Fungal laccases are remarkable green catalysts that have a broad substrate specificity and many potential applications in bioremediation, lignocellulose processing, organic synthesis, and more. However, most of these transformations must be carried out at high concentrations of organic cosolvents in which laccases undergo unfolding, thereby losing their activity. We have tailored a thermostable laccase that tolerates high concentrations of cosolvents, the genetic product of five rounds of directed evolution expressed in Saccharomyces cerevisiae. This evolved laccase--R2 variant--was capable of resisting a wide array of cosolvents at concentrations as high as 50% (v/v). Intrinsic laccase features such as the redox potential and the geometry of catalytic copper varied slightly during the course of the molecular evolution. Some mutations at the protein surface stabilized the laccase by allowing additional electrostatic and hydrogen bonding to occur.

The generation of diversity for directed protein evolution experiments shows an important bottleneck in the in vitro random mutagenesis protocols. Most of them are biased towards specific changes that eventually confer a predicted and conservative mutational spectrum, limiting the exploration of the vast protein space. The current work describes a simple methodology to in vivo recombine mutant libraries with different nucleotide bias created by in vitro methods. This in vivo assembly was based on the accurate physiology of Saccharomyces cerevisiae, which as host, provided its high homologous recombination frequency to shuffle the libraries in a non-mutagenic way. The fungal thermophilic laccase from Myceliophthora thermophila expressed in S.cerevisiae was submitted to this protocol under the selective pressure of high concentrations of organic solvents. Mutant 2E9 with 3-fold better kinetics than parent type showed two consecutive amino acid changes (G614D -GGC/GAC-and E615K -GAG/AAG-) due to the in vivo shuffling of the mutant libraries. Both mutations are located in the C-terminal tail that is specifically processed at the Golgi during the maturation of the protein by the Kex2 protease. Notoriously, the oxygen consumption at the T2/T3 trinuclear copper cluster was altered and the catalytic copper at the T1 site was perturbed showing differences in its redox potential and geometry. The change in the isoelectric point of C-terminal extension upon mutations seems to affect the folding of the protein at the posttranslational processing steps providing new insights in the significance of the C-terminal tail for the functionality of the ascomycete laccases.

RL5, a gene coding for a novel polyphenol oxidase, was identified through activity screening of a metagenome expression library from bovine rumen microflora. Characterization of the recombinant protein produced in Escherichia coli revealed a multipotent capacity to oxidize a wide range of substrates (syringaldazine > 2,6-dimethoxyphenol > veratryl alcohol > guaiacol > tetramethylbenzidine > 4-methoxybenzyl alcohol > 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) Ͼ Ͼ phenol red) over an unusually broad range of pH from 3.5 to 9.0. Apparent K m and k cat values for ABTS, syringaldazine, and 2,6-dimetoxyphenol obtained from steady-state kinetic measurements performed at 40°C, pH 4.5, yielded values of 26, 0.43, and 0.45 M and 18, 660, and 1175 s ؊1 , respectively. The K m values for syringaldazine and 2,6-dimetoxyphenol are up to 5 times lower, and the k cat values up to 40 times higher, than values previously reported for this class of enzyme. RL5 is a 4-copper oxidase with oxidation potential values of 745, 400, and 500 mV versus normal hydrogen electrode for the T1, T2, and T3 copper sites. A three-dimensional model of RL5 and site-directed mutants were generated to identify the copper ligands. Bioinformatic analysis of the gene sequence and the sequences and contexts of neighboring genes suggested a tentative phylogenetic assignment to the genus Bacteroides.Kinetic, electrochemical, and EPR analyses provide unequivocal evidence that the hypothetical proteins from Bacteroides thetaiotaomicron and from E. coli, which are closely related to the deduced protein encoded by the RL5 gene, are also multicopper proteins with polyphenol oxidase activity. The present study shows that these three newly characterized enzymes form a new family of functional multicopper oxidases with laccase activity related to conserved hypothetical proteins harboring the domain of unknown function DUF152 and suggests that some other of these proteins may also be laccases.Laccases are multicopper oxidoreductases (benzenediol:oxygen oxidoreductases, EC 1.10.3.2) able to oxidize a wide variety of phenolic and nonphenolic compounds, including industrial dyes, polycyclic aromatic hydrocarbons, pesticides, and alquenes, but also capable of performing polymerization, depolymerization, methylation, and demethylation reactions (Refs.