Oxidation of 4-bromophenol by the recombinant fused protein cellulose-binding domain-horseradish peroxidase immobilized on cellulose.

A fused protein consisting of cellulose-binding domain (CBD) and horseradish peroxidase (HRP) was constructed and expressed in Escherichia coli. Refolded recombinant CBD-HRP (95% recovery yield) was bound to microcrystalline cellulose and applied for the oxidation of a model toxic phenol, 4-bromophenol (BP). Oxidation of BP by CBD-HRP resulted in the formation of dimers to pentamers as evidenced by mass spectrometry analysis. When immobilized, the vast majority of the oxidation products adsorbed to the cellulose matrix. CBD-HRP (0.75 pyrogallol units) bound to 0.1 g cellulose was packed in a column, connected to an HPLC pump and monitoring system, and column performance and capacity were studied under various operating conditions. When performance was studied as a function of BP loading rate at a constant H(2)O(2) loading rate of 1500 nmol/min, V(app) (max) and K(m) (app) were calculated to be 5.29 +/- 0.46 micromol mL min and 644.9 +/- 114.3 microM, respectively. Immobilized CBD-HRP exhibited enhanced stability to H(2)O(2) and oxidized considerably more BP than free CBD-HRP. Inclusion of gelatin, which suppresses product-dependent inactivation, further increased the amount of BP oxidation. These findings may have potential impact in terms of enzyme supply in high-rate treatment of wastewater contaminated with toxic phenols, since the susceptibility of peroxidases to both H(2)O(2) - and product-dependent inactivation demands continuous supply of fresh enzyme.