Correlative Light and Electron Microscopy for the Study of the Structural Arrangement of Bacterial Microcrystalline Cellulose Microfibrils

Processive Enzymes Kept on a Leash: How Cellulase Activity in Multienzyme Complexes Directs Nanoscale Deconstruction of Cellulose

“…The amorphous nanodomains were the preferred common initiation sites for substrate degradation by the cellulases irrespective of their assembly state. Although widely used, the idea of “amorphous material” in enzymatic cellulose deconstruction is vague. − Our findings define it, structurally and functionally, in terms of nanoscale defects of cellulose chain organization in the microfibril, ,, notably distinct from larger-scale deformations/dislocations of the fiber’s microfibril composite structure. , We notice that the results have relevance to inform cellulose pretreatment for facilitated enzymatic degradation. ,, Disintegrating the fiber structure solely in terms of organization of the different microfibrils seems not enough for optimum efficiency. The microfibril nanostructure is identified as additional target for the pretreatment.…”

“…Although widely used, the idea of "amorphous material" in enzymatic cellulose deconstruction is vague. 88−90 Our findings define it, structurally and functionally, in terms of nanoscale defects of cellulose chain organization in the microfibril, 21,22,70 notably distinct from larger-scale deformations/dislocations of the fiber's microfibril composite structure. 27,59 We notice that the results have relevance to inform cellulose pretreatment for facilitated enzymatic degradation.…”

“…Although instrumental to unravel the single-molecule behavior of certain cellulases, near-perfect crystallites 55 , 56 of cellulose lack “amorphous domains”, that is, areas of organizational disorder of the polysaccharide chains. 19 , 21 , 22 , 27 , 57 Such amorphous domains represent recurrent features of nanostructure distinctive of natural cellulose fibrils. 58 , 59 The crystallites therefore represent only partial substrates for cellulase multienzyme systems and have limited purview for assessing synergy between individual enzyme types in substrate deconstruction.…”

“…However, most polymer materials are solid. Their nanoscale morphology typically shows ordered (crystalline) phases interspersed with regions of irregular structure. − Spatial irregularities result from orientational and directional disorder in polymer chain organization into solid material. − Only a limited fraction of the enzyme-accessible external surface is usually available for polymer chain cleavage by the individual types of hydrolases. − For an enzyme system confined to working at the surface of such solids, there thus arises the fundamental challenge of a locally focused usage of enzyme synergy. Moreover, there is the additional challenge of perpetuation of enzyme synergy as the solid surface evolves because of the substrate’s ongoing deconstruction.…”

“…Struggling with exactly this dilemma, earlier studies of related purport − and our own ,− were limited in the scope of observation and interpretation. Although instrumental to unravel the single-molecule behavior of certain cellulases, near-perfect crystallites , of cellulose lack “amorphous domains”, that is, areas of organizational disorder of the polysaccharide chains. ,,,, Such amorphous domains represent recurrent features of nanostructure distinctive of natural cellulose fibrils. , The crystallites therefore represent only partial substrates for cellulase multienzyme systems and have limited purview for assessing synergy between individual enzyme types in substrate deconstruction. We show that single fibers (bundles of microfibrils) of bacterial cellulose, , released carefully from the fiber network of the native material, met the challenging functional-analytical requirements of investigation comprehensively.…”