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“Isolation & characterization of Thermus aquaticus and production of Taq DNA polymerse enzyme”

KrishiKosh

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Title “Isolation & characterization of Thermus aquaticus and production of Taq DNA polymerse enzyme”
 
Creator GUPTA, MONIKA
 
Contributor SHIRKOT, POONAM
 
Subject ---
Thermophiles,enzymes,hot water springs,Himachal Pradesh
 
Description 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.
 
Date 2017-01-20T11:41:29Z
2017-01-20T11:41:29Z
2011
 
Type Thesis
 
Identifier http://krishikosh.egranth.ac.in/handle/1/97303
 
Language en
 
Relation 47312;