Hsu, S.-Y.; Hsieh, H.-Y.; Stewart, G. C.; Lin, C.-H.

The contamination of the drinking water sources with atrazine (ATR), a widely used herbicide in the United States, has raised public health concerns. A green technology, Bacillus thuringiensis spore-based display system, has been developed to express a high density of enzymes on the spore surface. The B. thuringiensis spore can serve as a bioparticle to deliver enzymes to different environments and enhance the stability of enzymes expressed on their surface. A cluster of enzymes, AtzA, AtzB, AtzC, AtzD, AtzE, and AtzF, found in Pseudomonas sp. strain ADP, catalyze the degradation of ATR to CO2 and NH3. AtzA-bearing spores have successfully demonstrated the capability of converting ATR to its metabolite, hydroxyatrazine (HA). In this study, AtzB, a hydroxyatrazine N-ethylaminohydrolase that hydrolyzes HA to N-isopropylammelide (NiPA), was also successfully expressed on the Bacillus spores by fusing atzB to the gene encoding the attachment domain of the BclA spore surface nap layer protein. The optimal ratio of 1:2 for AtzA-bearing to AtzB-bearing spores was applied and successfully converted 90% ATR to NiPA in the water after 96 hours of incubation. The surface water with the addition of 34.5 nM (7.5 {micro}g L-1) of ATR was treated with a combination of AtzA-bearing and AtzB-bearing spores in a 96-hour time course study. Over 80% of applied ATR in surface water was degraded within 24 hours. At the end of 96 hours. ATR in surface water was completely converted to NiPA. We have successfully demonstrated the application of multienzyme bioremediation of ATR using this delivery system.