PROCESS FOR THE ENZYMATIC REGENERATION OF REDOX COFACTORS
The present invention relates to a process for the enzymatic regeneration of the redox cofactors NAD+/NADH and NADP+/NADPH in a one-pot reaction, wherein, as a result of at least two further enzymatically catalyzed redox reactions proceeding in the same reaction batch (=product-forming reactions), one of the two redox cofactors accumulates in its reduced form and, respectively, the other one in its oxidized form. Enzymatically catalyzed redox reactions are used in industrial operations, for example, in the production of chiral alcohols, α-amino acids and α-hydroxy acids. The majority of enzymes employed in industrial redox reactions use cofactors such as NADH or NADPH. Among enzymatic redox reactions, those are particularly interesting wherein redox cofactors are restored by in situ cofactor regeneration systems. The reason therefor is that it is possible to use only catalytic amounts of the expensive cofactors (NAD(P)+/NAD(P)H). The availability of suitable dehydrogenases and other enzymes has resulted in the development of various cofactor regeneration systems. The regeneration systems described up to now may be classified as: enzyme-linked, substrate-linked, in vivo (natural cofactor regeneration systems in living organisms), photochemical, chemichal or electro-enzymatic. The process herein described relates to an enzyme-linked regeneration system. Advantages of enzyme-linked systems are high selectivity, applicability for the production of various products and a high reuse rate of the cofactor (total turnover number, TTN). In the mid-90ies, a first industrial process using an enzyme-linked cofactor regeneration system was employed on a ton scale. In said process, formate dehydrogenase from Processes wherein two or more enzymatic redox reactions which are involved in the formation of the product and two enzymatic systems for the cofactor regeneration (simultaneously or sequentially) are proceeding in one reaction batch without an intermediate being isolated must be distinguished therefrom. Recently, such enzymatic cascade reactions—herein referred to as one-pot reactions—have drawn significant attention, since they effectively reduce operating costs, operating time and environmental impacts. In addition, enzymatic cascades of redox reactions facilitate transformations which are not easy to implement by conventional chemical methods. It is, however, a challenge to perform several reactions (oxidation and reduction) simultaneously in one one-pot reaction with a parallel cofactor regeneration, since highly divergent reaction conditions are often required for the individual transformations. So far, only a very small number of one-pot trials comprising oxidation and reduction reactions with associated cofactor regeneration systems have been performed. In the literature (Advanced Synth. Catal., 2008, Volume 351, Issue 9, p. 1303-1311), the experiment of a one-pot reaction using 7α-hydroxysteroid dehydrogenase (HSDH), 7β-HSDH and 12α-HSDH has been described. In said process, an oxidation, both regioselective and stereoselective, was performed at positions 7 and 12 of cholic acid, followed by a regio- and stereoselective reduction at position 7. In that process both, a lactate dehydrogenase (NAD+-dependent) and a glucose dehydrogenase (NADP+-dependent) were used as a cofactor regeneration system. Pyruvate and glucose were used as cosubstrates. Although this process was originally aimed at a true one-pot process, at the end oxidation and reduction reactions were performed separately. In doing so, the partitioning of oxidative and reductive steps occurred either in a so-called “tea bag”-reactor or in a membrane reactor. Said partitioning was necessary in order to avoid the production of byproducts due to the low cofactor selectivity of NADPH-glucose dehydrogenase. However, in the one-pot reaction, the glucose dehydrogenase NADP+ converted partly also NAD+, which impeded the oxidation. In the process described, only 12.5 mM (˜0.5%) of the substrate cholic acid was used, which renders the process uninteresting from an ecological point of view. Furthermore, an attempt to perform the deracemization of racemates of secondary alcohols via a prochiral ketone as an intermediate using a one-pot system has been described (J. Am. Chem. Soc., 2008, Volume 130, p. 13969-13972). The