Background: Lytic polysaccharide monooxygenase (LPMO) has recently been discovered to depolymerize cellulose.Results: Dynamic imaging was applied to reveal the effects of LPMO and cellulase activity on solid cellulose surface.Conclusion: Critical features of surface morphology for LPMO synergy with cellulases are recognized.Significance: Direct insights into cellulose deconstruction by LPMO alone and in synergy with cellulases are obtained.
Background: Synergistic interplay of cellulases is key for efficiency of cellulose hydrolysis.Results: In situ observation of individual and synergistic action of endo- and exo-cellulases on a polymorphic cellulose substrate reveals specificity of individual enzyme components for crystalline or amorphous regions.Conclusion: Cellulase synergism is governed by mesoscopic morphological characteristics of the cellulose substrate.Significance: Advanced knowledge basis for rational optimization of cellulose saccharification.
Highly heterogeneous and usually weakly defined substrate morphologies complicate the study of enzymatic cellulose hydrolysis. The cellulose surface has a non-uniform shape in particular, with consequent impacts on cellulase adsorption and activity. We have therefore prepared a cellulosic model substrate which is shown by atomic force microscopy (AFM) to display a completely smooth surface, the residual squared mean roughness being 10 nm or lower, and applied it for kinetic analysis of cellulase action. The substrate consists of an amorphous cellulose matrix into which variably sized crystalline fibers are distributed in apparently irregular fashion. Its conversion into soluble sugars by Trichoderma sp. cellulase at 50°C proceeded without apparent limitation up to 70% completion and was paralleled by a steady increase in cellulase adsorption to the cellulose. Individual cellulase components (CBH I, CBH II, EG) also showed strongly enhanced adsorption with progressing cellulose conversion, irrespective of their preference for degrading the amorphous or crystalline substrate parts as revealed by AFM. The specific activity of the adsorbed cellulases, however, decreased concomitantly. Cellulose surface morphologies evolving as a consequence of cellulase action were visualized by AFM. Three-dimensional surface degradation by the cellulases resulted in a large increase in cellulose surface area for enzyme adsorption. However, the decline in enzyme specific activity during conversion was caused by factors other than surface ablation and disruption. Based on kinetic evidence for enzymatic hydrolyses of the smooth-surface model substrate and microcrystalline cellulose (Avicel), we hypothesize that, due to gradual loss of productive dynamics in their interactions with the cellulose surface, individual cellulases get progressively confined to substrate parts where they are no longer optimally active. This eventually leads to an overall slow-down of hydrolysis.
Commercial exploitation of lignocellulose for biotechnological production of fuels and commodity chemicals requires efficient-usually enzymatic-saccharification of the highly recalcitrant insoluble substrate. A key characteristic of cellulose conversion is that the actual hydrolysis of the polysaccharide chains is intrinsically entangled with physical disruption of substrate morphology and structure. This "substrate deconstruction" by cellulase activity is a slow, yet markedly dynamic process that occurs at different length scales from and above the nanometer range. Little is currently known about the role of progressive substrate deconstruction on hydrolysis efficiency. Application of advanced visualization techniques to the characterization of enzymatic degradation of different celluloses has provided important new insights, at the requisite nano-scale resolution and down to the level of single enzyme molecules, into cellulase activity on the cellulose surface. Using true in situ imaging, dynamic features of enzyme action and substrate deconstruction were portrayed at different morphological levels of the cellulose, thus providing new suggestions and interpretations of rate-determining factors. Here, we review the milestones achieved through visualization, the methods which significantly promoted the field, compare suitable (model) substrates, and identify limiting factors, challenges and future tasks.
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