Biological deconstruction
of polymer materials gains efficiency
from the spatiotemporally coordinated action of enzymes with synergetic
function in polymer chain depolymerization. To perpetuate enzyme synergy
on a solid substrate undergoing deconstruction, the overall attack
must alternate between focusing the individual enzymes locally and
dissipating them again to other surface sites. Natural cellulases
working as multienzyme complexes assembled on a scaffold protein (the
cellulosome) maximize the effect of local concentration yet restrain
the dispersion of individual enzymes. Here, with evidence from real-time
atomic force microscopy to track nanoscale deconstruction of single
cellulose fibers, we show that the cellulosome forces the fiber degradation
into the transversal direction, to produce smaller fragments from
multiple local attacks (“cuts”). Noncomplexed enzymes,
as in fungal cellulases or obtained by dissociating the cellulosome,
release the confining force so that fiber degradation proceeds laterally,
observed as directed ablation of surface fibrils and leading to whole
fiber “thinning”. Processive cellulases that are enabled
to freely disperse evoke the lateral degradation and determine its
efficiency. Our results suggest that among natural cellulases, the
dispersed enzymes are more generally and globally effective in depolymerization,
while the cellulosome represents a specialized, fiber-fragmenting
machinery.
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.
This study investigates flexible (polyamide 6.6 PA-6.6, polyethylene terephthalate PET, Cu, Al, and Ni foils) and, for comparison, stiff substrates (silicon wafers and glass) differing in, for example, in surface free energy and surface roughness and their ability to host cellulose-based thin films. Trimethylsilyl cellulose (TMSC), a hydrophobic acid-labile cellulose derivative, was deposited on these substrates and subjected to spin coating. For all the synthetic polymer and metal substrates, rather homogenous films were obtained, where the thickness and the roughness of the films correlated with the substrate roughness and its surface free energy. A particular case was the TMSC layer on the copper foil, which exhibited superhydrophobicity caused by the microstructuring of the copper substrate. After the investigation of TMSC film formation, the conversion to cellulose using acidic vapors of HCl was attempted. While for the polymer foils, as well as for glass and silicon, rather homogenous and smooth cellulose films were obtained, for the metal foils, there is a competing reaction between the formation of metal chlorides and the generation of cellulose. We observed particles corresponding to the metal chlorides, while we could not detect any cellulose thin films after HCl treatment of the metal foils as proven by cross-section imaging using scanning electron microscopy (SEM).
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