Purification and characterization of the alpha-agarase from Alteromonas agarlyticus (Cataldi) comb. nov., strain GJ1B.
The phenotypic features of strain GJ1B, an unidentified marine bacterium that degrades agar [Young, K. S. Bhattacharjee, S. S. & Yaphe, W. (1978) Carbohydr. Res. 66, 207-212], were investigated and its agarolytic system was characterized using 13C-NMR spectroscopy to analyse the agarose degradation products. The bacterium was assigned to the genus Alteromonas and the new combination A. agarlyticus (Cataldi) is proposed. An alpha-agarase, i.e. specific for the alpha(1-->3) linkages present in agarose, was purified to homogeneity from the culture supernatant by affinity chromatography on cross-linked agarose (Sepharose CL-6B) and by anion-exchange chromatography (Mono Q column). The major end product of agarose hydrolysis using the purified enzyme was agarotetraose. Using SDS/PAGE, the purified alpha-agarase was detected as a single band with a molecular mass of 180 kDa. After the affinity-chromatography step, however, the native molecular mass was approximately 360 kDa, suggesting that the native enzyme is a dimer which is dissociated to active subunits by anion-exchange chromatography. The isolectric point was estimated to be 5.3. Enzyme activity was observed using agar as the substrate over the pH range 6.0-9.0 with a maximum value at pH 7.2 in Mops or Tris buffer. The enzyme was inactivated by prolonged treatment at a pH below 6.5, or by temperatures over 45 degrees C or by removing calcium. In addition, a beta-galactosidase specific for the end products of the alpha-agarase was present in the alpha-agarase affinity-chromatography fraction, probably as part of a complex with this enzyme. The degradation of agarose by this agarase complex yielded a mixture of oligosaccharides in the agarotetraose series and the agarotriose series, the latter consisting of oligosaccharides with an odd number of galactose residues.
Centre d'Etudes d'Océanologie et de Biologie Marine, Centre National de la Recherche Scientifique, France.
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Potin
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Richard
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Rochas
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Kloareg
D002236:Carbohydrate Conformation; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006090:Gram-Negative Bacteria / Q000145:classification / Q000201:enzymology* / Q000254:growth & development; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007526:Isoelectric Point; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy; D008970:Molecular Weight; D012685:Sepharose / Q000378:metabolism; D013379:Substrate Specificity
D046911:Macromolecular Substances; D012685:Sepharose; D006026:Glycoside Hydrolases; C019624:agarase