deracemization of secondary alcohols was achieved via two alcohol dehydrogenases (S- and R-specific) with different cofactor specificities. In said system, NADP was regenerated by NADPH oxidase (hydrogen peroxide producing) and NADH was regenerated by formate dehydrogenase. Formate and oxygen were used as cosubstrates. In that system 4 enzymes were used without partitioning of oxidative and reductive steps. A drawback of the process is the very low concentration of the substrate used of 0.2-0.5%, which is inappropriate for industrial purposes. A further one-pot system has been described in WO 2009/121785 A2. In said process, a stereoisomer of an optically active secondary alcohol was oxidized to the ketone and then reduced to the corresponding optical antipode, wherein two alcohol dehydrogenases having opposite stereoselectivities and different cofactor specificities were used. The cofactors were regenerated by means of a so-called “hydride-transfer system”, using only one additional enzyme. For regenerating the cofactors, various enzymes such as formate dehydrogenase, glucose dehydrogenase, lactate dehydrogenase were used. A drawback of said process is the low concentration of the substrates used. A drawback of the enzymatic one-pot methods involving cofactor regeneration systems yet known is altogether the very low substrate concentration, which is inefficient for industrial processes. In contrast to that, many individual enzymatic redox reactions are already known in which cofactor regeneration systems are used. The experiments were described with whole microorganisms, cell lysates or isolated enzymes with concurrent NAD(P)H or NAD(P)+ regeneration. Known enzymatic cofactor regeneration systems for individual redox reactions comprise, for example, formate dehydrogenase for NADH (formate as a cosubstrate), alcohol dehydrogenase from An example of use of such individual redox reactions is the production of chiral hydroxy compounds, starting from appropriate prochiral keto compounds. In said process, the cofactor is regnerated by means of an additional enzyme. These methods have in common that they constitute an isolated reduction reaction and regenerate NAD(P)H (see e.g. EP 1 152 054). Enzymatic processes using hydroxysteroid dehydrogenases, coupled with a cofactor regeneration system, which proceed at higher substrate concentrations (approx. >1%), have been described (EP 1 731 618; WO 2007/118644; Appl. Microbiol. Biotechnol., 2011 Volume 90 p. 127-135). In said processes, the cofactors NAD(P)H or NAD(P) were regenerated by means of different enzymes such as, e.g., lactate dehydrogenase (pyruvate as a cosubstrate), alcohol dehydrogenase from A cofactor regeneration system for NADH using malate dehydrogenase (“malate enzyme”) has already been described (Can. J. Chem. Eng. 1992, Volume 70, p. 306-312). In said publication, it was used for the reductive amination of pyruvate by alanine dehydrogenase. The pyruvate emerging during the cofactor regeneration was subsequently used in the product-forming reaction. In WO 2004/022764, it is likewise described to regenerate NADH by malate dehydrogenase. Differently to the previously described publication the pyruvate emerging during the oxidative decarboxylation of malate was not used further. An example of an enzymatic reduction of D-xylose to xylitol involving a cofactor regeneration system has been described (FEBS J., 2005, Volume 272, p. 3816-3827). An NADPH-dependent mutant of phosphite dehydrogenase from Further examples of an enzymatic production of chiral enantiomerically enriched organic compounds, e.g., alcohols or amino acids, have been described (Organic Letters, 2003, Volume 5, p. 3649-3650; U.S. Pat. No. 7,163,815; Biochem. Eng. J., 2008, Volume 39(2) p. 319-327; EP 1 285 962). In said systems, an NAD(P)H-dependent oxidase from In WO 2011/000693, a 17beta-hydroxysteroid dehydrogenase as well as a process are described enabling the execution of redox reactions at position 17 of 4-androstene-3,17-dione. Again, this is an isolated reduction reaction. The above-mentioned individually proceeding oxidation or reduction reactions lack the advantages of a one-pot reaction, such as for example cost-effectiveness as a result of time and material savings as well as a better turnover due to enzymatic cascade reactions. The object of the present invention was to provide a process