Paul-Gerhard Rieger

BASIC KNOWLEDGE AND PERSPECTIVES ON BIODEGRADATION OF 2,4,6-TRINITROTOLUENE AND RELATED NITROAROMATIC COMPOUNDS IN CONTAMINATED SOIL

Although a few aromatic compounds bearing one nitro group as a substituent are produced as secondary metabolites by microorganisms (31, 44, 45, 49) the majority of nitroaromatic compounds in the environment are due to anthropogenic activities. Nitrations are important reactions for the large-scale production of aminoaromatic structures that are synthons for pesticides, dyes, polymers, and pharmaceuticals. Nitroaromatic compounds such as nitrobenzene are used as solvents, whereas polynitroaromatic compounds serve as explosives. According to Hartter (16) 2,4,6-trinitrotoluene (TNT) is produced in amounts of 2 million pounds per year. Nitroaromatic compounds are therefore abundantly present in industrial waste streams and surface waters. 2,4,6-Trinitrotoluene is commonly found as the main contaminant of soil and ground water originating from facilities for manufacturing, processing, and disposing of explosives. Often these contaminants have leached from disposal lagoons into the surrounding soil, and in the case of military burdens of World War I and II, have contaminated the groundwater (13). Consequently, in Germany large areas of highly contaminated soils at former production plants must be remediated. TNT, its metabolites, and related compounds represent an environmental hazard because they exhibit considerable toxicity to humans, fish, algae, and microorganisms (39, 43, 50). Since incineration, the only proven technology for the destruction of explosives, is prohibitively costly, bioremediation represents an important alternative approach, which deserves to be considered.

Xenobiotics in the environment: present and future strategies to obviate the problem of biological persistence.

Sustainable chemistry aims at an improved efficiency of using natural resources which are used to meet human needs for chemical products. Chemists in science and industry, have become aware of the importance to design environmentally benign chemicals. One aspect is the biological persistence and the present paper reviews work in this field focussing on the degradation of xenobiotics in the environment. Different structural reasons for chemical and biological persistence are described and strategies to use single bacterial isolates or microbial communities for the elimination of xenobiotic pollutants in the environment are summarized. Perspectives and limitations to evolve and use this catabolic potential are critically discussed with respect to the complexity of mixtures of xenobiotics often found in practice. An interdisciplinary approach for the prospective design of environmentally benign substances is presented and examples for new commodity chemicals that better fit the naturally existing catabolic potential are included.

Anaerobic Transformation of 2,4,6-Trinitrotoluene and Other Nitroaromatic Compounds

Considerations about transformation of nitroaromatic compounds in soil must include anaerobic reactions, because “the soil” is a heterogenous medium that consists of microhabitats of different oxygen partial pressures. Anaerobic conditions are created by bacteria that consume oxygen during degradation of soil organic matter and respiration. As a consequence, aerobic and anaerobic habitats coexist in or around soil particles within a very small space (Fig. 1). Therefore, it is important to pay attention to transformation reactions occurring under anaerobic conditions. There have been many publications on the reductive transformation of nitroaromatic compounds by strictly anaerobic bacteria. Table 1 indicates the variety of strictly anaerobic bacteria that carry out nitro reduction and the nitro compounds that can be transformed by these organisms.

Biodegradation of All Stereoisomers of the EDTA Substitute Iminodisuccinate by Agrobacterium tumefaciens BY6 Requires an Epimerase and a Stereoselective C-N Lyase

ABSTRACT Biodegradation tests according to Organization for Economic Cooperation and Development standard 301F (manometric respirometry test) with technical iminodisuccinate (IDS) revealed ready biodegradability for all stereoisomers of IDS. The IDS-degrading strain Agrobacterium tumefaciens BY6 was isolated from activated sludge. The strain was able to grow on each IDS isomer as well as on Fe2+-, Mg2+-, and Ca2+-IDS complexes as the sole carbon, nitrogen, and energy source. In contrast, biodegradation of and growth on Mn2+-IDS were rather scant and very slow on Cu2+-IDS. Growth and turnover experiments with A. tumefaciens BY6 indicated that the isomer R,S-IDS is the preferred substrate. The IDS-degrading enzyme system isolated from this organism consists of an IDS-epimerase and a C-N lyase. The C-N lyase is stereospecific for the cleavage of R,S-IDS, generating d-aspartic acid and fumaric acid. The decisive enzyme for S,S-IDS and R,R-IDS degradation is the epimerase. It transforms S,S-IDS and R,R-IDS into R,S-IDS. Both enzymes do not require any cofactors. The two enzymes were purified and characterized, and the N-termini were sequenced. The purified lyase and also the epimerase catalyzed the transformation of alkaline earth metal-IDS complexes, while heavy metal-IDS complexes were transformed rather slowly or not at all. The observed mechanism for the complete mineralization of all IDS isomers involving an epimerase offers an interesting possibility of funneling all stereoisomers into a catabolic pathway initiated by a stereoselective lyase.

