Enantioselective degradation of racemic epoxides is an interesting method to obtain optically pure epoxides. In this review, an overview is presented on the bioconversion of epoxides in microorganisms. Both the degradation and biosynthesis routes involving epoxides are discussed as well as their usefulness in enantioselective degradation.

We investigated the effect of carnitine analogues on the physiology of Lactobacillus plantarum subjected to salt stress. Salt stressed cells of L. plantarum accumulated exogenously provided carnitine and its structural analogues acetylcarnitine and propionylcarnitine to maximum concentrations of 466, 122 and 75 μmol (g dry weight of cells)−1, respectively. Addition of these carnitines to osmotically stressed medium increased growth rate. Furthermore, the intracellular amino acid pool, consisting of mainly aspartate and glutamate, was reduced when carnitine, acetylcarnitine or propionylcarnitine were included in the medium. This is the first study demonstrating a role for β-substituted acylcarnitine esters in osmoadaptation of a lactic acid bacterium.

Aryl metabolites are known to have an important role in the ligninolytic system of white rot fungi. The addition of known precursors and aromatic acids representing lignin degradation products stimulated the production of aryl metabolites (veratryl alcohol, veratraldehyde, p-anisaldehyde, and 3-chloro-p-anisaldehyde) in the white rot fungus Bjerkandera sp. strain BOS55. The presence of manganese (Mn) is known to inhibit the biosynthesis of veratryl alcohol (T. Mester, E. de Jong, and J.A. Field, Appl. Environ. Microbiol. 61:1881-1887, 1995). A new finding of this study was that the production of the other aryl metabolites, p-anisaldehyde and 3-chloro-p-anisaldehyde, was also inhibited by Mn. We attempted to bypass the Mn-inhibited step in the biosynthesis of aryl metabolites by the addition of known and suspected precursors. Most of these compounds were not able to bypass the inhibiting effect of Mn. Only the fully methylated precursors (veratrate, p-anisate, and 3-chloro-p-anisate) provided similar concentrations of aryl metabolites in the presence and absence of Mn, indicating that Mn does not influence the reduction of the benzylic acid group. The addition of deuterated benzoate and 4-hydroxybenzoate resulted in the formation of deuterated aryl metabolites, indicating that these aromatic acids entered into the biosynthetic pathway and were common intermediates to all aryl metabolites. Only deuterated chlorinated anisyl metabolites were produced when the cultures were supplemented with deuterated 3-chloro-4-hydroxybenzoate. This observation combined with the fact that 3-chloro-4-hydroxybenzoate is a natural product of Bjerkandera spp. (H. J. Swarts, F. J. M. Verhagen, J. A. Field, and J. B. P. A. Wijnberg, Phytochemistry 42:1699-1701, 1996) suggest that it is a possible intermediate in chlorinated anisyl metabolite biosynthesis.

The ability of Dutch grassland soil and Dutch peat soil for methane oxidation was investigated. The kinetics of methane oxidation by soil from different depths were determined in batch cultures incubated with 1; 10; 100; and 10,000 ppmv methane, respectively. All 4 applied concentrations of methane were degraded by both types of soil. Thereby, the highest oxidative activities were observed between 5 and 10 cm soil depth. Most importantly, these experiments demonstrated that this soil acts as a sink for methane even at concentrations well below 1 ppmv. Especially at higher methane concentrations (100 - 10,000 ppmv) much higher degradation rates were found in the peat soil. This also correlates with the higher methane production rates which had been observed in peat soil.

The effect of osmotic stress, given as decreased water activity (aw), on growth and the accumulation of potassium and the compatible solute betaine by Pseudomonas putida S12 was investigated. Reduced aw was imposed by addition of sodium chloride, sucrose, glycerol or polyethylene glycol to the growth medium. Accumulation of potassium and betaine was established when sodium chloride and sucrose were used to cause osmotic stress. No accumulation of these solutes was found in the presence of glycerol. Addition of polyethylene glycol to the medium strongly decreased the growth rate in comparison with the other osmolytes tested at the corresponding aw. Although polyethylene glycol did decrease the aw, neither potassium nor betaine was accumulated by the cells.

