Immunohistochemical staining was applied together with the multicolor fluorescent scheme to demonstrate the
amylase activity for
polysaccharide hydrolysis in stored or starved aerobic granules that are in substrate deficiency. If sufficient nutrients were present,
alpha-amylase and
beta-amylase were found close to the surface layer of the original granules. Following storage or
starvation during which most external nutrients were depleted, the
alpha-amylase and
beta-amylase were distributed over the entire granule interior, suggesting endogenous respiration at the core of the granule. In particular, the fluorescent intensities of
alpha-amylase and
beta-amylase were enriched 5-20 microm from the edge of the internal cavity, suggesting the strong correlation between
polysaccharide hydrolysis and the formation of interior cavities. The secreted
amylase was located near the living cells, suggesting that the
polysaccharide hydrolysis is restricted to local environment that occurs near the functional strains. Internal hydrolysis within the core, for the case of both
proteins and
polysaccharides should correspond in principle to the loss of granule stability.