David J. Hardman

Dehalogenases in Soil Bacteria

Sixteen bacterial strains isolated from soil were able to grow on either 2-monochloropropionic acid or monochloroacetic acid as the sole carbon and energy source. The isolates were divided into five groups on the basis of differences in their dehalogenase activities towards four substrates — monochloroacetic acid, dichloroacetic acid, 2-monochloropropionic acid and 2,2-dichloropropionic acid. Disc gel electrophoresis of crude extracts identified four distinct dehalogenases with different electrophoretic mobilities: three isolates contained one, three or four dehalogenases, respectively, and the remaining 13 isolates contained different combinations of two dehalogenases. In some cases, dehalogenases with the same mobilities from different isolates appeared to be identical enzymes. In others, enzymes from different isolates with the same electrophoretic mobility had different substrate activity profiles. Pseudomonas putida PP3 was shown to contain one enzyme which was comparable with one of the dehalogenases detected in several of the newly isolated soil bacteria. The second enzyme was not found in soil bacteria and represented a fifth dehalogenase. The significance of these results in terms of the evolution of dehalogenase activity is discussed.

A monobromoacetate dehalogenase from Pseudomonas cepacia MBA4

Pseudomonas cepacia MBA4 able to utilize monobromoacetic acid as a sole source of carbon and energy was isolated from soil by enrichment culture. In batch culture the ability to utilize the substrate was conferred by a single halidohydrolase-type dehalogenase which demonstrated a high activity towards the enrichment substrate. The purified enzyme, designated as dehalogenase IVa by activity-stain polyacrylamide gel electrophoresis, had a relative molecular weight of 45,000 and was comprised of two electrophoretically identical subunits with relative molecular weights of 23,000. Dehalogenase IVa demonstrated isomer specificity, being active towards the L-isomer of 2-monochloropropionic acid only. The significance of activity-stain polyacrylamide gel electrophoresis in characterizing dehalogenases and their ubiquitous distribution among bacterial genera are discussed.

Microbial Dehalogenation of Halogenated Alkanoic Acids, Alcohols and Alkanes

Publisher Summary This chapter focuses on the catabolism of halogenated aliphatic compounds in the series: alkanoic acids, alcohols, and alkanes. Microorganisms remove halogens from halogenated aliphatic compounds by the activity of enzymes generally called “dehalogenases.” A scheme for classifying dehalogenases is provided to accommodate the current range of known enzymes and their properties. The classification and naming of dehalogenases are based on catalytic properties, with subgroups based on other factors, such as substrate specificities, and nucleotide or amino-acid sequence information. On this basis four classes of 2HAA (halogenated alkanoic acids substituted in the C2 position) hydrolytic dehalogenases are recognized: Class IL 2HAA, Class ID 2HAA, Class 2I 2HAA, and Class 2R 2HAA. Dehalogenation of haloalcohols showed that 3-bromopropanol was biodegradable. Two main pathways for the mineralization of haloalcohols are discussed. The first pathway is mediated by enzymes that accept halosubstituted molecules as their substrates, with dehalogenation taking place only after the haloalcohols have been oxidized to the corresponding haloalkanoic acids. In the complete pathway, the epoxide resulting from the dehalogenation reaction is hydrolyzed by an epoxide hydrolase to the corresponding alcohol. Haloalcohol dehalogenases showed a restricted range of activities being active only when the halogen is vicinal to a hydroxyl group or to a keto group, such as in chloroacetone. Haloalkanes are significant environmental compounds, occurring as both natural products and as xenobiotic compounds. There seems to be a much wider diversity of systems and mechanisms involved in haloalkane dehalogenation than for either halogenated alkanoic acids or alcohols. Haloalkane degradation involving NADH-linked reactions, oxygenases, glutathione (GSH)-dependent reactions, as well as hydrolytic mechanisms have been proposed and demonstrated.