We investigated the mechanisms behind the organic-solvent resistance of the solvent-tolerant strain Pseudomonas putida S12. By use of 14C-labeled toluene, we obtained evidence that an energy-dependent export system may be responsible for this resistance to toluene.

The aim of this study was to identify the compatible solutes accumulated by Pseudomonas putida S12 subjected to osmotic stress. In response to reduced water activity, P. putida S12 accumulated Nalpha-acetylglutaminylglutamine amide (NAGGN) simultaneously with a novel compatible solute identified as mannitol (using 13C- and 1H-nuclear magnetic resonance, liquid chromatography-mass spectroscopy and high-performance liquid chromatography methods) to maximum concentrations of 74 and 258 micromol g (dry weight) of cells(-1), respectively. The intracellular amounts of each solute varied with both the type and amount of osmolyte applied to induce osmotic stress in the medium. Both solutes were synthesized de novo. Addition of betaine to the medium resulted in accumulation of this compound and depletion of both NAGGN and mannitol. Mannitol and NAGGN were accumulated when sucrose instead of salts was used to reduce the medium water activity. Furthermore, both compatible solutes were accumulated when glucose was substituted by other carbon sources. However, the intracellular quantities of mannitol decreased when fructose, succinate, or lactate were applied as a carbon source. Mannitol was also raised to high intracellular concentrations by other salt-stressed Pseudomonas putida strains. This is the first study demonstrating a principal role for the de novo-synthesized polyol mannitol in osmoadaptation of a heterotrophic eubacterium.

The membrane reactions of Pseudomonas putida S12 to environmental stress were investigated. Cells reacted to the addition of six different heavy metals with an increase in the ratio of trans to cis unsaturated fatty acids. A correlation among the increase in the trans/cis ratio, the toxic effects of the heavy metals, and nonspecific permeabilization of the cytoplasmic membrane, as indicated by an efflux of potassium ions, was measured. Cells previously adapted to toxic concentrations of toluene exhibited increased tolerance to all applied concentrations of zinc compared with nonadapted cells. Cells exposed to different temperatures grew optimally at 30(deg)C. The degree of saturation of the membrane fatty acids of these cells decreased with decreasing temperature. An increase in the trans/cis ratio of unsaturated fatty acids took place only at higher temperatures. Osmotic stress, expressed as reduced water activity, was obtained by using different types of solutes. Only in the presence of toxic concentrations of sodium chloride or sucrose did the trans/cis ratio increase. At no applied water activity a significant effect of glycerol on the trans/cis ratio was measured. When cells were exposed to different pHs, a distinct optimum cis/trans isomerase activity was measured at pHs between 4.0 and 5.0, whereas at higher or lower pHs no reaction occurred. This optimum coincided with a loss of viability between pH 4 and 5.

Epoxide degradation in cell extracts of Xanthobacter strain Py2 has been reported to be dependent on NAD+ and dithiols. This multicomponent system has now been fractionated. A key protein encoded by a DNA fragment complementing a Xanthobacter strain Py2 mutant unable to degrade epoxides was purified and analyzed. This NADP-dependent protein, a novel type of pyridine nucleotide-disulfide oxidoreductase, is essential for epoxide degradation. NADPH, acting as the physiological cofactor, replaced the dithiols in epoxide conversion.

The isomerization of cis to trans unsaturated fatty acid in Pseudomonas putida, a mechanism of this bacterium to adapt its membrane to toxic environmental influences, was tested as an indicator for toxicity of 10 organic compounds (aromatics and aliphatic alcohols). A direct correlation was observed between the hydrophobicity of the compounds (logP), concentration dependent growth inhibition, and the trans/cis ratio of unsaturated fatty acids. The application of this system is discussed in terms of an indicator for toxicity and environmental stress particularly during bioremediation processes.

