Secretion and export of amylolytic activities in Schwanniomyces alluvius

We define secretion as the passage from the cytoplasm, across the cell membrane, to the periplasmic space. By contrast, export is the passage across the cell wall into the medium. Operationally we distinguish the two processes by comparing the molecular species in the medium, in whole cells, and in spheroplasts. Two techniques make the task possible: complete spheroplast preparation and detection of activities in bands obtained by sodium dodecyl sulfate – polyacrylamide gel electrophoresis. The capability of Schwanniomyces alluvius to export α-amylase during stationary phase gradually increased with continual successive transfers from a slant culture to a liquid medium containing starch until a maximum was reached. Only cells which had developed full capability to export α-amylase were used in these studies. About 1 h after the end of the log phase of growth, α-amylase and glucoamylase start to be exported above constitutive levels and a concentration 10 times the constitutive level is reached 3 h later. Electrophoretic results show that at least three active molecular species of α-amylase appear in the cytoplasm at the end of log phase and that the smaller component (52 000 daltons) is secreted into the periplasm 0.5 h later and starts to be exported 1 h after that. The sequence of events suggests that the larger species are precursors of the 52 000 dalton molecules. Amylolytic activities in the cytoplasm and periplasm in late log phase are not detectable.