Bacterial dehalogenases: biochemistry, genetics, and biotechnological applications.

Considerable progress has been made in the last few years in understanding the mechanisms of microbial degradation of halogenated aromatic compounds. Much is already known about the degradation mechanisms under aerobic conditions, and metabolism under anaerobiosis has lately received increasing attention. The removal of the halogen substituent is a key step in degradation of halogenated aromatics. This may occur as an initial step via reductive, hydrolytic or oxygenolytic mechanisms, or after cleavage of the aromatic ring at a later stage of metabolism. In addition to degradation, several biotransformation reactions, such as methylation and polymerization, may take place and produce more toxic or recalcitrant metabolites. Studies with pure bacterial and fungal cultures have given detailed information on the biodegradation pathways of several halogenated aromatic compounds. Several of the key enzymes have been purified or studied in cell extracts, and there is an increasing understanding of the organization and regulation of the genes involved in haloaromatic degradation. This review will focus on the biodegradation and biotransformation pathways that have been established for halogenated phenols, phenoxyalkanoic acids, benzoic acids, benzenes, anilines and structurally related halogenated aromatic pesticides. There is a growing interest in developing microbiological methods for clean-up of soil and water contaminated with halogenated aromatic compounds.

Microbial breakdown of halogenated aromatic pesticides and related compounds

Biological dehalogenation and halogenation reactions.

Biodegradation of xenobiotic compounds is examined with s-triazines as an example and with biological treatment of wastewater containing s-triazines as an aim. s-Triazines have been termed recalcitrant, but examination of the literature indicates that a potential for biodegradation exists. Nitrogen-limited enrichment cultures yield organisms able to degrade by-products of the industrial synthesis of s-triazine herbicides as sources of nitrogen. The choice of inoculum for these enrichments is important and often allows for successful enrichment after simple batch culture, but organisms containing several degradative reactions could be obtained only after selection in extended culture. Routine, specific determinations of all s-triazines (by HPLC) were essential throughout the work. Molar growth yields show complete mass balances for the utilization of s-triazines. Kinetic experiments indicate that specific degradation rates of s-triazines in growing cells are about 0.4 mkat/kg of protein. Characterised biochemical pathways consist of a series of hydrolytic cleavages of chloro-, amino- and alkylamino-groups from the s-triazine ring. Pathways converge to cyanuric acid, which is subject to hydrolytic ring cleavage to CO2 and NH4+ via hydrolysis of biuret and urea. Our cultures degraded all significant s-triazines in real wastewater. But the system was not practicable because the specific activities of some enzymes were too low, because of inhibition by salt in the wastewater and because expensive carbon sources were necessary. Improved planning for enrichment cultures is seen to be necessary and this depends on adequate knowledge of the chemistry of the wastes.

http://kops.uni-konstanz.de/bitstream/handle/123456789/6885/Biodegradation_of_s_triazine.pdf?sequence=1

Biodegration of s-triazine xenobiotics

Bioremediation of organic compounds — putting microbial metabolism to work

Incorporation of [18O]water into 4-hydroxybenzoic acid in the reaction of 4-chlorobenzoate dehalogenase from Pseudomonas spec. CBS 3

Extracts of Pseudomonas sp. CBS3 converted 4-chlorobenzoate into 4-hydroxybenzoate. The enzyme responsible for this conversion was enriched by ammonium sulphate fractionation (30–60% saturation, 1.3-fold). The optimum conditions for the reaction were 30–35°C and pH 7–7.5. The enzyme was activated by Mn2+ (1 mM final concentration) up to 120-fold, and by Co2+ (1 mM final concentration) up to 60-fold. Other divalent ions had no effect. EDTA inhibited the enzyme. 4-Bromobenzoate and 4-iodobenzoate were substrates for the enzyme, but 4-fluorobenzoate was not converted.

Initial characterisation of 4-chlorobenzoate dehalogenase from Pseudomonas sp. CBS3

4-Chlorobenzoate dehalogenase from Pseudomonas sp. strain CBS3 converted 4-chloro-3,5-dinitrobenzoate to 3,5-dinitro-4-hydroxybenzoate and 1-chloro-2,4-dinitrobenzene to 2,4-dinitrophenol. The activities were 0.13 mU/mg of protein for 4-chloro-3,5-dinitrobenzoate and 0.16 mU/mg of protein for 1-chloro-2,4-dinitrobenzene compared with 0.5 mU/mg of protein for 4-chlorobenzoate.

Enzymatic dehalogenation of chlorinated nitroaromatic compounds.

4-Chlorobenzoate dehalogenase from Pseudomonas sp. CBS3 showed dehalogenating activity in various organic solvents. In alcohols like methanol (150%) or ethanol (120%) higher activities than in water (100%) were obtained. In apolar solvents like petroleum ether (5%) and nhexane (5%) only trace activities were observed. The solvents did not increase the stability of the enzyme. 4-Chlorobenzoic acid methylester, a substance not soluble in water, was not dehalogenated in organic solvents.

Enzymatic dehalogenation of 4-chlorobenzoate by 4-chlorobenzoate dehalogenase from Pseudomonas sp. CBS3 in organic solvents

Theactivities were 0.13mU/mgofprotein for4-chloro-3,5-dinitrobenzoate and0.16mU/mgofprotein for1-chloro-2,4dinitrobenzene compared with0.5mU/mgofprotein for4-chlorobenzoate. Nitroaromatic compounds arewidely usedinthesynthesis ofpesticides, drugs, dyes, andexplosives (3,16).Someof these compounds carrynotonlyanitro group, butalsoa chlorine substituent onthearomatic ring(10,11).A large numberofchlorinated nitroaromatic compounds areintermediates inthesynthesis ofchlorinated anilines. During the synthesis ofsuchproducts, sideproducts veryoftenare formed whichcarrymorethanthedesired nitro orchlorogroup.Thesehigher chlorinated ornitrated compounds usually endupinthewastewater ofproduction plants, where theycauseserious problems (1). Manyofthese compounds arehighly toxic tobacteria. Oxygenases whichusually initiate thedegradation ofother aromatic compounds suchas benzene ortoluene byanelectrophilic attack cannot attack these compounds since theelectron density atthearomatic ring inthese compounds isverylow.Thus,other reactions thatproceed viaanucleophilic attack atthearomatic ring havetobeconsidered forthedegradation ofthesesubstances. Inprevious papers wereported thereplacement of achlorine substituent from4-chlorobenzoate byahydroxy groupderived fromwatercatalyzed byanenzymefrom Pseudomonas sp.strain CBS3(9,14).Assuming thatthis reaction proceeds through anucleophilic attack, thereplacementofachlorine inchloroaromatics carrying anitro group intheortho- orpara-position should befacilitated. Sucha replacement wouldleadtonitrophenols, whosemicrobial degradation iswellunderstood (3,10,13,16,17).We therefore checked theactivity of4-chlorobenzoate dehalogenase fromPseudomonas sp.strain CBS3towards different nitroaromatic compounds.

Jürgen Thiele

Papers

Incorporation of [18O]water into 4-hydroxybenzoic acid in the reaction of 4-chlorobenzoate dehalogenase from Pseudomonas spec. CBS 3

Initial characterisation of 4-chlorobenzoate dehalogenase from Pseudomonas sp. CBS3

Enzymatic dehalogenation of chlorinated nitroaromatic compounds.

Enzymatic dehalogenation of 4-chlorobenzoate by 4-chlorobenzoate dehalogenase from Pseudomonas sp. CBS3 in organic solvents

Enzymatic Dehalogenation ofChlorinated Nitroaromatic Compounds