Four hot water springs viz., Manikaran, Vashisht, Khirganga and Tattapani were purposely selected for isolation of thermostable lipase producing bacterial isolates. The pH and temperature of the four thermal springs were ranged from 4.0-6.0 and 51-105oC respectively. Isolated forty two thermophilic bacterial isolates, were described as putative thermostable lipase producers on the basis of their ability to form zone of clearance on tributyrin agar medium. Quantitative screening led to the selection of MBW2 bacterial isolate showing maximum thermostable lipase activity of 4.83U/ml after 24 hrs of incubation time at 60° C temperature, was selected for morphological, biochemical and molecular characterization. Genomic DNA isolated from the selected MBW2 bacterial isolate was subjected to PCR amplification followed by sequencing using universal primers for 16S rrna gene. In silico molecular analysis identified MBW2 bacterial isolate as Aneurinibacillus thermoaerophilus strain MBW2. Optimum culture conditions for growth and thermostable lipase activity of selected isolate were used for the enzyme production which was purified to 3.08 fold with percent yield of 13.73% using ammonium sulphate precipitation technique, gel filteration chromatography and ion exchange chromatography technique. The molecular weight of the purified enzyme was found to be 42.5 kDa using SDS-PAGE.
Thermostable lipase, Thermophiles, Hot water spring.
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