Abstract
BackgroundA tannic acid-inducible and mycoviral-regulated laccase3 (lac3) from the chestnut blight fungus Cryphonectria parasitica has recently been identified, but further characterization was hampered because of the precipitation of protein products by tannic acid supplementation. The present study investigated the heterologous expression of the functional laccase3 using a yeast Saccharomyces cerevisiae.ResultsLaccase activity in the culture broth of transformants measured using a laccase-specific substrate suggested that the lac3 gene was successfully expressed and the corresponding protein product secreted into the culture media. In addition, activity staining and Western blot analysis of a native gel revealed that the enzyme activity co-existed with the protein product specific to anti-laccase3 antibody, confirming that the cloned lac3 gene is responsible for the laccase activity. When transformants were grown on plates containing tannic acid-supplemented media, brown coloration was observed around transformed cells, indicating the oxidation of tannic acid. However, the enzymatic activity was measurable only in the selective ura- media and was negligible in nonselective nutrient-rich culture conditions. This was in part because of the increased plasmid instability in the nonselective media. Moreover, the protein product of lac3 appears to be sensitive to the cultured nonselective nutrient-rich broth, because a rapid decline in enzymatic activity was observed when the cultured broth of ura- media was mixed with that of nonselective nutrient-rich broth. In addition, constitutive expression of the lac3 gene resulted in a reduced cell number of the lac3 transformants compared to that of vector-only transformed control. However, the presence of recombinant vector without lac3 induction did not affect the growth of transformants.ConclusionsThe results suggest that expression of the lac3 gene has an inhibitory effect on the growth of transformed S. cerevisiae and that the controlled expression of lac3 is appropriate for the possible application of recombinant yeast to the treatment of phenolic compounds.
Highlights
A tannic acid-inducible and mycoviral-regulated laccase3 from the chestnut blight fungus Cryphonectria parasitica has recently been identified, but further characterization was hampered because of the precipitation of protein products by tannic acid supplementation
Analysis of transformed S. cerevisiae From 10 to 20 transformants of S. cerevisiae representing each recombinant plasmid were selected on uramedium
Transformation of E. coli using DNA preparations from all selected transformants confirmed the presence of the recombinant plasmid in yeast
Summary
A tannic acid-inducible and mycoviral-regulated laccase (lac3) from the chestnut blight fungus Cryphonectria parasitica has recently been identified, but further characterization was hampered because of the precipitation of protein products by tannic acid supplementation. One electron at a time is removed from the substrate by a type-1 copper ion and is transferred to the type-2/type-3 copper site, where molecular oxygen is reduced to water [14]. Because of their low substrate specificity, industrial applications for laccases include delignification [15], the purification of colored waste water [16], textile dye decoloration [17], beverage and food treatment [18], the sulfurization and solublization of coal to their use in enzyme-based biosensors [19], and the transformation and inactivation of toxic environmental pollutants [20]. Given the versatility and broad spectrum of substrate specificity, laccases could become among the most important biocatalysts in fungal biotechnology [3]
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