Isolation and study of Lipase activity of an olive oil degrading Thermoactionomyces SP. (c2002)

Abstract

A ftlamentous, Gram-Positive, spore forming filamentous bacterium was isolated from soil (AI Koura - Lebanon) on rhodamine agar plates at 60°C. The isolate, HRK-l produced large quantities of an extra cellular thermostable lipase that degrades olive oil. It can be primarily classified as Thermoactynomyces putidus due to filamentous structure of its molecules that bear one spore each on an un-branched sporophore, the resistance of its spores to boiling, utilization of sucrose as a carbon source and production of dark pigments. The isolate grew optimally at a temperature of 60°C, a pH of 7.3, and an orbital shaking of 250 r.p.m. It showed an efficient olive oil degrading ability. No traces of triolein were detected after 36 hours of cultivation. A concentration of 10% (v/v) olive oil did not inhibit its growth. Lipase production was constitutive, and did not depend on the presence of olive oil. The optimal concentration of olive oil for lipase activity was 1 % (v /v), and activity was not enhanced at higher concentrations, but on the contrary, a decrease in enzyme activity was recorded. The lipase HRKL-1 was bio-chemically characterized. It has a molecular weight of 80 k-Daltons and an optimal activity at 60°C and pH 8.0. This lipolytic enzyme showed resistance to boiling and to a wide range of metallic 10ns and inhibitors. It was only slightly inhibited by PMSF (phenylmethylsulfonyl fluoride), while its activity increased in the presence of Ca ++ ions. The Km value for HRKL-1 (0.021 mM) indicated high affiniity of the enzyme to its substrate. The formation of this heat-stable lipase started in the early exponential growth phase, while maximal extracellular enzyme activity was detected at the end of the decline phase, when most of the cells appeared as spherical spores. The exceptionally high activity of lipase (2.37 U I ml) produced by HRK-1 measured in the cell free supernatant clearly indicated the commercial importance of this isolate, especially after it showed great stability at elevated temperatures.