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.

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大肠杆菌未探索的脂肪分解活性：对脂肪酶克隆的影响

我们实验室最近对克隆细菌脂肪酶的研究揭示了脂肪分解活性的存在，这不是由于克隆的脂肪酶编码基因，而是可能是大肠杆菌本身的内在活性的结果。为了证实这一假设，我们通过快速纸测试、酶谱图和荧光分光光度法测定了常用大肠杆菌菌株的活性。一组 Ca。18-20 kDa 显示对 MUF-丁酸盐的活性，在所有被分析的大肠杆菌菌株的粗细胞提取物的酶谱分析中检测到。此外，获得的荧光光谱结果证实了大肠杆菌中存在低但显着的脂肪分解活性，其中菌株 BL21 显示出最高的活性。使用大肠杆菌BL21 细胞提取物对这种脂解活性进行了详细表征，其中发现对 C7 底物的偏好，尽管较短的底物也有较小程度的水解。有趣的是，大肠杆菌脂肪分解活性显示出嗜热酶的特征，在 50 °C 和 pH 值 8 时显示出最大活性，这是前所未有的意外特征。还进行了动力学和抑制分析，结果表明活性可以被几种金属离子或 Triton X-100® 和 SDS 抑制，用于酶谱分析。这些特性——低活性、偏爱中等链长的底物和高操作温度——可能解释了为什么直到现在这种活性仍未被探索，即使过去许多脂肪酶和酯酶已经在大肠杆菌菌株中被克隆和表达。从现在开始，