Real-time Single Molecular Study of a Pretreated Cellulose Hydrolysis Mode and Individual Enzyme Movement

Background: The main challenges of large-scale biochemical conversion involve the high costs of cellulolytic enzymes and the inefficiency in enzymatic deconstruction of polysaccharides embedded in the complex structure of the plant cell wall, leading to ongoing interests in studying the predominant mode of enzymatic hydrolysis. In this study, complete enzymatic hydrolysis of pretreated biomass substrates was visualized in situ and in real time by atomic force microscopy (AFM) topography and recognition imaging. Throughout the entire hydrolytic process, a hydrolysis mode for exoglucanase (CBH I) consisting of a peeling action, wherein cellulose microfibrils are peeled from sites on the pretreated cellulose substrate that have cracks sufficiently large for CBH I to immobilize.

Results: We quantitatively monitored the complete hydrolytic process on pretreated cellulose. The synergetic effect among the different enzymes can accelerate the cellulose hydrolysis rate dramatically. However, the combination of CBH I and β-glucosidases (β-G) exhibited a similar degradation capacity as did whole enzyme (contains the cellobiohydrolases and endoglucanase as its major enzyme components). We developed a comprehensive dynamic analysis for individual cellulase acting on single pretreated cellulose through use of functional AFM topography and recognition imaging. The single crystalline cellulose was divided into different regions based on the cracks on the substrate surface and was observed to either depolymerize or to peel away by the jammed enzyme molecules. After the exfoliation of one region, new cracks were produced for the enzyme molecules to immobilize. The fiber width may have a relationship with the peeling mode of the fibers. We performed a statistical height measure of the generated peaks of the peeled fibers. The height values range from 11 to 24 nm. We assume that the CBH I enzymes stop progressing along the cellulose microfibril when the peeled microfibril height exceeds 11 nm.

Conclusion: The combination of CBH I and β-G can achieve an effective hydrolysis of the pretreated biomass substrates. The single-molecule study of the complete hydrolytic process indicates that the hydrolytic mode involves the peeling of the microfibrils and progressive depolymerization, which depend on the size of the cracks on the surface of the pretreated cellulose microfibrils.