Isolation and characterization of an endo-(1,4)-{beta}-glucanase secreted by Achlya ambisexualis

Models of wall loosening in fungi and other walled eukaryotes require the action of proteins able to reduce the degree of linkage between components of the wall. In the oomycete Achlya ambisexualis, such a role has been proposed for a suite of endoglucanases that are secreted during branching and during the measurable wall softening associated with osmotic stress. We report here the isolation and characterization of one of these isoenzymes. The enzyme has a molecular weight of 32 kDa, a pH optimum of 6.75, a pI of 4.5, and a temperature optimum of 35 C. It is partially inhibited by sulfhydryl-binding reagents and completely inhibited by the tryptophan-binding reagent NBS. The enzyme has an endohydrolytic mode of action with substrate specificity towards glucans that contain β-(1,4) linkages, either alone (carboxymethyl cellulose) or as mixed linkage (1,4-1,3)-β-glucans (e.g., Avena glucan). It does not, however, degrade amorphous insoluble (phosphoric acid swollen) cellulose. Most significantly, the enzyme can also hydrolyze linkages in an Achlya cell wall fraction previously shown to consist of a mixed-linkage (1,4-1,3)-β-glucan. This property is consistent with the long-standing hypothesis that the branching-related endoglucanases of oomycetes play a role in cell wall loosening.