Enhancement of mycolytic activity of an antagonistic Bacillus subtilis through ethyl methane sulfonate (EMS) mutagenesis

Bacillus subtilis, isolated from the chili rhizosphere, capable of producing 3 mycolytic enzymes (chitinase, beta-1,3-glucanase, and cellulase), was mutated by the chemical mutagen ethyl methane sulfonate (EMS). Mutants were screened based on their antifungal ability on dual plate assay against Colletotrichum gloeosporioides OGC1, the causal agent of anthracnose disease in fruit crops. EMS mutagenesis yielded 60 isolated mutants on NA plates, of which 3 (M3, M4, and M24) showed loss of antagonism against C. gloeosporioides OGC1 and 6 (M8, M21, M22, M57, M58, and M59) exhibited increased antagonism. The remaining mutants did not show any difference in their antagonistic property. In liquid culture, the mutants exhibited varied levels of the 3 enzymes. These mutants were studied for their mycolytic enzyme activities under shake flask conditions. Of all these mutants, M57 showed a 36-fold and 4.71-fold increase in beta-1,3-glucanase and cellulase activities, respectively, with a concomitant 1.95-fold increase in hydrolytic activity, followed by M59, with a 5.68-fold and 1.57-fold increase in beta-1,3-glucanase and cellulase activities, respectively, and a 2.23-fold increase in hydrolytic activity as compared to the wild type B. subtilis strain. M24 exhibited a complete loss of beta-1,3-glucanase and a decrease in chitinase, with a concomitant decrease in levels of hydrolytic activity with C. gloeosporioides mycelia as compared to the wild type strain. The study clearly indicated the mycolytic enzyme mediated antagonism of this strain and yielded 2 hyperproducing mutants for beta-1,3-glucanase and cellulase, 2 important enzymes for various agricultural and industrial uses.