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.
Levy
Ilan
I
The Institute of Plant Sciences and Genetics in Agriculture, The Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel.
Ward
Gary
G
Hadar
Yitzhak
Y
Shoseyov
Oded
O
Dosoretz
Carlos G
CG
D002480:Cellulase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002482:Cellulose / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006735:Horseradish Peroxidase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007220:Industrial Waste / Q000517:prevention & control; D046911:Macromolecular Substances; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D010636:Phenols / Q000737:chemistry*; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis / Q000737:chemistry; D014874:Water Pollutants, Chemical / Q000378:metabolism; D018508:Water Purification / Q000379:methods
D004800:Enzymes, Immobilized; D007220:Industrial Waste; D046911:Macromolecular Substances; D010636:Phenols; D011993:Recombinant Fusion Proteins; D014874:Water Pollutants, Chemical; D002482:Cellulose; D006735:Horseradish Peroxidase; D010544:Peroxidases; D002480:Cellulase; C032037:4-bromophenol