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 approximate to 50 and 85% sequence identity to bacterial pullulanases and rice starch debranching enzyme, respectively. By using H-1-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 cc-anomeric configuration. beta-Cyclodextrin competitively inhibits the LD activity with K-i of 40 mu M, while K-i is 1.9 mM and 2.4 mM for alpha-cyclodextrin and gamma-cyclodextrin, respectively.