Browsing by Subject "tyrosinaasi"

The literature part of the thesis discusses about Agaricus mushrooms and the structure and function of laccase and tyrosinase. Also the potential and existing applications of these enzymes were reviewed. In experimental part the activity levels of tyrosinase and laccase were measured in two 28 day cultivations. Medium in the cultivation was 2% (w/v) malt extract broth and in other cultivations the medium was 1.5% (w/v) soybean meal. In soybean meal medium seven white rot fungi were cultivated and in malt broth six Agaricus strains were cultivated. Agaricus bisporus FBCC396 strain was cultivated in pure malt broth and with 25 µM copper addition. The laccase activity from culture media and tyrosinase activity, both monophenolase and diphenolase activities, were measured in seven different time points (day 7, 11, 14, 18, 21, 24 and 28). Tyrosinase activity was measured with bound quinone method from all samples. Diphenolase activity from samples from soybean meal cultivations was measured also with free quinone method in addition to bound quinone method. Morphological identification of all strains was confirmed with the ITS region sequencing. Also attempts to isolate the copper binding regions of laccase and tyrosinase genes were made, and the sequences from isolated copper binding regions were compared to existing data in gene databases. The highest laccase activity was measured from Phlebiopsis gigantea FBCC730 culture broth in soybean meal cultivation and in malt broth cultivation the highest laccase activity was measured from A. bisporus FBCC396 strain with the addition of copper in the culture medium. The highest tyrosinase activities were measured from Agrocybe praecox and P. gigantea mycelium extracts, which had both highest mono- and diphenolase activities. Both used tyrosinase activity measurement methods had their own difficulties and uncertainties, and neither results are absolutely reliable, mainly due to overlapping activities of phenol oxidising enzymes