Biochemical Characterization of a Haloalcohol Dehalogenase from Arthrobacter erithii H10a

Arthrobacter erithii H10a possesses two enzymes capable of catalyzing the dehalogenation of vicinal halohydrins which have been designated as dehalogenases DehA and DehC. The DehA dehalogenase demonstrated greater activity toward 1,3-dichloro-2-propanol (1,3-DCP) while the DehC dehalogenase showed higher activity toward 3-chloro-1,2-propanediol (3-CPD) and brominated alcohols. The DehA dehalogenase was composed of two non-identical subunits (relative molecular mass of 31.5 and 34 kDa) which probably associate with other proteins to form a large catalytically active protein of 200 kDa. The two subunits were purified and the amino acid sequence of their tryptic digests determined. The DehA enzyme catalyzed the conversion of vicinal halohydrins to epoxides and the reverse reaction in the presence of an excess of halogen. This enzyme had maximum activity at 50 degrees C and a broad pH optimum over the range 8.5-10.5. The apparent K(m) and Vmax values for dehalogenation of 1,3-DCP and 3-CPD were 0.105 mM and 223 mumol min-1 mg-1; and 2.366 mM and 1.742 mumol min-1 mg-1, respectively. The enzyme was inhibited by 2-chloroacetic acid (MCA) and 2,2-dichloroacetic acid (DCA). The inhibition pattern suggested a mixed type inhibition which was predominantly uncompetitive. Amino acid modification experiments demonstrated that one or more cysteine and arginine residues are likely to be involved in catalysis or play an important role in the maintenance of the enzyme structure. The characteristics of the DehA enzyme are compared to those of previously reported haloalcohol dehalogenases and discussed in terms of diversity of this type of dehalogenase.

Synthesis of Chiral Epihalohydrins Using Haloalcohol Dehalogenase A from Arthrobacter Erithii H10a

Abstract Investigation of the epoxide enantiomers formed by the action of the haloalcohol dehalogenase from Arthrobacter erithii H10a revealed that ( r )-epichlorohydrin (ECH) was selectively produced from 1,3-dichloro-2-propanol (1,3-DCP). A maximum enantiomeric excess (e.e. > 95%) of ( r )-ECH was obtained when dehalogenation of 1,3-DCP occurred in the presence of an excess of KBr. During the reverse reaction, ( r )-ECH was stereoselectively halogenated to form 1,3-DCP if the halogen in the reaction mixture was chloride; however, if chloride was substituted by bromide, the ( s )-isomer was halogenated preferentially, resulting in the accumulation of the ( r )-isomer. ( r )-epibromohydrin (EBH) was formed as the result of transhalogenation. If the starting substrates were EBH and KCl, the ( r )-isomer was selectively chlorinated while the transhalogenation product was ( s )-ECH.

Dehalogenation of haloalkanes byRhodococcus erythropolis Y2

Phodococcus erythropolis Y2 produced two types of dehalogenase: a hydrolytic enzyme, that is an halidohydrolase, which was induced by C3 to C6 1-haloalkane substrates, and at least one oxygenase-type dehalogenase induced by C7 to C16 1-haloalkanes andn-alkanes. The oxygenase-type activity dehalogenated C4 to C18 1-chloroalkanes with an optimum activity towards 1-chlorotetradecane. The halidohydrolase catalysed the dehalogenation of a wide range of 1- and α,ω-disubstituted haloalkanes and α,ω-substituted haloalcohols. In resting cell suspensions of hexadecane-grownR. erythropolis Y2 the oxygenase-type dehalogenase had a specific activity of 12.9 mU (mg protein)−1 towards 1-chlorotetradecane (3.67 mU mg−1 towards 1-chlorobutane) whereas the halidohydrolase in 1-chlorobutane-grown batch cultures had a specific activity of 44 mU (mg protein)−1 towards 1-chlorobutane.The significance of the two dehalogenase systems in a single bacterial strain is discussed in terms of their contribution to the overall catabolic potential of the organism.Rhodococcus erythropolis Y2 produced two types of dehalogenase: a hydrolytic enzyme, that is an halidohydrolase, which was induced by C3 to C6 1-haloalkane substrates, and at least one oxygenase-type dehalogenase induced by C7 to C16 1-haloalkanes and n-alkanes. The oxygenase-type activity dehalogenated C4 to C18 1-chloroalkanes with an optimum activity towards 1-chlorotetradecane. The halidohydrolase catalysed the dehalogenation of a wide range of 1- and alpha,omega-disubstituted haloalkanes and alpha,omega-substituted haloalcohols. In resting cell suspensions of hexadecane-grown R. erythropolis Y2 the oxygenase-type dehalogenase had a specific activity of 12.9 mU (mg protein)-1 towards 1-chlorotetradecane (3.67 mU mg-1 towards 1-chlorobutane) whereas the halidohydrolase in 1-chlorobutane-grown batch cultures had a specific activity of 44 mU (mg protein)-1 towards 1-chlorobutane. The significance of the two dehalogenase systems in a single bacterial strain is discussed in terms of their contribution to the overall catabolic potential of the organism.

