Bioprospecting of Thermotolerant Bacteria from Hot Water Springs of Himachal Pradesh for the Production of Taq DNA Polymerase

Abstract

Thermophilic microorganisms are adapted to live at high temperatures and produce many thermostable enzymes such as Taq polymerase, amylase, lipase, protease, etc., which find a number of commercial applications because of their thermostability. Thermal springs of Manikaran, Vashisht, Khirganga, Tattapani and Jeory of districts Kullu, Mandi and Shimla respectively, were selected for the present study and forty two thermophilic bacterial isolates were isolated using Castenholz trypticase yeast extract medium. All these thermophilic bacterial isolates were screened using morphological and biochemical characters. For molecular characterization of the selected isolates, Polymerase chain reaction was carried out for the amplification of the DNA using genus specific primers for 16S ribosomal RNA gene. Nucleotide sequences obtained after sequencing were blasted and only two isolates were found to belong to genus Thermus and the strain MS3 was found to possess maximum homology of 99 % with Thermus aquaticus. The growth parameters optimized were found to be pH of 7.5, incubation temperature 65 °C and incubation time 96 h. Taq polymerase produced using selected strain MS3, was purified by ammonium sulphate precipitation, gel permeation chromatography and ion exchange chromatography. The Taq polymerase assay with 1.5 µl purified enzyme fraction obtained after ion exchange chromatography, yielded comparable results with 0.5 µl (3.0 U/µl) of commercial Taq DNA polymerase, indicating that the protein fraction has 1.0 U/µl of DNA polymerase activity. Molecular mass of the purified Taq DNA polymerase was found to be 94,000 daltons. Protein sequencing analysis of the purified enzyme product showed 99 % homology with A-Chain structure of Taq DNA polymerase with accession number 1TAQ A.