Publication no. C-1998-0603-01R |  VIEW ARTICLE

Large-Scale Purification and Characterization of Barley Limit Dextrinase, a Member of the Alpha-Amylase Structural Family.

Michael Kristensen (1), Véronique Planchot (2), Jun-ichi Abe (3), and Birte Svensson (1,4). (1) Department of Chemistry, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Valby, Denmark. Present address (MK): Danish Pest Infestation Laboratory, Skovbrynet 14, DK-2800 Lyngby. (2) Laboratoire de Biochimie et Technologie des Glucides, I.N.R.A., Rue de la Géraudière, B.P. 1627, 44316 Nantes cedex 03, France. (3) Department of Bioscience and Technology, Kagoshima University, Korimoto 1-21-24, Kagoshima 890, Japan. (4) Corresponding author. Phone: +45 3327 5345. Fax: +45 3327 4708. E-mail: <bis@crc.dk> Cereal Chem. 75(4):473-479. Accepted March 30, 1998. Copyright 1998 American Association of Cereal Chemists, Inc.

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 beta-cyclodextrin-Sepharose in the presence of 2M AMS. LD was eluted by 7 mM beta-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 beta-strand 2, 3, and 5 characteristic of a catalytic (beta/alpha)(8)-barrel domain of the alpha-amylase family of amylolytic enzymes. Barley LD has approximately 50 and 85% sequence identity to bacterial pullulanases and rice starch debranching enzyme, respectively. By using (^1)H-NMR spectroscopy, LD hydrolyzes specifically alpha-1,6-glucosidic linkages in pullulan and a branched oligodextrin, 6(^2)-O-alpha-maltotriosyl-maltotriose, with retention of the alpha-anomeric configuration. beta-Cyclodextrin competitively inhibits the LD activity with K(i) of 40 mM, while K(i) is 1.9 mM and 2.4 mM for alpha-cyclodextrin and gamma-cyclodextrin, respectively.