Molecular and functional characterization of unique thermo-halophilic thioredoxin from the metagenome of an exotic environment.

The lower convective layer (LCL) at Atlantis II brine pool of the Red sea represents one of the exceptional, unique ecosystems. Thioredoxin is a multi-functional antioxidant redox protein that has a crucial role in various vital cellular processes. In the current study, a functional metagenomics approach was used to isolate and characterize thioredoxin from the LCL of Atlantis II Deep brine pool (Trx-ATII). From the metagenomic DNA of the LCL, the thioredoxin gene was directly retrieved and sequenced. Sequence analysis showed that the gene belonged to thioredoxin-like superfamily with classical Trx motif (-CXXC-). Phylogenetic analysis revealed that Trx-ATII was closely related to Trx of Prochlorococcus marinus with a maximum identity of 86%. Successfully, Trx-ATII was cloned and expressed in E. coli, where the purified protein had M.wt of 16 kDa. Characterization studies revealed that Trx-ATII protein is halophilic; can tolerate up to 2.5 M NaCl and thermostable, where 90% of its activity was retained at 60 °C. Trx-ATII can reduce both DTNB and insulin disulfide- containing substrates. In conclusion, a unique thioredoxin protein was isolated from a harsh environment that can maintain its activity under extreme conditions of salinity and temperature as a promising redox protein for biotechnological applications.