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6 1 Directed Evolution of Ligninolytic Oxidoreductases

• The use of lignocellulosic materials (e.g., agricultural wastes) in the production of
second-generation biofuels (bioethanol, biobutanol) or the manufacture of new
cellulose-derived and lignin-derived value-added products.
• The organic synthesis of drugs and antibiotics, cosmetics and complex polymers,
and building blocks.
• In nanobiotechnology as (i) biosensors (for phenols, oxygen, hydroperoxides,
azides, morphine, codeine, catecholamines, or flavonoids) for clinical and envi-
ronmental applications; and (ii) biofuel cells for biomedical applications.
• In bioremediation: oxidation of polycyclic aromatic hydrocarbons (PAHs),
dioxins, halogenated compounds, phenolic compounds, benzene derivatives,
nitroaromatic compounds, and synthetic organic dyes.
• The food industry: drink processing and bakery products.
• The paper industry: pulp biobleaching, pitch control, manufacture of mechanical
pulps with low energy cost, and effluent treatment.
• The textile industry: remediation of dyes in effluents, textile bleaching (e.g.,
jeans), modification of dyes and fabrics, detergents.
A few years ago, the engineering and improvement of ligninolytic oxidoreductases
was significantly hampered by the lack of suitable heterologous hosts to carry out
directed evolution studies. Fortunately, things have changed and several reliable
platforms for the directed evolution of ligninolytic peroxidases, peroxygenases,
and several medium-redox potential laccases and high-redox potential laccases
(HRPLs) have been developed using the budding yeast Saccharomyces cerevisiae.
These advances have allowed us, for the first time, to specifically tailor ligninolytic
oxidoreductases to address new challenges.

1.4
Directed Evolution of Laccases

Laccases (EC 1.10.3.2) are extracellular glycoproteins that belong to the blue
multicopper oxidase family (along with ascorbate oxidase, ceruloplasmin, nitrite
reductase, bilirubin oxidase, and ferroxidase). Widely distributed in nature, they
are present in plants, fungi, bacteria, and insects [27, 28]. Laccases are green
catalysts, which are capable of oxidizing dozens of compounds using O from
2
air and releasing H O as their sole by-product [29–31]. These enzymes harbor
2
one type I copper (T1), at which the oxidation of the substrates takes place,
and a trinuclear copper cluster (T2/T3) formed by three additional coppers,
oneT2and twoT3s,atwhich O is reduced to H O. The reaction mechanism
2 2
resembles a battery, storing electrons from the four monovalent oxidation reactions
of the reducing substrate required to reduce one molecule of oxygen to two
molecules of H O. Laccases catalyze the transformation of a wide variety of
2
aromatic compounds, including ortho-and para-diphenols, methoxy-substituted
phenols, aromatic amines, benzenothiols, and hydroxyindols. Inorganic/organic
metal compounds are also substrates of laccases, and it has been reported that
3+
Mn 2+ is oxidized by laccase to form Mn , and organometallic compounds such