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.
Similar content being viewed by others
References
Brock TD (1967) Life at high temperatures. Science 158:1012–1019
Brock TD, Freeze H (1969) Thermus aquaticus gen. n and sp. n. a nonsproulating extreme thermophile. J Bacteriol 98(1):289–297
Ramaley RF, Hixson (1970) Isolation of a non-pigmented, thermophilic bacterium similar to Thermus aquaticus. J Bacteriol 103:527–528
Hugenholtz P, Pitulle C, Hershbergerand KL, Pace NR (1998) Novel division level bacterial diversity in a Yellowstone hot spring. J Bacteriol 180:366–376
Guo C, Wang T, Zhu W, Zhang D, Cui X, Xu L, Peng Q (2003) The phylotype of Thermus from Rehai geothermal area, Tengchong, China. J Microbiol 41(2):152–156
Kurosawa N, Itoh YH, Itoh T (2005) Thermus kawarayensis sp. nov., a new member of the genus Thermus, isolated from Japanese hot springs. Extremophiles 9:81–84
Imperio T, Viti C, Marri L (2008) Alicyclobacillus pohliae sp. nov., a thermophilic, endospore forming bacterium isolated from geothermal soil of the north–west slope of Mount Melbourne (Antarctica). Int J Syst Evol Microbiol 58(1):221–225
Khalil A (2011) Screening and characterization of thermophilic bacteria (lipase, cellulose and amylase producers) from hot springs in Saudi Arabia. J Food Agric Environ 9(2):672–675
Obeidat M, Horani HK, Zoubi A, Otri I (2012) Isolation, characterization, and hydrolytic activities of Geobacillus species from Jordanian hot springs. Afr J Biotechnol 11(25):6763–6768
Sharma M, Sharma NN, Bhalla TC (2012) Purification studies on a thermo-active amidase of Geobacillus pallidus BTP-5x MTCC 9225 isolated from thermal springs of Tatapani, Himachal Pradesh. Appl Biochem Biotechnol 10:9945–9958
Saha P, Mondal AK, Mayilraj S, Krishnamurthi S, Bhattacharya A, Chakrabarti S (2005) Paenibacillus assamensis sp nov, a novel bacterium isolated from a warm spring in Assam, India. Int J Syst Evol Microbiol 55:2577–2581
Acharya S, Chaudhary A (2012) Alkaline cellulase produced by a newly isolated thermophilic Aneurinibacillus thermoaerophilus WBS2 from hot spring, India. Afr J Microbiol Res 6(26):5453–5458
Bisht SS, Panda AK (2011) Biochemical characterization and 16S rRNA sequencing of few lipase producing thermophilic bacteria from Taptapani hot water spring, Orissa, India. Biotechnol Res Int 11:1–5
Trivedi S, Gehlot HS, Rao SR (2006) Protein thermostability in Archaea and Eubacteria. Genet Mol Res 5(4):816–827
Mullis K, Faloona F (1986) Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harb Symp Quant Biol 51:263–273
Lawyer FC, Stoffel S, Saiki RK (1993) High-level expression, purification and enzymatic characterization of full-length Thermus aquaticus DNA polymerase. PCR Methods Appl 2(4):275–287
Cline J, Braman J, Hogrefe H (1996) PCR fidelity of Pfu DNA polymerase and other DNA polymerases. Nucleic Acids Res 24:3546–3551
Srinivasan MC (1994) Microbial biodiversity and its relevance to screening for novel industrially useful enzymes. Curr Sci 66:137–140
Castenholz RW (1969) Thermophilic blue-green algae and the thermal environment. Bacteriol Rev 33:476–504
Kristjansson JK, Hreggvidson, Alfredsson GA (1986) Isolation of halotolerant Thermus spp. from submarine hot springs in Iceland. Appl Environ Microbiol 52:1313–1316
Aneja KR (2003) Experiments in microbiology, plant pathology and biotechnology, 4th edn. New Age International Limited, New Delhi
Altschul SF, Thomas LM, Alejandro AS, Jinghui Z, Zhang Z, Webb M, David JL (1997) Gaped BLAST and PSIBLAST: a new generation of protein database search program. Nucleic Acid Res 25:3389–3402
Scopes RK (1982) Ammonium sulfate precipitation table. Protein purification: principal and practice. Springer, New York
Roayaei M, Galehdari H (2008) Cloning and expression of Thermus aquaticus DNA polymerase in Escherichia coli. Jundishapur J Microbiol 1(1):1–5
Chien A, Edgar DB, Trela JM (1976) Deoxyribonucleic acid polymerase from the extreme thermophile, Thermus aquaticus. J Bacteriol 127(3):1550–1557
Sato S, Harris JI (1977) Isolated and characterized superoxide dismutase from the extreme thermophile Thermus aquaticus. Eur J Biochem 73(2):373–381
Ferrali P, Egan JD, Floyd LE (2007) Making Taq DNA polymerase in the undergraduate biology laboratory. Bios 78(2):69–74
Harell RA, Hart RP (1994) Rapid preparation of Thermus flavus DNA polymerase. Genome Res 3:372–375
Yang Z, Ding Y, Zhang Y, Liu F (2008) Rapid purification of truncated Taq DNA polymerase Stoffel fragments by boiling lysis of bacterial expression cultures. Biotechnol Appl Biochem 50:71–75
Acknowledgments
The first author gratefully acknowledges the financial help granted by the Department of Biotechnology, Dr YSP University of Horticulture and Forestry, Nauni, Solan, for carrying out the research work.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Verma, A., Dhiman, K., Gupta, M. et al. Bioprospecting of Thermotolerant Bacteria from Hot Water Springs of Himachal Pradesh for the Production of Taq DNA Polymerase. Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci. 85, 739–749 (2015). https://doi.org/10.1007/s40011-014-0412-x
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s40011-014-0412-x