Large‐Scale Purification and Characterization of Barley Limit Dextrinase, a Member of the α‐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 represents a 9,400‐fold purification and 29% recovery of the activity in a flour extract in 0.2 M NaOAc (pH 5.0) containing 5 m M 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 2 M AMS. LD was eluted by 7 m M β‐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 1 H‐NMR spectroscopy, LD hydrolyzes specifically α‐1,6‐glucosidic linkages in pullulan and a branched oligodextrin, 6 2 ‐ O ‐α‐maltotriosyl‐maltotriose, with retention of the α‐anomeric configuration. β‐Cyclodextrin competitively inhibits the LD activity with K i of 40 μ M , while K i is 1.9 m M and 2.4 m M for α‐cyclodextrin and γ‐cyclodextrin, respectively.