High-level extracellular secretion of organophosphorous hydrolase of Flavobacterium sp. in Escherichia coli BL21(DE3)pLysS.
Organophosphorus (OPs) compounds are widely used in many pesticides, insecticides, and chemical nerve agents. These compounds are hazardous for humans and the environment. There are many reports on detoxification of these compounds, among them enzymatic cleavage of these compounds with organophosphorus hydrolase (OPH) has been taken into more consideration. Several studies have been performed to improve OPH secretion in Escherichia coli by different signal peptides, but have not been successful. In this study, to achieve the extracellular secretion of OPH in E. coli, the complete opd gene along with its native signal peptide was codon optimized and expressed in E. coli BL21(DE3)pLysS. The culture medium showed OPH activity after 2, 4, and 6 H of induction time. The extracellular secretion of OPH was also confirmed by SDS-PAGE and Western blot analysis. The effects of different factors in growth medium were also investigated regarding expression and extracellular secretion of OPH. It appears that the secretion of OPH into the extracellular medium is highly affected by culture conditions. Therefore, our results revealed that the recombinant OPH was successfully secreted into the extracellular medium. This secretion system can be considered as a high efficiency biocatalyst for detoxification of OPs compounds.
Department of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran.|Department of Biotechnology, Graduate University of Advanced Technology, Kerman, Iran.
Soudeh Khanamani
SK
Falahati-Pour
Department of Clinical Biochemistry, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
Abbas Sahebghadam
AS
Lotfi
Department of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran.
Gholamreza
G
Ahmadian
Department of Biotechnology, Institute of Science and High Technology and Environmental Sciences, Graduate University of Advanced Technology, Kerman, Iran.
Amin
A
Baghizadeh
Department of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran.
Reza
R
Behroozi
Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran.
Fereshteh
F
Haghighi
D043303:Aryldialkylphosphatase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003035:Cobalt / Q000494:pharmacology; D003062:Codon / Q000235:genetics; D003470:Culture Media / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000166:cytology* / Q000187:drug effects / Q000235:genetics / Q000254:growth & development; D005110:Extracellular Space / Q000378:metabolism*; D005417:Flavobacterium / Q000201:enzymology* / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods*; D005998:Glycine / Q000494:pharmacology; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics; D021382:Protein Sorting Signals; D013997:Time Factors
D003062:Codon; D003470:Culture Media; D021382:Protein Sorting Signals; D007544:Isopropyl Thiogalactoside; D003035:Cobalt; D043303:Aryldialkylphosphatase; D005998:Glycine
Escherichia coli BL21(DE3)pLysS; Tat signal peptide; organophosphorus hydrolase; secretion