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The solvent-tolerant bacterium Pseudomonas putida S12, which adapts its membrane lipids to the presence of toxic solvents by a cis to trans isomerization of unsaturated fatty acids, was used to study possible in vivo regiospecificity of the isomerase. Cells were supplemented with linoleic acid (C18:2delta9-cis,delta12-cis), a fatty acid that cannot be synthesized by this bacterium, but which was incorporated into membrane lipids up to an amount of 15% of total fatty acids. After addition of 1-octanol, which was used as an activator of the cis-trans isomerase, the linoleic acid was converted into the delta9-trans,delta12-cis isomer, while the delta9-cis,delta12-trans and delta9-trans,epsilon12-trans isomers were not synthesized. Thus, for the first time, regiospecific in vivo formation of novel, mixed cis/trans isomers of dienoic fatty acid chains was observed. The maximal conversion (27-36% of the chains) was obtained at 0.03-0.04% (v/v) octanol, after 2 h. The observed regiospecificity of the enzyme, which is located in the periplasmic space, could be due to penetration of the enzyme to a specific depth in the membrane as well as to specific molecular recognition of the substrate molecules.

The active efflux system contributing to the solvent tolerance of Pseudomonas putida S12 was characterized physiologically. The mutant P. putida JK1, which lacks the active efflux system, was compared with the wild-type organism. None of 20 known substrates of common multi-drug-resistant pumps had a stronger growth-inhibiting effect on the mutant than on the wild type. The amount of [14C]toluene accumulating in P. putida S12 increased in the presence of the solvent xylene and in the presence of uncouplers. The effect of uncouplers confirms the proton dependency of the efflux system in P. putida S12. Other compounds, potential substrates for the solvent pump, did not affect the accumulation of [14C]toluene. These results show that the efflux system in P. putida S12 is specific for organic solvents and does not export antibiotics or other known substrates of multi-drug-resistant pumps.

We cloned and characterized the epoxide hydrolase gene, EPH1, from Rhodotorula glutinis. The EPH1 open reading frame of 1230 bp was interrupted by nine introns and encoded a polypeptide of 409 amino acids with a calculated molecular mass of 46.3 kDa. The amino acid sequence was similar to that of microsomal epoxide hydrolase, which suggests that the epoxide hydrolase of R. glutinis also belongs to the alpha/beta hydrolase fold family. EPH1 cDNA was expressed in Escherichia coli and resting cells showed a specific activity of 200 nmol min(-1)(mg protein)(-1) towards 1,2-epoxyhexane.

Limonene-1,2-epoxide hydrolase (LEH) from Rhodococcus erythropolis DCL14, an enzyme involved in the limonene degradation pathway of this microlorganism, has a narrow substrate specificity. Of the compounds tested, the natural substrate, limonene-1,2-epoxide, and several alicyclic and 2-methyl-1,2-epoxides (e.g. 1-methylcyclohexene oxide and indene oxide), were substrates for the enzyme. When LEH was incubated with a diastereomeric mixture of limonene-1,2-epoxide, the sequential hydrolysis of first the (1R,2S)- and then the (1S,2R)-isomer was observed. The hydrolysis of (4R)- and (4S)-limonene-1,2-epoxide resulted in, respectively, (1S,2S,4R)- and (1R,2R,4S)-limonene-1,2-diol as the sole product with a diastereomeric excess of over 98%. With all other substrates, LEH showed moderate to low enantioselectivities (E ratios between 34 and 3).

Large-scale resolution of epoxides by the yeast Rhodotorula glutinis was demonstrated in an aqueous/organic two-phase cascade membrane bioreactor. Due to the chemical instability and low solubility of epoxides in aqueous phases, an organic solvent was introduced into the reaction mixture in order to enhance the resolution of epoxide. A cascade hollow-fiber membrane bioreactor was used (1) to minimize the toxicity of organic solvents towards the epoxide hydrolase of R. glutinis, and (2) to remove inhibitory amounts of formed diol from the yeast cell containing aqueous phase. Dodecane was selected as a suitable solvent and 1,2-epoxyhexane as a model substrate. By use of this membrane bioreactor, highly concentrated (0.9 M in dodecane) enantiopure (> 98% ee) (S)-1,2-epoxyhexane (6.5 g, 30% yield) was obtained from the racemic mixture.

