The Red Sea harbors a number of deep-sea brine pools with diverse chemical, physical, and geological conditions. One of the environments, the Atlantis II Brine Pool, is unique to the Red Sea. The deepest part of Atlantis II Brine Pool â the Lower Convective Layer (LCL) â is 2060 meters below the sea surface water, with 26% salinity, and a temperature of about 68 Â°C. Moreover, it is almost anoxic, and contains a very high concentration of heavy metals. We are interested in identifying subtilisin-like serine proteases, which are an important group of proteolytic enzymes with a broad range of use in biotechnological applications. Toward this goal, and in collaboration with King Abdullah University for Science and Technology (KAUST) and Woods Hole Oceanographic Institute (WHOI), we collected water samples from this environment. Microbial DNA was isolated, and using pyrosequencing technology (454 Roche platform) we established a metagenome dataset composed of 1.34 millions reads with average length of around 390 nucleotides/read. In addition, we constructed a fosmid library composed of around 11,000 clones, representing 440 Mbp. Using annotated sequence information from the metagenomic sequencing dataset and a PCR-based method for fast screening, we identified a clone containing a sequence of 1194 base pairs comprising an open reading frame with high similarity to proteins of the predicted function. Further computational analysis revealed the presence of a ribosomal binding site, transcription regulatory elements in a promoter region and translation start/stop codons. In addition, the presence of a signal peptide and predictions of the protein localization allowed us to expect that the protein is likely to be adapted to extreme conditions such as high temperatures and salinity levels. Functional characterization of the protein and examination of activity and stability under a variety of conditions may lead to a beneficial biotechnological enzyme adapted to extreme environments.
School of Sciences and Engineering
MS in Biotechnology
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(2021).Mining and identification of a potential subtilisin-like serine protease from the lower convective layer of the Atlantis II brine pool in the Red Sea [Thesis, the American University in Cairo]. AUC Knowledge Fountain.
Shibl, Ahmed. Mining and identification of a potential subtilisin-like serine protease from the lower convective layer of the Atlantis II brine pool in the Red Sea. 2021. American University in Cairo, Thesis. AUC Knowledge Fountain.