Characterization of a laccase present in the conidial mucilage of Colletotrichum graminicola and factors influencing the production of phenol oxidizing enzymes by the fungus

The maize and sorghum hosts of Collectotricham graminicola produce phenylpropanoids and anthocyanidins that are toxic to the fungus. The purpose of this research was to determine if the conidial mucilage of C. graminicola, isolate CgM2, conmins an enzyme capable of oxidizing these compounds. A enzyme identified as a laccase based on substrate specificity was found in the conidial mucilage. The fungus also produced a laccase in liquid cultures which appears to be identical to the laccase found in the conidial mucilage. Anion exchange and molecular sieve column chromatography were used to partially purify laccase produced by the fungus in liquid cultures. This resulted in at least a 39 fold purification of the activity present in the culture medium, or a 820 fold increase in specific activity over that contained in the conidial mucilage. The enzyme had an estimated molecular weight of 85 kDa based on SDS PAGE results. This enzyme has a copper absorbance band at 593 nm characteristic of laccase. Kinetic analysis of enzyme activity with syringaldazine as the substrate gave a V$\sb{\rm max}$ of 1.01 mM liter$\sp{-1}$ min$\sp{-1}$ and a K$\sb{\rm m}$ of $2.14 \times 10\sp{-4}$ M. Phenolic compounds with hydroxyl groups para or ortho to each other were oxidized more readily by this laccase than compounds with meta hydroxyl groups. This laccase can readily oxidize two of the phenylpropanoids, caffeic and ferulic acids, produced by maize that had been previously shown to be toxic to the fungus. It can also oxidize p-coumaric acid, the third toxic phenylpropanoid, produced by corn but at a much slower rate. This enzyme can also oxidize luteolinidin and apigeninidin, the two phytoalexins produced by sorghum. The fungus produces conidial mucilage containing laccase when grown on corn leaves. Another goal of this research was to identify factors influencing the production of phenol-oxidizing enzymes by the fungus. Three bands of phenol oxidizing enzymes were detected when the intracellular proteins produced in the fungal hypha were separated on native polyacrylamide gels. One of these bands corresponded to the laccase present in conidial mucilage. A second band containing laccase was found, while the third band had activity consistent with polyphenol oxidase or tyrosinase. Light was required for the production of all three of these enzymes. Assays of material harvested from cultures of different ages indicated that the production of these enzymes is developmentally controlled. Light was also required for melanization of hyphae and the production of conidia by isolate CgM2. CgM2-N5, a photoinsensitive strain C. graminicola, produced melanized hyphae, conidia, and the three bands of enzyme activity when grown in the dark. Laccase activity was also found inside the conidia