Open AccessGene Cloning and Nucleotide Sequencing and Properties of a Cocaine Esterase fromRhodococcussp. Strain MB1
Author(s) -
Matthew M. Bresler,
Susan J. Rosser,
Amrik Basran,
Neil C. Bruce
Publication year - 2000
Publication title -
applied and environmental microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.552
H-Index - 324
eISSN - 1070-6291
pISSN - 0099-2240
DOI - 10.1128/aem.66.3.904-908.2000
Subject(s) - esterase , biochemistry , open reading frame , nucleic acid sequence , microbiology and biotechnology , rhodococcus , molecular cloning , biology , serine hydrolase , recombinant dna , chemistry , serine , peptide sequence , gene , enzyme
A strain ofRhodococcus designated MB1, which was capable of utilizing cocaine as a sole source of carbon and nitrogen for growth, was isolated from rhizosphere soil of the tropane alkaloid-producing plantErythroxylum coca . A cocaine esterase was found to initiate degradation of cocaine, which was hydrolyzed to ecgonine methyl ester and benzoate; both of these esterolytic products were further metabolized byRhodococcus sp. strain MB1. The structural gene encoding a cocaine esterase, designatedcocE , was cloned fromRhodococcus sp. strain MB1 genomic libraries by screening recombinant strains ofRhodococcus erythropolis CW25 for growth on cocaine. The nucleotide sequence ofcocE 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 esterase has a region of similarity with the active serine consensus of X-prolyl dipeptidyl aminopeptidases, suggesting that the cocaine esterase is a serine esterase. ThecocE coding sequence was subcloned into the pCFX1 expression plasmid and expressed inEscherichia coli . The recombinant cocaine esterase was purified to apparent homogeneity and was found to be monomeric, with anM r of approximately 65,000. The apparentKm of the enzyme (mean ± standard deviation) for cocaine was measured as 1.33 ± 0.085 mM. These findings are of potential use in the development of a linked assay for the detection of illicit cocaine.