Diversity of cutinases from plant pathogenic fungi: Purification and characterization of two cutinases from Alternaria brassicicola

Abstract

Previous evidence indicated a relationship between the pH optima of fungal cutinases and the tissue specificities of respective pathogens. Cutinases with slightly acidic pH optima were associated with leaf pathogens, whereas stem base pathogens produced cutinases most active under alkaline conditions. Alternaria brassicicola secreted both types of cutinases and caused disease on all above-ground host tissues. Two cutinases, A c and B a, which were expressed after growth of the pathogen on polymer cutin and in the presence of cutin monomers as cutinase inducers, were purified to apparent homogeneity. Cutinase A c had a cutinolytic pH optimum of 6·5 and cutinase B a had a cutinolytic pH optimum of 8·5. Their molecular weights were 23·0 kDa and 21·0 kDa, respectively. Other enzymatic and structural parameters were similar for both enzymes. The V- and K m-values for p-nitrophenyl esters with acyl chains ranging from C 4 to C 16 decreased for both enzymes with increasing chain length. The esterase inhibitors ebelactones A and B were equally active competitive inhibitors of both cutinases. Amino acid compositions were similar but not identical, and the separation pattern of tryptic peptides indicated structural differences between the two isozymes. Although both cutinases could be distinguished by their pH dependency of cutinolytic activities and their size, the similarities indicate a close evolutionary relationship.