BIOPROSPECTING OF THERMOTOLERANT BACTERIA FROM HOT WATER SPRINGS OF H.P. FOR PRODUCTION OF THERMOSTABLE PROTEASE ENZYME
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Date
2013
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ABSTRACT
Thermophiles are the organisms which are adapted to live at high temperatures. The survival of these organisms at high temperature is possible due to the thermostability of their enzymes. Many thermostable enzymes such as Taq DNA polymerase, aldolase, amylase, lipase, protease, cellulase, RNA polymerase etc. find a number of commercial applications because of their thermostability. Therefore, the isolation of thermophilic bacteria from natural sources and their identification are very important in terms of discovering new industrial enzymes. Keeping in view, hot water springs of Himachal Pradesh could serve as a good source for new thermophilic microorganisms with novel industrially important properties. Therefore aim of the present study was the isolation and characterization of thermotolerant bacteria from hot water springs of Himachal Pradesh for production of thermostable protease enzyme. Four hot water springs viz., Tattapani , Manikaran, Khirganga and Vashisht were purposely selected for the present studies. Samples consisted soil, water, rock matting and pebbles were collected from each hot water spring. The pH and temperature of the four thermal springs were ranged from 4.1-6.0 and 51-105oC respectively. The chloride, sulphate, total hardness, calcium hardness, CaCO3 hardness and magnesium content ranged from 490-2673, 12-50, 147-483, 30.0-144.2, 65-264 and 7.53-26.73 mg/l respectively. Forty seven bacterial isolates were isolated from the samples using skim milk medium. All the bacterial isolates were studied for various morphological characters and on the basis of ability of formation of zone of clearance on skim milk medium, forty bacterial isolates were selected, which were further investigated for biochemical characters. Quantitative screening was done to select one bacterial isolate showing maximum thermostable protease activity. MCW220 bacterial isolate was found to show maximum thermostable protease activity of 33.4 U/l and was selected further for molecular characterization. Genomic DNA was isolated from the selected bacterial isolate MCW220. PCR amplification of the isolated DNA was carried out using universal primers for 16S rDNA gene and an amplicon of 1200 bp was obtained. Sequencing of the PCR product was done using same primers. Sequence of the MCW220 bacterial isolate so obtained was found to be 1469 bp. In silico analysis using BLASTn showed 97% homology of the query sequence with Aneurinibacillus thermoaerophilus strain L420-91. A total of 20 sequences were mined and these sequences were used to compare the 16S rDNA sequence of the test isolate MCW220. Alignment score was highest for Aneurinibacillus thermoaerophilus strain L420-91 16S ribosomal RNA, partial sequence (NR_029303.1). Phylogenetic analysis based on nucleotide sequences using NJ method was achieved via phylip 3.68 and EXOMETM HORIZON. Aneurinibacillus thermoaerophilus strain MCW220 was studied further for enzymatic studies. Optimization of culture conditions for growth and thermostable protease activity of isolate Aneurinibacillus thermoaerophilus strain MCW220 was carried out. The crude extracellular extract was produced using optimized conditions and further partially purified using ammonium sulphate precipitation technique. The procedure yielded 0.435 g protein with 0.70 fold purification with a percent yield 67.5 %. Molecular weight of the partially purified enzyme was found to be 40 kDa using SDS-PAGE gel.
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enzymes, bacteria, surface water, irrigation, inorganic acid salts, productivity, sampling, proteins, animal husbandry, selection, Thermophiles