The Dehalogenase Complement of a Soil Pseudomonad Grown in Closed and Open Cultures on Haloalkanoic Acids

Pseudomonas sp. strain E4 was grown in continuous-flow culture with either monochloroacetate (MCA) or 2-monochloropropionate (2MCPA) as the growth-limiting substrate. In contrast to previous observations made for this organism grown in closed culture on 2MCPA, a third dehalogenase was detected under certain growth conditions, as well as dehalogenases I and II. The response of Pseudomonas sp. strain E4, in terms of its enzyme complement, varied depending on the organisms growth rate and the nature of the growth-limiting substrate. With MCA as the growth-limiting substrate, dehalogenases II and III were only detected in slowly growing organisms. With 2MCPA as growth-limiting substrate, the responses were more variable and complex. The overall dehalogenase activities also depended on the growth rate and substrate limitation, with higher dehalogenation rates found in organisms growing slowly with MCA limitation compared with 2MCPA limitation. In fast growing organisms the relative rates of dehalogenation were reversed for the two limitations.

Biodehalogenation of low concentrations of 1,3-dichloropropanol by mono- and mixed cultures of bacteria

Abstract The degradation of low concentrations of 1,3-dichloro-2-propanol (1,3-DCP) and related halohydrins by whole cells and cell-free extracts of soil bacteria has been investigated. Three bacteria (strains A1, A2, A4), isolated from the same soil sample, were distinguished on the basis of cell morphology, growth kinetics and haloalcohol dehalogenase profiles. Strain A1, probably an Agrobacterium sp., dehalogenated 1,3-DCP with the highest specific activity (0.33 U mg protein−1) and also had the highest affinity for 1,3-DCP (Km, 0.1 mM). Non-growing cells of this bacterium dehalogenated low concentrations of 1,3-DCP with a first-order rate constant (k1) of 1.13 h−1 . The presence of a non-dehalogenating bacterium, strain G1 (tentatively identified as Pseudomonas mesophilius), did not enhance the dehalogenation rate of low 1,3-DCP concentrations. However, the mixed-species consortium of strains A1 and G1 had greater stability than the mono-species culture at DCP concentrations above 1.0 gl−1.

Generation of environmentally enhanced products: Clean technology for paper chemicals

The modification of existing chemical manufacturing processes to selectively remove unwanted chemicals in products, offers a realistic approach to novel clean technologies. Adjunct biotechnological processing offers a means to achieve the manufacture of new environmentally enhanced products (EEPs). This paper describes the development and implementation of a bioprocess for the manufacture of an enhanced paper chemical. The process was integrated into existing manufacturing plants involved in the production of neutral curing poly(aminoamide) chemicals which are used commercially to impart wet-strength to paper products such as tissues and towels (e.g. Kymene® wet-strength resins). A consequence of the epichlorohydrin chemistry involved in the polymers manufacture, haloalcohols (predominantly, 1,3-dichloropropan-2-ol (DCP) and 1-chloropropanediol (3-CPD)) contaminate the product. The objective was to reduce the concentration of the two haloalcohols in Kymene®-SLX wet-strength resins (c. 8000 ppm db) without affecting the performance of the product. A two-membered bacterial consortium was used in an aerobic stirred tank bioreactor system which was capable of rapidly reducing the concentrations of DCP and CPD in an aqueous solution of the wet-strength resin to less than 1 ppm and 5 ppm respectively. A 3000 dm 3 bioreactor was integrated into two established manufacturing plants, generating a reliable and predictable process to enhance the value of the neutral curing wet-strength chemical.

Performance of a 2-haloalkanoate dehalogenase immobilized in a hollow fiber reactor

Abstract A hollow fiber reactor for the specific dehalogenation of 2-haloalkanoic acids has been developed. Cell-free extracts of Pseudomonas cepacia MBA4 containing dehalogenase IVa were immobilized on hollow fibers contained in a glass reactor, and the reactors efficiency assessed in comparison with free enzyme activity. At all protein loadings examined, the reactor was under diffusional control. While the V max of the enzyme was decreased by immobilization, the sensitivity of the observed reaction rates to temperature also decreased. These findings suggest that this immobilization procedure is an effective method for entrapping the biocatalyst for processes such as the resolution of racemic mixtures of 2-haloalkanoic acids.