Biocatalytic Buffering System for Bioremediation of Nerve Agents

In a number of enzyme-catalyzed hydrolytic reactions, the production of acid and resulting pH drop can significantly alter enzyme activity. While conventional buffers can often be used to maintain pH during hydrolysis, the buffering capacity will eventually be exceeded in the presence of large amounts of substrate. An alternative method to controlling pH in enzymatic hydrolysis reactions involves incorporation of a base producing enzyme/substrate pair in which the base-producing enzyme has a distinctly lower pH optimum in comparison to the acid-producing enzyme.


The pH dependence profile of the acid (profile a) and base (profile b) generating enzymes. Increasing the relative concentration of the base producing enzyme, represented by profile c, results in a new pHeq.

The pH of the system is a function of the relative rates of the two enzymes and will equilibrate (pHeq) when the rate of proton generation and release of hydroxide ions are equivalent. Variation of the ratio of the enzymes enables controlled shifting of the pHeq to a desired set point.

We have applied this theory as a means of overcoming enzyme deactivation as a result of acid production during the aqueous enzymatic hydrolysis of organophosphorous (OP) nerve agents. In specific, we are studying the OPH-catalyzed hydrolysis of paraoxon in the presence of urease-catalyzed urea hydrolysis, a base producing reaction.


OPH-catalyzed hydrolysis of paraoxon and urease-catalyzed hydrolysis of urea.


We have further developed a theoretical model based on Michaelis-Menton kinetics capable of predicting the pHeq and paraoxon conversion as a function of time from a given set of initial conditions including concentrations and ratio of the hydrolytic enzymes, initial substrates concentrations, and initial pH. The model accounts for changing pH and substrate concentrations over the course of agent hydrolysis.


Biocatalytic buffering for paraoxon degradation in aqueous solution. The solid and dashed lines illustrate measured pH in the presence and absence of urease and urea respectively while the dotted line represents the model predicted pH. The circles and triangles illustrate the measured paraoxon converion in the presence and absence of urease and urea while the dash-dot line corresponds to the model predicted paraoxon conversion.

Having demonstrated the ability of the concept of biocatalytic buffering to successfully maintain pH in aqueous systems, we are investigating its applicability in low water environments including reversed micelle microemulsions, fire-fighting foams and sprays, nanoemulsions, and aircraft deicing solutions.

With the current growing concern over the threat of use of chemical weapons, we have applied the theory of biocatalytic buffering to the development of a positive-response sensor capable of detecting OP agents. OP toxins are well characterized as inhibitors of choline esterase activity towards acetylcholine, its natural substrate. When active, the choline esterase-catalyzed hydrolysis of acetylcholine generates acid.


Positive-response OP agent sensor based on the inhibition of choline esterase. The color of the sensor in the absence of an agent remains yellow as shown in the sensor on the left. When in the presence of an agent, the sensor turns red within 1 minute creating a positive response as demonstrated by the sensor on the right.

Therefore, in theory, the simultaneous urease-catalyzed hydrolysis of urea would neutralize the generated acid resulting in a constant pH while inhibition of choline esterase would result in a distinct increase in pH due to urease activity. Through co-immobilization of choline esterase and acetylcholine, urease and urea, and a pH sensitive dye within a polyurethane foam, the sensor will undergo a color transformation in the presence of an agent.

In addition to the use for nerve agent degradation, we are studying the potential use of the biocatalytic buffering concept in a variety of biosensors for medical applications.