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Limonene-1,2-epoxide hydrolase from Rhodococcus erythropolis DCL14, an enzyme involved in the limonene metabolism of this microorganism, catalyzes the enantioselective hydrolysis of 1-methylcyclohexene oxide. (1R,2S)-1- Methylcyclohexene oxide was the preferred substrate and it was mainly hydrolyzed to (1S,2S)-1-methylcyclohexane-1,2-diol, while (1S,2R)-1- methylcyclohexene oxide was converted more slowly and mainly yielded (1R,2R)- 1-methylcyclohexane-1,2-diol. The reaction proceeded with a high regioselectivity (C1:C2, 85:15). H218O-labelling experiments confirmed that the nucleophile was mainly incorporated at the most substituted carbon atom, suggesting that limonene-1,2-epoxide hydrolase uses an acid-catalyzed enzyme mechanism.

The toxicity of fine chemicals to the producer organism is a problem in several biotechnological production processes. In several instances, an organic phase can be used to extract the toxic product from the aqueous phase during a fermentation. With the discovery of solvent-tolerant bacteria, more solvents can now be used in such two-liquid water-solvent systems. We are gaining new insights into the mechanisms of bacterial solvent tolerance, such as the active efflux of solvents from the cytoplasmic membrane and solvent-impermeable outer membranes.

Bacteria able to grow in aqueous:organic two-phase systems have evolved resistance mechanisms to the toxic effects of solvents. One such mechanism is the active efflux of solvents from the cell, preserving the integrity of the cell interior. Pseudomonas putida S12 is resistant to a wide variety of normally detrimental solvents due to the action of such an efflux pump. The genes for this solvent efflux pump were cloned from P. putida S12 and their nucleotide sequence determined. The deduced amino acid sequences encoded by the three genes involved show a striking resemblance to proteins known to be involved in proton-dependent multidrug efflux systems. Transfer of the genes for the solvent efflux pump to solvent-sensitive P. putida strains results in the acquisition of solvent resistance. This opens up the possibilities of using the solvent efflux system to construct bacterial strains capable of performing biocatalytic transformations of insoluble substrates in two-phase aqueous:organic medium.

This paper reports on the properties of methyl-3-chloro-2-methyl propionate (MeCMPA) esterase from Rhodococcus sp. ME6. This enzyme catalyzes the hydrolysis of MeCMPA to 3-chloro-2-methyl propionate (CMPA). From the experimentally derived kinetic data, an E value (ratio of specificity constants) of9 (±1) was obtained. This showed good agreement with respect to an E value predicted from the relationship between the percentage enantiomeric excess ( eeS ) and the percentage conversion ( c ). The enzyme showed good stability, with a half-life of about 40 h at 20°C. The pH and temperature optima were 8.0 and 50°C, respectively. Using a crude preparation of this enzyme it was possible to resolve 1 g of the racemate; after 24 h incubation the racemic mixture was resolved [ c = 47%; eeS = 59%].

Enantioselectivity in the degradation of racemic 1,2-epoxypropane in select bacteria from a culture collection and in newly isolated strains was detected in only very few cases. Strain Nocardia H8 by means of enantioselective degradation gave various epoxyalkanes in high optical purity from racemic mixtures, but yields were low. The best yield was 19% in the case of 1-chloro-2,3-epoxypropane (98% ee).

The propylene-utilizing Mycobacterium Py1 has been shown to oxidize gaseous alkenes, including ethylene, to the corresponding epoxides through an NAD (P)H-requiring monooxygenase. The organism could not metabolize ethylene oxide and as a consequence excreted this epoxide when fed with ethylene. Prolonged formation of ethylene oxide by cells immobilized in alginate gel or fixed on sand was dependent on a cosubstrate for regeneration of NAD(P)H used in the monooxygenase reaction. Experiments were performed in a gas-solid reactor to prevent accumulation of the toxic ethylene oxide in the immediate vicinity of the biocatalyst.

A Pseudomonas sp. able to grow on benzene as sole carbon and energy source was isolated from a mixture of soil and water samples. Experiments with whole cells and enzyme studies showed that benzene was metabolized via cis-1,2-dihydroxycyclohexa-3,5-diene (cis-benzeneglycol) to catechol. A mutant of this strain, which lacked a functional cis-benzeneglycol dehydrogenase, converted benzene almost quantitatively to cis-benzeneglycol. Addition of benzene to a carbon-limited chemostat in which this mutant was growing on succinic acid, resulted in an accumulation of cis-benzeneglycol in the medium. Under the conditions used cis-benzeneglycol was produced at a rate of about 1.4 10−6 mmol s−1 (mg protein)−1. In order to predict the cis-benzeneglycol concentration at various times, a mathematical model is described that fitted rather well for both benzene-transport-limited and kinetically limited production conditions. However, during prolonged continuous production under carbon-limited conditions, the mutant cells were outcompeted by revertants after about three days. To circumvent this problem, the chemostat was operated under nitrogen-limited conditions, which resulted in a very stable continuous cis-benzeneglycol production process for 10 days.

Resting cells of ethene grown Mycobacterium 2W produced 1,2-epoxypropane stereospecifically from propene as revealed by optical rotation, 1H n.m.r. using a chiral shift reagent, and also by complexation gas chromatography involving a glass capillary column coated with an optically active metal chelate. The gas-liquid chromatography method allowed the rapid screening of 11 strains with regard to stereospecific formation of 1,2-epoxypropane, 1,2-epoxybutane and 1-chloro-2,3-epoxypropane. Bacteria grown on either ethene, propene or butadiene all predominantly produced the R form of 1,2-epoxypropane from propene and 1,2-epoxybutane from 1-butene while the strains tested for 1-chloro-2,3-epoxypropane production from 3-chloro-1-propene predominantly accumulated the S enantiomer.