Surface density of cellobiohydrolase on crystalline celluloses - A critical parameter to evaluate enzymatic kinetics at a solid-liquid interface

The enzymatic kinetics of glycoside hydrolase family 7 cellobiohydrolase (Cel7A) towards highly crystalline celluloses at the solid-liquid interface was evaluated by applying the novel concept of surface density (rho) of the enzyme, which is defined as the amount of adsorbed enzyme divided by the maximum amount of adsorbed enzyme. When the adsorption levels of Trichoderma viride Cel7A on cellulose I-alpha from Cladophora and cellulose I-beta from Halocynthia were compared, the maximum adsorption of the enzyme on cellulose I-beta was similar to 1.5 times higher than that on cellulose I-alpha, although the rate of cellobiose production from cellulose I-beta was lower than that from cellulose I-alpha. This indicates that the specific activity (k) of Cel7A adsorbed on cellulose I-alpha is higher than that of Cel7A adsorbed on cellulose I-beta. When k was plotted versus rho, a dramatic decrease of the specific activity was observed with the increase of surface density (rho-value), suggesting that overcrowding of enzyme molecules on a cellulose surface lowers their activity. An apparent difference of the specific activity was observed between crystalline polymorphs, i.e. the specific activity for cellulose I-alpha was almost twice that for cellulose I-beta. When cellulose I-alpha was converted to cellulose I-beta by hydrothermal treatment, the specific activity of Cel7A decreased and became similar to that of native cellulose I-beta at the same rho-value. These results indicate that the hydrolytic activity (rate) of bound Cel7A depends on the nature of the crystalline cellulose polymorph, and an analysis that takes surface density into account is an effective means to evaluate cellulase kinetics at a solid-liquid interface.