Méthodes de qualification des immobilisations chimiques d'une enzyme sur un support solide

Abstract

Carbonic anhydrase was immobilized on a polymer thermoplastic to promote gaseous CO2hydration into bicarbonate ions. Catalyst immobilization was realized through a series of chemical reactions enabling enzyme covalent binding to polyamide support. Different initial enzyme concentrations of 0.25, 0.50, 0.75, 1, and 2 mg/ml were verified. Different techniques were developed to assess enzyme attachment. The amount of bound protein was determined using Bradford dosage of proteins remaining in solution following CA II incubation with solid support. ELISA has given a qualitative evaluation of the protein, enabling a follow up of enzyme binding robustness as a function of time. p-nitrophenyl acetate hydrolysis and CO2hydration were assessed by spectrophotometry and electrometry, respectively. The percentage of active enzyme following immobilization was measured using an esterase linear model. Catalytic transformation rates for gaseous CO2hydration were calculated for each type of immobilization.Key words: immobilization, enzyme, esterase, hydration.[Journal translation]