Extracellular Location of Thermobifida fusca Cutinase Expressed in Escherichia coli BL21(DE3) without Mediation of a Signal Peptide

• DOI: 10.1128/aem.00239-13
• Publication Date: 2013-04-20T03:18:42Z
• Authors: Lingqia Su; Ronald W. Woodard; Jian Chen; Jing Wu
• Keywords: Cutinase

Cutinase is a multifunctional esterase with potential industrial applications. In the present study, truncated version of extracellular Thermobifida fusca cutinase without signal peptide (referred to as NS ) was heterologously expressed in Escherichia coli BL21(DE3). The results showed that majority activity located culture medium. 3-liter fermentor, medium reached 1,063.5 U/ml (2,380.8 mg/liter), and productivity 40.9 U/ml/h. Biochemical characterization purified it has enzymatic properties similar those wild-type enzyme. addition, E. cells producing inactive S130A were constructed, found enzyme cytoplasm. Furthermore, T. confirmed have hydrolytic toward phospholipids, an important component cell membrane. Compared expressing S130A, increased membrane permeability irregular morphology. Based on these results, hypothesis “cell leakage induced by limited phospholipid hydrolysis ” proposed explain underlying mechanism for release .