A STUDY OF THERMOSTABLE AND UV RESISTANT LIPASE PRODUCING BACILLUS SPECIES

ABSTRACT The objective of present study is to identify, screen and isolate a lipase producing bacterium which is stable at different temperatures and which is UV resistant. Screening and isolation of lipase producing strains of bacteria was carried out from eleven different soil samples collected from various places in Andhra Pradesh and Hyderabad. The isolates were positive on tribuytrin agar media and thus were selected as lipase producing strain. The strain was identified and characterised by the microscopic and biochemical tests as Bacillus.sps, a lipase producing organism. The optimisation of various cultural conditions was carried out by which the lipase production was enhanced with the optimal parameters being incubation period of 48 hours (5.9U/ml), palm oil as carbon source (5.9/ml), peptone as nitrogen source (6.2U/ml), initial pH of 7.0 and incubation temperature of 36ºc (6.7U/ml).The optimum agitation speed of 160 rpm produced lipase having (7.9 U/ml) activity. Finally, the enzyme lipase was purified by ammonium sulphate fractionation, dialysis and column chromatography. The ammonium sulphate precipitation and dialysis showed an increased specific activity of 1.71 U/ml and 6.17 U/ml when compared to crude enzyme which showed specific activity of 0.45 U/mg. Further purification was carried out by ion-exchange chromatography using DEAE column. The purified enzyme showed higher specific activity (15.24 U/mg) with a purification fold of 33. The molecular mass of purified lipase was estimated to be approximately 40.14 kDa by SDS-PAGE. Six cultures have shown tolerance to various organic solvents (acetone, benzene, toulene, ethyl benzene, 1-butanol, n-hexane and nheptane) with hydrolytic zones when flooded on tribuytrin agar. It is evident from the results that the mutants LUV-8, LUV-9 and LUV-10 showed higher lipase activity than the parent strain-26, with the mutant LUV-9 showing the maximum activity of 9.5 U/ml after uv-irradiation. The sequence was deposited in NCBI and was identified to be a novel strain with an accession number which can be beneficial for industries