Gene cloning and nucleotide sequencing and properties of a cocaine esterase from Rhodococcus sp strain MB1

A strain of Rhodococcus designated MB1, which was capable of utilizing coca ine as a sole source of carbon and nitrogen for growth, was isolated from r hizosphere soil of the tropane alkaloid-producing plant Erythroxylum coca, A cocaine esterase was found to initiate degradation of cocaine, which was hydrolyzed to ecgonine methyl eater and benzoate; both of these esterolytic products were further metabolized by Rhodococcus sp. strain MB1, The struc tural gene encoding a cocaine esterase, designated cocE, was cloned from Rh odococcus sp, strain MB1 genomic libraries by screening recombinant strains of Rhodococcus erythropolis CW25 for growth on cocaine. The nucleotide seq uence of cocE corresponded to an open reading frame of 1,724 bp that codes for a protein of 574 amino acids. The amino acid sequence of cocaine estera se has a region of similarity with the active serine consensus of X-prolyl dipeptidyl aminopeptidases, suggesting that the cocaine esterase is a serin e esterase. The cocE coding sequence was subcloned into the pCFX1 expressio n plasmid and expressed in Escherichia coli, The recombinant cocaine estera se was purified to apparent homogeneity and was found to be monomeric, with an M-r of approximately 65,000, The apparent K-m of the enzyme (mean +/- s tandard deviation) for cocaine was measured as 1.33 +/- 0.085 mM, These fin dings are of potential use in the development of a linked assay for the det ection of illicit cocaine.