The exopolysaccharides produced by Lactobacillus delbrueckii subsp. bulgaricus NCFB 2772 grown in defined medium were investigated. At equal cell densities, the strain produced 95 mg l−1 exopolysaccharides with glucose and 30 mg l−1 with fructose as the carbohydrate source. High-performance size-exclusion chromatography of the exopolysaccharides produced on glucose showed the presence of two fractions with relative molecular masses (M r) of 1.7 × 106 and 4 × 104 in almost equal amounts. The exopolysaccharides produced on fructose contained mainly a fraction of low M r of 4 × 104. The high-M r fraction of the purified exopolysaccharides produced on glucose appeared to have a sugar composition of galactose, glucose and rhamnose in the molar ratio of 5:1:1, whereas the low-M r weight fraction contained galactose, glucose and rhamnose in the molar ratio of approximately 11:1:0.4. The purified exopolysaccharide fractions produced on fructose showed comparable ratios. The high-molecular-mass fractions contained terminally linked galactose, 1,2,3-linked galactose, 1,3,4-linked galactose, 1,3-linked glucose and terminally linked rhamnose. The low-molecular-mass fractions contained mainly 1,3-linked galactose and 1,6-linked galactose and lower amounts of other sugar linkages. The production of the high-M r fractions appeared to be dependent on the carbohydrate source, whereas the low-M r fractions were produced more continuously.

The influence of calcium alginate entrapment on the physiology of Mycobacterium sp. E3 is reported. As a model system the NADH-requiring conversion of propene to 1,2-epoxypropane in the presence and absence of glucose as co-substrate was selected. The co-factor-dependent reaction was used as a measure of the physiological status of the resting cells. Initial kinetic experiments established a system free from diffusional limitations. In the presence of glucose there were no differences between the physiology of the free and immobilized cells. The apparent differences observed in the absence of co-substrate were demonstrated to be caused by calcium ions and to a lesser degree alginate; the addition of calcium, alginate or calcium alginate beads containing no cells to the free cells gave similar data to that obtained with immobilized cells. The results presented highlight the high concentrations of calcium to which cells immobilized in calcium alginate beads can be exposed.

A number of bacteria and yeast was screened for asymmetric reduction of prochiral chloroacetone into chiral 1-chloro-2-propanol, which is chemically convertible into chiral 1,2-epoxypropane. In this way Rhodotorula glutinis produced optically pure S-1,2-epoxypropane with 98% enantiomeric excess and in a relatively high final concentration. The enzyme that catalysed the asymmetric reduction was an NAD(P)H-dependent alcohol dehydrogenase. Reduction of racemic 3-chloro-2-butanone resulted in mixtures of cis and trans-2,3-epoxybutane, indicating that no enantioselective reduction of this haloketone occurred.

Both stereoisomers of 3-chloro-2-methylpropionic acid (CMPA) and its methyl esters (MeCMPA) serve as growth substrates for a bacterial isolate (Xanthobacter sp. CIMW 99) when supplied as sole source of carbon and energy. Biodegradation of DL-CMPA and DL-MeCMPA was shown to be via a common pathway; an initial, constitutive, esterase converted the methyl ester to the corresponding carboxylic acid. Further metabolism required the activation of CMPA involving a CoA-, ATP-, Mg(2+)-dependent chloroacyl-CoA synthetase. Most noteworthy, it was the product of this reaction (3-chloro-2-methylpropionyl-CoA) that underwent hydrolytic dehalogenation to give 3-hydroxy-2-methylpropionyl-CoA (3-hydroxyisobutyryl-CoA). Further biodegradation proceeded by the action of a dehydrogenase on the CoA derivative to give methylmalonate-CoA-semialdehyde. Cells of CIMW 99 also contained a stable, constitutive, highly active 3-hydroxyisobutyrate dehydrogenase that was specific for the L(+) isomer. However, evidence is presented suggesting that this enzyme was not involved in the catabolism of the chlorinated substrates.

