Purification and characterization of an alkaline amylopullulanase with both α-1,4 and α-1,6 hydrolytic activity from alkalophilic Bacillus sp. KSM-1378
| Autor: | Kazuaki Igarashi, Katsuhisa Saeki, Hiroshi Hagihara, Katsutoshi Ara, Susumu Ito, Shuji Kawai, Takaaki Uemura, Mikio Takaiwa |
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| Rok vydání: | 1995 |
| Předmět: |
Glycoside Hydrolases
Amylopectin Molecular Sequence Data Biophysics Bacillus Biochemistry Substrate Specificity chemistry.chemical_compound Bacterial Proteins Enzyme Stability Carbohydrate Conformation Maltotriose Amino Acid Sequence Isoelectric Point Amylase Pullulanase activity Glucans Molecular Biology chemistry.chemical_classification Cyclodextrins Pullulanase biology Molecular mass Hydrolysis Temperature Mercury Hydrogen-Ion Concentration Peptide Fragments Molecular Weight Isoelectric point Enzyme chemistry biology.protein Amylose Trisaccharides Glycogen |
| Zdroj: | Biochimica et Biophysica Acta (BBA) - General Subjects. 1243:315-324 |
| ISSN: | 0304-4165 |
| DOI: | 10.1016/0304-4165(94)00148-q |
| Popis: | 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 alpha-1,6 linkages in pullulan to generate maltotriose as the major end product, as well as the alpha-1,4 linkages in amylose, amylopectin and glycogen to generate various oligosaccharides. The pH and temperature optima for the pullulanase and alpha-amylase activities were pH 9.5 and 50 degrees C and pH 8.5 and 50 degrees 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 Hg2+ ions, alpha-cyclodextrin and beta-cyclodextrin while the amylase activity was strongly inhibited by EDTA and EGTA, although inhibition could be reversed by Ca2+ ions. It is suggested that the single alkaline amylopullulanase protein has two different active sites, one for the cleavage of alpha-1,4-linked substrates and one for the cleavage of alpha-1,6-linked substrates. |
| Databáze: | OpenAIRE |
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