Enzyme Synergy in Transient Clusters of Endo- and Exocellulase Enables a Multilayer Mode of Processive Depolymerization of Cellulose

Abstract

Biological degradation of cellulosic materials relies on the molecular-mechanistic principle that internally chain-cleaving endocellulases work synergistically with chain end-cleaving exocellulases in polysaccharide chain depolymerization. How endo–exo synergy becomes effective in the deconstruction of a solid substrate that presents cellulose chains assembled into crystalline material is an open question of the mechanism, with immediate implications on the bioconversion efficiency of cellulases. Here, based on single-molecule evidence from real-time atomic force microscopy, we discover that endo- and exocellulases engage in the formation of transient clusters of typically three to four enzymes at the cellulose surface. The clusters form specifically at regular domains of crystalline cellulose microfibrils that feature molecular defects in the polysaccharide chain organization. The dynamics of cluster formation correlates with substrate degradation through a multilayer-processive mode of chain depolymerization, overall leading to the directed ablation of single microfibrils from the cellulose surface. Each multilayer-processive step involves the spatiotemporally coordinated and mechanistically concerted activity of the endo- and exocellulases in close proximity. Mechanistically, the cooperativity with the endocellulase enables the exocellulase to pass through its processive cycles ∼100-fold faster than when acting alone. Our results suggest an advanced paradigm of efficient multienzymatic degradation of structurally organized polymer materials by endo–exo synergetic chain depolymerization.

Impact of Synergy Partner Cel7B on Cel7A Binding Rates: Insights from Single-Molecule Data

“…Next, we increased the illumination power to detect cellulase cluster formation reported by others. 42 At higher illumination, the bleaching time of single fluorophores was, on average, comparable to or even shorter than the residence time of the enzyme (Figure S11). However, we were able to calculate the k OFF of the enzymes by correcting for fluorophore bleaching (Figure 5a).…”

“…It was recently shown using AFM that the presence of Cel7B and Cel7A in a mixture leads to the formation of transient clusters of, on average, three enzymes. 42 In the scenario where enzymes form clusters when both types of enzymes are present, the measured reduction in the frequency of new binding events might be due to the fact that a spot now contains several Cel7A enzymes. This does not imply that the total amount of enzymes adsorbed is diminished.…”

“…This could be the direct result of CBH cluster formation in synergistic mixtures as shown previously by high-speed AFM. 42 ■ METHODS Enzyme Variant Design, Expression, and Purification. The enzymes Cel7A and Cel7B are the main CBH and EG from T. reesei (Genbank ID CAH10320.1 and AAA34212.1, respectively).…”

“…These results show great variation but are only focused on one type of enzyme, namely, the CBH Cel7A and therefore provide no single molecule data on the synergy between different classes of cellulases. To address this, Zajki-Zechmeister et al used high-speed AFM to show that clusters of enzymes are formed in mixtures of exo-and endoglucanases as they synergistically act on the cellulose surface 42 and that the exoglucanases' processive cycle accelerates about 100 times compared to that when acting alone.…”

Ab-initio modeling of bend-losses in silicon nitride waveguides from visible to near-infrared

“…The nanoscale precision provided by AFM allows for a detailed examination of the surface topography, identifying irregularities that could otherwise go unnoticed with less sensitive techniques. 18 AFM enables a more comprehensive and precise assessment of waveguide performance by providing a means to quantify and visualise the roughness accurately. 12 The sample's roughness is evaluated using the Anton Paar Tosca 400 Atomic Force Microscope.…”