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 cutinase NS ) 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 cutinase NS showed that it has enzymatic properties similar to those of the wild-type enzyme. In addition, E. coli cells producing inactive cutinase NS S130A 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 cutinase NS S130A, the cells expressing cutinase NS showed increased membrane permeability and irregular morphology. Based on these results, a hypothesis of "cell leakage induced by the limited phospholipid hydrolysis of cutinase NS " was proposed to explain the underlying mechanism for the extracellular release of cutinase NS . Cutinase not only catalyzes the cleavage of the ester bonds of cutin, but is also capable of hydrolyzing soluble esters, insoluble triglycerides, and a variety of polyesters (1). In addition to its hydrolytic capability, cutinase is also used in ester synthesis (2, 3) and transesterification (4). Therefore, as a multifunctional enzyme, cutinase has potential uses in the food, chemical, textile, and other industries (5).Enzymes with cutinase activity have been found in both fungi and bacteria, such as Fusarium solani pisi and Thermobifida fusca, respectively (5-7). It has been demonstrated that cutinases from both groups belong to the ␣/␤-hydrolase superfamily, share similar spatial structures, and display similar catalytic properties, as well as substrate specificities. In addition, consistent with their ability to accommodate large substrates like cutin, they contain an open active site, and the lid structure normally seen in most lipases is absent in cutinase. Despite these similarities, there does not appear to be significant sequence homology between fungal and bacterial cutinases; thus, it has been suggested that they be classified into prokaryotic and eukaryotic cutinase subfamilies, respectively (6).To date, all the studied cutinases from microorganisms have been found to be secretory enzymes (6). Therefore, for heterologous expression, signal peptides are usually utilized to mediate the secretion of recombinant cutinase. Using this approach, F. solani cutinase has been expressed in a variety of host cells, such as Escherichia coli (8), Aspergillus awamori (9), Fusarium venenatum (10), Saccharomyces cerevisiae (11, 12), and Pichia pastoris (13). The highest yield of extracellu...

Extracellular expression of natural cytosolic arginine deiminase from Pseudomonas putida and its application in the production of l-citrulline

“…At the same time, the co-expressed cutinase was also measured to have an activity of 63.8 U mL À1 at its highest level in the culture medium, which accounts for 70.3% of its total cutinase activity. This cutinase expression level represented only about 30% of that when it was expressed alone (Su et al, 2013a), and it is also lower than that was found in the co-expression system in which other recombinant proteins were co-expressed with cutinase in previous studies.…”

“…In addition, the extracellular cutinase expression level was also increased significantly to 223.3 U mL À1 , which was a 3.5-fold higher compared to that of the original one and accounted for 95.0% of its total activity. This high concentration of cutinase generated improved hydrolysis of membrane phospholipids, the activity found in the previous study (Su et al, 2013a), and consequently increased membrane permeability (Fig. 2) that probably allows release of ADI in large quantities.…”

“…The permeability of inner and outer membranes was assessed using the b-galactosidase substrate ONPG and the hydrophobic fluorescent probe NPN, respectively, as previously described (Su et al, 2013a).…”

“…Due to the modification to the membrane, cutinase is released into the culture medium in large quantities. It should be noted that this process did not cause obvious physiological damage to the cells, and cell lysis was not observed (Su et al, 2013a). More importantly, other cytosolic proteins could also be ''secreted'' via the increased membrane permeability, which provided a novel approach for extracellular expression of recombinant cytosolic proteins in E. coli (Su et al, 2013b).…”

Common Non-classically Secreted Bacterial Proteins with Experimental Evidence

“…Although a truncated version of T. fusca cutinase does not contain a signal peptide and other known or secretion signals, as high as 92.5 % of the cutinase was found in the culture medium and the cutinase activity reached 1063.5 U/ml when it was heterologously expressed in Escherichia coli BL21(DE3) ( Table 1). When an inactive version with the mutation of Ser 130 to Ala was expressed, the majority of the mutant cutinase was located in the cytoplasm [42]. These authors using elegant experiments further found that the extracellular yield was improved when the secretory proteins were co-expressed with cutinase, and more than half of the native cytosolic proteins were ''secreted'' into the culture medium when they were co-expressed with cutinase.…”