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.

Background: The exact mechanism by which cellulases degrade cellulose is still elusive.Results: An empirical model of the structural dynamics of cellulose degradation is shown.Conclusion: Enzymatic cellulose hydrolysis is subjected to deceleration and acceleration caused by periodically emerging internal limitations and overcoming them.Significance: Understanding structural dynamics of enzymatic cellulose disintegration is pivotal for making biofuel production from lignocellulosic feedstock economic.

Efforts to boost crop yield and meet global food demands while striving to reach sustainability goals are hindered by the increasingly severe impacts of abiotic stress, such as drought. One strategy for alleviating drought stress in crops is to utilize rootassociated bacteria, yet knowledge concerning the relationship between plant hosts and their microbiomes during drought remain under-studied. One broad pattern that has recently been reported in a variety of monocot and dicot species from both native and agricultural environments, is the enrichment of Actinobacteria within the drought-stressed root microbiome. In order to better understand the causes of this phenomenon, we performed a series of experiments in millet plants to explore the roles of drought severity, drought localization, and root development in provoking Actinobacteria enrichment within the root endosphere. Through 16S rRNA ampliconbased sequencing, we demonstrate that the degree of drought is correlated with levels of Actinobacterial enrichment in four species of millet. Additionally, we demonstrate that the observed drought-induced enrichment of Actinobacteria occurs along the length of the root, but the response is localized to portions of the root experiencing drought. Finally, we demonstrate that Actinobacteria are depleted in the dead root tissue of Japanese millet, suggesting saprophytic activity is not the main cause of observed shifts in drought-treated root microbiome structure. Collectively, these results help narrow the list of potential causes of drought-induced Actinobacterial enrichment in plant roots by showing that enrichment is dependent upon localized drought responses but not root developmental stage or root death.

The structure of Pseudomonas fluorescens mannitol 2-dehydrogenase with bound NAD + leads to the suggestion that the carboxylate group of Asp 69 forms a bifurcated hydrogen bond with the 2 0 and 3 0 hydroxyl groups of the adenosine of NAD + and contributes to the 400-fold preference of the enzyme for NAD + as compared to NADP + . Accordingly, the enzyme with the Asp 69 fi Ala substitution was found to use NADP(H) almost as well as wild-type enzyme uses NAD(H). The Glu 68 fi Lys substitution was expected to enhance the electrostatic interaction of the enzyme with the 2 0 -phosphate of NADP + . The Glu 68 fi Lys:Asp 69 fi Ala doubly mutated enzyme showed about a 10-fold preference for NADP(H) over NAD(H), accompanied by a small decrease in catalytic efficiency for NAD(H)-dependent reactions as compared to wild-type enzyme.

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