Detoxificatibn Of toxic heavy. metals by serine protease inhibitor isolated from polluted soil

Various anthropogenic activities attribute to the addition and accumulation of heavy metals in soil which creates an environment unsuitable for all organisms. Despite such conditions, some microbial populations are able to survive and grow due to functioning of certain genes. Investigating the role of eukaryotic microbes in metal contaminated soil was the focus of this study. Culture independent approach was employed and size fractionated eukaryotic cDNA libraries (library A < 0.5 kb, library B 0.5-1.0 kb, and library C 1.0-4.0 kb) of soil were constructed from soil RNA and were screened for cadmium (Cd) tolerance by functional complementation assay by using Cd sensitive yeast mutant ycf1(Delta). Screening of the library C yielded clones capable of growing in presence of Cd amended media. In the present study, one of the clones, PLCg49 was selected because of its ability to grow at high concentrations of Cd (40-100 mu M). PLCg49 was also found to be highly inducible in presence of Cd where the highest fold expression was observed at 60 mu M of Cd. Sequence analysis of PLCg49 showed homology with Serine Protease Inhibitor (Serpin) of the eukaryote Hypsibius dujardini. Serpins are a broad family of protease inhibitors which are majorly stress-inducible genes usually triggered by biotic and abiotic stresses and help in maintaining cell viability. Copper-sensitive (cup1(Delta)), Zn-sensitive (zrc1(Delta)) and Cobalt-sensitive (cot1(Delta)) yeast mutants rescued their sensitivity when transformed with PLCg49, indicating its tolerance to different heavy metals. The protease sensitive yeast mutant (pbi2(Delta)) transformed with PLCg49 protected from the cell lysis when grown in presence of trypsin and proteinase K confirming the role of PLCg49 as an effective serpin. Present study results suggested that PLCg49 serves as a potential metal tolerant gene, helping us to understand new pathways participating in metal tolerance.