PARTIAL PURIFICATION OF A BASIC GUAIACIOL PEROXIDASE FROM FAVA BEAN (VICIA FABA L.): CHARACTERIZATION OF ENZYME STABILITY FOLLOWING ELICITOR TREATMENT

Abstract

Previous research in our lab investigated the stimulatory effects of phenolic elicitors on plant seed development and the production of phenolics for lignification and nutraceutically relevant antioxidants. Metabolic flux of metabolites through the pentose-phosphate pathway towards eventual lignin formation might be driven by regulated activity of the rate-limiting enzyme glucose-6-phosphate dehydrogenase (G6PDH) in response to phenolic-scavenging peroxidase activity. Through regulated activity of such enzymes, the metabolism for plant recovery and defense may be optimized under stressful conditions. As of yet, no direct correlation between stress-inducing treatments in plant cultures and possible stimulatory effects on purified enzymes involved inlignification and phenolic antioxidant pathways (specifically, peroxidases) has been reported.

In order to investigate our hypothesis of an elicitor-induced stimulatory effect on peroxidase activity, we studied partially-purified guaiacol peroxidase (GPX) enzyme extracts from fava bean (Vicia faba L.) following treatment with proline and a proline analogue, azetidine-2-carboxylic acid (A2C), and six days of dark-germination. Using preparative isoelectric focusing, GPX enzyme was purified from crude extract to a purification factor of 11.1 and a specific activity of 1.25 U/mg protein at pH 10.39. The purified basic peroxidase (pI<9.6) showedsignificant changes in an acidic shift of the enzyme's pH optimum (pH 6.1, broad, to pH 4,6, and 8), as well as a decrease in temperature stability by 25% activity following elicitor treatment in comparison to untreated extracts. These results may support the hypothesis that an ability of the peroxidase enzyme, and other enzymes perhaps, to tolerate a stress-inducedintracellular acidification may help the plant to survive periods of metabolic instability and generate phenolic compounds during stress responses.

ACKNOWLEDGMENTS

We would like to thank Professor John H. Nordin (Department of Biochemistry & Molecular Biology, University of Massachusetts, Amherst) and Preethi Shetty (Department of Nutrition, University of Massachusetts, Amherst) for helpful discussions during this work.