Visualizing cellulase activity

Abstract

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.

Visualization of structural changes in cellulosic substrates during enzymatic hydrolysis using multimodal nonlinear microscopy

“…High resolution imaging techniques such as electron microscopy and scanning probe microscopy are widely used to study cellulose structure (Bubner et al 2013). These techniques can provide nanoscale information about cellulose structure, but lack chemical specificity, thus revealing mostly topographic details of the specimen.…”

Recombinant Trichoderma harzianum endoglucanase I (Cel7B) is a highly acidic and promiscuous carbohydrate-active enzyme

“…Besides, most of the published work deals with exoglucanases, particularly celobiohydrolase I (CBHI) acting on pure cellulose substrates. A comprehensive review including important contributions to the area can be found in (Bubner et al 2013).…”

Interrelationships between cellulase activity and cellulose particle morphology

“…Whole pretreated biomass has a complex architecture across multiple length scales, and while this architecture has been studied extensively for many different substrates (Antal 1985;Ciesielski et al 2014;Donohoe and Resch 2015), only in rare cases have structural effects of enzymatic degradation of these substrates been reported (Resch et al 2014). In contrast numerous studies on enzyme induced changes to the morphology of pure cellulose substrates like BMCC ribbons, Valonia fibrils and Avicel have been published over the years (for an extensive review see Bubner et al 2013). The general observation is that cellobiohydrolases (CBHs)-notably CBH1-target crystalline regions while endoglucanases (EGs) target amorphous regions, and that the combined action of the two types of enzymes has a profoundly different effect than each of them alone (Bubner et al 2013;Payne et al 2015).…”

“…In contrast numerous studies on enzyme induced changes to the morphology of pure cellulose substrates like BMCC ribbons, Valonia fibrils and Avicel have been published over the years (for an extensive review see Bubner et al 2013). The general observation is that cellobiohydrolases (CBHs)-notably CBH1-target crystalline regions while endoglucanases (EGs) target amorphous regions, and that the combined action of the two types of enzymes has a profoundly different effect than each of them alone (Bubner et al 2013;Payne et al 2015). Furthermore it has been found that CBHs degrades cellulose fibrils from the ends and cause narrowing and sharpening of the fibril while EGs have no apparent spatial preference and cause general surface disruption or fibrillation (Chanzy and Henrissat 1985;Chanzy et al 1983).…”