for regenerating the redox cofactors NAD+/NADH and/or, e.g. and, NADP+/NADPH in order to perform therewith two or more enzymatically catalyzed redox reactions in one reaction batch in an economical fashion. According to the present invention, said object is achieved in a process of the kind initially mentioned, in that a process for the enzymatic regeneration of the redox cofactors NAD+/NADH and/or, e.g. and, NADP+/NADPH in a one-pot reaction is provided, wherein, as a result of at least two further enzymatically catalyzed redox reactions proceeding in the same reaction batch (product-forming reactions), one of the two redox cofactors accumulates in its reduced form and, respectively, the other one in its oxidized form, which process is characterized in that
A process provided according to the present invention is herein also referred to as “process according to (of) the present invention”. In a further aspect, the present invention provides a process according to the present invention for the enzymatic regeneration of the redox cofactors NAD+/NADH and/or, e.g. and, NADP+/NADPH in a one-pot reaction, wherein, as a result of at least two further enzymatically catalyzed redox reactions proceeding in the same reaction batch (=product-forming reactions), one of the two redox cofactors accumulates in its reduced form and, respectively, the other one in its oxidized form, which process is characterized in that
In a further aspect, in a process according to the present invention, R2and R3independently of each other are selected from the group consisting of H, (C1-C6) alkyl, wherein alkyl is linear-chain or branched, (C1-C6) alkenyl, wherein alkenyl is linear-chain or branched and comprises one to three double bonds, aryl, in particular C6-C12aryl, carboxyl, or (C1-C4) carboxy alkyl. Compared to the prior art, a process according to the present invention constitutes a significant improvement of processes in which compounds are both enzymatically oxidized and reduced, since it is enabled to run the required oxidation and reduction reactions as well as the associated reactions for the cofactor regeneration in one reaction batch and, at the same time, to use significantly higher substrate concentrations than according to prior art. In a process according to the present invention, the cofactors NADH and/or NADPH are used. Therein, NAD+ denotes the oxidized form and NADH denotes the reduced form of nicotinamide adenine dinucleotide, whereas NADP+ denotes the oxidized form and NADPH denotes the reduced form of nicotinamide adenine dinucleotide phosphate. Enzymatically catalyzed redox reactions which are not part of the cofactor regeneration and, in a process according to the present invention, are involved in the formation of the product are herein referred to as “oxidation reaction(s)” and “reduction reaction(s)”. “Oxidation reaction(s)” and “reduction reaction(s)” are summarized under the term “product-forming reactions”. The product-forming reactions in a process according to the present invention comprise, in each case, at least one oxidation reaction and at least one reduction reaction. If NAD+ is used as a cofactor for the oxidation reaction(s), NADPH is the cofactor for the reduction reaction(s). If NADP+ is used as a cofactor for the oxidation reaction(s), NADH is the cofactor for the reduction reaction(s). In a process according to the present invention, oxidation reaction(s) and reduction reaction(s) can be performed either chronologically parallel or in chronological succession, preferably chronologically parallel in the same reaction batch. Compounds which are used with the objective of forming a product are herein referred to as substrates. Compounds which are reacted during the cofactor regeneration are herein referred to as cosubstrates. In a process according to the present invention, one substrate as well as several substrates can be used. In doing so, reduction and/or oxidation reaction(s) can take place on the same substrate (molecular backbone) and also on different substrates, preferably on the same substrate. Furthermore, in a process according to the present invention, reduction and/or oxidation reactions can take place on the same or on different functional groups. A process according to the present invention is suitable for a plurality of reactions, for example for the inversion of configuration of