Comparison of Action Patterns of Debranching Amylases

Pseudomonas amyloderamosa isoamylase and Klebsiella pneumoniae pullulanase were crystalline preparations obtained from Hayashibara Biochemical Laboratory, Inc., Okayama, Japan. Bacillus acidopullulyticus pullulanase was purified from Promozyme 200 L (Novo Nordisk Bioindustry, Ltd., Copenhagen, Denmark) by the method of Kusano et al. (Agric. Biol. Chem., 52, 2293). These three enzyme preparations showed a single band on PAGE. Optimum pH of Pseudomonas isoamylase for amylopectin was 3.5 with a shoulder near pH 5. The optimum pH for Br-CDs shifted from 3.5 for amylopectin to 4.5-4.7 (Br-α- and -β-CDs) and 4.0 (G3-, G4-γ-CDs). The pH curve for Br-γ-CDs had a shoulder near pH 5.0. Optimum pHs of Klebsiella and Bacillus pullulanases for pullulan and Br-γ-CDs were 5.5 and 5. 0, but those for Br-α-CDs were 6.0 and 4. 0, respectively. Kinetic parameters of these enzymes for Br-CDs indicated that (1) all of them cleaved more easily the a(1→6) linkages of G3-G5-CDs than those of G2-CDs, (2) they split more easily the a(1→6) linkages of Br-r-CDs than those of the other Br-CDs, (3) Pseudomonas isoamylase hydrolyzed readily the a(1→6) linkage of G2-7-CD and (4) Br-r-CDs were better substrates for kinetical analysis of debranching amylase than amylopectin and pullulan.