Developing a plate test screening method and screening for fungal sulfhydryl oxidases: Master's thesis

Thesis work is part of the European Union project HIPERMAX, which is aiming at the improvent of textile fibres by enzymatic treatment to develop high quality fabrics for the textile and medical purposes. By the mean of crosslinking enzymes novel functionalities can be attached onto fibres. Such desired properties could be anti-odour or anti-microbial properties in sportwear or medical materials. Modification could be possible in protein matrices e.g. in wool, silk and leather. For example SOX can make disulphide bonds between the fibre bound cystein residues and small molecule compounds containing thiol groups. Fibres can also be strengthened by additional disulphide bonds. Use of enzymes in the textile industry is also environmentally friendly, because less chemical pollutants are used. Sulfhydryl oxidases (SOXs) catalyze the oxidation of thiol groups to disulfides with molecular oxygen as the electron acceptor. A traditional plate test screening method for SOX activity based on Ellman's reagent was developed. Ellman's reagent reacts with free sulfhydryl groups forming a colored end-product, which can be detected at 412 nm. Commercial Saccharomyces cerevisiae SOX, Erv1p, was successfully used to develop plate test screening method. The detection limit of Erv1p with dithiothreitol (DTT) as substrate was 15 ƒÝg protein or 0.08 nkat. The method was miniaturized for high throughput screening (HTS) purposes. The assay can be applied in a microtiter plate format for an activity screening. However, systematic analyses are needed to estimate reliable detection limit of the assay. In addition, genome mining for searching SOX activity from Trichoderma reesei and Phanerochaete chrysoporium was used. The plate test was successfully used for screening of SOX activity from fungi in VTT Culture Collection. Out of 32 tested fungal strains 13 SOX-positive fungi were discovered. A SOX was a common enzyme among the tested strains, especially among Aspergillus strains. All A. niger strains were SOX-positive and one out of five A. oryzae strains. SOX-positive fungi were also found from genera belonging to Penicillium, Coniophora, and Melanocarpus. DTT, glutathione and D-cystein were used as substrates in the screens. The substrate specificity of the SOX-positive strains varied remarkably. Genome mining results implied that also T. reesei could be SOX-positive fungus. In order to find more SOX-positive strains more screening should be carried out. Besides filamentous fungi also yeast and bacterial strains should be included. A metagenomic library in Escheria coli that was recently generated at VTT could also be an interesting source for screening SOX activity. Plate test and robotic screening would be the most suitable methods for screening library in the HTS format.