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1.4 Directed Evolution of Laccases 7

[Fe(CN) ] 2− are also accepted by the enzyme [32]. The range of reducing substrates
6
can be further expanded to nonphenolic aromatic compounds, otherwise difficult
to oxidize, by including redox mediators from natural or synthetic sources. Upon
oxidation by the enzyme, such mediators act as diffusible electron carriers in the
so-called laccase-mediator systems [24].
Later we summarize the main advances made in the directed evolution of this
interesting group of oxidoreductases, paying particular attention to fungal laccases.

1.4.1
Directed Evolution of Low-Redox Potential Laccases

Several directed evolution studies of bacterial laccase CotA have successfully
improved its substrate specificity and functional expression, modifying its specifici-
ties by screening mutant libraries through surface display [33–37]. The advantages
of some bacterial laccases include high thermostability and activity at neu-
tral/alkaline pH, although a low-redox potential at the T1 site often precludes
their use in certain sectors.

1.4.2
Directed Evolution of Medium-Redox Potential Laccases

The first successful example of the directed evolution of fungal laccase involved
the laccase from the thermophile ascomycete Myceliophthora thermophila laccase
(MtL). This study led to subsequent directed evolution experiments in S. cerevisiae
with several high-redox potential ligninolytic oxidoreductases (see below). MtL was
subjected to 10 cycles of directed evolution to enhance its functional expression
in S. cerevisiae [38]. The best performing variant of this process (the T2 mutant
that harbored 14 mutations) exhibited a total improvement of 170-fold in activity:
its expression levels were enhanced 8-fold and the k /K around 22-fold. The
cat m
H(c2)R mutation at the C-terminal tail of MtL introduced a recognition site for
the KEX2 protease of the Golgi compartment, which facilitated its appropriate
maturation and secretion by yeast. Using this laccase expression system as a
departure point, five further cycles of evolution were performed to make the laccase
both active and stable in the presence of organic co-solvents, a property that makes
it suitable for many potential applications in organic syntheses and bioremediation
[39–42]. The stability variant (the R2 mutant) functioned in high concentrations
of co-solvents of different chemical natures and polarities (a promiscuity toward
co-solvents that was promoted during the directed evolution [40]). Most of the
mutations introduced in the evolutionary process were located at the surface of the
protein, establishing new interactions with surrounding residues, which resulted
in structural reinforcement. In the course of these 15 generations of evolution
for functional expression in yeast [38] and stabilization in the presence of organic
co-solvents [40], the laccase shifted its optimum pH toward less acidic values.
Fungal laccases that are active at neutral/alkaline pHs are highly desirable for
many applications, such as detoxification, pulp biobleaching, biomedical uses,