Properties of yeast debranching enzyme and its specificity toward branched cyclodextrins

Authors

  • Shiro TABATA,

    Corresponding author
    1. Department of Chemistry, Nara Medical University, Japan
      Correspondence to S. Tabata, Department of Chemistry, Nara Medical University, Kashihara Nara, Japan 634
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  • Susumu HIZUKURI

    1. Department of Biochemical Science and Technology, Faculty of Agriculture, Kagoshima University, Japan
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Correspondence to S. Tabata, Department of Chemistry, Nara Medical University, Kashihara Nara, Japan 634

Abstract

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.

Abbreviations
Glc-cGlc6

6-O-α-d-glucosyl cyclomaltohexaose

Glc-cGlc7

6-O-α-d-glucosyl cyclomaltoheptaose

Glc-cGlc8

6-O-α-d-glucosyl cyclomalto-octaose

Glc2-cGlc7

6-O-α-maltosyl cyclomaltohetaose

Glc3-cGlc7

6-O-α-maltotriosyl cyclomaltoheptaose

Glc4-cGlc7

6-O-α-maltotetraosyl cyclomaltoheptaose

ϕ-dextrin

glycogen phosphorylase limit dextrin

transferase/glucosidase

oligo-1,4 → 1,4-glucantransferase and amylo-1,6-glucosidase

Enzymes
 

Amylo-1,6-glucosidase (EC 3.2.1.33)

 

oligo-1,4→1,4-glucantransferase, 1,4-α-d-glucan: 1,4-d-glucan 4-α-d-glucosyl-transferase (EC 2.4.1.25)

 

isoamylase, (glycogen 6-glucanohydrolase (EC 3.2.1.68)

 

pullulanase, α-dextrin 6-glucanohydrolase (EC 3.2.1.41)

 

phosphorylase (EC 2.4.1.1)

 

glucoamylase (EC 3.2.1.3)

 

hexokinase (EC 2.7.1.1)

 

glucose-6-phosphate dehydrogenase (EC 1.1.1.49)