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Sulfhydryl oxidases (SOX) are FAD-dependent enzymes capable of oxidising free thiol groups and forming disulphide bonds. Although the quantity of scientific papers and suggested applications for SOX is constantly increasing, only a limited number of microbial SOX have been reported and are commercially available. Hence, the aim of this study was to develop a fast and reliable qualitative plate test for screening novel secreted fungal SOX. The screening was based on the Ellman's reagent, i.e. 5,5'-dithiobis[2-nitrobenzoic acid]. Altogether, 32 fungal strains from an in-house culture collection were screened. A total of 13 SOX-producing strains were found positive in the plate test screen. The novel SOX producers were Aspergillus tubingensis, Chaetomium globusum, Melanocarpus albomyces, Penicillium aurantiogriseum, Penicillium funiculosum, Coniophora puteana and Trametes hirsuta. Six of the discovered SOX were partially characterised by determination of isoelectric point, pH optimum and substrate specificity. A. tubingensis was identified as the most efficient novel SOX producer.

Enzymatic crosslinking provides valuable means for modifying functionality and structural properties of different polymers. Tyrosinases catalyze the hydroxylation of various monophenols to the corresponding o-diphenols, and the subsequent oxidation of o-diphenols to the corresponding quinones, which are highly reactive and can further undergo non-enzymatic reactions to produce mixed melanins and heterogeneous polymers. Tyrosinases are also capable of oxidizing protein- and peptide-bound tyrosyl residues, resulting in the formation of inter- and intra-molecular crosslinks. Tyrosinases from apple (AT), potato (PT), the white rot fungus Pycnoporus sanguineus (PsT), the filamentous fungus Trichoderma reesei (TrT) and the edible mushroom Agaricus bisporus (AbT) were compared for their biochemical characteristics. The enzymes showed different features in terms of substrate specificity, stereo-specificity, inhibition, and ability to crosslink the model protein, alpha-casein. All enzymes were found to produce identical semiquinone radicals from the substrates as analyzed by electron spin resonance spectroscopy. The result suggests similar reaction mechanism between the tyrosinases. PsT enzyme had the highest monophenolase/diphenolase ratio for the oxidation of monophenolic L-tyrosine and diphenolic L-dopa, although the tyrosinases generally had noticeably lower activity on monophenols than on di- or triphenols. The activity of AT and PT on tyrosine was particularly low, which largely explains the poor crosslinking ability of the model protein alpha-casein by these enzymes. AbT oxidized peptide-bound tyrosine, but was not able to crosslink alpha-casein. Conversely, the activity of PsT on model peptides was relatively low, although the enzyme could crosslink alpha-casein. In the reaction conditions studied, TrT showed the best ability to crosslink alpha-casein. TrT also had the highest activity on most of the tested monophenols, and showed noticeable short lag periods prior to the oxidation.

Aims: To discover novel laccases potential for industrial applications., Methods and Results: Fungi were cultivated on solid media containing indicator compounds that enabled the detection of laccases as specific colour reactions. The indicators used were Remazol Brilliant Blue R (RBBR), Poly R-478, guaiacol and tannic acid. The screening work resulted in isolation of 26 positive fungal strains. Liquid cultivations of positive strains confirmed that four efficient laccase producers were found in the screening. Biochemical characteristics of the four novel laccases were typical for fungal laccases in terms of molecular weight, pH optima and pI. The laccases showed good thermal stability at 60 degrees C., Conclusions: Plate-test screening based on polymeric dye compounds, guaiacol and tannic acid is an efficient way to discover novel laccase producers. The results indicated that screening for laccase activity can be performed with guaiacol and RBBR or Poly R-478., Significance and Impact of the Study: Laccases have many potential industrial applications including textile dye decolourization, delignification of pulp and effluent detoxification. It is essential to find novel, efficient enzymes to further develop these applications. This study showed that relatively simple plate test screening method can be used for discovery of novel laccases., (Copyright 2004 The Society for Applied Microbiology)

A strain of Peniophora species was shown to produce laccase, which is a new feature for this species. Peniophora secreted several laccase isoforms with pI-values 3.7–4.2 when grown on glucose and soya meal medium. The Peniophora laccases were typical for Basidiomycetes with respect to pH-dependence, specific activity and inhibitors. The interesting exceptional feature of Peniophora laccases was their good thermostability. At 60 °C, the half-life of the isoforms in the culture filtrate was approximately 5 h and at 70 °C for 15 min. Also, the isolated main isoform with pI-value 4.1 and molecular mass 63 kDa was similarly thermostable.The N-terminal amino acid sequence, altogether 22 amino acid residues, of the separated isoform was 50–63% identical with those of the Basidiomycete laccases analyzed recently. [Copyright &y& Elsevier]

Abstract: Growth of Irpex lacteus on polyurethane solid support led to secretion of various manganese-dependent peroxidase (MnP) isoenzymes with pI in the range of 3.8–6.7. The highest MnP activity was obtained at 8.9mmoll−1 Mn2+ when predominantly a pI 4.8 isoenzyme was produced. Optimization of anion exchange chromatography (Mono Q) allowed the separation of MnP isoenzymes and collection of extensive sequence information by tandem LC–MS. The pI 4.8 isoenzyme was purified 109-fold (6% activity yield) by ion exchange chromatography, its specific activity of Mn2+ oxidation was 2800nkatmg−1, molecular mass 37kDa and pH optimum 5. The other properties were: respective half-lives (pH 4.5) of 575, 325 and 7min at 40, 50 and 60°C, K m values 46.7; 9.5; 21.4μmoll−1 and k cat values 69.0; 64.2; 15.7s−1 for Mn2+, H2O2 and DMP with Mn2+, respectively. The pI 4.8 isoenzyme was shown to be a true MnP affecting secondary substrates via the oxidation of Mn2+. Its biochemical properties and production by I. lacteus make the enzyme a candidate for biotechnological applications. [Copyright &y& Elsevier]