Biochemical, structural and functional characterization of two novel antifungal endoglucanases from Anabaena laxa

An investigation was undertaken to analyse the various biochemical and structural facets of two purified endoglucanases (End1 and End2) exhibiting antifungal activity from Anabaena laxa. SDS-PAGE analyses indicated that End1 and End2 proteins were 38 and 74 kDa, respectively. Both the purified End proteins showed activities (high β-1,4 in both End1 and End2 and low β-1,3-endoglucanase activity only in End2) in the zymograms. Thin layer chromatography (TLC) analyses of the hydrolyzed products using different substrates confirmed endo and exo/endo-type nature of End1 and End2, respectively. The highest endoglucanase activity of both the purified End proteins was recorded at pH 6.0 and temperature 30 °C, however, stabilities of End1 and End2 were recorded over a wide range of pH (5.0–7.0 and 5.0–9.0, respectively) up to 12 h incubation; and temperature of 40 and 50 °C, up to 3 h incubation. The predicted three dimensional structures of End1 and End2 were found to be distorted beta-barrel and beta-barrel, respectively. The binding sites of both End proteins were identified and cellotetraose substrate was found to completely span over the active site in the three dimensional cartoon and surface models for End2, confirming the biochemical and kinetic analyses. Site-directed mutagenesis of the end1 and end2 led to the identification of Glu-23 and Cys-11 (present at signal peptide regions) as catalytic residues critical to antifungal activity, respectively. To our knowledge, this study represents a first time in-depth investigation into the structural/functional aspects of purified endoglucanases from cyanobacteria.