Europäisches Patentamt *EP000649465B1* (19) European Patent Office Office européen des brevets (11) EP 0 649 465 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.7: C12N 9/02, C12P 17/18, of the grant of the patent: C12Q 1/32 24.07.2002 Bulletin 2002/30 (86) International application number: (21) Application number: 93913236.1 PCT/GB93/01129 (22) Date of filing: 28.05.1993 (87) International publication number: WO 94/00565 (06.01.1994 Gazette 1994/02) (54) MORPHINONE REDUCTASE FOR THE PREPARATION OF HYDROMORPHONE AND HYDROCODONE MORPHINONE REDUKTASE ZUR HERSTELLUNG VON HYDROMORPHONE UND HYDROCODONE MORPHINONE REDUCTASE DESTINEE A LA PREPARATION D’HYDROMORPHONE ET D’HYDROCODONE (84) Designated Contracting States: (74) Representative: Perry, Robert Edward AT BE CH DE DK ES FR GB IE IT LI NL PT SE GILL JENNINGS & EVERY Broadgate House (30) Priority: 25.06.1992 GB 9213524 7 Eldon Street London EC2M 7LH (GB) (43) Date of publication of application: 26.04.1995 Bulletin 1995/17 (56) References cited: WO-A-90/13634 (73) Proprietor: MACFARLAN SMITH LIMITED Edinburgh EH11 2QA (GB) • THE BIOCHEMICAL JOURNAL vol. 274, no. 3, 15 March 1991, pages 875 - 880 NEIL C. BRUCE ET (72) Inventors: AL. ’Microbial degradation of the morphine • HAILES, Anne, Maria 59 Lower Road alkaloids. Purification and characterization of Kent DA8 1AY (GB) morphine dehydrogenase from Pseudomonas • FRENCH, Christopher, Edward putida M10’ Palmerston North (NZ) • BRUCE, Neil, Charles Cambridge CB2 1ND (GB) Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 0 649 465 B1 Printed by Jouve, 75001 PARIS (FR) EP 0 649 465 B1 Description Field of the Invention 5 [0001] This invention relates to a new enzyme and to its use. Background of the Invention [0002] Current synthesises of the analgesic hydromorphone (dihydromorphinone) and the antitussive hydrocodone 10 (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 plat- inum 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 °C in concentrated hydrochloric acid until sulphur dioxide evolution ceases. 15 [0003] 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. [0004] GB-A-2231332 describes an acetylmorphine carboxylase that catalyses the hydrolysis of heroin, 3-acetyl- morphine and 6-acetylmorphine to morphine, and also a morphine dehydrogenase that oxidises morphine to morphi- none, utilising NADP as a cofactor. These enzymes are obtained from a novel strain of Pseudornones putida designated 20 as "M10", NCIMB 40119. They can be used for the detection of heroin and morphine. Summary of the Invention [0005] The present invention is based on the discovery of an enzyme which can be used as a biocatalyst in the 25 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. 30 Description of the Invention [0006] 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. [0007] Alternatively, or in addition, the morphinone reductase of the invention can be characterised by any of the 35 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 elec- 40 trospray 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. [0008] 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 45 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. [0009] 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, enzym- 50 ically 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. [0010] The enzyme of the invention can be produced by culturing the known M10 microorganism on a source of carbon and nitrogen. Any conventional sources can be used but it is preferred to grow P. putida M10 on a carbon source comprising morphine. The morphinone reductase is produced constitutively and, therefore, the P. putida M10 55 can be cultured on glucose to produce a highly active preparation of morphinone reductase. Cultivation of the organism is generally aerobic. Any usual temperature, e.g. within the 20 - 40 °C range, preferably 25 to 35 °C, can be used. To obtain the enzyme, the cells can be disrupted in any conventional way. Preferably a cell-free extract is made. The enzyme is then recovered from cells or extract. 2 EP 0 649 465 B1 [0011] Instead of the precise starting organism deposited, a mutant thereof, e.g. derived from gamma-ray irradiation or use of a chemical mutagen, induction by culture on another medium etc. or a transconjugant thereof with another bacterium or an artificially-produced variant can be used. The ability of any such organism to give the novel enzyme can be determined by the skilled man. 5 [0012] The enzyme or some modification thereof can also be made by recombinant technology using methods well recognised in that art. These may entail producing the enzyme in another host organism. [0013] The enzyme of the present invention is useful primarily as an industrial biocatalyst in the synthesis of hydro- morphone and hydrocodone. This may involve using purified preparations of the enzyme. Alternatively, the enzyme could be used in an appropriate blocked mutant of the organism. 10 [0014] The enzyme of the invention is also applicable to the detection and quantitation of morphinone and codeinone in biological fluids, especially in urine and blood. The detection may be conducted spectrophotometrically, in various ways. For example, the reduction of morphinone may be used to drive a redox reaction in which a change in UV absorption occurs. Thus, the cofactor itself can be used to detect the reaction by observing the oxidation of NADH to NAD+, particularly as a decrease in absorbance at 340 nm. 15 [0015] As indicated above, the morphinone reductase enzyme may be obtained from the same deposited bacterium as that disclosed in GB-A-2231332 as a source of acetylmorphine carboxylesterase and morphine dehydrogenase enzymes. Those enzymes are respectively capable of catalysing the hydrolysis of heroin, 3-acetylmorphine and 6-acetylmorphine to morphine, and the oxidation of morphine (to morphinone), codeine and ethylmorphine. The N- terminus of the second of these enzymes has been sequenced. 20 [0016] The combination of the two known enzymes, and of the morphinone reductase of the present invention, all isolated from the same organism, provides a pathway from, say, heroin to hydromorphone. [0017] The gene for the novel enzyme, optionally together with genes for the enzymes described in GB-A-2231332, may be isolated and transformed into a suitable host, by conventional recombinant technology. The transformed mi- croorganism may be used to prepare hydromorphone or hydrocodone from a variety of precursors. The novel enzyme, 25 optionally together with one or both of the known enzymes, may be used to assay a specimen for the presence of, say, morphinone or an earlier substrate in the pathway. [0018] The following Examples illustrate the invention. Example 1 refers in particular to the purification and charac- terisation of morphinone reductase, Example 2 to its utility in the generation of hydrocodone, and Example 3 to a coupled assay. 30 Example 1 1. Organism 35 [0019] Pseudomonas putida (M10) was isolated from industrial waste liquors (Bruce et al, (1990). Arch. Microbiol. 154:465-470) which were enriched with morphine as the sole source of carbon and energy. 2. Growth conditions 40 [0020] Cultures of Pseudomon putida (M10) were grown in 2 1 Erlenmeyer flasks containing 750 ml of sterile defined mineral medium consisting of (NH4)2SO4 (0.5 g/l), K2HPO4 (2.0 g/l), KH2PO4 (0.2 g/l) and MgSO4 (0.05 g/l).