Dangerous organophosphorus (OP) compounds have been used as insecticides in agriculture and in chemical warfare. Because exposure to OP could create a danger for humans in the future, butyrylcholinesterase (BChE) has been developed for prophylaxis to these chemicals. Because it is impractical to obtain sufficient quantities of plasma BChE to treat humans exposed to OP agents, the production of recombinant BChE (rBChE) in milk of transgenic animals was investigated. Transgenic mice and goats were generated with human BChE cDNA under control of the goat ␤-casein promoter. Milk from transgenic animals contained 0.1-5 g/liter of active rBChE. The plasma half-life of PEGylated, goat-derived, purified rBChE in guinea pigs was 7-fold longer than non-PEGylated dimers. The rBChE from transgenic mice was inhibited by nerve agents at a 1:1 molar ratio. Transgenic goats produced active rBChE in milk sufficient for prophylaxis of humans at risk for exposure to OP agents.organophosphorus nerve agent ͉ recombinant protein expression ͉ transgenic production H uman plasma butyrylcholinesterase (huBChE) (EC 3.1.1.8) is a globular, tetrameric serine esterase with a molecular mass of Ϸ340 kDa that is stable in plasma with a half-life of Ϸ12 days (1, 2). Although the physiological function of huBChE is unclear, the enzyme prevents intoxication of animals exposed to organophosphorus (OP) compounds (3, 4). The huBChE enzyme also hydrolyzes many ester-containing drugs, such as cocaine and succinylcholine (5). The toxicity of OP agents is due to irreversible inhibition of acetylcholinesterase and the subsequent continuous stimulation of neurons by acetylcholine (6). Administration of exogenous huBChE, which irreversibly binds OP agents to prevent inactivation of acetylcholinesterase and continuous cholinergic stimulation, is a potential strategy for preventing toxicity from OP agents (4). Although huBChE has been obtained from human plasma by a large scale purification technique, this procedure is severely limited by the volume of human plasma needed (7). It is unlikely that a sufficient amount of enzyme could be purified commercially by this technique. Because of the 1:1 stoichiometry required for protection against exposure to OP agents (8), large quantities of huBChE are needed for effective prophylaxis and treatment of exposure. Compared with other potential enzymatic bioscavengers of OP agents, huBChE has a broad spectrum of activity, a relatively long half-life, and limited, if any, physiological side effects (9). Producing recombinant BChE (rBChE) is an alternative to purification of the enzyme from human plasma. Recombinant huBChE has been expressed in Escherichia coli (10), albeit in a nonfunctional form; mammalian 293T (11); and CHO (12) cells. However, these expression systems cannot economically produce sufficient quantities of rBChE with a residence time similar to native huBChE that would allow development of the enzyme as an agent for prophylaxis against OP poisoning.The production of recombinant proteins by the mammary g...

Enterobacter cloacae strain EM was isolated from a commercial dietary mineral supplement stabilized by a mixture of methylparaben and propylparaben. It harbored a high-molecular-weight plasmid and was resistant to high concentrations of parabens. Strain EM was able to grow in liquid media containing similar amounts of parabens as found in the mineral supplement (1,700 and 180 mg of methyl and propylparaben, respectively, per liter or 11.2 and 1.0 mM) and in very high concentrations of methylparaben (3,000 mg liter ؊1 , or 19.7 mM). This strain was able to hydrolyze approximately 500 mg of methyl-, ethyl-, or propylparaben liter ؊1 (3 mM) in less than 2 h in liquid culture, and the supernatant of a sonicated culture, after a 30-fold dilution, was able to hydrolyze 1,000 mg of methylparaben liter ؊1 (6.6 mM) in 15 min. The first step of paraben degradation was the hydrolysis of the ester bond to produce 4-hydroxybenzoic acid, followed by a decarboxylation step to produce phenol under aerobic conditions. The transformation of 4-hydroxybenzoic acid into phenol was stoichiometric. The conversion of approximately 500 mg of parabens liter ؊1 (3 mM) to phenol in liquid culture was completed within 5 h without significant hindrance to the growth of strain EM, while higher concentrations of parabens partially inhibited its growth.

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Most North American pulp and paper mills now biologically treat (biotreat) their liquid effluent. However, treated water still contains effluent‐derived recalcitrant organic material (EROM), measured as chemical oxygen demand (COD), for which emission limits exist in Europe and are being considered in the U.S. Production of microbially resistant, dissolved natural organic material (NOM) typically found in Canadian stream and lake waters occurs slowly under gentle conditions, while mill EROM is generated from lignocellulosics by faster and harsher processes. Similarity of the environmental effects of NOM and pulp and paper mill EROM are examined. Changes occurring over 4 months in biologically treated effluent from two modern Canadian mills and lake NOM when sealed in gas‐ and light‐permeable bags and placed in a pristine Quebec lake are reported. Addition of microbial co‐metabolites significantly improved the dark mineralization of organochlorines surviving mill biological treatment. Mill EROM was light sensitive, nonacutely toxic in the Microtox assay, and similar to NOM in the surrounding lake in most bulk properties. There was no evidence to suggest that placing specific limits on mill EROM (COD) emissions would be environmentally beneficial.

To counter the claim that all biotreated organochlorine (AOX) emissions from modern kraft mills are unnatural and inherently hazardous, it has been argued that over 2400 different AOX compounds are now known to be produced by living organisms. This is an invalid rationale. These 2400 natural compounds are mostly specific halometabolites — each is produced as a large number of identical molecules by a specific enzyme-mediated mechanism. In contrast, in a kraft mill bleachery, heterogeneous wood derivatives are non-specifically chlorinated by hypochlorous acid (HOCl) or hypochlorite ion (OCl-) to produce mixtures containing hundreds of different AOX species. It is therefore much more reasonable to compare mill-derived AOX to other human and natural sources of OCl-/HOCl-generated non-specific AOX. Chloroperoxidase (CPO)-type enzymes naturally produce HOCl and OCl-from common chloride ions and hydrogen peroxide (H2O2). They have now been demonstrated in bacteria, fungi, algae, lichens, higher plants and animals. We demonstrate that a common fungal CPO can, when applied to soil extracts, lignins, cellulosics, tannins and natural lake water organics, rapidly generate nonspecific AOX. In agreement with earlier work done in other geographic areas, AOX was found in all of a wide range of samples from the Montreal region, both from urban and relatively remote pristine lakes and woodlands. Much of it is almost certainly produced via nonspecific HOCl/OCl- chlorination. Finally, we demonstrate that nonspecific AOX is produced by a CPO in mammalian blood as the natural result of the body's immune system fighting infections. Thus, HOCl/OCl- based production of AOX from mixtures of organic molecules is seen to be a common occurrence in nature as well as a product of human technology.