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    • 专利摘要:
    PROCESS FOR ENZYMATIC REGENERATION OF REDOX COFACTORS. The present invention relates to a process for enzymatic regeneration of the NAD+/NADH and NADP+/NADPH redox cofactors in a one-pot reaction, in which, as a result of at least two other enzymatically catalyzed redox reactions that process in the same reaction charge (product formation reactions), one of the redox cofactors accumulates in its reduced form and, respectively, the other cofactor in its oxidized form, characterized by the fact that a) in the regeneration reaction that reconverts the reduced cofactor into its original oxidized form, oxygen or a compound of the general formula R1C(O)COOH is reduced, and b) in the regeneration reaction which reconverts the oxidized cofactor to its original reduced form, a compound of the general formula R2CH(OH)R3 is oxidized and wherein R 1 , R 2 and R 3 in the compounds have different meanings.
    公开号:BR112014019287B1
    申请号:R112014019287-1
    申请日:2013-02-06
    公开日:2021-07-06
    发明作者:Ortwin Ertl；Nicole Staunig；Marta Sut；Bernd Mayer
    申请人:Annikki Gmbh；
    IPC主号:
    专利说明:

    Background of the invention
    [001] The present invention relates to a process for enzymatic regeneration of the NAD+/NADH and NADP+/NADPH redox cofactors in a one-pot reaction, in which, as a result of at least two other enzymatically catalyzed redox reactions that process at the same reaction charge (= product formation reactions), one of the redox cofactors accumulates in its reduced form and, respectively, the other cofactor in its oxidized form. prior technique
    [002] Enzymatically catalyzed redox reactions are used in industrial operations, for example, in the production of chiral alcohols, a-amino acids and a-hydroxy acids. Most enzymes employed in industrial redox reactions use cofactors such as NADH or NADPH. Among enzymatic redox reactions, those are particularly interesting in which redox cofactors are restored by in situ cofactor regeneration systems. The reason for this purpose 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 several cofactor regeneration systems.
    [003] The regeneration systems described so far can be classified as: enzyme-linked, substrate-bound, in vivo (natural cofactor regeneration systems in living organisms), photochemical, chemical or electro-enzymatic. The process described here refers to an enzyme-linked regeneration system. Advantages of enzyme-linked systems have high selectivity, applicability for the production of various products and a high rate of cofactor reuse (total change number, TTN).
    [004] In the mid-1990s, a first industrial process using an enzyme-linked cofactor regeneration system was employed on a ton scale. In said process, Candida boidinii formate dehydrogenase was used. Known industrial processes typically use one redox enzyme for product synthesis and another enzyme for cofactor regeneration.
    [005] Processes in which two or more enzymatic redox reactions that are involved in product formation and two enzymatic systems for cofactor regeneration (simultaneously or sequentially) are processing in a reaction charge without an intermediate being isolated must be distinguished from it. Recently, such enzymatic cascade reactions - here called one-pot reactions - have attracted significant attention as they effectively reduce operating costs, operating time and environmental impacts. Furthermore, enzymatic cascades of redox reactions facilitate transformations that are not easy to implement by conventional chemical methods.
    [006] It is therefore a challenge to carry out several reactions (oxidation and reduction) simultaneously in a one-pot reaction with a parallel cofactor regeneration, since highly divergent reaction conditions are often required for the individual transformations. Until now, only a very small number of one-pot experiments comprising oxidation and reduction reactions with associated cofactor regeneration systems have been performed.
    [007] In the literature (Advanced Synth. Catal., 2008, Volume 351 Issue 9, p. 1303-1311), the experience of a one pot reaction using 7a-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. Because both processes, a lactate dehydrogenase ( NAD+ dependent) and a glucose dehydrogenase (NADP+ dependent) were used as a cofactor regeneration system. Pyruvate and glucose were used as co-substrates. Although this process was originally aimed at in a true one pot process, the final oxidation and reduction reaction were carried out separately. In doing so, the separation of oxidative and reductive steps took place in a reactor or in a so-called "tea bag" reactor or in a membrane reactor. Said division was necessary in order to avoid the production of by-products due to the low selectivity of the NADPH-glucose cofactor dehydrogenase. Therefore, in the one-pot reaction, glucose dehydrogenase NADP+ also partially covered NAD+, which prevented oxidation. In the described process, only 12.5 mM (~0.5%) of the substrate cholic acid was used, which makes the process uninteresting from an ecological point of view.
    [008] Furthermore, an attempt to carry out the deracemization of secondary alcohol racemates 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). Deracemization of secondary alcohols was achieved via two alcohol dehydrogenases (S- and R-specific) with cofactor specificities. In said system, NADP was regenerated by NADPH oxidase (production of methyl peroxide) and NADH was regenerated by formate dehydrogenase. Format and oxygen were used as co-substrates. In those 4 system enzymes were used without separating oxidative and reductive steps. A disadvantage of the process is the very low concentration of used substrate of 0.2-0.5%, which is unsuitable for industrial purposes.
