SHIRKOT, POONAMMANJUL, ANSHUL SHARMA2017-05-152017-05-152015http://krishikosh.egranth.ac.in/handle/1/5810011191ABSTRACT Thermophiles are the organisms which are adapted tolive 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, pullulanase, 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 pullulanase enzyme. Six hot water springs viz., Manikaran, Vashisht, Khirganga, Kasol, Kalath and Tattapani 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 six thermal springs ranged from 5.7-9.5 and 35-106 o C respectively. The chloride, sulphate, total hardness, calcium hardness, CaCO 3 and magnesium content ranged from 578-1978, 09-54,32.9-141.3, 22-134, 52-283 and 3.15-26.75 mg/l respectively. Fifty eight bacterial isolates were isolated from the samples using nutrient agar medium. Bacterial population in water was highest (3.45×10 5 cfu/ml) at S1 site of Tattapani and minimum (0.76 ×10 5 cfu/ml) bacterial population was noted at S2 site of Kalath. Similarly Bacterial population in soil was highest (3.54×10 5 cfu/g) at S3 site of Vashisht and minimum (0.54 ×10 5 cfu/g) at S3 site of Kalath. All the bacterial isolates were studied for various morphological characters and on the basis of ability to grow on KWF medium seven bacterial isolates were selected, which were further investigated for biochemical characters. Quantitative screening was done to select one bacterial isolate showing maximum thermostable pullulanase activity. KS2W bacterial isolate was found to show maximum thermostable pullulanase activity of 3.96 U/l and was selected further for molecular characterization. Genomic DNA was isolated from the selected bacterial isolate KS2W. PCR amplification of the isolated DNA was carried out using universal primers for 16S rrnagene and an amplicon of 1500 bp was obtained. Sequencing of the PCR product was done using same primers. Sequence of the KS2W bacterial isolate so obtained was found to be 556 bp. In silico analysis using BLASTn showed 96% homology of the query sequence with Bacillus licheniformisstrain BCRC 11702. A total of 20 sequences were mined and these sequences were used to compare the 16S rrnagene sequence of the test isolate KS2W. Alignment score was highest for Bacillus licheniformisstrain BCRC 11702, 16S ribosomal RNA, partial sequence (NR_116023.1).Phylogenetic analysis based on nucleotide sequences using NJ method was achieved via MEGA 5.0. Bacillus licheniformis strain ASM3 was studied further for enzymatic studies. Optimization of culture conditions for growth and thermostable pullulanase activity of isolate Bacillus licheniformisstrain ASM3 was carried out. The crude extracellular extract was produced using optimized conditions and further partially purified using ammonium sulphate precipitation, gel chromatography and ion exchange chromatography. The procedure yielded 3.96 g protein with 29.04 folds of purification with a percent yield 15.48%. Molecular weight of the partially purified enzyme was found to be ~96 kDa using SDS-PAGE gel.ennullThesis