The Red Sea Atlantis II brine pool is an extreme environment that displays multiple harsh conditions such as high temperature, high salinity and high concentrations of multiple, toxic heavy metals. The survival of microbes in such an environment by utilizing resistant enzymes makes them an excellent source of extremophilic enzymes. We constructed a fosmid metagenomic library using DNA isolated from the deepest and most secluded layer of this pool. We report the isolation and biochemical characterization of an unusual esterase: EstATII. EstATII is thermophilic (optimum temperature, 656C), halotolerant (maintains its activity in up to 4.5 M NaCl) and maintains at least 60% of its activity in the presence of a wide spectrum of heavy metals. The combination of biochemical characteristics of the Red Sea Atlantis II brine pool esterase, i.e., halotolerance, thermophilicity and resistance to heavy metals, makes it a potentially useful biocatalyst.U ntil recently and despite its uniqueness, the Red Sea has received little attention compared with other marine environments. The Red Sea formed 3-5 million years ago when the Arabian and African plates began to split 1 , and it is characterized by high temperature and salinity due to the high rate of evaporation, lack of major river inflows and low rate of rainfall. The Red Sea is characterized by the presence of deep-sea, hypersaline anoxic basins called brine pools, which are large bodies of water at the bottom of the ocean that are characterized by high temperature and salinity. To date, 25 brine pools have been found in the Red Sea 1,2 . Atlantis II Deep (Figure 1) is the largest brine pool in the Red Sea, and it has the highest temperature and is the most dynamic 1,3 . This brine pool has a maximum depth of 2,194 m and is stratified into several layers that increase in temperature and salinity with increasing depth: the brine-seawater interface, the upper convective layer, the middle convective layer and the lower convective layer (LCL) 1,3 . The lowest layer, LCL, is characterized by a temperature of 68.2uC, pH value of 5.3 and salinity of 270 psu, which is 7.5 times that of normal seawater 1,3 . Atlantis II Deep is nearly anoxic and contains high concentrations of iron, zinc, copper and other heavy metals 1,3 . Together, these extreme conditions make the Atlantis II brine pool an attractive site for mining for biocatalysts, such as lipolytic enzymes, which are predicted to possess desirable traits that include but are not limited to thermo-tolerance, halo-tolerance, pH plasticity and resistance to inhibition by heavy metals.Industrialized societies are moving toward white (industrial) biotechnology, which has proven to be environmentally sound and commercially efficient 4 . Doing so poses a continuous demand for novel biocatalysts, preferably biocatalysts that demonstrate high activity over a wide range of conditions such as temperature, salinity, pH and metal concentrations. Biocatalysts of microbial origin represent the majority of biocatalysts that are used in indu...
