Abstract:Background: Wood rotting white-rot and litter-decomposing basidiomycetes form a huge reservoir of oxidative enzymes, needed for applications in the pulp and paper and textile industries and for bioremediation.Objective: The aim was (i) to achieve higher throughput in enzyme screening through miniaturization and automatization of the activity assays, and (ii) to discover fungi which produce efficient oxidoreductases for industrial purposes.Methods: Miniaturized activity assays mostly using dyes as substrate were carried out for lignin peroxidase, versatile peroxidase, manganese peroxidase and laccase. Methods were validated and 53 species of basidiomycetes were screened for lignin modifying enzymes when cultivated in liquid mineral, soy, peptone and solid state oat husk medium.Results: Manganese peroxidases were the most common enzymes produced by 96% of the species. They typically had acidic pH optima, although Hyphodontia sp., Pleurotus pulmonarius and Trametes ochracea produced enzymes highly active at pH 7. Versatile peroxidase was produced by 66% of the fungi with efficient production from Phlebia radiata, P. pulmonarius and Galerina marginata. Novel lignin peroxidase producing fungi Cylindrobasidium evolvens and Daedaleopsis septentrionalis were found among the 26% of the species showing here lignin peroxidase production. Laccase was shown in 92% of the species. Several fungi produced laccase active at pH 7, which is noteworthy because usually laccases of white-rot fungi are efficient and relevant for many industrial applications. Conclusion:Automated screening allowed us to monitor many specific enzyme activities and extend the range of assay conditions from relatively small fungal cultivation sample volumes.
Effects of gadolinium (Gd) and tin (Sn) on the growth and production of oxidative enzymes with five basidiomycetous fungi were tested. For this study we have selected well-known white-rot fungi Obba rivulosa and Kuehneromyces mutabilis, in addition to this we have tested three new isolates, the white-rot fungus Phlebia subochracea, the litter-degrading fungus Gymnopus dryophilus and the brown-rot fungus Heliocybe sulcata. This approach allowed us to find possible new sources for oxidative enzymes, such as laccases and versatile peroxidases (VPs). All five tested fungi grew in the presence of Gd (0-200 mg/l) or Sn (0-200 mg/l) on ABTS (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) containing plates. The growth rate of H. sulcata was tolerant to Gd and Sn (0-200 mg/l). The growth rates of P. subochracea and G. dryophilus were sensitive to Gd (5-200 mg/l) and Sn (5-200 mg/l). O. rivulosa, K. mutabilis, P. subochracea and G. dryophilus formed colour zones on the ABTS plates indicating that these fungi produced oxidative enzymes, most probably laccases. The brown-rot fungus H. sulcata did not form colour zone on the ABTS plate indicating that this fungus did not produce laccase. The production of laccase with G. dryophilus and K. mutabilis was tolerant to Gd (0-200 mg/l) and Sn (0-200 mg/l). The production of laccase with P. subochracea was sensitive to Gd (5-200 mg/l) and Sn (5-200 mg/l). P. subochracea decolorized the dye Reactive Black 5 without or with Gd and Sn (0-200 mg/l) indicating the production of VP. O. rivulosa, K. mutabilis, G. dryophilus and H. sulcata did not produce VP. The production of VP by P. subochracea was sensitive to 200 mg/l Gd and Sn.
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