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1.5 Directed Evolution of Peroxidases and Peroxygenases 11

laccases, demonstrating the importance of accumulating neutral mutations to
create an artificial genetic drift that is beneficial to stabilize the protein structure.
Other laccase chimeragenesis experiments have been performed using laccase
isoenzymes from Trametes sp. C30, but employing a low-redox potential laccase
backbone to construct the chimeric libraries [55].
PcL and PM1L evolution aside, the lcc1 gene from Trametes versicolor laccase
(TvL) was evolved in the yeast Yarrowia lipolytica, demonstrating the potential
for directed evolution in this host [56]. More recently, the lcc2 gene from TvL
expressed by S. cerevisiae was subjected to two rounds of random mutagenesis for
improved ionic liquid resistance [57]. In addition, directed evolution experiments
have been carried out with HRPLs from Pleurotus ostreatus to enhance the laccase
activity in combination with computational approaches [58–60], and with HRPLs
from Rigidoporus lignosus to increase functional expression in Pichia pastoris [61].
Recently, the evolved PM1L was analyzed using a computational algorithm to
elucidate the physical forces that govern the thermostability of the variant [62].
Indeed, the combination of in silico computational methods (based on Monte
Carlo simulations and molecular dynamics) and directed evolution may offer new
directions to study evolved enzymes.

1.5
Directed Evolution of Peroxidases and Peroxygenases

Ligninolytic peroxidases (EC 1.11.1) are high-redox potential oxidoreductases
belonging to Class II of the plant-fungal-prokaryotic peroxidase superfamily, and
they correspond to fungal secreted heme-containing peroxidases. These enzymes
contain ∼300 amino acids distributed in 10–12 α-helix and 4–5 short β-structures
that are located in two domains. The heme-prosthetic group contains an Fe 3+ in
the resting state, and the overall structure is supported by four or five disulfide
bridges and two structural Ca 2+ ions that confer stability to the protein. The
general catalytic cycle of ligninolytic peroxidases begins with the oxidation of the
enzyme by one molecule of H O . This activates the enzyme to Compound I (a
2 2
two-electron-deficient intermediate), which under turnover conditions is reduced
back to the resting state via two successive one-electron oxidation steps. Ligninolytic
peroxidases are divided into three types [12, 13, 15, 26, 63, 64]:

(i) Lignin peroxidases (LiP, EC 1.11.1.13) are capable of directly oxidizing model
lignin dimers and nonphenolic aromatic compounds, as well as other high-
redox potential substrates (including dyes) using VA as redox mediator,
through a catalytic tryptophan located at the surface of the protein.
2+ 3+
(ii) Mn peroxidases (MnP, EC 1.11.1.14) oxidize Mn to form Mn ,which upon
chelation with organic acids can act as a diffusible oxidant for the oxidation
of phenolic compounds.
(iii) Versatile peroxidases (VP, EC 1.11.1.16) combine the catalytic properties
of LiP and MnP, and they exhibit great versatility and biotechnological
potential. VP oxidizes typical LiP substrates (e.g., VA, methoxybenzenes, and