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Characterization of partially purified extracellular lipase fractions from Pseudomonas fragi CRDA 037
Author(s) -
Wahab Aliaa Abdul,
Kermasha Selim,
Bisakowski Barbara,
Morin André
Publication year - 1999
Publication title -
journal of the american oil chemists' society
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.512
H-Index - 117
eISSN - 1558-9331
pISSN - 0003-021X
DOI - 10.1007/s11746-999-0143-y
Subject(s) - triolein , lipase , triacetin , chromatography , chemistry , tripalmitin , hydrolysis , triacylglycerol lipase , size exclusion chromatography , ammonium sulfate precipitation , biochemistry , enzyme
The purification of extracellular lipases from the culture medium of Pseudomonas fragi CRDA 037 was obtained by ammonium sulfate precipitation, followed by ion‐exchange chromatography and then by size exclusion chromatography, and re‐size exclusion chromatography, which resulted in two enzymatic fractions, FIVa′ and FIVb′. The fractions FIVa′ and FIVb′ had specific activities of 105.5 and 121.6 U/mg, respectively, with purification folds of 169.3 and 195.2, respectively, using triacetin as a substrate. The two purified fractions showed optimal activities at pH 9.5 and 10.0, respectively, at 80°C. Three bands were found in fraction FIVa′ and two bands in fraction FIVb′ by native polyacrylamide gel electrophoresis; these results indicated that homogeneity of the purified fractions was not achieved. The enzyme efficiency values, calculated as the ratio of V max to K m value for fractions FIVa′ and FIVb′, were 72.16 × 10 −2 and 38.15 × 10 −2 , respectively. The lipase activity of fraction FIVa′ was more specific for the hydrolysis of fatty acid esters with fatty acid chain lengths of C12 to C18, whereas that of fraction FIVb′ showed a relatively broader range of specificity. The lipase activity of fraction FIVa′ showed higher specificity toward triacetin, tristearin, and tripalmitin as the substrate, whereas that of fraction FIVb′ exhibited higher affinity toward triacetin, trimyristin, and triolein. The effect of selected salts and detergents on the lipase activity of the purified fractions was also investigated. The lipase activity of the purified lipase fractions was completely inhibited by 10 mM of FeCl 2 , FeCl 3 , and Ellman’s reagent. However, 10 mM of CaCl 2 and EDTA activated the two purified lipase fractions by 20 to 50%.