5-03: Substrate-Based Limitations in the Enzymatic Hydrolysis of Cellulose: Crystallinity, Reactivity and Adsorption

The enzymatic hydrolysis of crystalline cellulose encounters various limitations that are both substrate- and enzyme-related. They directly impact the rate of the reaction and lead to its dramatic slowdown, observed especially at high degrees of conversion. Although the initial crystallinity of cellulose plays a major role in determining the rate of hydrolysis, it was shown not to evolve over the time of conversion by cellulases, implying other reasons for the decrease in rate. Using increasing concentration of phosphoric acid to generate acid-swollen Avicel, samples with intermediate crystallinity indexes were obtained and their subsequent enzymatic hydrolysis gave a clearer overview of the relevance of the initial degree of crystallinity on reaction rate. Change in adsorption capacity and decrease in reactivity along conversion were also confirmed to be involved. Reactivity (measured as of glucose production rate from restart experiments) experienced a serious drop already after 5% conversion, supporting the hypothesis that the cellulose surface has been modified by the action of the enzymes. Both X-ray diffraction and solid state CP/MAS ¹³C-NMR were employed and gave insight into molecular changes occurring with cellulose along the conversion. The (021) face was shown to be converted first by pure cellobiohydrolase. Strategies to improve the overall reaction rate will be presented.