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Oxidation of 4‐bromophenol by the recombinant fused protein cellulose‐binding domain‐horseradish peroxidase immobilized on cellulose
Author(s) -
Levy Ilan,
Ward Gary,
Hadar Yitzhak,
Shoseyov Oded,
Dosoretz Carlos G.
Publication year - 2003
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.10562
Subject(s) - chemistry , horseradish peroxidase , cellulose , chromatography , microcrystalline cellulose , peroxidase , bromophenol blue , immobilized enzyme , nuclear chemistry , organic chemistry , enzyme
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 μmol mL min and 644.9 ± 114.3 μ M, 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. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 82: 223–231, 2003.