    [009] Another 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 give the ketone and then reduced to the corresponding optical antipodes, in which two alcohol dehydrogenases having opposite stereoselectives 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 the regeneration of the cofactors, various enzymes such as formate dehydrogenase, glucose dehydrogenase, lactate dehydrogenase were A disadvantage of said process is the low concentration of the substrates used.
    [0010] A disadvantage of one pot enzymatic methods involving known cofactor regeneration systems altogether is the very low substrate concentration, which is inefficient for industrial processes.
    [0011] In contrast to this, many individual enzymatic redox reactions are already known in which cofactor regeneration systems are used. Experiments have been 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 to NADH (format as a co-substrate), alcohol dehydrogenase from Pseudomonas sp. for NADH (2-propanol as a co-substrate), dehydrogenase for NADH and NADPH (H2 as a co-substrate), L. mesenteroides glucose-6-phosphate dehydrogenase for NADPH (glucose-6-phosphate as a co-substrate), glucose dehydrogenase to NADH and NADPH (glucose as a co-substrate), NADH oxidase to NADH (O2 as a co-substrate) and phosphite dehydrogenase to NADH (phosphite as a co-substrate).
    [0012] An example of use of such individual redox reactions is the production of chiral hydroxy compounds, starting from appropriate pro-chiral keto compounds. In said process, the cofactor is regenerated by means of an additional enzyme. These methods have in common that they constitute an isolated reduction reaction and regenerate NAD(P)H (see eg EP 1 152 054).
    [0013] Enzymatic processes using hydroxysteroid dehydrogenases, coupled with a cofactor regeneration system, which proceed at higher substrate concentrations (about >1%), have been described (EP 1 731 618; WO 2007/118644 ; Appl. Microbiol. Biotechol., 2011 Volume 90 p. 127-135). In said process, the cofactors NAD(P)H or NAD(P) were regenerated by means of different enzymes such as, for example, lactate dehydrogenase (pyruvate as a co-substrate), alcohol dehydrogenase from T. brockii (isopropanol) as a co-substrate), alcohol dehydrogenase from L. brevis, L. minor, Leuconostoc carnosum, T. ethanolicus, Clostridium beijerinckii. Therefore, these known processes refer merely to isolated simple reactions for the oxidation of a hydroxy compound or for the reduction of an oxo compound.
    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 amidation of pyruvate by alanine dehydrogenase. The pyruvate that emerges during cofactor regeneration was subsequently used in the product formation reaction.
    [0015] In WO 2004/022764, it is likewise described to regenerate NADH by malate dehydrogenase. Unlike the previously described publication, pyruvate that emerges during the oxidative decarboxylation of malate has not been further used.
    [0016] An example of an enzymatic reduction of D-xylose to xylitol that a cofactor regeneration system has been described (FEBS J., 2005, Volume 272, p. 3816-3827). An NADPH-dependent mutant of phosphite dehydrogenase from Pseudomonas sp. was used as the cofactor regeneration enzyme. This is also a simple reaction to product formation.
    [0017] Other 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; US patent no. 7,163,815; Biochem. Eng. J., 2008, Volume 39(2) p. 319-327; EP 1 285 962). In said systems, a NAD(P)H-dependent oxidase from Lactobacillus brevis pi Lactobacillus sanfranciscensis was used as the cofactor regeneration enzyme. Experiments likewise constitute simple reactions to the formation of a product.
    [0018] In WO 2011/000693 a 17beta-hydroxysteroid dehydrogenase as well as a process are described which allow to carry out redox reactions at the 17-position of 4-androstene-3,17-dione. Again, this is an isolated reduction reaction. The above mentioned individual processing 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 better switching due to cascade reactions enzymatic. Purpose and description of the process
    [0019] The objective of the present invention was to provide a process for the regeneration of the redox cofactors NAD+/NADH and/or, for example, e, NADP+/NADPH in order to carry out with them two or more redox reactions enzymatically catalyzed in a reaction charge in an economical way.
    [0020] According to the present invention, said objective is achieved in a process of the species mentioned above, by the fact that a process for enzymatic regeneration of the redox cofactors NAD+/NADH and/or, for example, e, NADP+ /NADPH in a one-pot reaction is provided, whereby, as a result of at least two other enzymatically catalyzed redox reactions that process at the same reaction charge (product formation reactions), one of the redox cofactors accumulates in its reduced form and, respectively, the other cofactor in its oxidized form,
    [0021] this process is characterized by the fact that
    [0022] in the regeneration reaction that reconverts the reduced cofactor to its original oxidized form, oxygen or a compound of the general formula

