A highly stable laccase obtained by swapping the second cupredoxin domain

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.

Aspects Determining the Dominance of Fomitopsis pinicola in the Colonization of Deadwood and the Role of the Pathogenicity Factor Oxalate

“…In contrast, both the completely dissociated HCl (pK a = −8.0) and the partially HC 2 O 4 − dissociated oxalate (pK a1 = 1.23; pK a2 = 4.9) inactivated laccase definitively at pH around 2.0 within 5-8 days of pre-incubation in the respective buffer solution (Section 3.3, Figure 3). Activity losses down to 3-9.5% are reported for Trametes versicolor and Pycnoporus cinnabarinus laccases incubated for 4-6 h in citrate and B&R buffer of pH 2.0 and 2.2, respectively [108,109], resembling the data of Figure 3. A 92 kDa laccase from the pH insensitive F. pinicola was stable from pH 1.5-11 [86].…”

Mapping the diversity of microbial lignin catabolism: experiences from the eLignin database

“…Specifically, the database content includes microorganisms, substrates, pathways, genes, metabolic reactions, and enzymes related to the topic (Table 1 ). So far, its prime focus has been on collecting data on microbial diversity and intracellular events; however, the database can later be expanded with extracellular enzymes and reactions (such as laccases and peroxidases), as these play an important role in microbial degradation of native lignin and can be applied for enzymatic depolymerzation of technical lignins (Bourbonnais et al 1995 ; Pardo et al 2018 ; Zhao et al 2016 ).…”

Applications of Microbial Laccases: Patent Review of the Past Decade (2009–2019)

“…The chimeric laccase from parent Pycnoporus cinnabarinus and Coriolopsis sp. laccases also showed improved stability in organic solvents [70]. The GreeDo and Lc OB-1 mutants of fungal laccase were obtained by combining computational design and directed evolution methods; these are recent examples of the improvement of activity and stability in high redox potential laccases [71,72].…”