Electron microscopic investigation of the diffusion of Bacillus licheniformis alpha-amylase into corn starch granules

A method for the direct electron microscopic observation of amylases in interaction with starch granules is presented. The technique involves immuno-gold labeling of the enzymes and cross-sectioning of hydrated starch granules. This approach allows the analysis of the internal degradation of starch with a concomitant visualization of enzymes at the sites of hydrolysis. The visualization of enzymes at the surface, inside the channel and inside the core of the degraded granules shows that the alpha-amylase molecules first proceed from the surface toward the center (centripetal hydrolysis). Then the core is completely degraded from within by erosion of its periphery (centrifugal hydrolysis). In the first case (centripetal hydrolysis), the enzymes act by progressing along the polysaccharide chains. By contrast, the centrifugal hydrolysis leads to even erosion, indicative of a more diffusive motion of the enzymes.