Debranching enzyme was purified from Saccharomyces cerevisiae by DEAE-cellulose, ω-aminobutyl agarose and hydroxyapatite column chromatography. The activity of the eluent was monitored by the iodine-staining method which detects both the direct and indirect debranching enzymes. The elution profiles at every step showed a single peak with no shoulder. The crude and the purified enzyme preparations gave a single activity band with the same mobility on PAGE. The crude product produced 80% glucose compared to reducing sugar from glycogen-phosphorylase-limited dextrin while the partially purified and purified preparations produced 100% glucose.
The activity of the purified enzyme was characterized and compared with that of the rabbit muscle enzyme by using various branched cyclodextrins as substrates. Both enzymes hydrolyzed 6-O-α-d-glucosyl cyclodextrins to glucose and cyclodextrins, but did not act on 6-O-α-maltosyl cyclomaltoheptaose. The yeast enzyme gave rise to glucose as a sole reducing sugar from 6-O-α-maltotriosyl cyclomaltoheptaose and 6-O-α-maltotraosyl cyclomaltoheptaose, indicating that maltosyl and maltotriosyl transfers, respectively, had occurred, prior to the action of amylo-1,6-glucosidase.
6-O-α-d-Glucosyl cyclomaltoheptaose and 6-O-α-d-glucosyl cyclomalto-octaose, respectively, were better substrates than glycogen-phosphorylase-limited dextrin for the yeast and muscle enzymes. The yeast enzyme released glucose at a similar rate from 6-O-α-maltotriosyl cyclomaltoheptaose as from 6-O-α-maltotetraosyl cyclomaltoheptaose, but considerably lower rates than that from limit dextrin.
The yeast debranching enzyme appears to be exclusively oligo-1,4→1,4-glucantransferase-amylo-1,6-glucosidase and does not have isoamylase.
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glycogen phosphorylase limit dextrin
oligo-1,4 → 1,4-glucantransferase and amylo-1,6-glucosidase
Amylo-1,6-glucosidase (EC 126.96.36.199)
oligo-1,4→1,4-glucantransferase, 1,4-α-d-glucan: 1,4-d-glucan 4-α-d-glucosyl-transferase (EC 188.8.131.52)
isoamylase, (glycogen 6-glucanohydrolase (EC 184.108.40.206)
pullulanase, α-dextrin 6-glucanohydrolase (EC 220.127.116.11)
phosphorylase (EC 18.104.22.168)
glucoamylase (EC 22.214.171.124)
hexokinase (EC 126.96.36.199)
glucose-6-phosphate dehydrogenase (EC 188.8.131.52)