Specific adhesion to cellulose and hydrolysis of organophosphate nerve agents by a genetically engineered Escherichia coli strain with a surface-expressed cellulose-binding domain and organophosphorus hydrolase

Abstract

A genetically engineered Escherichia coli cell expressing both organophosphorus hydrolase (OPH) and a cellulose-binding domain (CBD) on the cell surface was constructed, enabling the simultaneous hydrolysis of organophosphate nerve agents and immobilization via specific adsorption to cellulose. OPH was displayed on the cell surface by use of the truncated ice nucleation protein (INPNC) fusion system, while the CBD was surface anchored by the Lpp-OmpA fusion system. Production of both INPNC-OPH and Lpp-OmpA-CBD fusion proteins was verified by immunoblotting, and the surface localization of OPH and the CBD was confirmed by immunofluorescence microscopy. Whole-cell immobilization with the surface-anchored CBD was very specific, forming essentially a monolayer of cells on different supports, as shown by electron micrographs. Optimal levels of OPH activity and binding affinity to cellulose supports were achieved by investigating expression under different induction levels. Immobilized cells degraded paraoxon rapidly at an initial rate of 0.65 mM/min/g of cells (dry weight) and retained almost 100% efficiency over a period of 45 days. Owing to its superior degradation capacity and affinity to cellulose, this immobilized-cell system should be an attractive alternative for large-scale detoxification of organophosphate nerve agents.

FIG. 1.

Western blot analysis of protein fractions from XL1-Blue/pUCBD/pPNCO33 with anti-CBD serum (A), anti-OPH serum (B), and anti-INPNC serum (C). Anti-CBD serum, anti-OPH serum, and anti-INP serum were used at dilutions of 1:1,000, 1:1,000, and 1:3,000, respectively. Lanes 1, 2, and, 3 indicate total protein, soluble fraction, and membrane fraction, respectively. The desired fusion proteins are marked with arrows.

FIG. 2.

Immunofluorescence micrographs of E. coli cells harboring different plasmids.E. coli XL1-Blue (I), XL1-Blue/pET38b (II), XL1-Blue/pJK33 (III), XL1-Blue/pUCBD (IV), XL1-Blue/pPNCO33 (V), and XL1-Blue/pUCBD/pPNCO33 (VI) were probed with either anti-OPH serum (A) or anti-CBD_cex serum (B) and fluorescently stained with goat anti-rabbit IgG-FITC conjugate.

FIG. 3.

Comparison of binding capacities of XL1-Blue cells to cellulose fiber. The amount of cells immobilized or washed out is expressed in milligrams of cells (dry weight) per support. XL1-Blue/pPNCO33 and XL1-Blue/pUCBD/pPNCO33 cells are designated OPH and OPH/CBD, respectively. E. coli strain XL1-Blue was used as a control, with no protein anchored on the cell surface. Data are mean values ± standard deviations from four experiments.

FIG. 4.

Scanning electron micrographs of cellulose fiber showing immobilized XL1-Blue/pK184 (A), XL1-Blue/pPNCO33 (B), and XL1-Blue/pUCBD/pPNCO33 (C) cells after 24 h of washing. (D) Immobilization of XL1-Blue/pUCBD/pPNCO33 on cellulose beads.

FIG. 5.

(A) Expression levels of Lpp-OmpA-CBD at increasing concentrations of IPTG. Total proteins were probed with anti-CBD serum as described in the legend to Fig. 1. (B) Binding capacities of XL1-Blue/pUCBD/pPNCO33 cells under different levels of induction. The amount of cells immobilized is expressed in milligrams of cells (dry weight) per support. (C) Whole-cell activities of XL1-Blue/pUCBD/pPNCO33 under different levels of induction. Data are mean values ± standard deviations from four experiments.

FIG. 6.

Degradation of paraoxon by immobilized XL1-Blue/pUCBD/pPNCO33 cells in repeated-batch operations. Degradation experiments were conducted at 25°C. (A) Degradation of 0.25 mM paraoxon by immobilized cells on day 1. (B) Percentage of paraoxon degraded after 1 h by immobilized cells. (C) Whole-cell activity for cell suspensions in phosphate-citrate buffer. Data are mean values ± standard deviations from four experiments.