Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Patent
1994-01-26
1995-10-31
Naff, David M.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
4352533, 435874, 435 19, C12N 916, C12N 120, C12Q 144
Patent
active
054628687
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
This invention relates to a new enzyme isolated from a microorganism, the microorganism that produces this enzyme, the use of this enzyme in catalysing the degradation of cocaine and a method and apparatus for the detection of cocaine using this enzyme.
DESCRIPTION OF THE PRIOR ART
There is an urgent need for a better method of detection of cocaine in particulate form and in body fluids. In relation to particulate cocaine, although many different analytical systems have been proposed, most are based on large pieces of equipment such as mass spectrometry, and require specially trained laboratory technicians. Such systems include thin layer chromatography, gas chromatography and high pressure liquid chromatography (HPLC). In relation to body fluids they require extraction of the sample to remove interfering compounds. Labelled assays have been used but these also require specialist skills to carry out. Some of the above or other prior methods, e.g. mass spectrometry, also require bulky and expensive equipment.
Portable detecting devices for cocaine have recently been developed. Thus, Einceman et al. (Anal. Chem. (1990) 62 1374-1379) have developed a portable ion mobility spectrometer, but it relies heavily on the volatility of the drug. A similar drawback exists with the piezoelectric crystal coated detecting device of Ngeh-Ngwainbi et al. (Blosensors & Biomechanics (1990) 5 13-26). A potentiometric sensor has been developed by K. Vytras et al., (Mikrochlmica Acta (1984) [Wein] III 139-148) but this is very unspecific as it forms a complex with the tertiary amine group present in many illicit drugs.
SUMMARY OF THE INVENTION
We have now found an enzyme which can be used in the detection of cocaine. The enzyme is a cocaine esterase (hereinafter CE) which catalyses the debenzoylation of cocaine that is to say its hydrolysis to produce ecgonine methyl ester and benzoic acid. This enzyme could thus be used to detect cocaine, by reacting the enzyme with cocaine and detecting the occurrence of the enzyme-catalysed reaction. One such method of detection would be conductimetric, as the CE reaction will bring about a change in conductance of the solution. Alternatively, the method of detection could be potentiometric, the CE reaction bringing about a change in the pH of the solution. Other methods could include optical, color,metric, thermometric or amperometric detection of the reaction products, such methods being well known in the art. Accordingly the Invention includes sensors, especially of the conductrimetric or potentiometric type for cocaine esterase. These and similar sensors can be used as the basis for convenient portable sensors for detecting cocaine in body fluids, luggage, clothing etc. of smugglers, traffickers and cocaine users. Accordingly the invention provides an important advance in the fight against and control of use of drugs.
The term "cocaine" used throughout the specification comprises the free base and salts thereof, unless the context requires a more specific meaning.
In a first aspect the invention provides the cocaine esterase enzyme. The cocaine esterase enzyme catalyses the debenzoylation of cocaine into ecgonine methyl ester and benzoic acid; thus it attacks the benzoate ester linkage of cocaine. There is little or no activity at the methyl ester linkage. Thus, ability to specifically attack the benzoate ester linkage of cocaine is a distinctive feature of the cocaine esterase of the invention, that is not possessed by commercially available esterases, e.g. porcine liver esterase, horse serum butyryl-cholinesterase or other esterases such as microbial atropinesterase.
Another distinctive feature of the CE is that it has a native molecular weight in its unaggregated form of about 120,000 Daltons and when aggregated of about 420,000 Daltons (both as determined by elution from a gel filtration column calibrated with protein markers). By the term "about" we mean to encompass variations which are usual in the determination of high molecular weights by this
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Britt Adrian J.
Bruce Neil C.
Lowe Christopher R.
British Technology Group Limited
Meller Mike
Naff David M.
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