Gene cloning and nucleotide sequencing and properties of a cocaine esterase from Rhodococcus sp strain MB1
Gene cloning and nucleotide sequencing and properties of a cocaine esterase from Rhodococcus sp strain MB1
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DOI：
10.1128/aem.66.3.904-908.2000
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发表时间：
2000-03-01
影响因子：
4.4
通讯作者：
Bruce, NC
中科院分区：
文献类型：
--
作者：
Bresler, MM;Rosser, SJ;Bruce, NC
从产生托烷生物碱的植物古柯古柯的根际土壤中分离出一种红球菌菌株，命名为 MB1，它能够利用可卡因作为生长的唯一碳源和氮源。发现可卡因酯酶启动可卡因的降解，可卡因被水解为芽子碱甲酯和苯甲酸酯；这两种酯解产物均被红球菌进一步代谢。菌株MB1。通过筛选在可卡因上生长的红平红球菌 CW25 重组菌株，从红球菌菌株 MB1 基因组文库中克隆了编码可卡因酯酶的结构基因（命名为 cocE）。 cocE的核苷酸序列对应于1,724 bp的开放阅读框，编码574个氨基酸的蛋白质。可卡因酯酶的氨基酸序列与X-脯氨酰二肽基氨基肽酶的活性丝氨酸共有区域具有相似性，表明可卡因酯酶是丝氨酸酯酶。将cocE编码序列亚克隆到pCFX1表达质粒中并在大肠杆菌中表达。重组可卡因酯酶被纯化至表观同质性，并被发现是单体，M-r 约为 65,000。可卡因酶的表观 K-m（平均值 +/- 标准偏差）经测量为 1.33 +/- 0.085 mM。这些发现对于开发检测非法可卡因的关联测定具有潜在的用途。
A strain of Rhodococcus 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 plant Erythroxylum 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 by Rhodococcus sp. strain MB1. The structural gene encoding a cocaine esterase, designated cocE, was cloned from Rhodococcus sp, strain MB1 genomic libraries by screening recombinant strains of Rhodococcus erythropolis CW25 for growth on cocaine. The nucleotide sequence 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 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. The cocE coding sequence was subcloned into the pCFX1 expression plasmid and expressed in Escherichia coli. The recombinant cocaine esterase 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 +/- 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.