Fundamental features of microbial cellulose utilization are examined at successively higher levels of aggregation encompassing the structure and composition of cellulosic biomass, taxonomic diversity, cellulase enzyme systems, molecular biology of cellulase enzymes, physiology of cellulolytic microorganisms, ecological aspects of cellulase-degrading communities, and rate-limiting factors in nature. The methodological basis for studying microbial cellulose utilization is considered relative to quantification of cells and enzymes in the presence of solid substrates as well as apparatus and analysis for cellulose-grown continuous cultures. Quantitative description of cellulose hydrolysis is addressed with respect to adsorption of cellulase enzymes, rates of enzymatic hydrolysis, bioenergetics of microbial cellulose utilization, kinetics of microbial cellulose utilization, and contrasting features compared to soluble substrate kinetics. A biological perspective on processing cellulosic biomass is presented, including features of pretreated substrates and alternative process configurations. Organism development is considered for "consolidated bioprocessing" (CBP), in which the production of cellulolytic enzymes, hydrolysis of biomass, and fermentation of resulting sugars to desired products occur in one step. Two organism development strategies for CBP are examined: (i) improve product yield and tolerance in microorganisms able to utilize cellulose, or (ii) express a heterologous system for cellulose hydrolysis and utilization in microorganisms that exhibit high product yield and tolerance. A concluding discussion identifies unresolved issues pertaining to microbial cellulose utilization, suggests approaches by which such issues might be resolved, and contrasts a microbially oriented cellulose hydrolysis paradigm to the more conventional enzymatically oriented paradigm in both fundamental and applied contexts.

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Microbial cellulose utilization: fundamentals and biotechnology.

Information pertaining to enzymatic hydrolysis of cellulose by noncomplexed cellulase enzyme systems is reviewed with a particular emphasis on development of aggregated understanding incorporating substrate features in addition to concentration and multiple cellulase components. Topics considered include properties of cellulose, adsorption, cellulose hydrolysis, and quantitative models. A classification scheme is proposed for quantitative models for enzymatic hydrolysis of cellulose based on the number of solubilizing activities and substrate state variables included. We suggest that it is timely to revisit and reinvigorate functional modeling of cellulose hydrolysis, and that this would be highly beneficial if not necessary in order to bring to bear the large volume of information available on cellulase components on the primary applications that motivate interest in the subject.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/bit.20282

Toward an aggregated understanding of enzymatic hydrolysis of cellulose: noncomplexed cellulase systems.

Outlook for cellulase improvement: screening and selection strategies.

The enzymatic degradation of recalcitrant plant biomass is one of the key industrial challenges of the 21st century. Accordingly, there is a continuing drive to discover new routes to promote polysaccharide degradation. Perhaps the most promising approach involves the application of “cellulase-enhancing factors,” such as those from the glycoside hydrolase (CAZy) GH61 family. Here we show that GH61 enzymes are a unique family of copper-dependent oxidases. We demonstrate that copper is needed for GH61 maximal activity and that the formation of cellodextrin and oxidized cellodextrin products by GH61 is enhanced in the presence of small molecule redox-active cofactors such as ascorbate and gallate. By using electron paramagnetic resonance spectroscopy and single-crystal X-ray diffraction, the active site of GH61 is revealed to contain a type II copper and, uniquely, a methylated histidine in the copper's coordination sphere, thus providing an innovative paradigm in bioinorganic enzymatic catalysis.

https://www.pnas.org/content/pnas/108/37/15079.full.pdf

Insights into the oxidative degradation of cellulose by a copper metalloenzyme that exploits biomass components

The ability and, consequently, the limitations of various microbial enzyme systems to completely hydrolyze the structural polysaccharides of plant cell walls has been the focus of an enormous amount of research over the years. As more and more of these extracellular enzymatic systems are being identified and characterized, clear similarities and differences are being elucidated. Although much has been learned concerning the structures, kinetics, catalytic action, and interactions of enzymes and their substrates, no single mechanism of total lignocellulosic saccharification has been established. The heterogeneous nature of the supramolecular structures of naturally occurring lignocellulosic matrices make it difficult to fully understand the interactions that occur between enzyme complexes and these substrates. However, it is apparent that the efficacy of enzymatic complexes to hydrolyze these substrates is inextricably linked to the innate structural characteristics of the substrate and/or the modifications that occur as saccharification proceeds. This present review is not intended to conclusively answer what factors control polysaccharide biodegradation, but to serve as an overview illustrating some of the potential enzymatic and structural limitations that invariably influence the complete hydrolysis of lignocellulosic polysaccharides.

Substrate and Enzyme Characteristics that Limit Cellulose Hydrolysis

The action of cellobiohydrolases I and II (CBHI and CBHII) and endoglucanases I and II (EGI and EGII) purified from Tri-choderma reesei was evaluated against various substrates. CBHI degraded the β-D-glucan from barley in a typical endo pattern. With cellulose substrates, the synergism between CBHI and endoglucanase I or II depended on the structural and ultrastructural features of the substrate. This effect, unrelated to endo-exo cooperation, was found with substrates of intermediate crystallinity whereas weak or no synergism was recorded with cellulose microcrystals or the soluble carboxy-methyl cellulose derivative. Synergistic degradation of cellulose was also recorded with mixtures of CBHI and CBHII. On the other hand, synergism between endoglucanases and CBHII followed the pattern expected for an endo-exo cooperation. These results presented support evidence for multiple types of cooperation between the cellulolytic enzymes.

C. Viet

Papers

Synergism of Cellulases from Trichoderma reesei in the Degradation of Cellulose