Imaging the enzymatic digestion of bacterial cellulose ribbons reveals theendo character of the cellobiohydrolase Cel6A from Humicola insolens and its mode of synergy with cellobiohydrolase Cel7A

Dispersed cellulose ribbons from bacterial cellulose were subjected to dige stion with cloned Cel7A (cellobiohydrolase [CBH] I) and Cel6A (CBH II) from Humicola insolens either alone or in a mixture and in the presence of an e xcess of beta-glucosidase. Both Cel7A and Cel6A were effective in partially converting the ribbons into soluble sugars, Cel7A being more active than C el6A In combination, these enzymes showed substantial synergy culminating w ith a molar ratio of approximately two-thirds Cel6A and one-third Cel7A, Ul trastructural transmission electron microscopy (TEM) observations indicated that Cel7A induced a thinning of the cellulose ribbons, whereas Cel6A cut the ribbons into shorter elements, indicating an endo type of action. These observations. together with the examination of the digestion kinetics, ind icate that Cel6A ran be classified as an endo-processive enzyme, whereas Ce l7A is essentially a processive enzyme, Thus, the synergy resulting from th e mixing of Cel6A and Cel7A can be explained by the partial endo character of Cel6A. A preparation of bacterial cellulose ribbons appears to be an app ropriate substrate, superior to Valonia or bacterial cellulose microcrystal s, to visualize directly by TEM the endo-processivity of an enzyme such as Cel6A.