stereoisomeric hydroxy compounds via oxidation to the corresponding ketone and subsequent reduction to the opposite stereospecific hydroxy compound. A process in which two or more enzymatic redox reactions involved in the formation of a product and two enzymatic systems for cofactor regeneration proceed in one reaction batch without an intermediate being isolated is herein referred to as a “one-pot reaction”. The mentioning of an acid or a salt of an acid includes herein the respective unmentioned term. Likewise, the mentioning of acids, in particular of bile acids, includes herein all esters derived therefrom. Furthermore, compounds (partly) provided with protective groups are included in the mentioning of the underlying substances. In a preferred embodiment of the present invention, a process according to the present invention is characterized in that the oxidation reaction and the reduction reaction proceed chronologically parallel. In a preferred embodiment of the present invention, a process according to the present invention is characterized in that both the oxidation reaction and the reduction reaction occur on the same molecular backbone. In a preferred embodiment of the present invention, a process according to the present invention is characterized in that, as a compound of formula I (2-oxo acid), pyruvate (cosubstrate) is used which is reduced to lactate by means of a lactate dehydrogenase, which means that, in the regeneration reaction which reconverts the reduced cofactor into its original oxidized form, pyruvate is reduced to lactate by means of a lactate dehydrogenase. In a preferred embodiment of the present invention, a process according to the present invention is characterized in that, as a compound of formula II (secondary alcohol), 2-propanol (isopropyl alcohol, IPA) (cosubstrate) is used which is oxidized to acetone by means of an alcohol dehydrogenase, which means that, in the regeneration reaction which reconverts the oxidized cofactor into its original reduced form, 2-propanol is oxidized to acetone by means of an alcohol dehydrogenase. In a preferred embodiment of the present invention, a process according to the present invention is characterized in that oxygen is used which is reduced by means of an NADH oxidase. In a preferred embodiment of the present invention, a process according to the present invention is characterized in that, as a secondary alcohol, malate (cosubstrate) is used which is oxidized to pyruvate and CO2by means of an oxaloacetate-decarboxylating malate dehydrogenase (“malate enzyme”), e.g., that in the regeneration reaction which reconverts the oxidized cofactor into its original reduced form, malate is oxidized to pyruvate and CO2by means of a malate dehydrogenase. In this embodiment, the nascent pyruvate is reacted in a further redox reaction which does not serve for the formation of a product, but constitutes the second cofactor regeneration reaction. In a preferred embodiment of the present invention, a process according to the present invention is characterized in that it is used for performing at least one oxidation reaction and at least one reduction reaction, respectively, in the same reaction batch on compounds of general formula wherein
denotes a benzene ring or a ring comprising 6 carbon atoms and 0, 1 or 2 C—C-double bonds; wherein the substrate(s) is/are preferably provided at a concentration of <5% (w/v) in the reaction batch for the reduction reaction(s) involved in the formation of the product. In a preferred embodiment of the present invention, a process according to the present invention is characterized in that an enzymatic conversion of dehydroepiandrosterone (DHEA) of formula into testosterone of formula takes place. In a preferred embodiment of the present invention, a process according to the present invention is characterized in that an enzymatic epimerization of the hydroxysteroid compound 3α,7α-dihydroxy-5β-cholanic acid (chenodeoxycholic acid, CDC) of formula occurs via oxidation to ketolithocholic acid (KLC) of formula and reduction to 3α,7β-dihydroxy-5β-cholanic acid (ursodeoxycholic acid, UDC) of formula e.g. using two opposite stereospecific hydroxysteroid dehydrogenases. In a preferred embodiment of the present invention, a process according to the present invention is characterized in that it is used for the enzymatic epimerization of 3α,7α,12α-trihydroxy-5β-cholanic acid (cholanic acid) of formula either
