2.3 Etherases
As b-O-4 ether bonds account for up to 80% of the lignin intermolecular bonds [7,
8], intracellular b-etherases in the b-aryl ether degradation pathway have recently
gained attention as candidates for lignin degradation process. Several b-etherases
belonging to the glutathione-S-transferase superfamily from bacterial species such
as Sphingobium sp., Novosphingobium sp. and recently, one from the white-rot
fungi Dichomitus squalens, have been characterized [7, 17, 72]. Currently, the
number of characterized b-etherases is still limited, and the knowledge about the
role and potential of these enzymes in lignin degradation is somewhat lacking.
A recent study by Voß et al. [73] has shed more light on the diversity of bacterial
b-etherases, and conserved motifs among the enzymes were identified. Using a
peptide pattern recognition (PPR) approach for database mining, almost 100 novel
putative b-etherase sequences were found by the authors, many of them outside the
Sphingomonadaceae and Erythrobacteraceae families. A subset of 13 new
b-etherases with confirmed etherase activity were further biochemically characterized [73]. This study has thereby triggered new possibilities for finding new
b-etherases from genomic and metagenomics sequences based on sequence/motif
homology searches using, for example, profile Hidden Markov Models (HMMs).
3 Discovery of New Ligninolytic Enzymes
Several routes are available and have been applied so far to discover ligninolytic
enzymes from microbial biodiversity (Fig. 4). As presented above, fungi and bacteria have been the main source of such enzymes. In fact, most of the discoveries and
enzyme descriptions reported to date are derived from single fungal or bacterial
strain isolates or microbial consortia obtained from relevant natural environments
(e.g., rotting trees, termite guts, compost piles, etc.), showing growth and/or ligninolytic activity on or in the presence of lignin (Table 1). In addition, strain collections existing in many research groups and containing taxa later described to
be active on lignin (e.g., Actinobacteria), can be valuable sources to discover new
ligninolytic enzymes. Suitable approaches in their discovery range from simple
growth experiments (often long-term due to slow growth) on a broad spectrum of
commercially available lignin substrates to advanced conversion assays of culture
supernatants using lignin (native or technical) or lignin model substrates (e.g.,
veratryl alcohol, guaiacylglycerol-b-guaiacyl ether (GGE)) and detection of breakdown products by HPLC (increase in the low molecular weight fraction) or LC–MS
(identification of specific aromatic mono- and oligomers) [137–147]. Through
genome sequencing of active isolates and co-cultures, followed by
bioinformatics-based assembly, annotation and prediction, gene candidates putatively encoding enzymes of known lignin-active enzyme families can be identified in
silico. These are then (often in codon-optimized form) cloned in suitable expression
systems for enzyme production, purification and functional assessment (see
Recent Advances in Enzymatic Conversion of Lignin …
455
As b-O-4 ether bonds account for up to 80% of the lignin intermolecular bonds [7,
8], intracellular b-etherases in the b-aryl ether degradation pathway have recently
gained attention as candidates for lignin degradation process. Several b-etherases
belonging to the glutathione-S-transferase superfamily from bacterial species such
as Sphingobium sp., Novosphingobium sp. and recently, one from the white-rot
fungi Dichomitus squalens, have been characterized [7, 17, 72]. Currently, the
number of characterized b-etherases is still limited, and the knowledge about the
role and potential of these enzymes in lignin degradation is somewhat lacking.
A recent study by Voß et al. [73] has shed more light on the diversity of bacterial
b-etherases, and conserved motifs among the enzymes were identified. Using a
peptide pattern recognition (PPR) approach for database mining, almost 100 novel
putative b-etherase sequences were found by the authors, many of them outside the
Sphingomonadaceae and Erythrobacteraceae families. A subset of 13 new
b-etherases with confirmed etherase activity were further biochemically characterized [73]. This study has thereby triggered new possibilities for finding new
b-etherases from genomic and metagenomics sequences based on sequence/motif
homology searches using, for example, profile Hidden Markov Models (HMMs).
3 Discovery of New Ligninolytic Enzymes
Several routes are available and have been applied so far to discover ligninolytic
enzymes from microbial biodiversity (Fig. 4). As presented above, fungi and bacteria have been the main source of such enzymes. In fact, most of the discoveries and
enzyme descriptions reported to date are derived from single fungal or bacterial
strain isolates or microbial consortia obtained from relevant natural environments
(e.g., rotting trees, termite guts, compost piles, etc.), showing growth and/or ligninolytic activity on or in the presence of lignin (Table 1). In addition, strain collections existing in many research groups and containing taxa later described to
be active on lignin (e.g., Actinobacteria), can be valuable sources to discover new
ligninolytic enzymes. Suitable approaches in their discovery range from simple
growth experiments (often long-term due to slow growth) on a broad spectrum of
commercially available lignin substrates to advanced conversion assays of culture
supernatants using lignin (native or technical) or lignin model substrates (e.g.,
veratryl alcohol, guaiacylglycerol-b-guaiacyl ether (GGE)) and detection of breakdown products by HPLC (increase in the low molecular weight fraction) or LC–MS
(identification of specific aromatic mono- and oligomers) [137–147]. Through
genome sequencing of active isolates and co-cultures, followed by
bioinformatics-based assembly, annotation and prediction, gene candidates putatively encoding enzymes of known lignin-active enzyme families can be identified in
silico. These are then (often in codon-optimized form) cloned in suitable expression
systems for enzyme production, purification and functional assessment (see
Recent Advances in Enzymatic Conversion of Lignin …
455