Browsing by Subject "Laccase"

Filamentous basidiomycete fungi are an abundant source of laccase (benzedio:oxygen oxidoreductases, E.C.1.10.3.2) enzymes that oxidize phenolic compounds by a one electron removal resulting in free radicals with concomitant reduction of oxygen to water. Because of this, laccases are regarded as “green catalysts”, making them of great interest for industrial applications. Phenolic compounds of plant origin are of special interest to the food industry because of their low toxicity and high antioxidant, antimicrobial activities. Because of large number of laccase candidates present in fungal genomes and time demanding methods for their biochemical characterization, in silico methods for the prediction of their activity towards phenolic compounds are needed to be developed to better use the potential of fungal laccases in applications. The goal of this study was to utilize in silico predictions on 19 preselected basidiomycete laccases to determine their activities towards six selected phenolic compounds that are used in food related applications. Based on the structural and functional predictions, two laccases with the best docking characteristics, Pycnoporus cinnabarinus laccase 4273 and Trametes cingulata laccase 1498596, were then selected for recombinant production in the methylotrophic yeast Pichia pastoris, and their biochemical characteristics and activity towards the phenolic compounds were determined experimentally. The recombinant laccases presented optimal working pH in the acidic range from pH 2.0 to 6.0. Both laccases also presented good solvent stability in ethanol and DMSO. The T. cingulata laccase showed thermal tolerance up to 60°C while the P. cinnabarinus laccase was stable up to 40°C. Both recombinant laccases had activity towards 2,6-DMP and the phenolic compound sinapic acid, low activity (<1.9 µkat/L) was observed towards caffeic acid, and no activity (<1.1 µkat/L) was observed towards cinnamic acid, ferulic acid and p-couamaric acid. Experimental and in silico results were compared to evaluate if it is possible to accurately predict laccase activity towards the selected phenolic compounds using in silico approaches. From the in-silico predictions, it was suggested that hydrogen bonding between a substrate molecule and amino acid residue Ser-113 of P. cinnabarinus laccase 4273 and His-111 of T. cingulata laccase 1498596 may be important for the activity towards phenolic compounds. Formation of hydrogen bonds between other amino acid residues and the substrates might be detrimental for laccase activity. The predicted substrate binding site of the laccases differ from yet very close to that of crystal structures of Melanocarpus albomyces laccase. However, the activity towards selected phenolic compounds were somewhat consistent with predictions indicating a possible applicability of structural and functional predictions in the study of fungal laccase activities.