or
In a preferred embodiment of the present invention, a process according to the present invention is characterized in that a C5- or C6-sugar is used as a substrate, e.g., that the process is used for the isomerization of C5- or C6-sugars. In a preferred embodiment of the present invention, a process according to the present invention is characterized in that an isomerization of glucose occurs via reduction to sorbitol and oxidation to fructose, e.g., that the process is used for the isomerization of glucose via reduction to sorbitol and subsequent oxidation to fructose. A process according to the present invention is preferably carried out in an aqueous system, wherein it is possible that the substrate for the oxidation and reduction reaction is partly provided in an undissolved state in the form of a suspension and/or as a second liquid phase. In a particular embodiment, a process according to the present invention is characterized in that the substrate(s) for the oxidation reaction(s) involved in the formation of a product is/are provided in the reaction batch at a concentration of at least 5% (w/v) and more, preferably 7% (w/v) and more, particularly preferably 9% (w/v) and more, e.g. 5% (w/v) to 20% (w/v), such as 5% (w/v) to 15% (w/v), e.g. 5% (w/v) to 12% (w/v), such as 5% (w/v) to 10% (w/v). In a particular embodiment, a process according to the present invention is characterized in that, on the whole, a turnover of ≧70%, in particular ≧90%, is achieved in the product-forming reactions. In a process according to the present invention, a buffer can be added to the aqueous system. Suitable buffers are, for example, potassium phosphate, Tris-HCl and glycine with a pH ranging from 5 to 10, preferably from 6 to 9. Furthermore or alternatively, ions for stabilizing the enzymes, such as Mg2+ or other additives such as glycerol, can be added to the system. In a process according to the present invention, the concentration of the added cofactors NAD(P)+ and NAD(P)H is usually between 0.001 mM and 10 mM, preferably between 0.01 mM and 1 mM. Depending on the enzymes used, the process according to the present invention can be performed at a temperature ranging from 10° C. to 70° C., preferably from 20° C. to 45° C. Hydroxysteroid dehydrogenases (HSDH) are understood to be enzymes which catalyze the oxidation of hydroxy groups to the corresponding keto groups or, conversely, the reduction of keto groups to the corresponding hydroxy groups at the steroid skeleton. Appropriate hydroxysteroid dehydrogenases which can be used for redox reactions on hydroxysteroids are, for example, 3α-HSDH, 3β-HSDH, 7α-HSDH, 7β-HSDH or 17β-HSDH. Appropriate enzymes with 7α-HSDH activity can be obtained, for example, from Clostridia ( Appropriate enzymes with 7β-HSDH activity can be obtained, for example, from Appropriate lactate dehydrogenases can be obtained, for example, from Appropriate alcohol dehydrogenases can be obtained, for example, from An appropriate xylose reductase can be obtained, for example, from Appropriate sorbitol dehydrogenases can be obtained, for example, from sheep liver, Appropriate NADH oxidases can be obtained, for example, from In a process according to the present invention, enzymes are preferably used as proteins recombinantly overexpressed in In the figures the following abbreviations are used:
In the following examples, all temperature data are given in degrees Celsius (° C.). The following abbreviations are used:
A 0.5 ml charge contains 50 mg chenodeoxycholic acid, 12 U of recombinant 7α-hydroxysteroid dehydrogenase from A 0.5 ml charge contains 50 mg chenodeoxycholic acid, 20 U of recombinant 7α-hydroxysteroid dehydrogenase from A 0.5 ml charge contains 50 mg chenodeoxycholic acid, 12 U of recombinant 7α-hydroxysteroid dehydrogenase from Upon completion of reactions as described in Examples 1 to 3, the reaction mixture is extracted with EtOAc. Subsequently, the solvent is removed by evaporation. The evaporation resisue is dissolved in a mixture of MeOH:acetonitrile:sodium phosphate buffer pH=3, 0.78 g/l (40:30:37) and the conversion of chenodeoxycholic acid into ursodeoxycholic acid is monitored by HPLC. Thereby, a reversed-phase separation column (ZORBAX® Eclipse® XDB C18, flow 0.8 ml/min) and a light-refraction detector (RID), Agilent 1260 