    [0023] wherein R1 represents a branched or straight-chain (C1-C4) alkyl group or a (C1-C4) carboxy alkyl group, is reduced, and
    [0024] in the regeneration reaction that reconverts the oxidized cofactor to its original reduced form, a (C4-C8) cycloalkanol or a compound of the general formula

    [0025] wherein R2 and R3 are independently selected from the group consisting of H, (C1-C6) alkyl, wherein alkyl is branched or straight-chain, (C1-C6) alkenyl, wherein alkenyl is branched or straight-chain linear and comprises one to three double bonds, aryl, in particular C6-C12 aryl, carboxyl, or (C1-C4) carboxy alkyl, in particular also cycloalkyl, for example C3-C8 cycloalkyl, is oxidized.
    [0026] A process provided in accordance with the present invention and herein also called "process according to (of) the present invention".
    [0027] In another aspect, the present invention provides a process according to the present invention for the enzymatic regeneration of the redox cofactors NAD+/NADH and/or, for example, e, NADP+/NADPH in a reaction of a pot, in which, as a result of at least two other enzymatically catalyzed redox reactions that process at the same reaction charge (= product formation reactions), one of the redox cofactors accumulates in its reduced form and, respectively, the another cofactor in its oxidized form,
    [0028] this process is characterized by the fact that
    [0029] during the regeneration of the oxidized cofactor, a compound of the general formula

    [0030] wherein R1 represents a substituted or unsubstituted C1-C4 alkyl group, is reduced, and
    [0031] during the regeneration of the reduced cofactor, a compound of the general formula

