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

Ilan Levy, Gary Ward, Yitzhak Hadar, Oded Shoseyov, Carlos G. Dosoretz*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

35 Scopus citations

Abstract

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 H2O2 loading rate of 1500 nmol/min, Vmaxapp and Kmapp were calculated to be 5.29 ± 0.46 μmol mL min and 644.9 ± 114.3 μM, respectively. Immobilized CBD-HRP exhibited enhanced stability to H2O2 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 H2O2 - and product-dependent inactivation demands continuous supply of fresh enzyme.

Original languageAmerican English
Pages (from-to)223-231
Number of pages9
JournalBiotechnology and Bioengineering
Volume82
Issue number2
DOIs
StatePublished - 20 Apr 2003

Keywords

  • 4-bromophenol
  • Cellulose binding domain
  • Horseradish peroxidase
  • Immobilization
  • Oxidation
  • Recombinant fused protein

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