Rhodococcus sp. strain AD45 was isolated from an enrichment culture on isoprene (2-methyl-1,3-butadiene). Isoprene-grown cells of strain AD45 oxidized isoprene to 3,4-epoxy-3-methyl-1-butene,cis-1,2-dichloroethene tocis-1,2-dichloroepoxyethane, andtrans-1,2-dichloroethene totrans-1,2-dichloroepoxyethane. Isoprene-grown cells also degraded cis-1,2-dichloroepoxyethane andtrans-1,2-dichloroepoxyethane. All organic chlorine was liberated as chloride during degradation ofcis-1,2-dichloroepoxyethane. A glutathione (GSH)-dependent activity towards 3,4-epoxy-3-methyl-1-butene, epoxypropane,cis-1,2-dichloroepoxyethane, andtrans-1,2-dichloroepoxyethane was detected in cell extracts of cultures grown on isoprene and 3,4-epoxy-3-methyl-1-butene. The epoxide-degrading activity of strain AD45 was irreversibly lost upon incubation of cells with 1,2-epoxyhexane. A conjugate of GSH and 1,2-epoxyhexane was detected in cell extracts of cells exposed to 1,2-epoxyhexane, indicating that GSH is the physiological cofactor of the epoxide-transforming activity. The results indicate that a GSHS-transferase is involved in the metabolism of isoprene and that the enzyme can detoxify reactive epoxides produced by monooxygenation of chlorinated ethenes.

Cultures of the newly isolated bacterial strains AD20, AD25, and AD27, identified as strains ofAncylobacter aquaticus, were capable of growth on 1,2-dichloroethane (DCE) as the sole carbon and energy source. These strains, as well as two other new DCE utilizers, were facultative methylotrophs and were also able to grow on 2-chloroethanol, chloroacetate, and 2-chloropropionate. In all strains tested, DCE was degraded by initial hydrolytic dehalogenation to 2-chloroethanol, followed by oxidation by a phenazine methosulfate-dependent alcohol dehydrogenase and an NAD-dependent aldehyde dehydrogenase. The resulting chloroacetic acid was converted to glycolate by chloroacetate dehalogenase. The alcohol dehydrogenase was induced during growth on methanol or DCE in strain AD20, but no activity was found during growth on glucose. However, in strain AD25 the enzyme was synthesized to a higher level during growth on glucose than on methanol, and it reached levels of around 2 U/mg of protein in late-exponential-phase cultures growing on glucose. The haloalkane dehalogenase was constitutively produced in all strains tested, but strain AD25 synthesized the enzyme at a level of 30 to 40% of the total cellular protein, which is much higher than that found in other DCE degraders. The nucleotide sequences of the haloalkane dehalogenase (dhUA) genes of strains AD20 and AD25 were the same as the sequence of dhL4 from Xanthobacter autotrophicus GJ10 and GJ11. Hybridization experiments showed that the dhlA genes of six different DCE utilizers were all located on an 8.3-kb EcoRI restriction fragment, indicating that the organisms may have obtained the dhlA gene by horizontal gene transmission.

Chlorinated hydrocarbons are widely used synthetic chemicals that are frequently present in industrial emissions. Bacterial degradation has been demonstrated for several components of this class of compounds. Structural features that affect the degradability include the number of chlorine atoms and the presence of oxygen substituents. Biological removal from waste streams of compounds that serve as a growth substrate can relatively easily be achieved. Substrates with more chlorine substituents can be converted co-metabolically by oxidative routes. The microbiological principles that influence the biodegradability of chlorinated hydrocarbons are described. A number of factors that will determine the performance of microorganisms in systems for waste gas treatment is discussed. Pilot plant evaluations, including economics, of a biological trickling filter for the treatment of dichloromethane containing waste gas indicate that at least for this compound biological treatment is cost effective.

