized by higher values of adsorption capacity (3.7 and 55.6 IU mL-1 gel) and adsorbed specific activity (2.0 and 6.1 IU mg-1 prot) than those observed under affinity conditions. The space arm of Minileak gel, shown to be fundamental to enzyme adsorption under affinity conditions, preferentially adsorbed proteins in relation to the enzyme under pseudo-affinity conditions. However, this effect was partially minimized when the gel was derivatized by the affinity ligands at concentrations higher than 6 mumol mL-1 gel. Ampicillin was the affinity ligand that presented the best results for specific adsorption of penicillin acylase under affinity and pseudo-affinity adsorption processes. The Sepharose 4B-CNBr derivatized gel also presented a good adsorption capacity of enzyme activity (26.8 IU mL-1 gel) under pseudo-affinity adsorption processes.
Fonseca
L P
LP
Laboratório de Engenharia Bioquímica, Instituto Superior Técnico, Lisboa, Portugal.
Cabral
J M
JM
D000327:Adsorption; D000345:Affinity Labels; D000658:Amoxicillin; D000667:Ampicillin; D002846:Chromatography, Affinity / Q000379:methods*; D004926:Escherichia coli / Q000201:enzymology; D019856:Ethanolamine; D004983:Ethanolamines; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D008956:Models, Chemical; D010397:Penicillanic Acid / Q000031:analogs & derivatives; D010399:Penicillin Amidase / Q000302:isolation & purification*; D010400:Penicillin G; D012685:Sepharose / Q000031:analogs & derivatives
C034962:4-phenylbutylamine-sepharose 4B; D000345:Affinity Labels; D004983:Ethanolamines; D008024:Ligands; D019856:Ethanolamine; D000667:Ampicillin; D000658:Amoxicillin; D010397:Penicillanic Acid; D012685:Sepharose; D010399:Penicillin Amidase; D010400:Penicillin G; C004408:aminopenicillanic acid