The enzymatic conversion of alpha-pinene to verbenols, verbenone, and minor volatile flavors was studied using submerged cultured cells, lyophilisate, and microsomal fractions of the edible basidiomycete Pleurotus sapidus . The similarity of the product range obtained by the bioconversions with the range of products found after autoxidation of alpha-pinene at 100 degrees C suggested similar initial pinene radicals. Extracts of the bioconversions were analyzed using thin layer chromatography with hydroperoxide staining and cool on-column capillary gas chromatography-mass spectrometry. Two isomer alpha-pinene hydroperoxides were identified as the key intermediates and their structures confirmed by comparison with synthesized reference samples and by microchemical reduction to (Z)- and (E)-verbenol. When the biocatalysts were supplemented with one of the verbenols, only the (Z)-isomer was oxidized, indicating the activity of a highly stereospecific monoterpenol dehydrogenase. The structural comparison of subunits shows that fungal oxifunctionalization reactions of some common terpene substrates, such as (+)-limonene or (+)-valencene, might likewise be catalyzed by dioxygenases rather than by CYP450 enzymes, as previously assumed.

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HRGC-MS, using split/splitless injection (230°C), showed that a dioxygenase from Pleurotus sapidus regio-selectively transformed (+)-car-3-ene to car-3-en-5-one as the major volatile product to minor amounts of the corresponding alcohol, and to some other volatiles. Thus, the reaction was assumed to be radical mediated and similar to the lipoxygenase catalyzed peroxidation of polyunsaturated fatty acids, but the expected car-3-ene-hydroperoxides were not detected. TLC of the reaction products, followed by hydroperoxide specific staining, visually indicated the presence of hydroperoxides. TLC spots were eluted and re-analyzed using cool on-column injection, but only tailing peaks showing a mixed mass spectrum of car-3-en-5-ol/one were obtained. An unequivocal identification of car-3-en-5-hydroperoxides was achieved only after using APCI +-LC-MS. Upon structural confirmation, the car-3-en-5-hydroperoxide was accumulated by preparative HPLC, re-injected cool on-column, and the continuing degradation of the hydroperoxide to monoterpene ketone and alcohol during chromatography was verified. It was concluded that terpene hydroperoxides may occur in essential oils more frequently than anticipated, but are not recognized due to the principal blindness of capillary gas chromatography techniques and UV/vis LC-detectors.