Recent investigations on cloned bacterial lipases performed in our laboratory revealed the presence of lipolytic activity that was not due to the cloned lipase-coding gene but was probably the result of an intrinsic activity of Escherichia coli itself. To confirm such a hypothesis, we assayed the activity of frequently used E. coli strains by fast paper tests, zymograms and spectrofluorometry. A band of Ca. 18-20 kDa showing activity on MUF-butyrate was detected in zymogram analysis of crude cell extracts in all E. coli strains assayed. Moreover, the spectrofluorometric results obtained confirmed the presence of low but significant lipolytic activity in E. coli, with strain BL21 showing the highest activity. Detailed characterization of such a lipolytic activity was performed using E. coli BL21 cell extracts, where preference for C7 substrates was found, although shorter substrates were also hydrolysed to a minor extent. Interestingly, E. coli lipolytic activity displays traits of a thermophilic enzyme, showing maximum activity at 50 °C and pH 8, an unexpected feature never described before. Kinetic and inhibition analysis were also performed showing that activity can be inhibited by several metal ions or by Triton X-100® and SDS, used in zymogram analysis. Such properties ‒ low activity, preference for medium chain-length substrates, and high operational temperature ‒ might justify why this activity had gone unexplored until now, even when many lipases and esterases have been cloned and expressed in E. coli strains in the past. From now on, lipase researchers should take into consideration the presence of such a basal lipolytic activity before starting their lipase cloning or expression experiments in E.coli.
Keywords: E. coli; Lipase; Lipase cloning.
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