Methane → (+)-Limonene

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Product analysis and other analytical methods—An in situ bioassay was developed to evaluate functional expression of (−)-limonene hydroxylase activity. Expression cultures were incubated in the presence of ˜300 μM (−)-(4S)-limonene, which was added to the culture medium immediately following infection. At zero and various time intervals, 50-100 ml culture samples were removed and cells were harvested by centrifugation, washed, and resuspended in 3-6 ml of sodium phosphate buffer as described above. Resuspended cell suspensions were chilled on ice and extracted twice with 3 ml portions of ice cold ether after the addition of 25 nmol camphor as internal standard. The extract was decolorized with activated charcoal, backwashed with water, and the organic phase containing the products was passed through a short column of anhydrous MgSO4 and activated silica. The purified extracts were then concentrated to ˜500 μl under N2 and analyzed by capillary GLC (Hewlett-Packard 5890). GLC was performed on 0.25 mm i.d×30 m of fused silica capillary columns coated with superox FA or AT-1000 using “on column” injection and flame ionization detection with H2 as carrier gas at 13.5 psi (programmed from 45° C. (5 min) to 220° C. at 10° C. per min). The identities of the products, (−)-trans-carveol from C-6 hydroxylation and (−)-trans-isopiperitenol from C-3 hydroxlyation, were confirmed by coincidence of retention times with the corresponding authentic standard. Peak quantitation was by electronic integration based on the internal standard.

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