Production of lipase in nutrient broth pH 9.5 was studied at laboratory level using Pseudomonas sp. L1 isolated from Lonar meteorite crater lake. The Pseudomonas sp. L1 was confirmed by partial 16s r-RNA sequencing & by growth on Pseudomonas isolation agar containing triclosan and by using biochemical tests according to Bergeys manual of Systematic Bacteriology. The confirmation of lipase production was done by clear zone on Tween 80 medium and Egg yolk medium. Production of lipase was carried out using different oils like olive oil, coconut oil, groundnut oil, soyabean oil, Sunflower oil, corn oil and effects of different Physical & chemical factors like incubation period, pH, temperature, metal ions, different organic solvents on activity of lipase was studied. The maximum production was obtained at 27°C and at pH 9.5 & the enzyme showed maximum of 28 U/ml/min activity at temperature 40ºC, 43 U/ml/min activity at pH 8.5, 47 U/ml/min activity in Na+(1mM NaCl) & 32 U/ml/min activity in organic solvent methanol. The enzyme was partially purified by organic solvent precipitation using different (v/v) concentrations of acetone. The activity was checked spectrophotometrically by using r-Nitrophenyl acetate ester. 34 U/ml/min activity was observed in 60% (v/v) acetone precipitate & it was further purified by using DEAE Cellulose column affinity chromatography with retention time of 1ml/4min. in which fraction 21 showed maximum activity of 25 U/ml/min.
Pseudomonas sp., Lonar lake, Lipase, Alkaliphilic, DEAE Cellulose
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