Application of three-stage approach to isolation and purification of Pseudozyma antarctica lipase B
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2018-01-01
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Abstract
Lipases are serine hydrolases defined as triacyl glycerol acyl hydrolases (EU 3.1.1.3). The unique characteristics and biotechnological potential of lipases have led to their use in food, detergents, pharmaceuticals, etc. industry. Lipolytic enzymes can be isolated from plants, animals, bacteria, fungi and yeasts. In this work we report the synthesis, isolation and purification of an extracellular lipase from Pseudozyma antarctica NBIMCC 8340 using a three-step process. The lipase activity is analyzed by potentiometric and colorimetric method at every stage of the isolation. In order to optimize the synthesis and isolation conditions two separate experiments of fermentation process, test and real one, are realized. The isolation and separation of the lipase in our case are carried out by fractional precipitation with (NH4)2SO4, followed by purification through ultrafiltration using micro concentration tubes Sartorius VIVASPIN 6. The resulting isolate of P. antarctica lipase B is detected by SDS PAGE and analyzed for lipase activity. Preparative isolation of the enzyme in native PAGE is further conducted as a third step aiming to obtain a high purity lipase. Thus a highly purified CalB of a specific activity of 92 U/mg is obtained.