As presented in the previous section, much progress has been made in the
discovery, characterization, heterologous production, and improvement of enzymes
active on lignin, and the recent developments in the fields of metagenomics and
bioinformatics, triggered by plunging prices for high throughput sequencing since
the millennium, can be expected to accelerate this development. Except for the
etherases, all ligninolytic enzyme families known today use oxidative radical
chemistry for their function. This in principle allows their application also on the
bulk of highly modified lignin from large-scale industrial applications, like pulp and
paper, if leaving out the key complicating aspects of high condensation level (low
share of cleavable ether bonds) and low solubility at enzyme relevant pH of these
substrates, as well as the presence of inhibitors of enzyme function, contributing to
low yields in enzymatic depolymerization. In any case and as also relevant for
chemo-catalytic approaches of lignin depolymerization, this comes with the challenge of highly reactive intermediates that tend to react immediately with other
fragments within the substrate mix, leading to re-polymerization in competition
with lignin breakdown [152]. Also here, the condensation through the formation of
highly resistive C–C linkages needs to be minimized to maintain the possibility of
high yield recovery of desired LMW products. Besides the need for preventing
similar effects already during biomass pre-treatment and lignin recovery alongside
preserving the native, ether-bond rich lignin structure as much as possible, this can
be achieved by chemical quenching or rapid physical removal of the reactive
intermediates [10]. Chemical quenching can theoretically be achieved by scavengers that convert reactive intermediates to stable molecules before they can
cross-link with other lignin fragments. Examples for scavengers explored in thermochemical lignin degradation are phenol, short-chain acids and alcohols, and
formaldehyde [153–157]. However, the compatibility of such compounds with
enzymatic lignin degradation processes needs to be carefully examined. Membrane
technology separating lignin monomers close to their formation provides another
option for achieving more efficient lignin depolymerization. In a recently published
study involving commercial laccase from Aspergillus sp. (Novozym, 51,003),
depolymerization into fragments below 1 kDa was effective in a bioreactor with
in-situ membrane extraction of reactive fragments of lignin from the bulk medium,
providing a first successful proof-of-concept of that approach against detrimental
repolymerization [158].
It is clear from the above mentioned that the issue of low conversion yields from
enzymatic depolymerization of lignin involves improvements on several levels.
One key is that lignocellulose processing in an integrated biorefinery setting needs
to take the quality of the lignin into consideration from the start and prevent as
much as possible condensation and formation of recalcitrant C–C bonds at the steps
of pre-treatment and fractionation. The resulting more native lignin then represents
a much better substrate for both chemo- and biocatalytic depolymerization into low
molecular weight compounds. This is particularly true for enzymes in environmental microorganisms that have evolved to efficiently attack lignin in its natural
form as a source of carbon for their metabolism (see Sect. 4). The efficiency of
natural enzymes already known or to be discovered in the future (obtained primarily
Recent Advances in Enzymatic Conversion of Lignin …
459
discovery, characterization, heterologous production, and improvement of enzymes
active on lignin, and the recent developments in the fields of metagenomics and
bioinformatics, triggered by plunging prices for high throughput sequencing since
the millennium, can be expected to accelerate this development. Except for the
etherases, all ligninolytic enzyme families known today use oxidative radical
chemistry for their function. This in principle allows their application also on the
bulk of highly modified lignin from large-scale industrial applications, like pulp and
paper, if leaving out the key complicating aspects of high condensation level (low
share of cleavable ether bonds) and low solubility at enzyme relevant pH of these
substrates, as well as the presence of inhibitors of enzyme function, contributing to
low yields in enzymatic depolymerization. In any case and as also relevant for
chemo-catalytic approaches of lignin depolymerization, this comes with the challenge of highly reactive intermediates that tend to react immediately with other
fragments within the substrate mix, leading to re-polymerization in competition
with lignin breakdown [152]. Also here, the condensation through the formation of
highly resistive C–C linkages needs to be minimized to maintain the possibility of
high yield recovery of desired LMW products. Besides the need for preventing
similar effects already during biomass pre-treatment and lignin recovery alongside
preserving the native, ether-bond rich lignin structure as much as possible, this can
be achieved by chemical quenching or rapid physical removal of the reactive
intermediates [10]. Chemical quenching can theoretically be achieved by scavengers that convert reactive intermediates to stable molecules before they can
cross-link with other lignin fragments. Examples for scavengers explored in thermochemical lignin degradation are phenol, short-chain acids and alcohols, and
formaldehyde [153–157]. However, the compatibility of such compounds with
enzymatic lignin degradation processes needs to be carefully examined. Membrane
technology separating lignin monomers close to their formation provides another
option for achieving more efficient lignin depolymerization. In a recently published
study involving commercial laccase from Aspergillus sp. (Novozym, 51,003),
depolymerization into fragments below 1 kDa was effective in a bioreactor with
in-situ membrane extraction of reactive fragments of lignin from the bulk medium,
providing a first successful proof-of-concept of that approach against detrimental
repolymerization [158].
It is clear from the above mentioned that the issue of low conversion yields from
enzymatic depolymerization of lignin involves improvements on several levels.
One key is that lignocellulose processing in an integrated biorefinery setting needs
to take the quality of the lignin into consideration from the start and prevent as
much as possible condensation and formation of recalcitrant C–C bonds at the steps
of pre-treatment and fractionation. The resulting more native lignin then represents
a much better substrate for both chemo- and biocatalytic depolymerization into low
molecular weight compounds. This is particularly true for enzymes in environmental microorganisms that have evolved to efficiently attack lignin in its natural
form as a source of carbon for their metabolism (see Sect. 4). The efficiency of
natural enzymes already known or to be discovered in the future (obtained primarily
Recent Advances in Enzymatic Conversion of Lignin …
459