Unexplored lipolytic activity of Escherichia coli: Implications for lipase cloning.

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.

Development of an antimicrobial bioactive paper made from bacterial cellulose.

Bacterial cellulose (BC) has emerged as an attractive adsorptive material for antimicrobial agents due to its fine network structure, its large surface area, and its high porosity. In the present study, BC paper was first produced and then lysozyme was immobilized onto it by physical adsorption, obtaining a composite of lysozyme-BC paper. The morphology and the crystalline structure of the composite were similar to that of BC paper as examined by scanning electron microscopy and X-ray diffraction, respectively. Regarding operational properties, specific activities of immobilized and free lysozyme were similar. Moreover, immobilized enzyme showed a broader working temperature and higher thermal stability. The composites maintained its activity for at least 80 days without any special storage. Lysozyme-BC paper displayed antimicrobial activity against Gram-positive and Gram-negative bacteria, inhibiting their growth by 82% and 68%, respectively. Additionally, the presence of lysozyme increased the antioxidant activity of BC paper by 30%. The results indicated that BC is a suitable material to produce bioactive paper as it provides a biocompatible environment without compromising the activity of the immobilized protein. BC paper with antimicrobial and antioxidant properties may have application in the field of active packaging.