Europäisches Patentamt *EP001181356B1* (19) European Patent Office Office européen des brevets (11) EP 1 181 356 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.7: C12N 9/02, C12P 7/00, of the grant of the patent: C12P 13/02, C12P 1/00 07.12.2005 Bulletin 2005/49 (86) International application number: (21) Application number: 00917839.3 PCT/US2000/006300 (22) Date of filing: 10.03.2000 (87) International publication number: WO 2000/053731 (14.09.2000 Gazette 2000/37) (54) Phosphine stabilizers for oxidoreductase enzymes Phosphine Stabilisatoren für oxidoreduktase Enzymen Phosphines stabilisateurs des enzymes ayant une activité comme oxidoreducase (84) Designated Contracting States: (56) References cited: DE FR GB NL US-A- 5 777 008 (30) Priority: 11.03.1999 US 123833 P • ABRIL O ET AL.: "Hybrid organometallic/enzymatic catalyst systems: (43) Date of publication of application: Regeneration of NADH using dihydrogen" 27.02.2002 Bulletin 2002/09 JOURNAL OF THE AMERICAN CHEMICAL SOCIETY., vol. 104, no. 6, 1982, pages 1552-1554, (60) Divisional application: XP002148357 DC US cited in the application 05021016.0 • BHADURI S ET AL: "Coupling of catalysis by carbonyl clusters and dehydrigenases: (73) Proprietor: EASTMAN CHEMICAL COMPANY Redution of pyruvate to L-lactate by dihydrogen" Kingsport, TN 37660 (US) JOURNAL OF THE AMERICAN CHEMICAL SOCIETY., vol. 120, no. 49, 11 October 1998 (72) Inventors: (1998-10-11), pages 12127-12128, XP002148358 • HEMBRE, Robert, T. DC US cited in the application Johnson City, TN 37601 (US) • OTSUKA K: "Regeneration of NADH and ketone • WAGENKNECHT, Paul, S. hydrogenation by hydrogen with the San Jose, CA 95129 (US) combination of hydrogenase and alcohol • PENNEY, Jonathan, M. dehydrogenase" JOURNAL OF MOLECULAR New York, NY 10025 (US) CATALYSIS, vol. 51, no. 1, 1989, pages 35-39, XP000946525 ISSN:0304-5102 cited in the (74) Representative: Wibbelmann, Jobst application Wuesthoff & Wuesthoff, • OIKONOMAKOS NIKOS S ET AL: "Activator Patent- und Rechtsanwälte, anion binding site in pyridoxal phosphorylase b: Schweigerstrasse 2 The binding of phosphite, phosphate and 81541 München (DE) fluorophosphate in the crystal." PROTEIN SCIENCE, vol. 5, no. 12, December 1996 (1996-12), pages 2416-2428, XP000978804 ISSN: 0961-8368 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 1 181 356 B1 Printed by Jouve, 75001 PARIS (FR) EP 1 181 356 B1 Description CROSS REFERENCE TO RELATED APPLICATIONS 5 [0001] This application claims priority to U.S. Provisional Patent Application Serial Number 60/123,833, filed March 11, 1999. FIELD OF THE INVENTION 10 [0002] This invention relates generally to a process for stabilizing the activity of an oxidoreductase enzyme, in par- ticular a stabilization process comprising mixing a phosphine or phosphite with an oxidoreductase enzyme. BACKGROUND OF THE INVENTION 15 [0003] The present invention belongs to the technical field of oxidoreductase enzymes, in particular to a process of stabilizing them. [0004] One broad class of enzymes capable of selective reduction and/or hydrogenation of unsaturated organic compounds are the nicotinamide dependent oxidoreductases, as discussed by Walsh (Enzymatic Reaction Mecha- nisms Freeman & Co., N.Y., 1979; pages 311-521). 20 [0005] Nicotinamide dependent oxidoreductases require the presence of nicotinamide cofactors. The structures of the naturally occurring nicotinamide cofactors, (NAD+, NADP+, NADH and NADPH) are shown below. 25 30 35 40 45 [0006] A reduced nicotinamide cofactor (NADH or NADPH) binds to the nicotinamide cofactor dependent enzyme, and transfers a "hydride" (two electrons and one hydrogen nucleus) to reduce a substrate that also binds to the enzyme. After the substrate is reduced, the enzyme releases the oxidized form of the nicotinamide cofactor (NAD+ or NADP+). Biological systems typically recycle the oxidized nicotinamide cofactors, by employing an external reducing agent, in combination with other enyzmes, to regenerate the reduced form of the nicotinamide cofactors. In these nicotinamide 50 cofactors the nicotinamide ring (shown schematically immediately below) is the reactive group. To regenerate the reduced cofactor, an external reducing agent must transfer the equivalent of a "hydride" to the oxidized (pyridinium) form of the cofactor, regioselectively to form the reduced (1,4-dihydropyridine) form of the cofactor. 