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Red Sea Atlantis II brine pool nitrilase with unique thermostability profile and heavy metal tolerance.

ABSTRACT: Nitrilases, which hydrolyze nitriles in a one-step reaction into carboxylic acids and ammonia, gained increasing attention because of the abundance of nitrile compounds in nature and their use in fine chemicals and pharmaceutics. Extreme environments are potential habitats for the isolation and characterization of extremozymes including nitrilases with unique resistant properties. The Red Sea brine pools are characterized by multitude of extreme conditions. The Lower Convective Layer (LCL) of the Atlantis II Deep Brine Pool in the Red Sea is characterized by elevated temperature (68 °C), high salt concentrations (250 ‰), anoxic conditions and high heavy metal concentrations.We identified and isolated a nitrilase from the Atlantis II Deep Brine Pool in the Red Sea LCL. The isolated 338 amino-acid nitrilase (NitraS-ATII) is part of a highly conserved operon in different bacterial phyla with indiscernible function. The enzyme was cloned, expressed and purified. Characterization of the purified NitraS-ATII revealed its selectivity towards dinitriles, which suggests a possible industrial application in the synthesis of cyanocarboxylic acids. Moreover, NitraS-ATII showed higher thermal stability compared to a closely related nitrilase, in addition to its observed tolerance towards high concentrations of selected heavy metals.This enzyme sheds light on evolution of microbes in the Atlantis II Deep LCL to adapt to the diverse extreme environment and can prove to be valuable in bioremediation processes.

SUBMITTER: Sonbol SA

PROVIDER: S-EPMC4751646 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Project description:BackgroundCancer and infectious diseases are problematic because of continuous emergence of drug resistance. One way to address this enormous global health threat is bioprospecting the unlikeliest environments, such as extreme marine niches, which have tremendous biodiversity that is barely explored. One such environment is the Red Sea brine pool, Atlantis II Deep (ATII). Here, we functionally screened a fosmid library of metagenomic DNA isolated from the ATII lower convective layer (LCL) for antibacterial and anticancer activities.ResultsSelected clones, 14-7E and 10-2G, displayed antibacterial effects on the marine strain Bacillus sp. Cc6. Moreover, whole cell lysates from 14-7E and 10-2G exhibited decreased cell viability against MCF-7 (39.1% ± 6.6, 42% ± 8.1 at 50% v/v) and U2OS cells (35.7% ± 1.9, 79.9% ± 5.9 at 50% v/v), respectively. By sequencing the insert DNA from 14-7E and 10-2G, we identified two putative orphan biosynthetic gene clusters. Both clusters harbored putative ATP-binding cassette (ABC) transporter permeases and S-adenosylmethionine-related genes. Interestingly, the biosynthetic gene cluster identified on 14-7E is of archaeal origin and harbors a putative transcription factor. Several identified genes may be responsible for the observed antibacterial and anticancer activities. The 14-7E biosynthetic gene cluster may be encoding enzymes producing a specialized metabolite (effect of detected genes involved in C-C bond formation and glycosylation). The bioactivity may also be due to predicted subtilases encoded by this cluster. The 10-2G cluster harbored putative glycosyltransferase and non-ribosomal peptide synthase genes; thus the observed activity of this clone could be caused by a bioactive peptide.ConclusionsThe ATII LCL prokaryotic metagenome hosts putative orphan biosynthetic gene clusters that confer antibiotic and anticancer effects. Further biochemical studies should characterize the detected bioactive components, and the potential use of 14-7E metabolite for antibiosis and 10-2G metabolite as a selective anti-breast cancer drug.

Project description:BackgroundThe search for novel antimicrobial agents is crucial as antibiotic-resistant pathogens continue to emerge, rendering the available antibiotics no longer effective. Likewise, new anti-cancer drugs are needed to combat the emergence of multi-drug resistant tumors. Marine environments are wealthy sources for natural products. Additionally, extreme marine environments are interesting niches to search for bioactive natural compounds. In the current study, a fosmid library of metagenomic DNA isolated from Atlantis II Deep Lower Convective Layer (ATII LCL), was functionally screened for antibacterial activity as well as anticancer effects.ResultsTwo clones exhibited antibacterial effects against the marine Bacillus Cc6 strain, namely clones 102-5A and 88-1G and they were further tested against eleven other challenging strains, including six safe relatives of ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.), a safe relative to Mycobacterium tuberculosis and four resistant clinical isolates. Clone 88-1G resulted in clear zones of inhibition against eight bacterial strains, while clone 102-5A resulted in zones of inhibition against five bacterial strains. The whole cell lysates of clone 88-1G showed 15% inhibition of Mtb ClpP protease -Mycobacterium tuberculosis drug target-, while whole cell lysates of clone 102-5A showed 19% inhibition of Mtb ClpP protease. Whole cell lysates from the selected clones exhibited anticancer effects against MCF-7 breast cancer cells (cell viability at 50% v/v was 46.2% ± 9.9 for 88-1G clone and 38% ± 7 for 102-5A clone), U2OS osteosarcoma cells (cell viability at 50% v/v was 64.6% ± 12.3 for 88-1G clone and 28.3% ± 1.7 for 102-5A clone) and 1BR hTERT human fibroblast cells (cell viability at 50% v/v was 74.4% ± 5.6 for 88-1G clone and 57.6% ± 8.9 for 102-5A clone). Sequencing of 102-5A and 88-1G clones, and further annotation detected putative proteases and putative biosynthetic genes in clones 102-5A and 88-1G, respectively.ConclusionsThe ATII LCL metagenome hosts putative peptidases and biosynthetic genes that confer antibiotic and anti-cancer effects. The tested clones exhibited promising antibacterial activities against safe relative strains to ESKAPE pathogens and Mycobacterium tuberculosis. Thus, searching the microbial dark matter of extreme environments is a promising approach to identify new molecules with pharmaceutical potential use.

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Red Sea Atlantis II brine pool nitrilase with unique thermostability profile and heavy metal tolerance.

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