Caracterización molecular de las hidrolasas producidas por la bacteria halófila extrema salicola SP. IC10

Resumen

Solar marine salterns constitute excellent models for studying the ecology of hypersaline environments where molar concentrations of salts are present. Extremely halophilic microorganisms, able to grow optimally in media containing 15-30% (w/v) NaCl, are evolved two different strategies for the osmoadaptation to saline habitats: salt-in accumulation of inorganic cations) and salt-out (synthesis and/or accumulation of compatible solutes). Extreme halophiles have potential biotechnological applications including the production of compatible solutes, enzymes, ß-carotene and the degradation of toxic chemicals or enhanced oil recovery. To our knowledge, no proteases and lipases from extremely halophilic bacteria have been characterized. Therefore, the detection, isolation and characterization of hydrolytic enzymes produced by extreme halophiles with optimal activity over a wide range of salt concentrations, constitute an interesting research topic. In this study we explored the presence of extremely halophilic Bacteria or Archaea producing hydrolytic enzymes (lipases, proteases, amylases and DNAses) in marine salterns in Huelva (South Spain). Based on its ability to produce proteolytic and lipolytic activity we selected and characterized the extremely halophilic bacterium Salicola marasensis IC10. Moreover, we determined the osmoadaptation mechanism of S. marasensis IC10 to balance the osmotic stress. For the achievement of these objectives, a screening program was performed in marine salterns of Huelva using saline media containing 20% (w/v) NaCl and the specific substrates for the hydrolytic enzymes. Under the restrictive conditions used, a total of 150 fresh cultures showing hydrolytic activities were isolated and from them, only 82 strains presented the typical growth salt requirement profile exhibited by the extreme halophiles. 43 strains were selected and characterized. Most hydrolase producers isolated were assigned to the family Halobacteriaceae, comprising species of the genera Halorubrum, Haloarcula and Halobacterium. Three strains were not phylogenetically assigned to the family Halobacteriaceae and were closely related to Salicola marasensis, Pseudomonas halophila and Salinibacter ruber. The most frequent hydrolytic activity was amylase (70%), followed by protease (17%) and lipase (13%). The majority of the amylase producers formed a clear branch with high affinity to the genus Halorubrum, the protease producers were closely related to the genus Halobacterium and the lipolytic producers had the major genera diversity. The strain IC10 showing proteolytic and lipolytic activity was selected for further studies. According to phylogenetic, phenotypic and genotypic data, this strain was assigned as S. marasensis IC10 and the optimum conditions for growth were 15% NaCl, pH 8.0 and 40ºC. Lipolytic activity was detected in the intracellular fraction corresponding to cytoplasmic proteins and the activity was stable in the temperature range from 37ºC to 45ºC, in presence of organic solvents such as 1-buthanol, 2-buthanol and acetone as well as the chelator EDTA and the salts of Ni2+, Ca2+ and Zn2+. The lipolytic fraction showed a high tolerance to saline conditions, up to even 4 M NaCl with an optimum at 1 M NaCl. The proteolytic activity was also detected in the intracellular fraction, showing optimal activity with egg albumin and gelatin as substrates. The gene coding the protease Salipro, designated protS, has been cloned by inverse PCR and encodes an 817 residues protein, showing high sequence similarity to serine proteases of the Lon family. The osmoadaptation mechanism to balance the osmotic stress of S. marasensis IC10 consists on the accumulation of betaine as the sole compatible solute. The accumulation of betaine is not affected by the salinity of the culture medium and this bacterium is able to synthesize betaine of de novo.