Operational stability of immobilised horseradish peroxidase in mini-packed bed bioreactors

Abstract

Mini-packed bed bioreactors containing horseradish peroxidase (HRP) immobilised on alkylamine controlled pore glass (CPG) were assembled for monitoring and quantification of hydrogen peroxide (H 2O 2), using a flow injection analysis (FIA) system. Samples (25 μl) were injected in a carrier stream containing the HRP reducing substrates, phenol-4-sulfonic acid (PSA) and 4-aminoantipyrine (4-AAP). A linear response of the flow system was obtained for concentrations of H 2O 2 lower than 11 mM. Different immobilisation protocols [e.g., covalent binding using glutaraldehyde and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimine (EDC) hydrochloride and adsorption followed by cross-linking] were tested in order to obtain high operational stabilities. High operational stabilities were obtained when HRP was covalently immobilised using glutaraldehyde (less than 3% of the initial conversion was lost after 24 h of continuous operation). EDC-bound HRP however showed a lower operational stability (40% of the initial conversion was already lost after 24 h of continuous operation). HRP was also adsorbed on the surface of CPG and further cross-linked with glutaraldehyde. When a washing step was included before the cross-linking step, the bioreactors rapidly lost their initial activity. The elimination of the washing step increased the amount of protein loaded and the initial conversion of the bioreactors. Furthermore, only 10% of the initial conversion was lost after 20 h of continuous operation at 32 °C. HRP glycans were oxidised with sodium periodate in order to introduce aldehyde groups, highly reactive towards primary amino groups. This technique allowed a direct coupling between the oxidised enzyme and the support, although EDC was also used to mediate this coupling. Both immobilised preparations showed high protein loadings (31 and 65 mg/g, respectively) and high operational stabilities (only 8% of the initial conversion loss during 24 h). This technique led to the formation of HRP homoconjugates (dimers and trimers).