Whole-cell immobilization and biodetoxification of organophosphate nerve agents by genetically engineered Escherichia coli with surface-expressed cellulose-binding domain and organophophorus hydrolase

A genetically engineered Escherichia coli whole cell system of innovative dual functions has been developed with both cellulose-binding domain (CBD) and organophosphorus hydrolase (OPH) expressed onto cell surface, enabling the simultaneous immobilization via specific adsorption to cellulose and hydrolysis of organophosphate nerve agents.; The specific adhesion of E. coli with surface-exposed cellulose-binding domain to cellulosic materials was first investigated. Whole-cell immobilization was very specific, forming essentially a monolayer of cells onto different supports as shown by electron micrographs. Cells with surface-exposed CBD bound specifically and tightly to cellulose supports at a wide range of pHs and temperatures. Optimal binding can be obtained under physiological conditions such as pH 7 and 37°C, demonstrating the utility of surface-exposed CBD as an efficient means of whole-cell immobilization.; Using two compatible translocation machineries, both OPH and CBD were then co-expressed onto cell surface. This was the first time that two functional proteins were expressed onto E. coli cell surface. An optimal level of OPH activity and binding affinity to cellulose supports was achieved by investigating expression under different induction levels. Immobilized cells degraded paraoxon rapidly and retained almost 100% efficiency over a period of 45 days. This result showed that the surface-engineered E. coli co-displaying CBD and OPH was endowed with the ability to rapidly degrade organophosphorus nerve agents and to bind tightly onto cellulose materials.; Owing to the superior stability and affinity to cellulose, this whole-cell system was immobilized in a cellulose hollow fiber bioreactor for the biodetoxification of paraoxon. The tightly immobilized biocatalysts maintained a stably high degradation capacity over a period of 48 days under operating and storage conditions. The bioreactor was also easily regenerated with 90 immobilization capacity and degradation efficiency recovery. In addition, the CBD-based specific immobilization provided operation with low pressure drop, low shear force and low energy requirement. The stably high degradation capacity and ability of easy regeneration adequately showed the promise of this dual functional biocatalyst in large scale application of immobilization and biodegradation of organophosphate nerve agents.