Amylolytic Activities Excreted by the Halophilic Archaeon Haloferax mediterranei to Assimilate Available Starch Depend on the Nitrogen Source

Several amylolytic activities have been isolated from a controlled growing media containing starch and nitrate or ammonium acetate as a carbon and energy source, excreted by the halophilic archaeon Haloferax mediterranei. These enzymes produced in nitrate-containing medium were different from those produced by the organism when cultured in ammonium acetate-containing medium. Haloferax mediterranei was able to grow optimally in both the media but not in a medium with ammonium chloride and starch as exclusive source of nitrogen and carbon, respectively. Growth was significantly lower when nitrate was replaced by ammonium, although there was significant amylolytic activity in the medium. At least six different activities were obtained in the nitrate-containing medium, but only five for the ammonium containing one. These enzymes displayed a different affinity for starch as a chromatographic matrix, when eluted with maltose in a range from 0.02 M to 0.2 M, and differed in their kinetic parameters for starch as a substrate. The average medium length of the products obtained from cracking starch was different for each amylolytic activity, ranging from glucose to larger polysaccharides. Moreover, they exhibited different molecular masses, from 15 to 80 kDa. On the other hand, all of them behaved as typical halophilic enzymes, requiring high salt concentrations from 2M to 4M NaCl for stability and activity. Also, it exhibited an optimal pH ranged from 7 to 8 and showed certain thermophilic behaviour, with maximal activity within 50°C to 60°C. The study of the presence and behaviour of this set of starch degrading enzymes will allow for a better understanding of how halophilic organism obtain the adequate carbohydrates to be incorporated and optimally used.