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

摘要

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 (25mul) 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 l-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 32degC.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).