Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving oxidoreductase
Patent
1994-11-30
1996-11-05
Gitomer, Ralph J.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving oxidoreductase
435 4, 435 26, 435122, 435189, C12Q 126, C12P 1712
Patent
active
055716857
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
This invention relates to a new enzyme and to its use.
BACKGROUND OF THE INVENTION
Current synthesises of the analgesic hydromorphone (dihydromorphinone) and the antitussive hydrocodone (dihydrocodeinone) use uneconomic reagents, chemical catalysts and activating groups, and have undesirable effects on the environment. For example, hydromorphone is made by catalytic reduction of morphine with finely-divided platinum or palladium in acidic media. The product is purified by the addition of sulphur dioxide gas to saturation point. This mixture is left to crystallise over a period of 4-5 days, the complex is filtered, dried, and then decomposed by heating at 90.degree. C. in concentrated hydrochloric acid until sulphur dioxide evolution ceases.
Biocatalysts can provide a cleaner technology. However, the range of enzyme activities presently available is rather limited, and these enzymes do not catalyse the reaction required.
GB-A-2231332 describes an acetylmorphine carboxylase that catalyses the hydrolysis of heroin, 3-acetylmorphine and 6-acetylmorphine to morphine, and also a morphine dehydrogenase that oxidises morphine to morphinone, utilising NADP as a cofactor. These enzymes are obtained from a novel strain of Pseudornones putida designated as "M10", NCIMB 40119. They can be used for the detection of heroin and morphine.
SUMMARY OF THE INVENTION
The present invention is based on the discovery of an enzyme which can be used as a biocatalyst in the synthesis of hydromorphone and hydrocodone. This is a highly specific NADH-dependent reductase that catalyses the reduction of the 7,8-unsaturated bond of morphinone and codeinone. The reaction of the enzyme, morphinone reductase, requires a cofactor such as reduced nicotinamide adenine dinucleotide (NADH) which is oxidised to NAD+ concurrently with the reduction of morphinone to hydromorphone.
DESCRIPTION OF THE INVENTION
The morphinone reductase of the invention can be defined in different several ways, but is probably best defined by its partial amino-acid sequence shown below, in the Sequence Listing.
Alternatively, or in addition, the morphinone reductase of the invention can be characterised by any of the following properties. With the aid of a cofactor, notably NADH, it reduces morphinone and codeinone to hydromorphone and hydrocodone, respectively. It also reduces neopinone to hydrocodone. It has no significant activity with other compounds of similar ring structure. Another distinguishing feature of the morphinone reductase is that it has a native molecular weight of 81,000 Dalton (as determined by elution from a gel filtration column calibrated with marker proteins). The enzyme is comprised of two identical subunits with a molecular weight of 41,100 Dalton (as determined by electrospray mass spectrometry). The optimal activity of the enzyme is exhibited at pH 7.5-8.0, with maximal activity in 50 mM phosphate buffer at pH 8.0.
Examples 1 to 3 below describe other features of the morphinone reductase, but it is expected that it will be possible to vary some of those by changing the conditions of growth of the microorganism which produces it or by recombinant DNA technology, which can be used to produce the enzyme. Accordingly, it is preferred not to rely on such characteristics in the most general definition of the enzyme. Any one or more of them can be considered (as the context permits) as alternative parameters for use in defining the enzyme, but they are best seen as one or more preferred characteristics, additional to one or more of those described above.
The novel enzyme converts morphinone and codeinone to hydromorphone and hydrocodone, respectively. Accordingly it is now possible to reduce the 7,8-unsaturated bond of morphinone, codeinone and neopinone, enzymically in the presence of morphinone reductase and a cofactor such as NADH. The enzyme can therefore be used in diagnostic assays for the detection and quantitation of morphinone and codeinone.
The enzyme of the invention can be produced by culturing the known
REFERENCES:
patent: 5298414 (1994-03-01), Bruce
patent: 5387515 (1995-02-01), Bruce
Pollock S., Dihydromorphinone Retone Reductases UFE Sciences vol. 17 (1975) pp. 465-476.
Bruce N., Microbial Degradation of the Morphine Alkaloids, Arch Microbiol, (1990) 154:465-470.
"Microbial degradation of the morphine alkaloids. Purification and characterization of morphine dehydrogenase from Pseudomonas putida M10" (Bruce et al.), The Biochemical Journal, Mar. 15, 1991, vol. 274, No. 3, pp. 875-880.
Bruce Neil C.
French Christopher E.
Hailes Anne M.
Gitomer Ralph J.
Macfarlan Smith Limited
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