Isolation & characterization of Thermus aquaticus and production of Taq DNA polymerse enzyme
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Date
2011
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ABSTRACT
Thermophiles are the organisms which are adapted to live at high temperatures. The enzymes from thermophiles find a number of commercial applications because of their thermostability and thermoactivity. Therefore, the isolation of thermophilic bacteria from natural sources and their identification are very important in terms of discovering new industrial enzymes. In keeping with this view, hot water springs of Himachal Pradesh could serve as a good source for new thermophilic microorganisms with novel industrially important properties. The aim of this research was therefore the isolation of Thermus aquaticus and production of Taq DNA polymerase enzyme. Eight hot water springs viz., Manikaran, Vashisht, Khirganga, Tattapani, Jeory and Rajgarh were purposely selected for the present studies. Ten samples from each hot water springs were collected. The pH and temperature of the eight thermal springs were recorded and ranged from 4.1-6.8 and 23oC-105oC respectively. The chloride, sulphate, total hardness, calcium hardness and magnesium content ranged from 198.0-2673.0, 12.0-70.0, 105.0-698.0, 33.66-258.0, 84.0-544.0 and 1.46-55.16 mg/l respectively. Forty two bacterial isolates were isolated from the selected eight hot water springs of Himachal Pradesh using different media. All the bacterial isolates were studied for various morphological characters and on the basis of morphological characterization, 20 isolates were screened out. The selected 20 isolates were further investigated for biochemical characters and five isolates of Thermus i.e, TMA5, TMA7, TVB8, TK10 & TT5 were selected for further enzymatic studies. The selected five bacterial isolates were screened for the intracellular production of Taq DNA polymerase enzyme. The maximum (0.03 U/mg protein) Taq polymerase was released by thermophilic bacterial isolate TMA5. Optimization of culture conditions for enzyme production was carried out. The intracellularly released Taq polymerase from thermophilic bacterial isolate TMA5 was partially purified by ammonium sulhate precipitation followed by gel permeation chromatography and SDS-PAGE. The Taq polymerase assay of the partially purified enzyme fraction revealed that although percent recovery of enzyme was low but it resulted in increasing the purity of Taq DNA polymerase by 1.67 folds. Genomic DNA was isolated from the selected isolate TMA5. PCR of the isolated DNA was carried out using genus specific primers for 16S rDNA gene and amplification of the DNA was carried out using primers. Sequencing of the PCR product was done using similar primers. Sequence of the TMA5 isolate so obtained was found to be 1325 bp. BLASTN analysis showed 95-98% homology of the query sequence with other isolates of Thermus aquaticus. A total of 17 Thermus aquaticus sequences were mined and these sequences were used to compare the 16S rDNA sequence of the test isolate TMA5. Alignment score was highest for Thermus aquaticus strain YT-1 16S ribosomal RNA, partial sequence (NR_025900.1). Phylogenetic analysis based on nucleotide sequences using NJ method was achieved via phylip 3.68 and EXOMETM HORIZON.
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Thermophiles,enzymes