Purification of peroxidase from Horseradish (Armoracia rusticana) roots.
Peroxidase (EC 1.11.1.7) from horseradish ( Armoracia rusticana ) roots was purified using a simple, rapid, three-step procedure: ultrasonication, ammonium sulfate salt precipitation, and hydrophobic interaction chromatography on phenyl Sepharose CL-4B. The preparation gave an overall yield of 71%, 291-fold purification, and a high specific activity of 772 U mg(-1) protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the purified enzyme was homogeneous and had a molecular weight of approximately 40 kDa. The isolated enzyme had an isoelectric point of 8.8 and a Reinheitszahl value of 3.39 and was stable when stored in the presence of glycerol at -20 degrees C, with >95% retention of original enzyme activity for at least 6 months. Maximal activity of purified horseradish peroxidase (HRP) was obtained under different optimized conditions: substrate (guaiacol and H(2)O(2)) concentrations (0.5 and 0.3 mM, respectively), type of buffer (50 mM phosphate buffer), pH (7.0), time (1.0 min), and temperature of incubation (30 degrees C). In addition, the effect of HRP and H(2)O(2) in a neutral-buffered aqueous solution for the oxidation of phenol and 2-chlorophenol substrates was also studied. Different conditions including concentrations of phenol/2-chlorophenol, H(2)O(2), and enzyme, time, pH, and temperature were standardized for the maximal activity of HRP with these substrates; under these optimal conditions 89.6 and 91.4% oxidations of phenol and 2-chlorophenol were obtained, respectively. The data generated from this work could have direct implications in studies on the commercial production of this biotechnologically important enzyme and its stability in different media.
Lavery
Christopher B
CB
Department of Chemistry, Cape Breton University, 1250 Grand Lake Road, Sydney, Nova Scotia, Canada B1P 6L2.
Macinnis
Morgan C
MC
Macdonald
M Jason
MJ
Williams
Joanna Bassey
JB
Spencer
Colin A
CA
Burke
Alicia A
AA
Irwin
David J G
DJ
D'Cunha
Godwin B
GB
D031215:Armoracia / Q000737:chemistry / Q000201:enzymology*; D002845:Chromatography; D004795:Enzyme Stability; D007526:Isoelectric Point; D007700:Kinetics; D008970:Molecular Weight; D009195:Peroxidase / Q000737:chemistry / Q000302:isolation & purification*; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification*; D018517:Plant Roots / Q000737:chemistry / Q000201:enzymology; D013379:Substrate Specificity
D010940:Plant Proteins; D009195:Peroxidase