P. chrysosporium, W171, has been shown to have a major role in the catalysis of
VA and other non-phenolic compounds through long-range electron transfer
(LRET). The tryptophan residue is conserved in all LiPs, and exchanging the amino
acid by site-directed mutagenesis (for example with a serine or phenylalanine),
rendered the enzyme inactive [21, 36, 39]. Regarding recombinant production of
LiPs, different LiP isoenzymes from P. chrysosporium have been expressed in
different hosts such as Escherichia coli (with additional refolding and activation
steps), Saccharomyces cerevisiae [40], and Pichia methanolica [24]. Using DNA
shuffling, Ryu et al. [41 achieved variants of lignin peroxidase from
P. chrysosporium with increased activity towards 2,4-dichlorophenol and an
89-fold increase in H 2 O 2 stability. More recently, flow cytometry was applied to
screen for a large mutant library (10
6 ) of P. chrysosporium lignin peroxidase H8 to
obtain variants with higher tolerance to H 2 O 2 [42]. One challenge of using LiPs for
lignin depolymerization in vitro is the reverse reaction of (re-)polymerization
happening at the same time. This could be overcome by methods for radical
quenching and/or rapidly removing the low molecular weight phenolic products
(see Sect. 4).
2.1.2 Manganese Peroxidases (MnPs) (EC 1.11.1.13)
MnP was originally discovered in the white-rot fungus P. chrysosporium and later
also found in other Basidiomycota (Panus tigrinus, Lenzites betulinus, Agaricus
bisporus, etc.), as well as in bacteria [21]. In the presence of H 2 O 2 , the enzyme
oxidizes Mn
2+ to Mn
3+ , which then departs from the enzyme and is complexed by
carboxylic acids (e.g., glycoxylate, malate, oxalate). The Mn
3+ complexes behave
like redox mediators and subsequently oxidize phenolic compounds from lignin
[17, 21, 24]. Having lower redox potentials than LiPs [24] (see above), MnPs
cannot oxidize non-phenolic compounds such as unsaturated fatty acids through the
process called lipid peroxidation without mediators [20]. MnPs and LiPs share
similarities in their tertiary structures as both are glycosylated two-domain globular
proteins comprised of 11–12 a-helices with the heme group located in the middle
and two Ca
2+ binding sites [21, 24]. While the structures of LiPs are stabilized by
four disulfide bridges, an additional disulfide bridge is found in MnPs in the
C-terminal region [17, 20, 43].
Fungal MnPs such as the ones from P. chrysosporium have been successfully
produced heterologously in E. coli and Pichia pastoris [24, 44]. Different attempts
in protein engineering of MnPs have been carried out to improve thermostability by
engineering a disulfide bond [45] or to increase the H 2 O 2 stability by combinatorial
mutagenesis [44].
2.1.3 Versatile Peroxidases (VPs) (EC 1.11.1.6)
A broad substrate scope, a high redox potential, and the combined molecular features of LiPs and MnPs have made the third family of ligninolytic heme peroxides
gain the name versatile peroxidases (VP). So far, VPs have only been found in the
species belonging to the genera Pleurotus and Bjerkandera, of which a VP from
Pleurotus eryngii was the first VP enzyme described [21, 24, 36]. The catalytic
450
G.-S. Nguyen et al.
VA and other non-phenolic compounds through long-range electron transfer
(LRET). The tryptophan residue is conserved in all LiPs, and exchanging the amino
acid by site-directed mutagenesis (for example with a serine or phenylalanine),
rendered the enzyme inactive [21, 36, 39]. Regarding recombinant production of
LiPs, different LiP isoenzymes from P. chrysosporium have been expressed in
different hosts such as Escherichia coli (with additional refolding and activation
steps), Saccharomyces cerevisiae [40], and Pichia methanolica [24]. Using DNA
shuffling, Ryu et al. [41 achieved variants of lignin peroxidase from
P. chrysosporium with increased activity towards 2,4-dichlorophenol and an
89-fold increase in H 2 O 2 stability. More recently, flow cytometry was applied to
screen for a large mutant library (10
6 ) of P. chrysosporium lignin peroxidase H8 to
obtain variants with higher tolerance to H 2 O 2 [42]. One challenge of using LiPs for
lignin depolymerization in vitro is the reverse reaction of (re-)polymerization
happening at the same time. This could be overcome by methods for radical
quenching and/or rapidly removing the low molecular weight phenolic products
(see Sect. 4).
2.1.2 Manganese Peroxidases (MnPs) (EC 1.11.1.13)
MnP was originally discovered in the white-rot fungus P. chrysosporium and later
also found in other Basidiomycota (Panus tigrinus, Lenzites betulinus, Agaricus
bisporus, etc.), as well as in bacteria [21]. In the presence of H 2 O 2 , the enzyme
oxidizes Mn
2+ to Mn
3+ , which then departs from the enzyme and is complexed by
carboxylic acids (e.g., glycoxylate, malate, oxalate). The Mn
3+ complexes behave
like redox mediators and subsequently oxidize phenolic compounds from lignin
[17, 21, 24]. Having lower redox potentials than LiPs [24] (see above), MnPs
cannot oxidize non-phenolic compounds such as unsaturated fatty acids through the
process called lipid peroxidation without mediators [20]. MnPs and LiPs share
similarities in their tertiary structures as both are glycosylated two-domain globular
proteins comprised of 11–12 a-helices with the heme group located in the middle
and two Ca
2+ binding sites [21, 24]. While the structures of LiPs are stabilized by
four disulfide bridges, an additional disulfide bridge is found in MnPs in the
C-terminal region [17, 20, 43].
Fungal MnPs such as the ones from P. chrysosporium have been successfully
produced heterologously in E. coli and Pichia pastoris [24, 44]. Different attempts
in protein engineering of MnPs have been carried out to improve thermostability by
engineering a disulfide bond [45] or to increase the H 2 O 2 stability by combinatorial
mutagenesis [44].
2.1.3 Versatile Peroxidases (VPs) (EC 1.11.1.6)
A broad substrate scope, a high redox potential, and the combined molecular features of LiPs and MnPs have made the third family of ligninolytic heme peroxides
gain the name versatile peroxidases (VP). So far, VPs have only been found in the
species belonging to the genera Pleurotus and Bjerkandera, of which a VP from
Pleurotus eryngii was the first VP enzyme described [21, 24, 36]. The catalytic
450
G.-S. Nguyen et al.