Purification and characterization of the alpha-agarase from Alteromonas agarlyticus (Cataldi) comb. nov., strain GJ1B

Eur J Biochem. 1993 Jun 1;214(2):599-607. doi: 10.1111/j.1432-1033.1993.tb17959.x.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Carbohydrate Conformation
  • Chromatography, Affinity
  • Chromatography, Ion Exchange
  • Glycoside Hydrolases / chemistry
  • Glycoside Hydrolases / isolation & purification*
  • Glycoside Hydrolases / metabolism
  • Gram-Negative Bacteria / classification
  • Gram-Negative Bacteria / enzymology*
  • Gram-Negative Bacteria / growth & development
  • Hydrogen-Ion Concentration
  • Hydrolysis
  • Isoelectric Point
  • Macromolecular Substances
  • Magnetic Resonance Spectroscopy
  • Molecular Weight
  • Sepharose / metabolism
  • Substrate Specificity


  • Macromolecular Substances
  • Sepharose
  • Glycoside Hydrolases
  • agarase