Reporter Group-Labeled Synthetic Cellulose: Structural Characterization and Utilization in Mapping the Cellulose Chain-Cleavage Modes of Cellulases

Iterative β-1,4-glucosylation of p-nitrophenyl (pNP)-β-cellobioside by cellodextrin phosphorylase was applied to synthesize reducing-end reporter group-labeled cellooligosaccharides (average degree of polymerization, DP: ∼5.7; DP range: 4-10) which, upon self-assembly in solution, precipitate as a cellulose allomorph II crystalline material. Atomic force microscopy revealed that the synthetic cellulose-pNP forms nanoscale sheet-like assemblies, with thickness ∼4 nm. The labeled cellulose was used to characterize the chain cleavage specificities of cellulose-degrading enzymes. Reactions were monitored by the release of soluble products as well as the change in oligosaccharide composition of the residual solid material, measured by mass spectrometry. The products formed by hydrolase systems (<i>Trichoderma reesei</i> cellulases; <i>Clostridium thermocellum</i> cellulosome) are controlled by processive chain cleavages from the chain ends. Atomic force microscopy shows that <i>T. reesei</i> Cel7A preferentially attacked sheet edges, with degradation efficiency and directionality dependent on local nanoscale structure. Collectively, we show synthesis of reporter group-labeled cellulose for probing enzymatic chain depolymerization activity on a crystalline solid substrate.