Page 36 - Cascade_Biocatalysis_Integrating_Stereoselective_and_Environmentally_Friendly_Reactions

12 1 Directed Evolution of Ligninolytic Oxidoreductases

nonphenolic model lignin compounds), as well as Mn 2+ (the classical MnP
substrate). VP contains a manganese binding site similar to that of MnP, and
a surface catalytic Trp similar to that of LiP that is involved in the oxidation of
high- and medium-redox potential compounds but that also oxidizes azo-dyes
and other nonphenolic compounds with high-redox potential in the absence
of mediators. VP also contains a third catalytic site, located at the entrance
to the heme channel, involved in the oxidation of low- to medium-redox
potential compounds (similar to generic (low-redox potential) peroxidases).
As described earlier for ligninolytic laccases, the directed evolution of high-redox
potential peroxidases has also been hindered by the absence of suitable heterologous
expression systems. Most attempts at directed peroxidase evolution have to date
been carried out using generic peroxidases. The Coprinopsis cinerea peroxidase
(CIP) was evolved to enhance its operational stability versus temperature, H O ,
2 2
and alkaline pH. S. cerevisiae was used as the expression system in the evolution
process, and the mutated variants were subsequently overexpressed in Aspergillus
oryzae [65]. These in vitro evolution studies were complemented by the resolution
of the crystal structures of both wild type and evolved CIP [66]. A few years later, the
evolution of horseradish peroxidase (HRP) for functional expression in S. cerevisiae
and overexpression in P. pastoris was described, using this system to improve
the thermal stability and resistance to H O [67]. A recent report described the
2
2
evolution of de novo designed proteins with peroxidase activity [68]. With regard to
ligninolytic peroxidases, using an in vitro expression system based on Escherichia
coli, preliminary attempts were made to enhance the oxidative stability of MnP [69].
Some years later, LiP was evolved to enhance its catalytic rate and stability by both
yeast surface display and secretion to the extracellular medium [70, 71].
VP was recently evolved for secretion, thermostabilization, and H O resistance
2
2
([72, 73] and Gonzalez-Perez, D., et al., unpublished material). First, a fusion gene
formed by the α-factor prepro-leader and the mature VP from Pleurotus eryngii
was subjected to four cycles of directed evolution to favor functional expression
−1
in S. cerevisiae, achieving secretion levels of ∼22 mg l . The secretion mutant
(R4 variant) harbored four mutations in the mature protein and increased its
total VP activity 129-fold relative to the parental type, together with a marked
improvement in catalytic efficiency at the heme channel. Although the catalytic
2+
Trp was unaltered after evolution, the Mn site was negatively affected by the
mutations. Notably, signal leader processing by the STE13 protease at the Golgi
compartment was altered as a consequence of the levels of VP expression, retaining
the additional N-terminal sequence EAEA (Glu-Ala-Glu-Ala, Figure 1.4). A similar
effect was detected with the evolved prepro-leader of the laccase OB-1 [74]. With
both enzymes, the engineered N-terminal truncated variants displayed similar
biochemical properties to those of their nontruncated counterparts, although their
secretion levels were negatively affected, probably owing to the modifications in the
acidic environment close to the KEX2 cleavage site. The R4 secretion mutant was
used as the departure point to improve thermostability [46, 72] and three additional
cycles of evolution led to a more thermostable variant (2-1B), harboring three