**1. Introduction**

As a neurotransmitter, acetylcholine (ACh) plays a vital role in brain and muscle function. Its function can be both excitatory and inhibitory. But in the central nervous system (CNS), ACh primarily plays the excitatory role, which means it can speed up nerve signals. Excess ACh is degraded by acetylcholinesterase (AChE) to maintain a balanced ACh level. Nerve agents are a group of organophosphorus (OP) compounds that are potent neurotoxins used as chemical warfare agents and insecticides. OP nerve agents disrupt the CNS by inhibiting AChE function. Inhibition of AChE results in ACh accumulation and ACh receptor overstimulation, leading to severe injuries and even death due to losing control of respiratory muscles. Those injured by nerve agents often express chronic health problems, such as visual impairment, dermatological conditions, neurological sequelae, and respiratory problems [1].

Weapons of mass destruction were first used in World War I. Rapid advances in chemistry during that time brought surging knowledge and constant growth in developing more effective chemical agents. The nerve agent, tabun, was first discovered

from an organophosphorus insecticide in 1936 by a German chemist, Dr. Gerhart Schrader. About two years later, Dr. Schrader developed another similar nerve agent, sarin [2]. Suitable delivery systems and large-scale production of nerve agents were also developed rapidly for their usage in warfare. Since nerve agents are stable, easily dispersed and work at low concentrations, the effects are long-lasting and increase with continued exposure. The threat from nerve agents was not confined to military battlefields. The Matsumoto sarin attack (1994) and Tokyo subway sarin attack (1995) exhibited the usage of nerve agents in terrorist attacks. Paraoxon, which is the active metabolite of the insecticide parathion, is also a potent nerve agent. As such, nerve agents pose a threat to armed forces, agricultural workers, and civilians.

Current medical countermeasures available include atropine and pralidoxime chloride (2-PAM Cl). Atropine competitively inhibits ACh binding to acetylcholine receptors, reducing receptor overstimulation. The therapeutic 2-PAM Cl re-cleaves AChE phosphorylation induced by nerve agents and reactivates AChE. These countermeasures target the down-stream pathways of OP; thus, none of them effectively eliminates OP agents.

A direct method that can hydrolyze OP agents before they enter the central nervous system is urgently needed. Organophosphorus hydrolase (OPH) is a bacterial enzyme that can detoxify a wide range of OP nerve agents and pesticides. The advantage of OPH over existing treatments is rapid hydrolysis of OP agents, which provides the potential to eliminate nerve agents in the circulatory system, before they penetrate the blood-brain barrier and exert effects in the CNS. Kinetic properties of OPH from various bacteria have been studied, with *k*cat about 103 /s–104 /s and Km between 80 μM and 2.5 mM [3, 4], significantly higher compared to nerve agent lethal levels (a few μM).

The OPH active site is dominated by histidine residues and stabilized by the stacking network formed among these histidine residues. This unique feature makes OPH a great candidate for aromatic unnatural amino acid (UAA) substitutions. In this chapter, we first describe a high-throughput cell-free protein synthesis and kinetic measurement method for rapid screening and selection of OPH variants with enhanced substrate binding. Then, we examine the possibility to apply cell-free protein synthesis systems for expression of OPH UAA substitutions. Lastly, we present a genetic code expansion (GCE) machinery used to examine the expression of OPH UAA substitutions. The results of kinetic studies of these mutants showed greatly improved OPH substrate binding affinity. Overall, this work uniquely demonstrates that UAA replacements can enhance enzyme properties significantly.
