Inactivation of enzymes within spores of Bacillus megaterium ATCC 19213 by hydroperoxides

The organic hydroperoxides t-butyl hydroperoxide, cumene hydroperoxide, and peracetic acid were found to act similarly to hydrogen peroxide in causing inactivation of enzymes within intact spores of Bacillus megaterium ATCC 19213 concomitant with mortality. Spores treated with lethal levels of the agents were germinated and permeabilized for enzyme assays. The hierarchy of sensitivities among enolase, glucose-6-phosphate dehydrogenase (G6Pdh), and pyruvate kinase to inactivation varied somewhat with the specific hydroperoxide used, possibly because of differences in the types of radicals generated. However, each agent inactivated each of the enzymes, albeit at different rates. Comparative assessments of enzyme inactivation by lethal levels of H2O2 or by moist heat showed that some enzymes, such as G6Pdh, are highly sensitive to inactivation, while others, such as ATPases, are much more resistant. The enzymes G6Pdh and aldolase were highly sensitive to hydroperoxide inactivation and also to moist heat, while pyruvate kinase was much more sensitive to hydroperoxides than to moist heat. Our overall interpretation of the findings is that hydroperoxides and moist heat can produce cumulative damage to sensitive enzymes within spores, which progressively diminishes the capacities of the cells to undergo the outgrowth required for return to vegetative life.