Rapid Identification of Ectoine-Producing Halophiles Using Newly Designed Degenerate Primers and LC-MS/MS
Makaleler > Tam Makale · 2026
Yazarlar: Ebru Tekin, Mustafa ATEŞ
Çanakkale Onsekiz Mart University Journal of Marine Sciences and Fisheries, Uluslararası, 2026, cilt 9, ss 64-78
Özet
Ectoine is a valuable compatible solute widely used in pharmaceutical, cosmetic, and biotechnological applications due to its protective and stabilizing properties. This study aimed to identify ectoine-producing bacterial strains isolated from diverse saline environments, including the İzmir Çamaltı Saltern, İzmir Bird Paradise, and commercial Himalayan salt. A PCR-based screening approach was applied using in silico primer design. Newly designed degenerate primers targeting the ectB-ectC gene region was used in this study. Primer design was performed for each strain by analyzing conserved genomic regions between the adjacent ectB and ectC genes using representative type strains retrieved from NCBI. Gradient PCR conditions, particularly annealing temperature and extension time, were optimized individually for each isolate to improve amplification efficiency and specificity. For confirmation of genetic screening, ectoine production was assessed in 15% salinity medium and quantified using LC–MS/MS analysis and ectoine standard. A total of eight bacterial isolates were purified and screened for the presence of the ectB-ectC region. Among them, three isolates -Kocuria sp. CT-6, Oceanobacillus sp. HT-9, and Halomonas sp. CS-5- were found positive for the target region. A representative ectB-ectC positive Halomonas sp. CS-5 strain was selected for further analysis as a study-specific model and cultivated under 15% salinity conditions. Ectoine production was confirmed as 0.395 g/L (mean±SD, n=3, SD=0) in the culture reached the stationary phase at 48.5 h, total dry cell weight was 1.77 g/L corresponding to 22.3% (w/w) of dry cell weight by LC-MS/MS analysis. The study demonstrates that the combined application of PCR-based screening and analytical confirmation provides a potentially useful preliminary screening approach for identifying ectoine-producing microorganisms. This approach supports the selection of promising strains for further biotechnological evaluation.