LARGE-SCALE PURIFICATION AND CHARACTERIZATION OF BARLEY LIMIT DEXTRINASE, A MEMBER OF THE ALPHA-AMYLASE STRUCTURAL FAMILY

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 represent s a 9,400-fold purification and 29% recovery of the activity in a flou r extract in 0.2M NaOAc (pH 5.0) containing 5 mM ascorbic acid. The pu rification protocol consists of precipitation from the extract at 20-7 0% saturated ammonium sulfate (AMS), followed by diethylaminoethyl (DE AE) 650S Fractogel anion-exchange chromatography, and affinity chromat ography on beta-cyclodextrin-Sepharose in the presence of 2M AMS. LD w as 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 f ragments, prepared from 2-vinylpyridinylated LD and purified by RP-HPL C, identified short motifs recognized in beta-strand 2, 3, and 5 chara cteristic of a catalytic (beta/alpha)(8)-barrel domain of the alpha-am ylase family of amylolytic enzymes. Barley LD has approximate to 50 an d 85% sequence identity to bacterial pullulanases and rice starch debr anching enzyme, respectively. By using H-1-NMR spectroscopy, LD hydrol yzes specifically alpha-1,6-glucosidic linkages in pullulan and a bran ched oligodextrin, 6(2)-O-alpha-maltotriosyl-maltotriose, with retenti on of the cc-anomeric configuration. beta-Cyclodextrin competitively i nhibits 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.