Page 27 - Cascade_Biocatalysis_Integrating_Stereoselective_and_Environmentally_Friendly_Reactions

1.3 The Ligninolytic Enzymatic Consortium 3

scientifically interesting and technologically useful enzymes to be designed [1–3].
Diversity is generated by introducing random mutations and/or recombination in
the gene encoding a specific protein [4, 5]. In this process, the best performers
in each round of evolution are selected and used as the parental types in a new
round, a cycle that can be repeated as many times as necessary until a biocatalyst
that exhibits the desired traits is obtained: for example, improved stability at high
temperatures, extreme pHs, or in the presence of nonconventional media such
as organic solvents or ionic fluids; novel catalytic activities; improved specificities
and/or modified enantioselectivities; and heterologous functional expression [6–8]
(Figure 1.1). Of great interest is the use of directed evolution strategies to engineer
ligninolytic oxidoreductases while employing rational approaches to understand
the mechanisms underlying each newly evolved property.

1.3
The Ligninolytic Enzymatic Consortium

Lignin is the most abundant natural aromatic polymer and the second most abun-
dant component of plant biomass after cellulose. As a structural part of the plant
cell wall, lignin forms a complex matrix that protects cellulose and hemicellulose
chains from microbial attack and hence from enzymatic hydrolysis. This recalci-
trant and highly heterogeneous biopolymer is synthesized by the dehydrogenative
polymerization of three precursors belonging to the p-hydroxycinnamyl alcohol
group: p-coumaryl, coniferyl, and sinapyl alcohols [9]. As one-third of the carbon
fixed as lignocellulose is lignin, its degradation is considered a key step in the
recycling of carbon in the biosphere and in the use of the plant biomass for
biotechnological purposes [10, 11]. Lignin is modified and degraded to different
extents by a limited number of microorganisms, mainly filamentous fungi and
bacteria. Lignin degradation by bacteria is somewhat limited and much slower
than that mediated by filamentous fungi [12, 13]. Accordingly, the only organisms
capable of completing the mineralization of lignin are the white-rot fungi, which
produce a white-colored material upon delignification because of the enrichment
in cellulose [14, 15].
Through fungal genome reconstructions, recent studies have linked the for-
mation of coal deposits during the Permo-Carboniferous period (∼260 million
years ago) with the nascent and evolution of white-rot fungi and their lignin-
degrading enzymes [16]. Lignin combustion by white-rot fungi involves a very
complex extracellular oxidative system that includes high-redox potential laccases
(HRPLs), peroxidases and unspecific peroxygenases (UPOs), H O -supplying oxi-
2 2
dases and auxiliary enzymes, as well as radicals of aromatic compounds and
oxidized metal ions that act as both diffusible oxidants and electron carriers [12, 13,
15, 17]. Although the role of each component of the consortium has been studied
extensively, many factors remain to be elucidated (Figure 1.2).
Laccases typically oxidize the phenolic units of lignin. Lignin peroxidases (LiPs)
oxidize both nonphenolic lignin structures and veratryl alcohol (VA), a metabolite
synthesized by fungi that helps LiP to avoid inactivation by H O and whose radical
2 2