Extracellular location of Thermobifida fusca cutinase expressed in Escherichia coli BL21(DE3) without mediation of a signal peptide

Abstract

Cutinase is a multifunctional esterase with potential industrial applications. in the present study, a truncated version of the extracellular thermobifida fusca cutinase without a signal peptide (referred to as cutinaseNS) was heterologously expressed in escherichia coli bl21(de3). the results showed that the majority of the cutinase activity was located in the culture medium. in a 3-liter fermentor, the cutinase activity in the culture medium reached 1,063.5 u/ml (2,380.8 mg/liter), and the productivity was 40.9 u/ml/h. biochemical characterization of the purified cutinaseNS showed that it has enzymatic properties similar to those of the wild-type enzyme. in addition, e. coli cells producing inactive cutinaseNSs130A were constructed, and it was found that the majority of the inactive enzyme was located in the cytoplasm. furthermore, t. fusca cutinase was confirmed to have hydrolytic activity toward phospholipids, an important component of the cell membrane. compared to the cells expressing the inactive cutinaseNSs130A, the cells expressing cutinaseNS showed increased membrane permeability and irregular morphology. based on these results, a hypothesis of "cell leakage induced by the limited phospholipid hydrolysis of cutinaseNS" was proposed to explain the underlying mechanism for the extracellular release of cutinaseNS. © 2013, American Society for Microbiology.