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Directed Evolution of Ligninolytic Oxidoreductases: from
Functional Expression to Stabilization and Beyond

Eva Garcia-Ruiz, Diana M. Mate, David Gonzalez-Perez, Patricia Molina-Espeja, Susana
Camarero, Angel T. Mart´ ınez, Antonio O. Ballesteros, and Miguel Alcalde

1.1
Introduction

The ligninolytic enzymatic consortium, formed mainly by nonspecific oxidoreduc-
tases (laccases, peroxidases, and H O -supplying oxidases), is a potentially powerful
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multipurpose tool for industrial and environmental biotechnology. In nature, these
enzymes are typically produced by basidiomycete white-rot fungi that are involved
in lignin decay. Thanks to their broad substrate specificity, high redox potential,
and minimal requirements, these enzymes have many potential applications in
the field of green chemistry, including the production of biofuels, bioremediation,
organic syntheses, pulp biobleaching, food and textile industries, and the design
of bionanodevices. The implementation of this enzymatic armoury in different
biotechnological sectors has been hampered by the lack of appropriate molecular
instruments (including heterologous hosts for directed evolution) with which to
improve their properties. Over the last 10 years, a wealth of directed evolution
strategies in combination with hybrid approaches has emerged in order to adapt
these oxidoreductases to the drastic conditions associated with many biotechnolog-
ical settings (e.g., high temperatures, the presence of organic co-solvents, extreme
pHs, the presence of inhibitors). This chapter summarizes all efforts and endeavors
to convert these ligninolytic enzymes into useful biocatalysts by means of directed
evolution: from functional expression to stabilization and beyond.

1.2
Directed Molecular Evolution
Enzymes are versatile biomolecules that exhibit a large repertory of functions
acquired over millions of years of natural evolution. Indeed, they are the fastest
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known catalysts (accelerating chemical reactions as much as 10 -fold) and are
environmentally friendly molecules, working efficiently at mild temperatures,
in water, and releasing few by-products. Moreover, they can exhibit high
enantioselectivity and chemoselectivity. Nonetheless, when an enzyme is removed

Cascade Biocatalysis: Integrating Stereoselective and Environmentally Friendly Reactions, First Edition.
Edited by Sergio Riva and Wolf-Dieter Fessner.
c 2014 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2014 by Wiley-VCH Verlag GmbH & Co. KGaA.