Assuntos
Aldrina/efeitos da radiação , Lipídeos , Efeitos da Radiação , Análise de Variância , Cromatografia Gasosa , Gorduras , Isomerismo , Nitrogênio , Óleos , Soluções , Temperatura , Fatores de Tempo , Zea maysRESUMO
The experimental conditions required to isolate a lipase from Pseudomonas fragi were determined. The organism was grown in a buffered tryptone medium for 4 to 5 days at 20 C. The lipase in the culture supernatant fluid was isolated by fractionation with ammonium sulfate at 60% saturation, followed by acetone precipitation at 30-60% concentration. Further purification was made by using Sephadex G-200 gel-filtration and diethylaminoethyl cellulose chromatography. Electrophoretic analysis of the purified lipolytic fraction showed apparent homogeneity by both cellulose polyacetate and disc electrophoresis. The specific activity of the purified enzyme was about 100 times that of the starting culture filtrate, and the yield was about 1.8% of the original activity.
RESUMO
The optimal pH value of a lipase from Pseudomonas fragi was between 7.5 and 8.9, and a high reaction rate was observed at 54 C. Heating the enzyme solution at 63 C for 30 min inactivated only 27.6% of its activity; however, total inactivation was observed at 66 C after 1 hr and at 71 C after 10 min. The lipase was inhibited strongly by Fe and Fe ions, and to a lesser extent by Co, Cu, Zn. No inhibition was observed with Ca or NaF. Ethylenediaminetetraacetate was effective in removing the toxicity of Fe. The activity of the enzyme was inhibited markedly by p-chloromercurobenzoate, but the effects of N-ethylmaleimide and iodoacetate were moderate. The enzyme was able to hydrolyze natural fats, synthetic triglycerides, and alcohol esters. The order of the rate of hydrolysis of some triglycerides under experimental conditions was, from the fastest to the lowest, trilaurin, tricaprin, tricaprylin, tripalmitin, tributyrin, tricaproin, and tristearin. The enzyme was capable of hydrolyzing methyl butyrate, but the rate of hydrolysis was about one-fifth that for triolein and one-thirteenth that for coconut oil. The enzyme lost its activity rapidly when held frozen, at 20 C, and at the extremes in pH. Glutathione, cysteine, and mercaptoethanol did not preserve the activity of the enzyme.