Infinity®, both from Agilent Technologies Inc., are used. A 0.5 ml charge contains 50 mg/ml glucose and 6 U/ml of recombinant xylose reductase from A 0.5 ml charge contains 50 mg/ml glucose, 6 U/ml of recombinant xylose reductase from The charge is incubated at 65° C. for 10 min for inactivating the enzymes and is subsequently centrifuged. The supernatant is then filtered over a 0.2 μM PVDF filter and analyzed by ligand-exchange HPLC (Agilent Technologies Inc.). In doing so, sugars and polyols are separated via a lead column of Showa Denko K.K. (Shodex® Sugar SP0810) with a flow of 0.5 ml/min water (VWR International GmbH, HPLC Grade) at 80° C. Detection occurs with the aid of a light-refraction detector (RID, Agilent 1260 Infinity®, Agilent Technologies Inc.). An inline filter of Agilent Technologies Inc. and, as precolumns, an anion-exchange column (Shodex® Axpak-WAG), a reversed-phase column (Shodex® Asahipak® ODP-50 6E) and a sugar precolumn (SUGAR SP-G) of Showa Denko K.K. are used. A 0.5 ml charge contains 25 mg of cholanic acid 12.5 U of recombinant 12α-hydroxysteroid dehydrogenase from Ein 0.5 ml charge contains 25 mg of cholanic acid, 12.5 U of recombinant 12α-hydroxysteroid dehydrogenase from An open system is further used in order to allow evaporation of acetone and to shift the reaction towards 3α,7β-dihydroxy-12-oxo-5β-cholanic acid. After 18 h and 24 h 2% of IPA (w/v) are dosed in additionally. After 48 h 70% of the cholanic acid used are reacted to 3α,7α-dihydroxy-12-oxo-5β-cholanic acid. A 0.5 ml charge contains 50 mg of chenodeoxy cholanic acid, 12 U of recombinant 7α-hydroxysteroiddehydrogenase aus A 0.5 ml charge contains 50 mg of chenodeoxy cholanic acid, 12 U of recombinant 7α-hydroxysteroid dehydrogenase from A 50 ml charge contains 5 g of chenodeoxy cholanic acid, 24 U/ml of recombinant 7α-hydroxysteroid dehydrogenase from After termination of the reaction as described in examples 8 to 12, the bile acids which are present in the trials may be anyalyzed via a method as described in example 4. A process for the enzymatic regeneration of the redox cofactors NAD+/NADH and NADP+/NADPH in a one-pot reaction, wherein, as a result of at least two further enzymatically catalyzed redox reactions proceeding in the same reaction batch (product-forming reactions), one of the two redox cofactors accumulates in its reduced form and, respectively, the other one in its oxidized form, characterized in that a) in the regeneration reaction which reconverts the reduced cofactor into its original oxidized form, oxygen or a compound of general formula R1C(O)COOH is reduced, and b) in the regeneration reaction which reconverts the oxidized cofactor into its original reduced form, a compound of general formula R2CH(OH)R3 is oxidized and wherein R1, R2 and R3 in the compounds have different meanings. 1. A process for the enzymatic regeneration of the redox cofactors NAD+/NADH and/or, in particular and, NADP+/NADPH in a one-pot reaction, wherein, as a result of at least two further enzymatically catalyzed redox reactions proceeding in the same reaction batch (product-forming reactions), one of the two redox cofactors accumulates in its reduced form and, respectively, the other one in its oxidized form, characterized in that
a) in the regeneration reaction which reconverts the reduced cofactor into its original oxidized form, oxygen or a compound of general formula wherein R1represents a linear-chain or branched (C1-C4)-alkyl group or a (C1-C4)-carboxy alkyl group, is reduced, and b) in the regeneration reaction which reconverts the oxidized cofactor into its original reduced form, a (C4-C8)-cycloalcanol or a compound of general formula wherein R2and R3are independently selected from the group consisting of H, (C1-C6) alkyl, wherein alkyl is linear-chain or branched, (C1-C6) alkenyl, wherein alkenyl is linear-chain or branched and comprises one to three double bonds, aryl, C6-C12aryl, carboxyl, (C1-C4) carboxy alkyl, cycloalkyl, or C3-C8cycloalkyl, is oxidized. 2. A process according to a) during the regeneration of the oxidized cofactor, a compound of general formula wherein R1represents a substituted or unsubstituted C1-C4 alkyl group, is reduced, and b) during the regeneration of the reduced cofactor, a compound of general formula wherein R2and R3independently of each other are selected from the group consisting of 1) —H, 2) —(C1-C6) alkyl, wherein alkyl is linear-chain or branched, 3) —(C1-C6) alkenyl, wherein alkenyl is linear-chain or branched and optionally comprises up to three double bonds, 4) -cycloalkyl, C3-C8cycloalkyl, 5) -aryl, C6-C12aryl, 6) —(C1-C4) carboxy alkyl, in case that the compound of formula I is pyruvate, optionally also carboxyl; is oxidized. 