A monoterpene -lactone hydrolase (MLH) from Rhodococcus erythropolis DCL14, catalyzing the ring opening of lactones which are formed during degradation of several monocyclic monoterpenes, including carvone and menthol, was purified to apparent homogeneity. It is a monomeric enzyme of 31 kDa that is active with (4R)-4-isopropenyl-7-methyl-2-oxo-oxepanone and (6R)-6-isopropenyl-3-methyl-2-oxo-oxepanone, lactones derived from (4R)-dihydrocarvone, and 7-isopropyl-4-methyl-2-oxo-oxepanone, the lactone derived from menthone. Both enantiomers of 4-, 5-, 6-, and 7-methyl-2-oxo-oxepanone were converted at equal rates, suggesting that the enzyme is not stereoselective. Maximal enzyme activity was measured at pH 9.5 and 30°C. Determination of the N-terminal amino acid sequence of purified MLH enabled cloning of the corresponding gene by a combination of PCR and colony screening. The gene, designated mlhB (monoterpene lactone hydrolysis), showed up to 43% similarity to members of the GDXG family of lipolytic enzymes. Sequencing of the adjacent regions revealed two other open reading frames, one encoding a protein with similarity to the short-chain dehydrogenase reductase family and the second encoding a protein with similarity to acyl coenzyme A dehydrogenases. Both enzymes are possibly also involved in the monoterpene degradation pathways of this microorganism.Lactones, internal cyclic monoesters, are ubiquitous in nature and have been identified in all major classes of foods, including fruits, vegetables, nuts, meat, milk products, and baked products, contributing to taste and flavor nuances (13). The organoleptically important lactones generally have ␥-or ␦-lactone structures (five-or six-membered ring structures), while a few are macrocyclic (29). Lactones are intermediates in microbial degradation pathways of alicyclic compounds (1, 5, 32) but have also been implicated in certain microbial aromatic degradation pathways (ortho cleavage pathways) (14,19). The degradation of lactones is accomplished by the activity of lactone hydrolase, which catalyzes the hydrolysis of lactones to the corresponding hydroxy acids (Fig. 1). Lactone hydrolases are commercially applied for the debittering of triterpenes present in citrus juices (15) and for the production of the vitamin D-pantothenate (T. Morikawa, K. Wada, S. Kita, K. Tuzaki, K. Sakamoto, M. Kataoka, S. Shimizu, and H. Yamada, Book Abstr. 6th Japanese-Swiss Meet. Bio/Technol., p. [34][35] 1998).The application of Baeyer-Villiger monooxygenases (BVMOs) seems an attractive option for the production of lactones (12, 31). BVMOs are enzymes catalyzing the insertion of one atom of oxygen next to an alicyclic keto group, thus forming lactones. However, BVMOs are relatively unstable enzymes that generally use the very expensive NADPH as the cofactor (31). Therefore, the application of whole cells of BMVO activity containing microorganisms is a prerequisite to ascertain in situ cofactor regeneration and to increase the stability of the enzyme. However, most microorganisms containing...