Urease Reutilization after Immobilization onto Magnetic Chitosan Beads

V. TUTTOLOMONDO; J. A. BERTINATTO; G. J. COPELLO; L. E. DÍAZ

Mar del Plata, Buenos Aires, Argentina

Congreso; XLIII Reunion Anual de la Sociedad Argentina de Investigacion Bioquimica y Biologia Molecular; 2007

The use of enzymes in clinical applications is often limited due to their high cost, instability and low availability. These enzymes are also soluble in aqueous media, being its recovery difficult and expensive. This restricts the use of soluble enzymes to batch operations, followed by disposal of the enzyme-containing solvent. This lead to an intense research in the field of immobilized biocatalysis. Even when immobilized enzymes often shows lower catalytic activity than free ones, they are reusable, more stable and, consequently, less costly. Our aim was to develop a versatile enzyme immobilization system for its recovery after a biological assay and its further reutilization. For this purpose urease was immobilized onto magnetic chitosan beads. Magnetic beads were obtained by coprecipitation of a chitosan, FeCl3 and FeCl2 mixture with NaOH. The beads thus generated were treated with glutaraldehyde, a bifunctional reagent that attaches to both chitosan and urease free NH2 groups. These enzyme-particle complexes are insoluble in aqueous solution and, therefore, recoverable by means of a magnet after an assay. The optimum enzyme/bead ratio was found to be 10 ¥ìg/mg. The immobilized enzyme maintains its activity during five consecutive reaction-recovery cycles. The reutilization of this system after one week storage at 4¨¬C without losing its activity demonstrated its stability.