Identification and Sequence Comparison of Carbohydrate Oxidase Enzyme from Plant Sources

Abstract

Plants produce hydrogen peroxide (H2O2) in response to wounding, pathogenic infection or other stresses, and it serves as a signal molecule that leads to the activation of plant defense genes. One enzyme capable of producing H2O2 in plants is carbohydrate oxidase. Previous work has provided information about highly homologous carbohydrate oxidase enzymes found in lettuce, sunflower and tobacco plants (Custers et al (2004) Plant J 39(2):147-160; Carter and Thornburg (2004) Plant Physiol. 134(1):460-469). This project looks to extend the knowledge base concerning the presence and function of plant carbohydrate oxidases. To begin, a number of plants from the greenhouse at The College of St. Scholastica as well as various food crops were tested for activity with a native in-gel assay. The in-gel assay probes for the presence of H2O2 using a horseradish peroxidase coupled reaction which produces a colored conjugate on any protein band that displays activity. Activity bands were obtained in-gel for plants including rabbit's foot fern, jade plant, tomato leaf and kale. Sequence data has been obtained by tandem mass spectrometry to confirm protein identity, and sequence comparison of enzymes from the literature and other databases will be presented. In future work, cloning target enzymes and overexpressing them in a recombinant system will provide a more robust way to access usable quantities of sample, allowing for kinetic characterization of individual enzymes. These studies will advance the understanding of plant defense mechanisms that utilize carbohydrate oxidase.