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

¹Department of Chemistry, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Valby, Denmark. Present address (MK): Danish Pest Infestation Laboratory, Skovbrynet 14, DK-2800 Lyngby. ²Laboratoire de Biochimie et Technologie des Glucides, I.N.R.A., Rue de la Géraudière, B.P. 1627, 44316 Nantes cedex 03, France. ³Department of Bioscience and Technology, Kagoshima University, Korimoto 1-21-24, Kagoshima 890, Japan. ⁴Corresponding author. Phone: +45 3327 5345. Fax: +45 3327 4708. E-mail: bis@crc.dk

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 β-cyclodextrin-Sepharose in the presence of 2M AMS. LD was eluted by 7 mMβ-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 β-strand 2, 3, and 5 characteristic of a catalytic (β/α)₈-barrel domain of the α-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 α-1,6-glucosidic linkages in pullulan and a branched oligodextrin, 6²-O-α-maltotriosyl-maltotriose, with retention of the α-anomeric configuration. β-Cyclodextrin competitively inhibits the LD activity with K_i of 40 μM, while K_i is 1.9 mM and 2.4 mM for α-cyclodextrin and γ-cyclodextrin, respectively.