Purification and characterization of an alkaline amylopullulanase with both α-1,4 and α-1,6 hydrolytic activity from alkalophilic Bacillus sp. KSM-1378

• Published in: Biochimica et biophysica acta Vol. 1243; no. 3; pp. 315 - 324
• Main Authors: Ara, Katsutoshi, Saeki, Katsuhisa, Igarashi, Kazuaki, Takaiwa, Mikio, Uemura, Takaaki, Hagihara, Hiroshi, Kawai, Shuji, Ito, Susumu
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 13.04.1995
• Subjects: Amino Acid Sequence; Amylase; Amylopectin - metabolism; Amylopullulanase; Amylose - metabolism; Bacillus; Bacillus - enzymology; Bacterial Proteins - chemistry; Bacterial Proteins - isolation & purification; Bacterial Proteins - metabolism; Carbohydrate Conformation; Cyclodextrins - pharmacology; Enzyme Stability; Glucans - metabolism; Glycogen - metabolism; Glycoside Hydrolases - chemistry; Glycoside Hydrolases - isolation & purification; Glycoside Hydrolases - metabolism; Hydrogen-Ion Concentration; Hydrolysis; Isoelectric Point; Mercury - pharmacology; Molecular Sequence Data; Molecular Weight; Peptide Fragments - chemistry; Pullulanase; Substrate Specificity; Temperature; Trisaccharides - metabolism; CD; Amylase; Bacillus; Amylopullulanase; p I; PAGE; DP; NBS; Pullulanase
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/0304-4165(94)00148-Q

The novel alkaline amylopullulanase produced by alkalophilic Bacillus sp. KSM-1378 was purified to an electrophoretically homogeneous state from culture medium. The purified enzyme was a glycoprotein with an apparent molecular mass of about 210 kDa and an isoelectric point of pH 4.8. The N-terminal amino acid sequence was Glu-Thr-Gly-Asp-Lys-Arg-Ile-Glu-Phe-Ser-Tyr-Glu-Arg-Pro and showed no homology to the N-terminal regions of other amylopullulanases reported to date. The enzyme was able to attack specifically the α-1,6 linkages in pullula.n to generate maltotriose as the major end product, as well as the α-1,4 linkages in amylose, amylopectin and glycogen to generate various oligosaccharides. The pH and temperature optima for the pullulanase and α-amylase activities were pH 9.5 and 50°C and pH 8.5 and 50°C respectively. Both activities were strongly inhibited by well characterized inhibitors, such as diethyl pyrocarbonate and N-bromosuccinimide. The pullulanase activity was specifically inactivated by Hg 2+ ions, α-cyclodextrin and β-cyclodextrin while the amylase activity was strongly inhibited by EDTA and EGTA, although inhibition could be reversed by Ca 2+ ions. It is suggested that the single alkaline amylopullulanase protein has two different active sites, one for the cleavage of α-1,4-linked substrates and one for the cleavage of α-1,6-linked substrates.