CATALYTIC BUFFERING: DEVELOPMENT OF THE FLUORIDE-RESISTANT UREASES OF Klebsiella pneumoniae

Catalytic buffering is an advanced method of pH control for the enzyme-based chemical agent decontamination technology Advanced Catalytic Enzyme System (ACES). Ammonia production from urea by urease neutralizes the production of Oalkylphosphonic acids resulting from OPAA and OPH catalytic hydrolysis of G-agents and VX. Unfortunately, ureases are inhibited by low levels of fluoride, another product of GB, GD and GF hydrolysis. To overcome this problem, the urease structural genes of the enteric bacterium Klebsiella pneumoniae were isolated from their accessory genes and randomly mutagenized to produce ureases with superior fluoride-resistance (FR). Mutagenized ureABC genes were co-transformed with another plasmid containing the accessory genes (ureDEFG) needed for nickel incorporation into E. coli to produce active urease in the clones. E. coli in vitro mutagenesis was followed by screening of the lysed, double-plasmid clones for fluoride-resistant urease activity. Several FR clones were isolated from this procedure with greatly enhanced activity in 0.1 M fluoride after successive generations of mutagenesis. Successive generations of urease mutants showed higher levels of fluoride resistance. These results indicate that in vitro mutagenesis can be used to successfully generate fluoride-resistant ureases needed for the catalytic buffering method of pH control.