variety of) relevant expression hosts likely to be able to express encoded genes and
produce functional enzymes [149, 150]. A range of functional assays for different
ligninolytic enzyme classes is available as described in the previous section, which
can be adapted for high throughput use, e.g., in microtiter plate format on robotic
HT screening workstations using colorimetric, absorbance or HT-MS analysis, or
droplet-based screening using FACS or microfluidics based on fluorescence,
absorption or light scattering as signals for sorting and isolating positive events
(hits). After hit confirmation, identification of the gene(s) responsible for the
observed phenotype is usually done by sequencing, sequence analysis, sub-cloning,
and heterologous expression of gene candidates in a suitable host strain that can
ideally also be used for the up-scaled production and purification of the new
ligninolytic enzyme for the purpose of full characterization. This usually involves
determination of the range and optimal conditions for enzyme function (e.g., pH,
temperature), full substrate spectrum and obtained reaction products, effects of
mediators and potential co-factors (also to be considered included in screening
assays), redox potential, and ultimately protein structure.
4 Challenges of Enzymatic Lignin Depolymerization
for Value-Added Products
As previously mentioned, lignin is a challenging substrate for targeted depolymerization by biocatalysis. In fact, in addition to providing structure and
mechanical stability in higher plants, lignin serves the purpose of exactly preventing
such decay from occurring in nature. Furthermore, technical processing of lignocellulose, like thermochemical pre-treatments or the pulping processes, have for a
long time targeted a high quality of the polysaccharide fraction of the feedstock for
the production of cellulose or later the generation of sugars for the biotechnological
production of biofuels and bio-based chemicals, leaving the lignin fraction condensed, largely insoluble and chemically modified as a low-value side-product that
can barely be used for anything else than thermochemical conversion into char, oil,
gases, heat, and power [151]. Exceptions are the soluble lignosulfonates from
sulfite pulping and derivable chemical products that play a role in value creation in
integrated biorefineries connected to pulping processes. More recently, sulfur-free
approaches, like Organosolv processes enable recovery of distinct fractions of
cellulose, hemicellulose, and lignin of higher quality and therewith may provide a
better basis for value creation beyond energy products from a larger fraction of
lignin in the future, including by using enzymes. However, as most of the lignin
produced today occurs in highly modified form, development and impact of
enzymatic processes in this field are challenged by the need for processing a
substrate that is highly recalcitrant and non-natural for enzymes that have not
evolved to efficiently degrade it [36].
458
G.-S. Nguyen et al.
produce functional enzymes [149, 150]. A range of functional assays for different
ligninolytic enzyme classes is available as described in the previous section, which
can be adapted for high throughput use, e.g., in microtiter plate format on robotic
HT screening workstations using colorimetric, absorbance or HT-MS analysis, or
droplet-based screening using FACS or microfluidics based on fluorescence,
absorption or light scattering as signals for sorting and isolating positive events
(hits). After hit confirmation, identification of the gene(s) responsible for the
observed phenotype is usually done by sequencing, sequence analysis, sub-cloning,
and heterologous expression of gene candidates in a suitable host strain that can
ideally also be used for the up-scaled production and purification of the new
ligninolytic enzyme for the purpose of full characterization. This usually involves
determination of the range and optimal conditions for enzyme function (e.g., pH,
temperature), full substrate spectrum and obtained reaction products, effects of
mediators and potential co-factors (also to be considered included in screening
assays), redox potential, and ultimately protein structure.
4 Challenges of Enzymatic Lignin Depolymerization
for Value-Added Products
As previously mentioned, lignin is a challenging substrate for targeted depolymerization by biocatalysis. In fact, in addition to providing structure and
mechanical stability in higher plants, lignin serves the purpose of exactly preventing
such decay from occurring in nature. Furthermore, technical processing of lignocellulose, like thermochemical pre-treatments or the pulping processes, have for a
long time targeted a high quality of the polysaccharide fraction of the feedstock for
the production of cellulose or later the generation of sugars for the biotechnological
production of biofuels and bio-based chemicals, leaving the lignin fraction condensed, largely insoluble and chemically modified as a low-value side-product that
can barely be used for anything else than thermochemical conversion into char, oil,
gases, heat, and power [151]. Exceptions are the soluble lignosulfonates from
sulfite pulping and derivable chemical products that play a role in value creation in
integrated biorefineries connected to pulping processes. More recently, sulfur-free
approaches, like Organosolv processes enable recovery of distinct fractions of
cellulose, hemicellulose, and lignin of higher quality and therewith may provide a
better basis for value creation beyond energy products from a larger fraction of
lignin in the future, including by using enzymes. However, as most of the lignin
produced today occurs in highly modified form, development and impact of
enzymatic processes in this field are challenged by the need for processing a
substrate that is highly recalcitrant and non-natural for enzymes that have not
evolved to efficiently degrade it [36].
458
G.-S. Nguyen et al.