title: Coexpression of molecular chaperone enhances activity and export of organophosphorus hydrolase in Escherichia coli.
abstract: Periplasmic secretion has been used in attempts to construct an efficient whole-cell biocatalyst with greatly reduced diffusion limitations. Previously, we developed recombinant Escherichia coli that express organophosphorus hydrolase (OPH) in the periplasmic space using the twin-arginine translocation (Tat) pathway to degrade environmental toxic organophosphate compounds. This system has the advantage of secreting protein into the periplasm after folding in the cytoplasm. However, when OPH was expressed with a Tat signal sequence in E. coli, we found that the predominant OPH was an insoluble premature form in the cytoplasm, and thus, the whole-cell OPH activity was significantly lower than its cell lysate activity. In this work, we, for the first time, used a molecular chaperone coexpression strategy to enhance whole-cell OPH activity by improving the periplasmic translocation of soluble OPH. We found that the effect of GroEL-GroES (GroEL/ES) assistance on the periplasmic localization of OPH was secretory pathway dependent. We observed a significant increase in the amount of soluble mature OPH when cytoplasmic GroEL/ES was expressed; this increase in the amount of mature OPH might be due to enhanced OPH folding in the cytoplasm. Importantly, the whole-cell OPH activity of the chaperone-coexpressing cells was ∼5.5-fold greater at 12 h after induction than that of cells that did not express the chaperone as a result of significant Tat-based periplasmic translocation of OPH in the chaperone-coexpressing cells. Collectively, these data suggest that molecular chaperones significantly enhance the whole-cell activity of periplasmic OPH-secreting cells, yielding an effective whole-cell biocatalyst system with highly reduced diffusion limitations.
authors.affiliation: Dept. of Chemical Engineering, Pohang University of Science and Technology, Pohang 790-784, Korea.
authors.forename: Dong Gyun
authors.initials: DG
authors.lastname: Kang
authors.forename: Chang Sup
authors.initials: CS
authors.lastname: Kim
authors.forename: Jeong Hyun
authors.initials: JH
authors.lastname: Seo
authors.forename: Im Gyu
authors.initials: IG
authors.lastname: Kim
authors.forename: Suk Soon
authors.initials: SS
authors.lastname: Choi
authors.forename: Jeong Hyub
authors.initials: JH
authors.lastname: Ha
authors.forename: Soo Wan
authors.initials: SW
authors.lastname: Nam
authors.forename: Geunbae
authors.initials: G
authors.lastname: Lim
authors.forename: Hyung Joon
authors.initials: HJ
authors.lastname: Cha
mesh_terms: D043303:Aryldialkylphosphatase / Q000235:genetics* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D005417:Flavobacterium / Q000201:enzymology*; D015870:Gene Expression; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C028715:GroE protein, E coli; D006360:Heat-Shock Proteins; D043303:Aryldialkylphosphatase