Figure 2.

Semipurification of wild-type and sta8 isoamylases. Crude extracts from 20-L cultures of wild-type strain A35 or the sta8-1 BafV13 strain were subjected to the purification procedure described previously by Dauvillée et al. (2000). The undenatured samples (80 μL from the fractions 52, 54, 56, 58, 60, 62, 64, and 66 of the S300 GPC) separated by PAGE under native conditions were blotted onto starch-containing gels according to Kakefuda and Duke (1984). The color of the bands observed after staining the gel with iodine will change according to the enzyme specificities. Branching enzymes will stain white or light red, amylases and glucosidases will stain white, and debranching enzymes (pullulanases or isoamylases) will stain blue. A, Zymogram obtained from wild-type isoamylase semi-purification. B, The same fractions analyzed from the reference sta8-1 mutant strain.