3. A process according to 4. A process according to 5. A process according to 6. A process according to 7. A process according to 8. A process according to 9. A process according to wherein R4denotes hydrogen, a methyl group, a hydroxy group or an oxo group, R5denotes hydrogen, a hydroxy group, an oxo group or a methyl group, R6denotes hydrogen or a hydroxy group, R7denotes hydrogen, —COR13, wherein R13is a C1-C4alkyl group which is unsubstituted or substituted with a hydroxy group, or a C1-C4carboxy alkyl group which is substituted, in particular with a hydroxy group, or unsubstituted, or R6and R7together denote an oxo group, R8denotes hydrogen, a methyl group, a hydroxy group or an oxo group, R9denotes hydrogen, a methyl group, a hydroxy group or an oxo group, R10denotes hydrogen, a methyl group or halogen, R11denotes hydrogen, a methyl group, a hydroxy group, an oxo group or halogen, and R12denotes hydrogen, a hydroxy group, an oxo group or a methyl group, wherein the structural element denotes a benzene ring or a ring comprising 6 carbon atoms and 0, 1 or 2 C—C-double bonds; in particular wherein the substrate(s) is/are provided at a concentration of <5% (w/v) in the reaction batch for the reduction reaction(s) involved in the formation of the product. 10. A process according to into testosterone of formula 11. A process according to into ketolithocholic acid of formula by oxidation, and into the stereoisomeric hydroxy compound 3α7β-dihydroxy-5β-cholanic acid (ursodeoxycholic acid) of formula by subsequent reduction, using two opposite stereospecific hydroxysteroid dehydrogenases, in particular whereby the oxidation reaction is catalyzed by a 7α-hydroxysteroid dehydrogenase from 12. A process according to either A) via oxidation to obtain 3α,7α-dihydroxy-12-oxo-5β-cholansäure (12-oxo-CDC) of formula which is further reacted to obtain 3α-hydroxy-7,12-dioxo-5β-cholanic acid (12oxo-KLC) of formula and subsequent reduction to the stereoisomeric hydroxy compound 3α,7β-dihydroxy-12-oxo-5β-cholanic acid (12-keto-ursodeoxycholanic acid) of formula or B) via oxidation to obtain 3α,12α-dihydroxy-7-oxo-5β-cholanic acid of formula followed by enzymatic oxidation to obtain 3α-hydroxy-7,12-dioxo-5β-cholanic acid(12oxo-KLC) of formula XI, and subsequent reduktion to obtain the stereoisomic hydroxy compound 3α,7β-dihydroxy-12-oxo-5β-cholansäure (12-keto-ursodeoxycholsäure) of formula XII, or C) via oxidation to obtain 3α,12α-dihydroxy-7-oxo-5β-cholanic acid of formula XIII, followed by enzymatic reduction to obtain 3α,7β,12α-triydroxy-5β-cholanic acid of formula and subsequent oxidation to obtain the stereoisomic hydroxy compound 3α,7β-dihydroxy-12-oxo-5β-cholanic acid (12-keto-ursodeoxycholanic acid) of formula XII; using 3 stereospecific hydroxysteroid dehydrogenases, 2 of which have opposite stereospecifity. 13. A process according to 14. A process according to 15. A process according to 16. A process according to 17. A process according to 18. A process according to BACKGROUND OF THE INVENTION
PRIOR ART
OBJECT AND DESCRIPTION OF THE PROCESS
R4denotes hydrogen, a methyl group, a hydroxy group or an oxo group,
R5denotes hydrogen, a hydroxy group, an oxo group or a methyl group,
R6denotes hydrogen or a hydroxy group,
R7denotes hydrogen, —COR13, wherein R13is a C1-C4alkyl group which is unsubstituted or substituted with a hydroxy group, or a C1-C4carboxy alkyl group which is substituted, in particular with a hydroxy group, or unsubstituted,
or R6and R7together denote an oxo group,
R8denotes hydrogen, a methyl group, a hydroxy group or an oxo group,
R9denotes hydrogen, a methyl group, a hydroxy group or an oxo group,
R10denotes hydrogen, a methyl group or a halogen,
R11denotes hydrogen, a methyl group, a hydroxy group, an oxo group or halogen, and
R12denotes hydrogen, a hydroxy group, an oxo group or a methyl group,
wherein the structural element
or
or
in any combination from A), B) and/or C)
e.g. using 3 stereospecific hydroxysteroid dehydrogenases, 2 of which have opposite stereospecifity.
