cycle of VPs is similar to that of LiPs. Due to having both, a manganese oxidation
site like the one in MnPs and a surface catalytic tryptophan like in LiPs in their
structures, VPs are able to oxidize not only lignin and compounds with
high/medium redox potentials but also dyes such as Reactive Black 5 [21, 36, 46].
Studies on different LRET pathways of VP isoenzymes from P. eryngii have
demonstrated the importance of the surface tryptophan residue, W164 in this case,
for the ability of the enzyme to oxidize high redox aromatic substrates [47].
Heterologous production of VPs from P. eryngii has been successfully carried
out in different types of hosts, including fungi (e.g., P. chrysosporium with the best
yield around 300 mg/L, and Pleurotus ostreatus), filamentous fungi (Emericella
nidulans and Aspergillus niger), yeasts (S. cerevisiae), and bacteria (E. coli BL21
(DE3), as thioredoxin fusion protein). Due to their substrate promiscuity, VPs are of
high interest for industrial applications, leading to efforts in protein engineering to
improve their stability and activity. To improve pH stability of the isoenzyme VPL2
from P. eryngii, a variant with the desired characteristic was obtained by
site-directed mutagenesis guided by sequence alignment analysis and comparison
of structures of the enzyme with the isoenzyme MnP4 from P. ostreatus [46].
A directed evolution strategy was employed to obtain variants of P. eryngii VP
expressed in S. cerevisiae with higher tolerance against H 2 O 2 and improved activity
[21, 48].
2.1.4 Dye-Decolorizing Peroxidases (DyPs) (EC 1.11.1.19)
DyPs belong to a new heme peroxidase family that shares no similarity in sequence
and structure with other families like LiPs, MnPs, and VPs. Though the first
identified DyP is from the fungus Bjerkandera adusta and similar enzymes have
later been found in other fungi such as Irpex lacteus and Termitomyces albuminosus, these enzymes are more commonly found in bacteria, notably in Proteobacteria (Pseudomonas spp.) and in Actinobacteria such as Rhodococcus
spp. and Thermobifida fusca [21, 49]. Recently, the crystal structure of a DyP-type
peroxidase from Thermobifida fusca (TfuDyP) was resolved. The enzyme itself
showed activity against kraft lignin and a b-aryl ether lignin model compound [50].
Currently, DyPs are divided into four subfamilies in which type A enzymes are
mostly extracellular bacterial enzymes having a Tat-dependent signal peptide. DyPs
belonging to types B and C are putative cytoplasmic bacterial or fungal enzymes
whose roles could be part of the intracellular metabolism, while type D DyPs are
primarily from fungi [21, 49]. Recently, a new classification of DyPs has been
suggested based on structural alignments that indicated only three classes, i.e., P
(primitive)—former class B, I (intermediate)—former class A, and V (advanced)—
former class C and D [34].
Owning their name to the ability of oxidizing various dyes, especially anthraquinone dyes, in addition to common peroxidase substrates at low pH, some DyPs
apparently displayed ligninolytic activity towards lignin model compounds even
though their role in the degradation of lignin in nature is still unclear [20, 49]. Some
DyPs even exhibit promiscuous hydrolyzing or oxygenase activity [49]. The catalytic cycle of DyPs is not yet fully understood, but there is evidence of conserved
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site like the one in MnPs and a surface catalytic tryptophan like in LiPs in their
structures, VPs are able to oxidize not only lignin and compounds with
high/medium redox potentials but also dyes such as Reactive Black 5 [21, 36, 46].
Studies on different LRET pathways of VP isoenzymes from P. eryngii have
demonstrated the importance of the surface tryptophan residue, W164 in this case,
for the ability of the enzyme to oxidize high redox aromatic substrates [47].
Heterologous production of VPs from P. eryngii has been successfully carried
out in different types of hosts, including fungi (e.g., P. chrysosporium with the best
yield around 300 mg/L, and Pleurotus ostreatus), filamentous fungi (Emericella
nidulans and Aspergillus niger), yeasts (S. cerevisiae), and bacteria (E. coli BL21
(DE3), as thioredoxin fusion protein). Due to their substrate promiscuity, VPs are of
high interest for industrial applications, leading to efforts in protein engineering to
improve their stability and activity. To improve pH stability of the isoenzyme VPL2
from P. eryngii, a variant with the desired characteristic was obtained by
site-directed mutagenesis guided by sequence alignment analysis and comparison
of structures of the enzyme with the isoenzyme MnP4 from P. ostreatus [46].
A directed evolution strategy was employed to obtain variants of P. eryngii VP
expressed in S. cerevisiae with higher tolerance against H 2 O 2 and improved activity
[21, 48].
2.1.4 Dye-Decolorizing Peroxidases (DyPs) (EC 1.11.1.19)
DyPs belong to a new heme peroxidase family that shares no similarity in sequence
and structure with other families like LiPs, MnPs, and VPs. Though the first
identified DyP is from the fungus Bjerkandera adusta and similar enzymes have
later been found in other fungi such as Irpex lacteus and Termitomyces albuminosus, these enzymes are more commonly found in bacteria, notably in Proteobacteria (Pseudomonas spp.) and in Actinobacteria such as Rhodococcus
spp. and Thermobifida fusca [21, 49]. Recently, the crystal structure of a DyP-type
peroxidase from Thermobifida fusca (TfuDyP) was resolved. The enzyme itself
showed activity against kraft lignin and a b-aryl ether lignin model compound [50].
Currently, DyPs are divided into four subfamilies in which type A enzymes are
mostly extracellular bacterial enzymes having a Tat-dependent signal peptide. DyPs
belonging to types B and C are putative cytoplasmic bacterial or fungal enzymes
whose roles could be part of the intracellular metabolism, while type D DyPs are
primarily from fungi [21, 49]. Recently, a new classification of DyPs has been
suggested based on structural alignments that indicated only three classes, i.e., P
(primitive)—former class B, I (intermediate)—former class A, and V (advanced)—
former class C and D [34].
Owning their name to the ability of oxidizing various dyes, especially anthraquinone dyes, in addition to common peroxidase substrates at low pH, some DyPs
apparently displayed ligninolytic activity towards lignin model compounds even
though their role in the degradation of lignin in nature is still unclear [20, 49]. Some
DyPs even exhibit promiscuous hydrolyzing or oxygenase activity [49]. The catalytic cycle of DyPs is not yet fully understood, but there is evidence of conserved
Recent Advances in Enzymatic Conversion of Lignin …
451