Three Xanthobacter Py2 mutants (M3, M8 and M10) lacking epoxyalkane-degrading activity were isolated and characterized. All mutants were able to grow on acetone, the degradation product of 1,2-epoxypropane conversions. Furthermore, they contained the unidentified 'low molecular mass fraction' (LMF) necessary for epoxyalkane-degrading activity. Three cosmids from a gene bank complemented the mutation in M10 and M8 but not in mutant M3. Epoxyalkane-degrading activity in crude extracts of 1,2-epoxypropane-grown complemented mutants was similar to the wild-type activity. Surprisingly, M10 transformed with complementing cosmid pEP9 showed a constitutively expressed epoxyalkane-degrading activity, which was not observed in the wild-type strain. The cosmid pEP9 was conjugated into Xanthobacter autotrophicus GJ10, which is not able to degrade 1,2-epoxypropane. In crude extracts of X. autotrophicus GJ10(pEP9), epoxyalkane-degrading activity was demonstrated, but only after the addition of the LMF from Xanthobacter Py2. Hybridization experiments demonstrated an overlap on complementing cosmids pEP1, pEP3 and pEP9. Subcloning revealed a 4.8 kb EcoRI-HindIII fragment to be necessary for complementing the mutant M10. In the sequence of this fragment four different ORFs were found.

Propene-grown Xanthobacter sp. strain Py2 cells can degrade trichloroethylene (TCE), but the transformation capacity of such cells was limited and depended on both the TCE concentration and the biomass concentration. Toxic metabolites presumably accumulated extracellularly, because the fermentation of glucose by yeast cells was inhibited by TCE degradation products formed by strain Py2. The affinity of the propene monooxygenase for TCE was low, and this allowed strain Py2 to grow on propene in the presence of TCE. During batch growth with propene and TCE, the TCE was not degraded before most of the propene had been consumed. Continuous degradation of TCE in a chemostat culture of strain Py2 growing with propene was observed with TCE concentrations up to 206 microns in the growth medium without washout of the fermentor occurring. At this TCE concentration the specific degradation rate was 1.5 nmol/min/mg of biomass. The total amount of TCE that could be degraded during simultaneous growth on propene depended on the TCE concentration and ranged from 0.03 to 0.34g of TCE per g of biomass. The biomass yield on propene was not affected by the cometabolic degradation of TCE.

The fungus Cladosporium sphaerospermum was isolated from a biofilter used for the removal of toluene from waste gases. This is the first report describing growth of a eukaryotic organism with toluene as the sole source of carbon and energy. The oxygen consumption rates, as well as the measured enzyme activities, of toluene-grown C. sphaerospermum indicate that toluene is degraded by an initial attack on the methyl group.

Common wood-degrading fungi produce chlorinated anisyl metabolites (CAM). These compounds, which are structurally related to xenobiotic chloroaromatics, occur at high concentrations of approximately 75 mg CAM kg-1 wood or litter in the environment. In contrast to the commmonly held belief that natural haloaromatics only fullfill an antimicrobial function, we were able to demonstrate a distinct physiological role of CAM. During wood biodegradation, they are substrates for extracellular aryl-alcohol oxidases, generating H2O2 for ligninolytic enzymes.

Microbial transformations of cyclic hydrocarbons have received much attention during the past three decades. Interest in the degradation of environmental pollutants as well as in applications of microorganisms in the catalysis of chemical reactions has stimulated research in this area. The metabolic pathways of various aromatics, cycloalkanes, and terpenes in different microorganisms have been elucidated, and the genetics of several of these routes have been clarified. The toxicity of these compounds to microorganisms is very important in the microbial degradation of hydrocarbons, but not many researchers have studied the mechanism of this toxic action. In this review, we present general ideas derived from the various reports mentioning toxic effects. Most importantly, lipophilic hydrocarbons accumulate in the membrane lipid bilayer, affecting the structural and functional properties of these membranes. As a result of accumulated hydrocarbon molecules, the membrane loses its integrity, and an increase in permeability to protons and ions has been observed in several instances. Consequently, dissipation of the proton motive force and impairment of intracellular pH homeostasis occur. In addition to the effects of lipophilic compounds on the lipid part of the membrane, proteins embedded in the membrane are affected. The effects on the membrane-embedded proteins probably result to a large extent from changes in the lipid environment; however, direct effects of lipophilic compounds on membrane proteins have also been observed. Finally, the effectiveness of changes in membrane lipid composition, modification of outer membrane lipopolysaccharide, altered cell wall constituents, and active excretion systems in reducing the membrane concentrations of lipophilic compounds is discussed. Also, the adaptations (e.g., increase in lipid ordering, change in lipid/protein ratio) that compensate for the changes in membrane structure are treated.

