Abstract
The robustness of a high-redox potential laccase has been enhanced by swapping its second cupredoxin domain with that from another fungal laccase, which introduced a pool of neutral mutations in the protein sequence without affecting enzyme functionality. The new laccase showed outstanding stability to temperature, pH (2–9) and to organic solvents, while maintaining the ability to oxidize high-redox potential substrates. By engineering the signal peptide, enzyme secretion levels in Saccharomyces cerevisiae were increased, which allowed to purify the engineered enzyme for further characterization. The purified domain-swap laccase presented higher activity in the presence of ethanol or methanol, superior half-lives at 50–70 °C, improved stability at acidic pH, and similar catalytic efficiency for DMP albeit a lower one for ABTS (due to a shift in optimum pH). A new N-glycosylation site and a putative new surface salt-bridge were evaluated as possible determinants for the improved stability by site-directed mutagenesis. Although neither seemed to be strictly responsible for the improved thermostability, the new salt bridge was found to notably contribute to the high stability of the swapped enzyme in a broad pH range. Finally, the application potential of the new laccase was demonstrated with the enzymatic treatment of kraft lignin, an industrially relevant lignin stream, at high temperature, neutral pH and short incubation times.
Highlights
Laccases, belonging to the superfamily of blue-multicopper oxidases, are enzymes capable of oxidizing a broad range of phenols, aromatic amines and other chemical compounds with the concomitant reduction of molecular oxygen to water
We have described the engineering of chimeric laccase variants by random DNA shuffling of two high-redox potential laccases actively expressed in Saccharomyces cerevisiae[22]
We decided to exchange D2 from OB1 for that of 3PO, as previous random chimeragenesis had shown that chimeric laccases more similar to OB1 were more active and stable[22]
Summary
Laccases, belonging to the superfamily of blue-multicopper oxidases, are enzymes capable of oxidizing a broad range of phenols, aromatic amines and other chemical compounds with the concomitant reduction of molecular oxygen to water They are typically monomeric proteins that consist of three cupredoxin domains (D1, D2 and D3) and present four catalytic copper ions: one T1 copper which accepts one electron from the reducing substrate; and one T2 and two T3 coppers that form a trinuclear cluster and transfer four electrons to O2 to render two molecules of H2O. The domain-swap laccase presents a remarkable increase in stability to high temperature, acidic pH and organic co-solvents Possible causes for this enhancement are evaluated, and the application potential of this new laccase is demonstrated with the enzymatic treatment of kraft lignin
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