Localization and Characterization of Lipolytic Enzymes Produced by Lysobacter enzymogenes

Abstract

Lipolysis by Lysobacter enzymogenes is due to the production of two major extracellular esterases. These were investigated using a turbidimetric assay with Tween 20 as the substrate. One esterase is cell-associated and found in the particulate fraction of cell-free extracts, the other esterase is secreted into the culture medium. In batch culture with 0.8% yeast extract as the medium the particulate esterase was produced mainly during the exponential growth phase and its yield was increased about twofold by the addition of olive oil and other substrates. The supernatant esterase was produced mainly after the exponential growth phase and was induced six-to tenfold by olive oil. PAGE, combined with activity staining using Tween 20 as the substrate, indicated that the two enzymes have different electrophoretic mobilities. Intact cells expressed 87 % of the particulate cell-associated esterase activity. The enzyme was released from cells or from the particulate fraction by incubation with 0.2% Zwittergent 3-14. Tween 20 (2%, v/v) or 1 mM-EDTA released little of the enzyme from whole cells. Separation of the inner and outer membranes of L. enzymogenes showed that the particulate esterase was localized in the outer membrane. Both esterases were very active with Tween 20 as the substrate, but they hydrolysed other compounds at very different rates. The relative activities with Tween 20, p-nitrophenyl palmitate, tributyrin and olive oil were 100,3, 11 and 0 for the particulate esterase and 100, 73, 0 and 28 for the secreted esterase. In addition to these two extracellular esterases, L. enzymogenes contains a hydrolase in the cytoplasm which is most active with tributyrin. INTRODUCTIONThe genus Lysubacter contains Gram-negative gliding bacteria which are noted for the production of many extracellular enzymes and for their role in biodegradation (Christensen & Cook, 1978;Reichenbach & Dworkin, 1981;Rosenberg & Varon, 1984). All species of the genus, except two strains of Lysobacter brunescens, were found to hydrolyse Tween when tested on solid media containing this detergent (Christensen & Cook, 1978). A number of the extracellular enzymes produced by Lysubacter species have been characterized (Jackson & Matsueda, 1970;Whitaker, 1970;Hedges & Wolfe, 1974;von Tigerstrom, 1980von Tigerstrom, , 1981von Tigerstrom, , 1984von Tigerstrom & Stelmaschuk, 1986, 1987, but the enzymes responsible for the lipase activity have not been investigated.Preliminary experiments indicated that L. enzymogenes produces two extracellular Tweendegrading hydrolases: one associated with the cells, the other secreted into the culture medium. Although the secreted enzyme can use olive oil as a substrate and might be called a lipase (Jensen, 1983), both enzymes prefer water-soluble substrates and for this reason we call the enzymes esterases rather than lipases (Sugiura, 1984). This paper describes the production of the two esterases by L . enzymogenes under different culture conditions, the major differences between the two enzymes and the localizat...

Estudio de la composición de la comunidad bacteriana de manantiales salinos ubicados en los departamentos de Risaralda y Boyacá

“…Tabla 27. Evaluación de la actividad lipolítica en nueve cepas aisladas Después de cuatro días de incubación a 30°C se detectó actividad lipolítica en cuatro cepas relacionadas con organismos de los géneros Lysobacter y Pseudomonas, lo cual ha sido previamente reportado para organismos de estos géneros (Von Tigerstrom & Stelmaschuk, 1989;Ramani et al, 2010).…”