Lactobacillus delbrueckii ssp. bulgaricus NCFB 2772 produced an extracellular polysaccharide when grown in a chemically defined medium with glucose or lactose as the substrate carbohydrate. The isolated extracellular polysaccharide had a sugar composition of glucose, galactose and rhamnose in a ratio of 1 : 6.8 : 0.7. The production of extracellular polysaccharides increased at higher temperatures, but the bacterium rapidly lost its polysaccharide producing ability at 47 degrees C. Production of polysaccharides was growth-related: no polysaccharide production was found after growth had ceased. An excess carbohydrate did not result in increased polysaccharide production.

The white rot fungus Bjerkandera sp. strain BOS55 produces veratryl, anisyl, 3-chloroanisyl, and 3,5-dichloroanisyl alcohol and the corresponding aldehydes de novo from glucose. All metabolites are produced simultaneously with the extracellular ligninolytic enzymes and have an important physiological function in the fungal ligninolytic system. Both mono- and dichlorinated anisyl alcohols are distinctly better substrates for the extracellular aryl alcohol oxidases than veratryl alcohol. The aldehydes formed are readily recycled by reduction by washed fungal mycelium, thus creating an extracellular H2O2 production system regulated by intracellular enzymes. Lignin peroxidase does not oxidize the chlorinated anisyl alcohols either in the absence or in the presence of veratryl alcohol. It was therefore concluded that the chlorinated anisyl alcohols are well protected against the fungus's own aggressive ligninolytic enzymes. The relative amounts of veratryl alcohol and the chlorinated anisyl alcohols differ significantly according to the growth conditions, indicating that production of veratryl alcohol and the production of the (chlorinated) anisyl metabolites are independently regulated. We conclude that the chlorinated anisyl metabolites biosynthesized by the white rot fungus Bjerkandera sp. strain BOS55 can be purposefully produced for ecologically significant processes such as lignin degradation.

Many processes in modern biotechnology, particularly biotransformations and environmental bioremediation, are hindered by the toxic effects of organic solvents on whole cells. These compounds dissolve in the cell membrane, disturbing its integrity and effecting specific permeabilization. The hydrophobicity of a compound, expressed as its log P value, is a good indicator of toxicity. Substances with a log P value in the range 1–5 are, in general, toxic to whole cells. However, in recent years, there have been several reports of bacteria exhibiting resistance to toxic solvents. The main adaptative reactions are alterations in the composition of the membrane, particularly changes in fatty-acid composition, phospholipid headgroups, and in the protein content. One of the key processes in the adaptation of some Pseudomonas strains, enabling them to tolerate organic solvents appears to be the isomerization of cis- into trans-unsaturated fatty acids. A greater understanding of these adaptations should eventually allow biotransformation reactions to be carried out in inhospitable two-phase systems incorporating an organic phase.

Many cyclic hydrocarbons, e.g. aromatics, cycloalkanes, and terpenes, are toxic to microorganisms. The primary site of the toxic action is probably the cytoplasmic membrane, but the mechanism of the toxicity is still poorly understood. The effects of cyclic hydrocarbons were studied in liposomes prepared from Escherichia coli phospholipids. The membrane-buffer partition coefficients of the cyclic hydrocarbons revealed that these lipophilic compounds preferentially reside in the membrane. The partition coefficients closely correlated with the partition coefficients of these compounds in a standard octanol-water system. The accumulation of hydrocarbon molecules resulted in swelling of the membrane bilayer, as assessed by the release of fluorescence self-quenching of fluorescent fatty acid and phospholipid analogs. Parallel to the expansion of the membrane, an increase in membrane fluidity was observed. These effects on the integrity of the membrane caused an increased passive flux of protons and carboxyfluorescein. In cytochrome c oxidase containing proteoliposomes, both components of the proton motive force, the pH gradient and the electrical potential, were dissipated with increasing concentrations of cyclic hydrocarbons. The dissipating effect was primarily the result of an increased permeability of the membrane for protons (ions). At higher concentrations, cytochrome c oxidase was also inactivated. The effective concentrations of the different cyclic hydrocarbons correlated with their partition coefficients between the membrane and aqueous phase. The impairment of microbial activity by the cyclic hydrocarbons most likely results from hydrophobic interaction with the membrane, which affects the functioning of the membrane and membrane-embedded proteins.