A stereoselective carbon-nitrogen lyase from Ralstonia sp. SLRS7 cleaves two of three isomers of iminodisuccinate.

Following biodegradation tests according to the OECD guidelines for testing of chemicals 301F different degradation rates were observed for the three stereoisomers of iminodisuccinate (IDS). A strain was isolated from activated sludge, which used two of three isomers, R,S-IDS and S,S-IDS, as sole source of carbon, nitrogen, and energy. The isolated strain was identified by 16S-rDNA and referred to as Ralstonia sp. SLRS7. An IDS-degrading lyase was isolated from the cell-free extract. The enzyme was purified by three chromatographic steps, which included anion-exchange chromatography, hydrophobic interaction chromatography and gel filtration. The lyase catalysed the non-hydrolytic cleavage of IDS without requirement of any cofactors. Cleavage of S,S-IDS led to the formation of fumaric acid and L-aspartic acid. Interestingly R,S-IDS yielded only D-aspartic acid besides fumaric acid. R,R-IDS was not transformed. Thus, the IDS-degrading enzyme is a carbon–nitrogen lyase attacking only the asymmetric carbon atom exhibiting the S-configuration. Besides S,S-IDS and R,S-IDS cleavage, the lyase catalysed also the transformation of certain S,S-IDS metal complexes, namely Ca2+-, Mg2+- and Mn2+-IDS. The maximum enzyme activity was found at pH 8.0–8.5 and 35 °C. SDS-PAGE analysis revealed a single 57-kDa protein band. The native enzyme was estimated to be around 240 kDa indicating a homotetramer enzyme.

Sequencing and Heterologous Expression of an Epimerase and Two Lyases from Iminodisuccinate-Degrading Bacteria

ABSTRACT Recently, degradation of all existing epimers of the complexing agent iminodisuccinate (IDS) in the bacterial strain Agrobacterium tumefaciens BY6 was proven to depend on an epimerase and a C-N lyase (Cokesa et al., Appl. Environ. Microbiol. 70:3941-3947, 2004). In the bacterial strain Ralstonia sp. strain SLRS7, a corresponding C-N lyase is responsible for the initial degradation step (Cokesa et al., Biodegradation 15:229-239, 2004). The ite gene, encoding the IDS-transforming epimerase, and the genes iclB and iclS, encoding the IDS-converting BY6-lyase and SLRS7-lyase, respectively, were cloned and sequenced. The epimerase gene encodes a protein with a predicted subunit molecular mass of 47.6 kDa. The highest degree of epimerase amino acid sequence identities was found with proteins of unknown function, indicating a novel protein. For the lyases, the deduced amino acid sequences show high similarity to enzymes of the fumarase II family. A classification into a new subfamily within the enzyme family is proposed. The subunit molecular masses of the lyases were calculated to be 54.4 and 54.7 kDa, respectively. In Agrobacterium tumefaciens BY6, the ite gene was on an approximately 180-kb circular plasmid, whereas the iclB gene was chromosomal like the corresponding iclS gene in Ralstonia sp. strain SLRS7. Heterologous expression in Escherichia coli and subsequent purification revealed recombinant enzymes with in vitro activity similar to that of the corresponding enzymes from the wild-type strains.

Purification, crystallization and preliminary crystallographic study of an IDS-epimerase from Agrobacterium tumefaciens BY6.

The initial degradation of all stereoisomers of the complexing agent iminodisuccinate (IDS) is enabled by an epimerase in the bacterial strain Agrobacterium tumefaciens BY6. This protein was produced in Escherichia coli, purified and crystallized by the hanging-drop vapour-diffusion method. Crystals of IDS-epimerase were obtained under several conditions. The best diffracting crystals were grown in 22% PEG 3350, 0.2 M (NH4)2SO4 and 0.1 M bis-Tris propane pH 7.2 at 293 K. These crystals belong to the monoclinic space group P2(1), with unit-cell parameters a = 55.4, b = 104.2, c = 78.6 angstroms, beta = 103.3 degrees, and diffracted to 1.7 angstroms resolution. They contain two protein molecules per asymmetric unit. In order to solve the structure using the MAD phasing method, crystals of the L-selenomethionine-substituted epimerase were grown in the presence of 20% PEG 3350, 0.2 M Na2SO4 and 0.1 M bis-Tris propane pH 8.5.