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来自红球菌属MB1菌株的可卡因酯酶的基因克隆、核苷酸测序及特性

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

作者信息

Bresler M M, Rosser S J, Basran A, Bruce N C

机构信息

Institute of Biotechnology, University of Cambridge, Cambridge CB2 1QT, United Kingdom.

出版信息

Appl Environ Microbiol. 2000 Mar;66(3):904-8. doi: 10.1128/AEM.66.3.904-908.2000.

Abstract

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.

摘要

从生产托烷生物碱的植物古柯(Erythroxylum coca)的根际土壤中分离出了一株红球菌(Rhodococcus),命名为MB1,它能够利用可卡因作为唯一的碳源和氮源进行生长。发现一种可卡因酯酶可启动可卡因的降解,可卡因被水解为芽子碱甲酯和苯甲酸盐;这两种酯解产物均被红球菌属菌株MB1进一步代谢。通过筛选在可卡因上生长的红平红球菌(Rhodococcus erythropolis)CW25重组菌株,从红球菌属菌株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一个1724 bp的开放阅读框,编码一个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序列有相似区域,表明可卡因酯酶是一种丝氨酸酯酶。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均一,发现其为单体,相对分子质量约为65,000。该酶对可卡因的表观米氏常数(平均值±标准差)测定为1.33±0.085 mM。这些发现可能有助于开发一种用于检测非法可卡因的联合检测方法。

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