Trametes Hirsuta Laccase Research Articles

Effect of Protein Structure on Laccase-Catalyzed Protein Oligomerization

Laccase-catalyzed oligomerization of proteins was studied using Trametes hirsuta laccase (ThL) and coactosin as a model system. The reaction mechanism was elucidated using free amino acids and the tripeptide Gly-Leu-Tyr as substrates. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and high-performance liquid chromatography (HPLC) as well as oxygen consumption measurements and SDS-PAGE were used to study the reactions. Of the 15 selected amino acids, ThL was found to oxidize tryptophan (Trp), tyrosine (Tyr), and cysteine (Cys), of which the reactions with Tyr and Cys have been described earlier. ThL was able to link four full-length coactosins, whereas coactosin that was truncated from its C-terminus remained unpolymerized. Of the four tyrosine residues present in coactosin, only the tyrosine in the C-terminus was found to be reactive. Polymerization between tyrosine side-chains was unambiguously shown using different oligomers of Gly-Leu-Tyr as parent ions in MALDI-TOF/TOF MS fragment ion analyses.

Direct Heterogeneous Electron Transfer Reactions of Trametes hirsuta Laccase at Bare and Thiol‐Modified Gold Electrodes

AbstractDirect electron transfer reactions of high redox potential Trametes hirsuta laccase on bare and 15 different thiol‐modified gold electrodes were studied using cyclic voltammetry and potentiometry. Well‐pronounced Faradaic processes were obtained for the enzyme adsorbed on bare gold, whereas reproducible and stable electrochemistry was obtained only when 4‐aminothiophenol was used for gold modification. Moreover, the laccase‐4‐aminothiophenol‐modified gold electrode showed the highest value of the steady‐state potential under aerobic conditions equal to 660 mV vs. NHE compared with the other 15 different thiol modified electrodes and also the bare electrodes with immobilized enzyme. However, this value is still too far away from the equilibrium potential of the oxygen electrode and Trametes hirsuta laccase‐modified graphite electrode, for which a well‐pronounced high potential process of oxygen bioelectroreduction is shown at 800 mV vs. NHE. Possible mechanisms of the enzyme function on bare and thiol‐modified gold electrodes are discussed in correlation with the structure and orientation of the enzyme on the surface of the various electrodes.