Blood Tolerant Laccase by Directed Evolution

Abstract

High-redox potential laccases are powerful biocatalysts with a wide range of applications in biotechnology. We have converted a thermostable laccase from a white-rot fungus into a blood tolerant laccase. Adapting the fitness of this laccase to the specific composition of human blood (above neutral pH, high chloride concentration) required several generations of directed evolution in a surrogate complex blood medium. Our evolved laccase was tested in both human plasma and blood, displaying catalytic activity while retaining a high redox potential at the T1 copper site. Mutations introduced in the second coordination sphere of the T1 site shifted the pH activity profile and drastically reduced the inhibitory effect of chloride. This proof of concept that laccases can be adapted to function in extreme conditions opens an array of opportunities for implantable nanobiodevices, chemical syntheses, and detoxification.

One-year stability for a glucose/oxygen biofuel cell combined with pH reactivation of the laccase/carbon nanotube biocathode

“…Despite the need of a laccase reactivation step at low pH, this work demonstrates the accessibility to a significant amount of power from a glucose/oxygen biofuel cell during one year. The continuous research, that especially involves the finding of novel stable enzymes for oxygen reduction at neutral pH in physiological conditions [21] or the use of local pH modification techniques [22], will certainly extend power delivery under continuous operation for in vivo applications.…”

Structure, functionality and tuning up of laccases for lignocellulose and other industrial applications

“…Laccases described in recent publications (Moreno et al, 2013;Ryu et al, 2013), are proposed to assist in the pretreatment of biomass by catalyzing the removal of inhibitors in lignocellulosic slurries, but the suggested applications of laccases range from paper and pulp bleaching (Galli et al, 2011), detergent and textile applications (bleaching purposes; Janssen et al, 2004), gelation and emulsion stabilization of food systems (Zaidel et al, 2012(Zaidel et al, , 2013, bioremediation (Galli et al, 2011) and even biomedical applications have been proposed in connection with development of a laccase tolerant to human blood components (Maté et al, 2013). Moreover, as described already 20-25 years ago, laccases can increase water-soluble degradation products by catalysis of ring opening of low molecular weight phenolic lignin model compounds (Kawai et al, 1988) as well as catalysis of C-C bond cleavage of ''synthetic lignin'' substances (Iimura et al, 1995).…”

Directed Evolution of Unspecific Peroxygenase from Agrocybe aegerita

“…S. cerevisiae is a particularly versatile vehicle for the functional expression and directed evolution of fungal genes involved in lignin modification (including laccases and peroxi-dases), and it has been used in the directed evolution of versatile peroxidases (VP) for functional expression and stabilization, whereby medium-redox-potential laccases have been engineered to confer high secretion levels and activity on organic cosolvents (14)(15)(16). More recently, this host has been used in the design of high-redox-potential laccases (HRPLs) that are active in human blood and to develop chimeric laccases with combined properties (17)(18)(19)(20). The number of protocols developed for the generation of DNA diversity in yeast is steadily increasing, and as such, the in vivo homologous-recombination machinery of the host can be used to enrich mutant libraries (21)(22)(23).…”