Part:BBa_K2244003
TorA-opdA
short description
This part is a functional gene that encodes organophosphate hydrolase (OPH) to degrade organophosphate pesticides. In order to export hydrolase to the periplasmic space of E.coli , We fused a TorA signal peptide to the N-terminal of opdA (BBa_K215090) that facilitates the exportation.
long description
Organophosphate hydrolase (OPH) is a homodimeric organophosphate triesterase that requires metal ion as a cofactor to degrade a wide range of toxic organophosphates. OPH can hydrolyze various phosphorus-ester bonds including P-O, P-F, P-CN, and P-S bonds. Recombinant E. coli expressing OPH can degrade a variety of organophosphate, however, due to low protein solubility, OPH production is at low yields. Also, as a gram-negative bacteria, E. coli. Cell membrane can be a substrate diffusion barrier affecting whole cell biocatalytic efficiency. Periplasmic secreting of target proteins via translocation across the cytoplasmic membrane can be a potential strategy to reduce the substrate diffusion barrier.
Among the pathways that mediate periplasmic secretion, twin-arginine translocation (Tat) pathway is the one that allows most proteins to be partially folded prior to export and without the requirement ATP hydrolysis. In addition, the Tat pathway has been successfully used for periplasmic secretion of several foreign proteins such as cofactor-containing, multimeric, and disulfide-containing proteinis. To express functional OPH molecules into the periplamic space, a twin-arginine signal peptide of E. coli trimethylamine N-oxide (TMAO) reductase (TorA) was added to the N-terminal of the opdA. TMAO reductase is a periplasmic enzyme that catalyzes reduction of TMAO to trimethylamine, and functions as a component of the anaerobic respiratory chain which provides energy for bacterial cell growth .
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 1123
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 220
Illegal AgeI site found at 415
Illegal AgeI site found at 754 - 1000COMPATIBLE WITH RFC[1000]
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