Surface density of cellobiohydrolase on crystalline celluloses

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Abstract
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 (ρ) 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 Iα from Cladophora and cellulose Iβ from Halocynthia were compared, the maximum adsorption of the enzyme on cellulose Iβ was ∼1.5 times higher than that on cellulose Iα, although the rate of cellobiose production from cellulose Iβ was lower than that from cellulose Iα. This indicates that the specific activity (k) of Cel7A adsorbed on cellulose Iα is higher than that of Cel7A adsorbed on cellulose Iβ. When k was plotted versus ρ, a dramatic decrease of the specific activity was observed with the increase of surface density (ρ-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 Iα was almost twice that for cellulose Iβ. When cellulose Iα was converted to cellulose Iβ by hydrothermal treatment, the specific activity of Cel7A decreased and became similar to that of native cellulose Iβ at the same ρ-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.