An enzyme capable of dehalogenating vicinal haloalcohols to their corresponding epoxides was purified from the 3-chloro-1,2-propanediol-utilizing bacterium Arthrobacter sp. strain AD2. The inducible haloalcohol dehalogenase converted 1,3-dichloro-2-propanol, 3-chloro-1,2-propanediol, 1-chloro-2-propanol, and their brominated analogs, 2-bromoethanol, as well as chloroacetone and 1,3-dichloroacetone. The enzyme possessed no activity for epichlorohydrin (3-chloro-1,2-epoxypropane) or 2,3-dichloro-l-propanol. The dehalogenase had a broad pH optimum at about 8.5 and a temperature optimum of 50°C. The enzyme followed Michaelis-Menten kinetics, and the Km values for 1,3-dichloro-2-propanol and 3-chloro-1,2-propanediol were 8.5 and 48 mM, respectively. Chloroacetic acid was a competitive inhibitor, with a Ki of 0.50 mM. A subunit molecular mass of 29 kDa was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With gel filtration, a molecular mass of 69 kDa was found, indicating that the native protein is a dimer. The amino acid composition and N-terminal amino acid sequence are given.Both epichlorohydrin (3-chloro-1,2-epoxypropane) and its precursor in chemical synthesis, 1,3-dichloro-2-propanol, are industrial chemicals that may enter the environment because of their volatile character or improper disposal. These compounds are considered important environmental pollutants (12), in part because epichlorohydrin has been shown to be mutagenic and carcinogenic in rats (18).Recently, three bacterial cultures able to grow on epichlorohydrin, 3-chloro-1,2-propanediol, or 1,3-dichloro-2-propanol were isolated (20). Two of these cultures, Pseudomonas sp. strain AD1 and Arthrobacter sp. strain AD2, were examined in more detail. Degradation of epichlorohydrin proceeded via 3-chloro-1,2-propanediol and glycidol (3-hydroxy-1,2-epoxypropane). Arthrobacter sp. strain AD2 was not capable of enzymatic opening of the epoxide ring in epichlorohydrin, but Pseudomonas sp. strain AD1 possessed an epoxide hydrolase, producing 3-chloro-1,2-propanediol., It was shown that both strains dehalogenated haloalcohols by a highly ihducible enzyme which we call haloalcohol dehalogenase (20). The dehalogenase catalyzed the conversion of vicinal haloalcohols to the corresponding epoxides with liberation of inorganic chloride or bromide. The same mechanism of dehalogenation of haloalcohols was found earlier in a Flavobacterium sp. isolated on 2,3-dibromo-1-propanol (1, 3).The products of dehalogenation and the substrate range of the enzyme suggest that haloalcohol dehalogenases do not belong to other established classes of dehalogenases, such as haloacid dehalogenases (4, 15), haloalkane dehalogenases (6-8, 10, 16, 22), and dichloromethane dehalogenases (11,17). Since no information on the biochemical characteristics of haloalcohol dehalogenases is available, we purified and studied the enzyme from Arthrobacter sp. strain AD2 in more detail. We also present evidence that the dehalogenase of Pseudomonas sp. strain AD1 differs from the...

With the pure bacterial cultures Ancylobacter aquaticus AD20 and AD25, Xanthobacter autotrophicus GJ1O, and Pseudomonas sp. strain AD1, Monod kinetics was observed during growth in chemostat cultures on 1,2-dichloroethane (AD20, AD25, and GJ1O), 2-chloroethanol (AD20 and GJ10), and 1,3-dichloro-2-propanol (AD1). Both the Michaelis-Menten constants (Ki) of the first catabolic (dehalogenating) enzyme and the Monod half-saturation constants (Ks) followed the order 2-chloroethanol, 1,3-dichloro-2-propanol, epichlorohydrin, and 1,2-dichloroethane. The Ks values of strains GJ10, AD20, and AD25 for 1,2-dichloroethane were 260, 222, and 24 ,M, respectively. The low Ks value of strain AD25 was correlated with a higher haloalkane