Purification and characterization of peroxidases from Withania somnifera (AGB 002) and their ability to oxidize IAA

Four types of class 111 peroxidases (EC 1.11.1.7) were detected in Withania somnifera (AGB 002) roots of 5 months old plant by zymogram. One of them showed relatively slow mobility on polyacrylamide gels under non-denaturating conditions in comparison to other three enzymes. All the four peroxidases designated as WS1, WS2, WS3 and WS4 were purified from cell free extract by FPLC using ion exchange, affinity and hydrophobic columns. The purity of peroxidases was ascertained by SDS-PAGE and spectral analysis. Purified peroxidases were shown to be monomer glycoproteins with molecular weights between 34 and 48 kDa and pI between 3.6 and 4.8. All the peroxidases were optimally active at pH 5.0 and highly thermotolerant and stable in the pH range of 3-9. Three of the four peroxidases exhibited indole-3-acetic acid oxidase activity with different rates and needed 2,4-DCP and manganese as cofactors but did not require H2O2 for the activity. In addition, all the peroxidases were capable of oxidizing general phenolic substrates like guaiacol, ABTS, o-dianisidine, aminoantipyrine, euginol and tyrosine.