Several bacteria, yeast and fungi selectively isolated from paper-mill waste-water grew on veratryl alcohol, a key intermediate of lignin metabolism. Penicillium simplicissimum oxidized veratryl alcohol via a NAD(P)+-dependent veratryl alcohol dehydrogenase to veratraldehyde, which was further oxidized to veratric acid in a NAD(P)+-dependent reaction. Veratric-acid-grown cells contained NAD(P)H-dependent O-demethylase activity for veratrate, vanillate and isovanillate. Protocatechuate was cleaved by a protocatechuate 3,4-dioxygenase.

A two-phase membrane bioreactor was developed to continuously produce enantiopure epoxides using the epoxide hydrolase activity of Rhodotorula glutinis. An aqueous/organic cascade, hydrophilic, hollow-fiber membrane bioreactor was used: (1) to carry out large-scale resolution of epoxides, (2) to continuously extract residual enantiopure epoxides from the aqueous phase, and (3) to separate inhibitory formed diol from the yeast cells contained in the aqueous phase. Dodecane was employed to dissolve-feed epoxide as well as to extract residual epoxide. 1,2-Epoxyhexane was used as a model substrate. By use of this membrane bioreactor, enantiopure (S)-1,2-epoxyhexane (98% enantiomeric excess) was obtained with a volumetric productivity of 3.8 g l(-1) h(-1). The continuous-production system was operated for 12 days and resulted in 38 g enantiopure (S)-1,2-epoxyhexane.

Nitroaromatic compounds are abundantly present in nature, but are in most cases highly toxic to living organisms. Several microorganisms, however, are capable of mineralizing or converting these compounds. Until now four pathways for the complete degradation of nitroaromatics have been described, which start with either the oxygenolytic or reductive removal of the nitro group from the aromatic ring or with this removal by means of replacement reactions. Besides these conversions many organisms are able to reduce nitroaromatics. The degradation of nitroaromatic compounds does not only occur in pure cultures but also in situ, for example in soil, water and sewage. However, several problems are associated with the application of microorganisms in the bioremediation of contaminated sites, as nitroaromatics or their conversion products may chemically interact with soil particles and cells. Besides the possibilities of applying microorganisms in the cleaning of sites contaminated with nitroaromatics, the use of microorganisms or enzymes in the biocatalytic production of industrially valuable products from nitroaromatics is also discussed.

Newly isolated and already available strains of alkene-utilizing bacteria were able to oxidize ethene, propene or 1-butene to the respective 1,2-epoxides. Resting-cell suspensions of organisms isolated on propene and butene, when grown on these substrates converted ethene quantitatively to epoxyethane. Some, but not all ethene-utilizing strains accumulated 1,2-epoxypropane or 1,2-epoxybutane when propene or butene was supplied, although not quantitatively because the epoxides produced were partially further metabolized. Suitable epoxide producers which eventually may be employed as biocatalysts in a biotechnological process were used for immobilization in calcium alginate and K-carrageenan; after immobilization, 60%–100% activity for epoxide production was retained.

A continuous culture study was made of the energetics of oxidation of various aldose sugars by Acinetobacter calcoaceticus LMD 79.41. The consumption of aldoses during carbon- and energy-limited growth of the organism on mixtures of acetate and an aldose was independent of the pH of the culture. Acid production, however, was strongly dependent on this parameter. It is shown that aldose consumption without concurrent acid production is due to formation of the corresponding lactone, the hydrolysis of which is pH-dependent. The cell yield of A. calcoaceticus on mixtures of acetate and glucose or xylose was much higher than during growth on acetate alone. This increase in cell yield was, however, dependent on the pH of the culture. Only at pH values which permitted a high rate of lactone hydrolysis an enhancement of the cell yield was observed. These results suggest that lactone hydrolysis has an important bearing on the efficiency of periplasmic oxidation of aldoses in bacteria.

Strain NTB-1, identified as a Alcaligenes denitrificans sp., was isolated from a mixture of soil and sewage samples using 4-chlorobenzoate as sole carbon and energy source. Simultaneous adaptation experiments and enzyme studies revealed that 4-chlorobenzoate was converted to 4-hydroxybenzoate which was further oxidized yielding 3,4-dihydroxybenzoate. Bioformation of 4-hydroxybenzoate from 4-chlorobenzoate when 4-chlorobenzoate-grown cells were incubated with 4-chlorobenzoate under conditions of low and controlled oxygen concentrations.