2008

Selinheimo, Emilia

Tyrosinases and laccases are copper-containing oxidoreductases, which catalyze oxidation of various mono- and polyphenolic compounds. Tyrosinases oxidize p-monophenols and o-diphenols to quinones, whereas laccases are capable of oxidizing a larger variety of aromatic compounds, such as substituted mono- and polyphenols, aromatic amines and thiol compounds, with subsequent production of radicals. Both tyrosinase and laccase generate reaction products which are prone to react further non-enzymatically, which may lead to polymerization. In addition to the low molecular mass phenolic compounds, the phenolic moieties present in certain biopolymers are susceptible to oxidation by tyrosinase and laccase, which enables crosslinking of the biopolymers. The biochemical properties of a novel fungal tyrosinase from Trichoderma reesei (TrT) were characterized in this work. The substrate specificity and protein crosslinking ability of TrT were compared to other tyrosinases of plant and fungal origin. Furthermore, the suitability of TrT and laccase from Trametes hirsuta (ThL) was examined for hetero-crosslinking of carbohydrates and proteins and for improving wheat breadmaking quality. TrT was over-expressed in its original host under a strong cbh1 promoter and purified with a three step purification procedure, consisting of desalting by gel filtration, and cation exchange and gel filtration chromatography. The purified TrT showed a molecular weight of 43.2 kDa as analyzed by mass spectrometry. TrT was found to be processed from the C-terminus by cleavage of a peptide fragment of about 20 kDa. TrT was active both on L-tyrosine and L-dopa, thus showing typical characteristics of a true tyrosinase. TrT had broad substrate specificity, and the enzyme showed the highest activity and stability in the neutral and alkaline pH range, with an optimum at pH 9. TrT retained its activity relatively well at temperatures of 40 ºC and below. When tyrosinases from apple (AT), potato (PT), the white rot fungus Pycnoporus sanguineus (PsT) and the edible mushroom Agaricus bisporus (AbT) were compared to TrT, it was found that the tyrosinases had clearly different features in terms of substrate specificity, inhibition and their ability to crosslink the model protein ?-casein. Generally the tyrosinases had lower activity on monophenols than on di- or triphenols. PsT had the highest monophenolase/diphenolase ratio for the oxidation of monophenolic L-tyrosine and diphenolic L-dopa. However, TrT had the highest activity on most of the tested monophenols, and showed clearly shorter lag periods prior to the oxidation of the monophenols than the other enzymes. The activity of AT and PT on tyrosine was undetectable which explains the poor crosslinking ability of ?-casein by these enzymes. AbT was also unable to crosslink ?-casein, although it could oxidize tyrosine of di- and tri-peptides. Conversely, the activity of PsT on the model peptides turned out to be relatively low, although the enzyme could crosslink ?-casein. Of the analyzed tyrosinases, TrT clearly had the best ability to directly crosslink ?-casein. However, by after addition of a small molecular weight phenolic compound, L-dopa, to the reaction mixture, the other tyrosinases were also able to crosslink ?-casein. It is assumed that L-dopa acted as a bridging compound between the ?-casein subunits. The capability of the two different fungal oxidative enzymes, the TrT tyrosinase and the ThL laccase, to catalyze formation of hetero-conjugates between tyrosine side-chains of ?-casein and phenolic acids of hydrolyzed oat spelt xylan (hOSX) was studied. TrT was able to crosslink ?-casein more efficiently than ThL, whereas only ThL was able to polymerize hOSX. The radical- and quinone-mediated protein crosslinking clearly differed, which was indicated by enhancement of cr