Haloalkane hydrolysis with an immobilized haloalkane dehalogenase.

Haloalkane dehalogenase from Rhodococcus rhodochrous was covalently immobilized onto a polyethyleneimine impregnated gamma-alumina support. The dehalogenating enzyme was found to retain greater than 40% of its original activity after immobilization, displaying an optimal loading (max. activity/supported protein) of 70 to 75 mg/g with an apparent maximum (max. protein/support) of 156 mg/g. The substrate, 1,2,3-trichloropropane, was found to favorably partition (adsorb) onto the inorganic alumina carrier (10 to 20 mg/g), thereby increasing the local reactant concentration with respect to the catalyst's environment, whereas the product, 2,3-dichloropropan-1-ol, demonstrated no affinity. Additionally, the inorganic alumina support exhibited no adverse effects because of solvent/component incompatibilities or deterioration due to pH variance (pH 7.0 to 10.5). As a result of the large surface area to volume ratio of the support matrix and the accessibility of the bound protein, the immobilized biocatalyst was not subject to internal mass transfer limitations. External diffusional restrictions could be eliminated with simple agitation (mixing speed: 50 rpm; flux: 4.22 cm/min). The pH-dependence of the immobilized dehalogenase was essentially the same as that for the native enzyme. Finally, both the thermostability and resistance toward inactivation by organic solvent were improved by more than an order of magnitude after immobilization.

Enzymatic dehalogenation of gas phase substrates with haloalkane dehalogenase.

Haloalkane dehalogenase is an enzyme capable of catalyzing the conversion of short-chained (C(2)-C(8)) aliphatic halogenated hydrocarbons to a corresponding primary alcohol. Because of its broad substrate specificity for mono-, di-, and trisubstituted halogenated hydrocarbons and cofactor independence, haloalkane dehalogenases are attractive biocatalysts for gas-phase bioremediation of pollutant halogenated vapor emissions. A solid preparation of haloalkane dehalogenase from Rhodococcus rhodochrous was used to catalyze the dehalogenation reaction of 1-chlorobutane or 1,3-dichloropropane delivered in the gas phase. For optimal gas-phase dehalogenase activity, a relative humidity of 100%, a(w) = 1, was desired. With a 50% reduction in the vapor-phase hydration level, an 80% decrease in enzymatic activity was observed. The enzyme kinetics for the gas-phase substrates obeyed an Arrhenius-"like" behavior and the solid haloalkane dehalogenase preparation was more thermally stable than its water-soluble equivalent. Triethylamine was added to the gaseous reaction environment in efforts to increase the rate of reaction. A tenfold increase in the dehalogenase activity for the vapor-phase substrates was observed with the addition of triethylamine. Triethylamine altered the electrostatic environment of haloalkane dehalogenase via a basic shift in local pH, thereby minimizing the effect of the pH-reducing reaction product on enzyme activity. Both organic phase and solid-state buffers were used to confirm the activating role of the altered ionization state.

Fighting nerve agent chemical weapons with enzyme technology.

The extreme toxicity of organophosphorous-based compounds has been known since the late 1930s. Starting in the mid-1940s, many nations throughout the world have been producing large quantities of organophosphorous (OP) nerve agents. Huge stockpiles of nerve agents have since developed. There are reportedly more than 200,000 tons of nerve agents in existence worldwide. There is an obvious need for protective clothing capable of guarding an individual from exposure to OP chemical weapons. Also, chemical processes that can effectively demilitarize and detoxify stored nerve agents are in great demand. The new and widely publicized Chemical Weapons Treaty requires such processes to soon be in place throughout the world. Biotechnology may provide the tools necessary to make such processes not only possible, but quite efficient in reducing the nerve agent dilemma. The following paper discusses some of the history in developing enzyme technology against nerve agents. Our laboratory has interest in enhancing the productivity and potential utility of these systems in both demilitarization and decontamination applications. Freeze-dried nerve agent-hydrolyzing enzyme preparations have been shown to be effective in decontaminating gaseous nerve agents. The direct incorporation of nerve agent-hydrolyzing enzymes within cross-linked polyurethane foam matrices during polymer synthesis has been shown to dramatically enhance the productivity of two different enzyme systems. The future goal of such work lies in building a bridge between the clinical application of nerve agent-hydrolyzing enzymes and practical processing techniques that may take advantage of the initial results already achieved in the laboratory.