Enhanced Peroxide Resistance of In Vitro Mutagenized Fluorideresistant Klebsiella pneumoniae Ureases for Catalytic Buffering of Agent Decontamination Reactions

摘要

Catalytic buffering is an advanced method of pH control for the enzyme-based Advanced Catalytic Enzyme System (ACES) and oxidative surety agent decontamination technologies. Ammonia production from urea by urease neutralizes the production of Oalkylphosphonic acids resulting from the hydrolysis of Nerve agents such as Sarin and VX. Fluoride production from Sarin hydrolysis inhibits native urease at low mM concentrations. Hydrogen peroxide, used in oxidative nerve agent treatment, is also potent inhibitor of urease activity. Fluoride-resistant (FR) ureases developed previously in our laboratory were greater than 95% inhibited by 1% hydrogen peroxide. To overcome this problem, the FR mutant urease structural genes of Klebsiella pneumoniae were mutagenized in vitro in an E. coli mutator strain to produce ureases with superior peroxide-resistance (PR). Mutagenized ureABC plasmids were recovered and cotransformed into an E. coli catalase mutant (UM2) with a plasmid bearing the urease accessory genes (ureDEFG) needed for nickel incorporation. The double-plasmid recombinant colonies were immobilized on nylon, lysed and screened in situ for peroxide-resistant urease activity. Several PR clones were isolated from this procedure with enhanced urease activity in 1% hydrogen peroxide. Several generations of in vitro mutagenesis of the PR ureABC plasmids resulted in ureases with many times the activity of their FR progenitors in 1% peroxide. These results show the promise of mutant peroxide-resistant ureases as catalytic buffering catalysts for agent decontamination reactions.