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Abstract

Castellaniella defragrans 65Phen mineralizes bicyclic monoterpenes with nitrate as electron acceptor. In previous studies 6 genes presumably involved in the monoterpene metabolism were identified by means of transposon mutagenesis. These genes were PCR amplified and five of them were cloned into the overexpression vector pET42a(+). To facilitate their insertion in the correct orientation specific restriction sites were included at the 5’-ends of the primers. After transformation into E. coli BL21 (DE3), the expression of the corresponding proteins was induced. Four proteins (Enol-CoA hydratase, 2-hydroxy-4-isopropenyl-cyclohexan-1-carboxyl-CoA dehydrogenase, 2-methylisocitrate lyase and Cystathionine beta-lyase) could be expressed in soluble form. Biomass from the induced cultures was tested for enzyme activity on the bicyclic monoterpenes α-pinene and sabinene and the monocyclic monoterpene limonene. Under the conditions used, no enzyme activity could be detected. Therefore, optimization for the enzyme activity assays, as well as for the cloning and expression of the remaining genes, were suggested.