1 Danish Pest Infestation Laboratory, Faculty of Agricultural Sciences, Aarhus University, Aarhus University2 Department of Agroecology - Entomology and Plant Pathology, Department of Agroecology, Science and Technology, Aarhus University3 Laboratoire de Biochimie et Technologie des Glucides, Nantes4 Dept. of Bioscience and Technology, Kagoshima University5 Dept. of Chemistry, Carlsberg Laboratory, Valby6 Department of Agroecology - Entomology and Plant Pathology, Department of Agroecology, Science and Technology, Aarhus University
Homogeneous barley limit dextrinase (LD) was isolated on a large scale in a yield of 9 mg/kg of 10-day germinated green malt. This represents a 9,400-fold purification and 29% recovery of the activity in a flour extract in 0.2M NaOAc (pH 5.0) containing 5 mM ascorbic acid. The purification protocol consists of precipitation from the extract at 20-70% saturated ammonium sulfate (AMS), followed by diethylaminoethyl (DEAE) 650S Fractogel anion-exchange chromatography, and affinity chromatography on b-cyclodextrin-Sepharose in the presence of 2M AMS. LD was eluted by 7 mM b-cyclodextrin and contains a single polypeptide chain of 105 kDa (SDS-PAGE) and pI 4.3. Sequence analysis of tryptic fragments, prepared from 2-vinylpyridinylated LD and purified by RP-HPLC, identified short motifs recognized in b-strand 2, 3, and 5 characteristic of a catalytic (b/a)8-barrel domain of the a-amylase family of amylolytic enzymes. Barley LD has »50 and 85% sequence identity to bacterial pullulanases and rice starch debranching enzyme, respectively. By using 1H-NMR spectroscopy, LD hydrolyzes specifically a-1,6-glucosidic linkages in pullulan and a branched oligodextrin, 62-O-a-maltotriosyl-maltotriose, with retention of the a-anomeric configuration. b-Cyclodextrin competitively inhibits the LD activity with Ki of 40 mM, while Ki is 1.9 mM and 2.4 mM for Υ-cyclodextrin and g-cyclodextrin, respectively.
Cereal Chemistry, 1998, Vol 75, Issue 4, p. 473-479