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.
Potin
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Centre d'Etudes d'Océanologie et de Biologie Marine, Centre National de la Recherche Scientifique, France.
Richard
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Rochas
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C
Kloareg
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B
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