aspartate or arginine residues involved in the formation of compound I. Despite
having low similarity among sequences of the family except for a highly conserved
GXXDG motif, the general structure of DyPs shares common features as a
two-domain protein with unique ferredoxin-like fold, comprising a-helices and
anti-parallel b-sheets with the heme ligand located in between the domains. LRET
was also suggested as the mechanism for DyPs to oxidize large substrates such as
lignin [49].
Bacterial DyPs have been heterologously expressed in E. coli, which enables the
improvement of expression yields and the possibility of protein engineering [49,
51]. A recent attempt of directed evolution of a bacterial DyP from Pseudomonas
putida MET94 has led to a variant with significant improvement in activity towards
2,6-dimethoxyphenol and lignin model compounds, as well as higher optimal pH
(pH 8.5) and increased tolerance to hydrogen peroxide [51].
2.2 Laccases (EC 1.10.3.2)
Laccases (benzenediol:oxygen oxidoreductases) belong to a diverse enzyme
superfamily called multicopper oxidase (MCO), also including ascorbate oxidase,
bilirubin oxidase, ferroxidase, nitrite reductase, and ceruloplasmin. A wide range of
substrates can be oxidized by laccases through the four-electron reduction of
dioxygen and the formation of two water molecules as byproducts [52, 53]. Biological functionalities of laccases are diverse, apart from lignin degradation,
including cell wall formation, detoxification, morphogenesis, etc. Laccase enzymes
are widespread and can be found in fungi, bacteria, plants, and insects [21, 52].
Ligninolytic fungal laccases have been abundantly found in Basidiomycetes,
notably in white-rot fungi [24], while a limited number of bacterial laccases has
been reported to be active towards lignin/depolymerized lignin and lignin model
compounds, including enzymes from Pseudomonas spp., Rhodococcus spp.,
Amycolatopsis sp., and Sphingobium sp.[54]. Laccases from the white-rot fungus T.
versicolor, CotA from Bacillus sp., and small laccase from Streptomyces coelicolor
(SLAC) are notable enzymes that have been extensively studied. The redox
potentials of laccases are lower than those of peroxidases (0.4–0.8 V instead of 0.8–
1.2 V, respectively, versus normal hydrogen electrode (NHE)), and phenolic substrates with lower redox potentials can be oxidized directly by laccases. In the case
of non-phenolic compounds with higher redox potentials or larger molecules such
as lignin and lignin fragments, laccases can oxidize the substrates through small
aromatic compounds acting as mediators (laccase-mediator-system—LMS). Fungal
laccases generally have higher redox potentials than bacterial ones and can oxidize
lignin substrates such as Kraft lignin without the need for mediators. The most
prominent examples are the laccase isoenzymes from T. versicolor [55]. A comprehensive survey of the substrate scope of laccases and laccase-like MCOs can be
found in a study by Reiss et al. [53], in which the activities of 11 laccases from
plant, fungi, and bacteria were tested against more than 90 compounds.
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G.-S. Nguyen et al.
having low similarity among sequences of the family except for a highly conserved
GXXDG motif, the general structure of DyPs shares common features as a
two-domain protein with unique ferredoxin-like fold, comprising a-helices and
anti-parallel b-sheets with the heme ligand located in between the domains. LRET
was also suggested as the mechanism for DyPs to oxidize large substrates such as
lignin [49].
Bacterial DyPs have been heterologously expressed in E. coli, which enables the
improvement of expression yields and the possibility of protein engineering [49,
51]. A recent attempt of directed evolution of a bacterial DyP from Pseudomonas
putida MET94 has led to a variant with significant improvement in activity towards
2,6-dimethoxyphenol and lignin model compounds, as well as higher optimal pH
(pH 8.5) and increased tolerance to hydrogen peroxide [51].
2.2 Laccases (EC 1.10.3.2)
Laccases (benzenediol:oxygen oxidoreductases) belong to a diverse enzyme
superfamily called multicopper oxidase (MCO), also including ascorbate oxidase,
bilirubin oxidase, ferroxidase, nitrite reductase, and ceruloplasmin. A wide range of
substrates can be oxidized by laccases through the four-electron reduction of
dioxygen and the formation of two water molecules as byproducts [52, 53]. Biological functionalities of laccases are diverse, apart from lignin degradation,
including cell wall formation, detoxification, morphogenesis, etc. Laccase enzymes
are widespread and can be found in fungi, bacteria, plants, and insects [21, 52].
Ligninolytic fungal laccases have been abundantly found in Basidiomycetes,
notably in white-rot fungi [24], while a limited number of bacterial laccases has
been reported to be active towards lignin/depolymerized lignin and lignin model
compounds, including enzymes from Pseudomonas spp., Rhodococcus spp.,
Amycolatopsis sp., and Sphingobium sp.[54]. Laccases from the white-rot fungus T.
versicolor, CotA from Bacillus sp., and small laccase from Streptomyces coelicolor
(SLAC) are notable enzymes that have been extensively studied. The redox
potentials of laccases are lower than those of peroxidases (0.4–0.8 V instead of 0.8–
1.2 V, respectively, versus normal hydrogen electrode (NHE)), and phenolic substrates with lower redox potentials can be oxidized directly by laccases. In the case
of non-phenolic compounds with higher redox potentials or larger molecules such
as lignin and lignin fragments, laccases can oxidize the substrates through small
aromatic compounds acting as mediators (laccase-mediator-system—LMS). Fungal
laccases generally have higher redox potentials than bacterial ones and can oxidize
lignin substrates such as Kraft lignin without the need for mediators. The most
prominent examples are the laccase isoenzymes from T. versicolor [55]. A comprehensive survey of the substrate scope of laccases and laccase-like MCOs can be
found in a study by Reiss et al. [53], in which the activities of 11 laccases from
plant, fungi, and bacteria were tested against more than 90 compounds.
452
G.-S. Nguyen et al.