Regio- and stereoselective allylic hydroxylation of D-limonene to (+)-trans-carveol with cellulosimicrobium cellulans EB-8-4

Abstract

Cellulosimicrobium cellulans EB-8-4 was discovered by screening of microorganisms as a pow-erful catalyst for the regio- and stereoselective allylic hydroxylation of D-limonene to (+)-trans-carveol that is a useful and valuable fragrance and flavour compound. Cells of strain EB-8-4 were easily obtained, demonstrated more than 99% regio- and stereoselectivity, showed a specific hydroxylation activity of 4.0 U/g cdw (cell dry weight), and accepted 62 mM D-limonene without inhibition. The hydroxylation was possibly catalyzed by an nicotinamide adenine dinucleotide (NADH)-dependent oxygenase involved in the degradation of aromatic ring during cell growth. 13.4 mM of (+)-trans-carveol were obtained by biohydroxylation of D-limonene with resting cells of C. cellulans EB-8-4, thus being 11 times higher than that obtained with the best biocatalyst known thus far. High conversion and high yield were obtained in the biohydroxylation of 11.6 mM of D-limonene with the resting cells as catalyst in a closed shaking flask, giving 10 mM of (+)-irans-carveol, and 0.30 mM of carvone as the only by-product. Thus, a unique biocatalyst for the regio- and stereoselective allylic hydroxylation of D-limonene and an efficient synthesis of natural identical (.+)-trans-carveol by biohydroxylation have been developed. © 2009 Wiley-VCH Verlag GmbH & Co. KGaA.