DESCRIPTION OF THE FIGURES
Example 1
Epimerization of chenodeoxycholic acid into ursodeoxycholic acid by 7α-hydroxysteroid dehydrogenase and 7β-hydroxysteroid dehydrogenase, using a lactate dehydrogenase- and alcohol dehydrogenase-dependent cofactor regeneration system
Example 2
Epimerization of chenodeoxycholic acid into ursodeoxycholic acid by 7α-hydroxysteroid dehydrogenase and 7β-hydroxysteroid dehydrogenase, using a lactate dehydrogenase- and malate dehydrogenase-dependent cofactor regeneration system
Example 3
Epimerization of chenodeoxycholic acid into ursodeoxycholic acid by 7α-hydroxysteroid dehydrogenase and 7β-hydroxysteroid dehydrogenase, using an NADH oxidase- and alcohol dehydrogenase-dependent cofactor regeneration system
Example 4
Reprocessing and Analytics of Bile Acids
Example 5
Conversion of Glucose into Fructose Via a Xylose Reductase and a Sorbitol Dehydrogenase, Using an Alcohol Dehydrogenase for Recycling the NADPH and a Lactate Dehydrogenase for Recycling the NAD+
Example 6
Conversion of Glucose into Fructose Via a Xylose Reductase and a Sorbitol Dehydrogenase, Using an Alcohol Dehydrogenase for Recycling the NADPH and a NADH Oxidase for Recycling the NAD+
Example 7
Reprocessing and Analytics of Sugars
Example 8
Bioconversion of cholanic acid to 3α,7β-dihydroxy-12-oxo-5β-cholanic acid by 12α-hydroxysteroiddehydrogenase, 7α-hydroxysteroiddehydrogenase and 7β-hydroxysteroiddehydrogenase using a lactate dehydrogenase and an alcohol dehydrogenase dependent cofactor regeneration system
Example 9
Bioconversion of cholanic acid to 3α,7β-dihydroxy-12-oxo-5β-cholanic acid by 12α-hydroxysteroid dehydrogenase, 7α-hydroxysteroid dehydrogenase and 7β-hHydroxysteroid dehydrogenase using a lactate dehydrogenase, NADH-oxidase and alcohol dehydrogenase dependent cofactor regeneration system
Example 10
Epimerization of chenodeoxy cholanic acid into ursodeoxy cholanic acid using 7α-hydroxysteroid dehydrogenase and 7β-hydroxysteroid dehydrogenase under use of a lactate dehydrogenase and alcohol dehydrogenase dependent cofactor regeneration system. Advantage of adding manganese chlorid (MnCl2)
Example 11
Epimerization of chenodeoxy cholanic acid to ursodeoxy cholanic acid by 7α-hydroxysteroid dehydrogenase and 7β-hydroxysteroid dehydrogenase under use of an alcohol dehydrogenase dependent cofaktor regeneration system as well as a combined lactate dehydrogenase and NADH oxidase dependent cofactor regeneration system
Example 12
Epimerization of chenodeoxy cholanic acid to ursodeoxy cholanic acid by 7α-hydroxysteroid dehydrogenase and 7β-hydroxysteroid dehydrogenase under use of an alcohol dehydrogenase dependent cofaktor regeneration system as well as a combined lactate dehydrogenase and NADH oxidase dependent cofactor regeneration system. Additiv effect of 2-pentanol and 2-propanol
Example 13
Workup and Analytics of Bile Acids










