Deep-sea anoxic brine pools of the Red Sea are considered one of the most remote and extreme environments on Earth while remaining one of the least studied. High salt concentrations (4.3 M), elevated temperatures (up to 68°C) and high metal content make them promising sources for novel enzymes with structures distinguished by evolutionary adaption. Reliable functional annotation of genomic data is the key-step in the discovery of novel enzymes. Therefore we developed a Profile & Pattern Matching algorithm to eliminate false positive annotations [1]. Based on scientific and industrial interest 13 genes were selected and are currently expressed in halophilic expression systems. One of the most interesting genes identified might open a novel class of Mn2+ dependent alcohol dehydrogenases. The enzyme is extremely tolerant to a broad range of salt concentrations (from millimolar to saturation) and high solvent concentrations, converts a divers spectrum of substrates and is stable at both, high temperatures (up to 80°C) and basic pH. More interesting is the altered activity by substitution with different metal ions, where in opposite to in silico predictions Fe2+ shows no effect on activity. The enzyme is slightly activated with Zn2+ but shows an activation boost with Mn2+. The activity profile at different temperatures, salt, pH, aggregation temperature, hydrodynamic radius and the substrate spectra changed significantly between Zn2+ or Mn2+ addition, representing the natural environment at the brine pool of origin. To optimize expression the halophilic host Haloferax volcanii was genetically modified and optimized creating the first halophilic expression system, which is applicable in a bioreactor [2]. This system achieves a 28-fold increase in total protein yield in comparison to shaking flasks. [1] Grötzinger, S. W.; et al., Frontiers in microbiology 2014, 5, 134 [2] Grötzinger, S.W.; et al., Appl. Microbiol. Biotechnol. DOI 10.1007/s00253-015-7007-1

Discovery, expression and characterization of novel extremozymes from the deep Red-Sea brine pools

251st National Meeting (2016)

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