Improvement of organophosphorus hydrolase activity toward nerve agents by amino acid substitutions

Immobilization of organophosphorus hydrolase for the degradation of organophosphorus nerve agents

“…The enzyme was cloned and has been biochemically studied to degrade pesticides and also other organophosphorus compounds such as nerve agents (Dumas et al, 1989;Hoskin et al, 1995;Lai et al, 1995;Mulbry et al, 1986;Vanhooke et al, 1996). Since the efficient decontamination of organophosphorus nerve agents, such as O-isopropyl methylphosphonofluoridate (sarin) and O-ethyl-S-(2-diisopropylaminoethyl) methylphosphonothiolate (VX), has been greatly needed as a countermeasure against chemical terrorisms such as the sarin gas attacks in Japan in 1994 and 1995 (Seto et al, 2000), many groups, including ourselves, have developed genetically manipulated OPH to degrade nerve agents (Gopal et al, 2000;Hill et al, 2003;Jeong et al, 2014;Nakayama et al, 2014;Reeves et al, 2008). Organophosphorus nerve agents are relatively unstable in alkaline conditions, and, therefore, strong alkaline liquids have been used for the decontamination.…”

“…In our previous study (Ohmori et al, 2013), we estab-from the N-terminal (the strain KGU0255) (Nakayama et al, 2014) was used in this study. The cells of KGU0255 were suspended in 20 mM Tris-HCl buffer (pH 8.0) and disrupted by sonication (Ultrasonic Homogenizer Model UH-300, SMT Ltd, Tokyo, Japan) for 10 min.…”

“…For the degradation assay for nerve agents, an aliquot of Co 2+ -activated OPH solution, or immobilized OPH, was mixed with a reaction mixture containing one of the nerve agents (Nakayama et al, 2014). In the case of immobilized OPH, the immobilized resin was mixed with the solution of the nerve agent, and incubated under the conditions described in the footnotes of Tables 2 and 3.…”

Expression of recombinant organophosphorus hydrolase in the original producer of the enzyme, Sphingobium fuliginis ATCC 27551

“…Since OPH was proved to exhibit the hydrolytic activity toward organophosphorus nerve agents such as sarin (O-isopropyl methylphosphonofluoridate) and VX (O-ethyl-S-(2-diisopropylaminoethyl) methylphosphonothiolate), it was expected to be applied to the decontamination of these nerve agents in the case of chemical terrorism, and the degrading activity toward VX has been improved. [12][13][14] We also constructed the mutant OPH for the degradation of VX 15) and established the purification method using the affinity between His-hexamer attached at N-terminal of OPH and Ni-chelating resin. The mutant OPH was further immobilized with porous silica or calcium alginate to use it as a bioreactor (submitted for publication).…”

“…The transformants of S. paucimobilis and S. fuliginis were disrupted by sonication to prepare crude enzyme solution, and they were purified by Ni-chelating resin. The OPH activity was measured by the method described previously 15) using paraoxon (O,O-Diethyl p-nitrophenylphosphate) as a substrate, and specific activity was calculated. As shown in Table 1, both transformants of S. paucimobilis and S. fuliginis showed clear activity of OPH, and the specific activity of their crude enzyme preparations are higher than that of E. coli stain KGU0255 harboring the same plasmid.…”