55 2 EP 1 181 356 B1 5 10 [0007] Although nicotinamide cofactor dependent enzymes and nicotinamide cofactors are present in all living or- ganisms at low concentrations, they tend to be chemically unstable under non-biological conditions, and are extremely expensive in purified form. Because of their high cost, most industrial processes that seek to employ a combination of 15 enzymes and nicotinamide cofactors must supply a method to regenerate the nicotinamide cofactors. [0008] A number of methods for cofactor regeneration are known, as discussed by Chenault and Whitesides (Appl. Biochem. Biotechnol. 1987, 14, 147-97), and in Enzymes in Organic Synthesis K. Drauz, H. Waldeman, Eds.; VCH: Weinheim, 1995; pages. 596-665. [0009] The activity of oxidoreductase enzymes, such as for example nicotinamide cofactor dependent enzymes, may 20 be enhanced or maintained by the presence of certain stabilizers such as certain sulfur containing stabilizers. However, there is still a need for further enzyme stabilizing compounds. SUMMARY OF THE INVENTION 25 [0010] The invention relates to a process for stabilizing the activity of an oxidoreductase enzyme, comprising mixing a phosphine or phosphite with an oxidoreductase enzyme. [0011] Additional advantages of the invention will be set forth in part in the description that follows, and in part will be obvious from the description, or may be learned by practice of the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. 30 It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. DESCRIPTION OF THE PREFERRED EMBODIMENTS 35 [0012] The present invention may be understood more readily by reference to the following detailed description of preferred embodiments of the invention and the Examples included therein and to the Figures and their previous and following description. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. [0013] It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an" and 40 "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an aromatic compound" includes mixtures of aromatic compounds. [0014] Often, ranges are expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be 45 understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. [0015] In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings: 50 A weight percent of a component, unless specifically stated to the contrary, is based on the total weight of the formulation or composition in which the component is included. A residue of a chemical species, as used in the specification and concluding claims, refers to the moiety that is the resulting product of the chemical species in a particular reaction scheme or subsequent formulation or chemical product, regardless of whether the moiety is actually obtained from the chemical species. Thus, an ethylene glycol 55 residue in a polyester refers to one or more -OCH2CH2O- repeat units in the polyester, regardless of whether ethylene glycol is used to prepare the polyester. Similarly, a sebacic acid residue in a polyester refers to one or more -CO(CH2)8CO- moieties in the polyester, regardless of whether the residue is obtained by reacting sebacic acid or an ester thereof to obtain the polyester. 3 EP 1 181 356 B1 [0016] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, the phrase "optionally substituted lower alkyl" means that the lower alkyl group may or may not be sub- stituted and that the description includes both unsubstituted lower alkyl and lower alkyl where there is substitution. 5 [0017] By the term "effective amount" of a compound or property as provided herein is meant such amount as is capable of performing the function of the compound or property for which an effective amount is expressed. As will be pointed out below, the exact amount required will vary from process to process, depending on recognized variables such as the compounds employed and the processing conditions observed. Thus, it is not possible to specify an exact "effective amount." However, an appropriate effective amount may be determined by one of ordinary skill in the art 10 using only routine experimentation. [0018] The Enzyme Commission of International Union of Biochemistry and Molecular Biology ("EC") has devised a well-known four digit numerical system for classifying enzymes, in terms of the type of reaction that the enzymes catalyze.