    [0032] where R2 and R3 independently of each other are selected from the group consisting of
    [0033] -H,
    [0034] -(C'-C6) alkyl, where alkyl is branched or straight chain,
    [0035] -(C1-C6) alkenyl, wherein alkenyl is branched or straight-chain and optionally comprises up to three double bonds,
    [0036] -cycloalkyl, in particular C3-C8 cycloalkyl,
    [0037] -aryl, and in particular C6-C12 aryl,
    [0038] -(C1-C4) carboxy alkyl, in the case of compound I it is pyruvate, optionally also carboxyl; is oxidized,
    [0039] In another aspect, in a process according to the present invention, R2 and R3 independently of each other are selected from the group consisting of H, (C1-C6) alkyl, wherein alkyl is branched or straight-chain, (C1-C6) alkenyl, wherein alkenyl is branched or straight-chained and comprises one to three double bonds, aryl, in particular C6-C12 aryl, carboxyl, or (C1-C4) carboxy alkyl.
    [0040] 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 allowed to perform the required oxidation and reduction reaction as well as the associated reactions for cofactor regeneration in a reaction charge and, at the same time, to use significantly higher substrate concentrations than according to the prior art.
    [0041] In a process according to the present invention, cofactors NADH and/or NADPH are used. There, NAD+ means the oxidized form and NADH means the reduced form of adenine dinucleotide of nicotinamide, while NADP+ means the oxidized form and NADPH means the reduced form of adenine dinucleotide of nicotinamide phosphate.
    [0042] Enzymatically catalyzed redox reactions which are not part of the cofactor regeneration and, in a process according to the present invention, are involved in product formation are here called "oxidation reaction(s)" and "reduction reaction" tion(s)". "Oxidation reaction(s)" and "reduction reaction(s)" are summarized under the term "product formation reactions". The product formation reactions in a process according to the present invention comprise, in each case, at least one oxidation reaction and at least one reduction reaction.
    [0043] If NAD+ is used as a cofactor for the oxidation reaction(s), NADPH is the cofactor for the reduction reaction(s). If NADP+form is used as a cofactor for the oxidation reaction(s), NADH is the cofactor for the reduction reaction(s).
    [0044] In a process according to the present invention, oxidation reaction(s) and reduction reaction(s) can be carried out either chronologically parallel or in chronological succession, preferably chronologically parallel in the same reaction charge.
    [0045] Compounds that are used for the purpose of forming a product are here called substrates. Compounds that are reacted during cofactor regeneration are here called co-substrates.
    [0046] In a process according to the present invention, a substrate as well as several substrates can be used. By doing this, reduction and/or oxidation reaction(s) can occur on the same substrate (molecular main chain) and also on different substrates, preferably on the same substrate. Furthermore, in a process according to the present invention, reduction and/or oxidation reactions may take place on the same or different functional groups.
    [0047] 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 give the corresponding ketone and subsequent reduction to the opposite stereospecific hydroxy compound.
    [0048] A process in which two or more enzymatic redox reactions involved in the formation of a product and two enzymatic systems for cofactor regeneration processed in a reaction charge without an intermediate being isolated is here called a reaction of a pot".
    [0049] The mention of an acid or a salt of an acid here includes the unnamed term thereof. Likewise, the mention of acids, in particular bile acids, here includes all esters derived from them. Furthermore, compounds (partially) provided with protective groups are included in the mention of the underlying substances.
    [0050] In a preferred embodiment of the present invention, a process according to the present invention is characterized by the fact that the oxidation reaction and the reduction reaction proceed chronologically parallel.
    [0051] In a preferred embodiment of the present invention, a process according to the present invention is characterized by the fact that both the oxidation reaction and the reduction reaction take place in the same molecular backbone.
    [0052] 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 (co-substrate) is used which is reduced to lactate by means of a lactate dehydrogenase, which means, in the regeneration reaction that reconverts the reduced cofactor to its original oxidized form, pyruvate is reduced to lactate by means of a lactate dehydrogenase.
    [0053] 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) (co-substrate ) is used that is oxidized to give acetone via an alcohol dehydrogenase, which means, in the regeneration reaction that reconverts the oxidized cofactor to its original reduced form, 2-propanol is oxidized to give acetone via an alcohol dehydrogenase .
    [0054] 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.
    [0055] In a preferred embodiment of the present invention, a process according to the present invention is characterized by the fact that, as a secondary alcohol, malate (co-substrate) is used which is oxidized to give pyruvate and CO2 through an oxaloacetate decarboxylating malate dehydrogenase ("malate enzyme"), for example, which in the regeneration reaction that reconverts the oxidized cofactor to its original reduced form, malate is oxidized to give pyruvate and CO2 by means of a dehydrogenase of malate.
    [0056] In this embodiment, the nascent pyruvate is reacted in another redox reaction that does not serve to form a product, but constitutes the second cofactor regeneration reaction.
    [0057] In a preferred embodiment of the present invention, a process according to the present invention is characterized in that it is used to carry out at least one oxidation reaction and at least one reduction reaction, respectively, at the same charge of reaction in the compounds of the general formula
    on what
    [0058] R4 means methyl, a methyl group, a hydroxy group or an oxo group,
    [0059] R5 means methyl, a hydroxy group, an oxo group or a methyl group,
    [0060] R6 means methyl or a hydroxy group,
    [0061] R7 means methyl, -COR13, wherein R13 is a C1-C4 alkyl group that is substituted or unsubstituted with a hydroxy group, or a C1-C4 carboxy alkyl group that is substituted, in particular with a hydroxy group, or is not replaced,
    [0062] or R6 and R7 together mean an oxo group,
    [0063] R8 means methyl, a methyl group, a hydroxy group or an oxo group,
    [0064] R9 means methyl, a methyl group, a hydroxy group or an oxo group,
    [0065] R10 means methyl, a methyl group or a halogen,
    [0066] R11 means methyl, a methyl group, a hydroxy group, an oxo or halogen group, and
    [0067] R12 means methyl, a hydroxy group, an oxo group or a methyl group,
    [0068] in which the structural element

    [0069] means a benzene ring or a ring comprising 6 carbon atoms and 0, 1 or 2 C-C double bonds;
    [0070] wherein the substrate(s) is/are preferably provided at a concentration of <5% (w/v) in the reaction load for the reduction reaction(s) involved( s) in the formation of the product.
    [0071] In a preferred embodiment of the present invention, a process according to the present invention is characterized by the fact that an enzymatic conversion of dehydroepiandrosterone (DHEA) of the formula
    in formula testosterone
    occurs.
    [0072] In a preferred embodiment of the present invention, a process according to the present invention is characterized by the fact that an enzymatic epimerization of the hydroxysteroid compound of 3α,7α-dihydroxy-5β-cholanic acid (chenodeoxy cholic acid, CDC) of the formula

    [0073] occurs via oxidation to give ketolite cholic acid (KLC) of the formula

    [0074] and reduction to 3α,7β-dihydroxy-5β-conic acid ( ursode-soxy colic acid, UDC) of the formula

    [0075] for example, using two opposite stereospecific hydroxysteroid dehydrogenases.
    [0076] 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-5e-cholanic acid (cholanic acid) of the formula
    or
    [0077] cholanic (12-oxo-CDC) of the formula

    [0078] which is further reacted to obtain 3α-hydroxy-7,12-dioxo-5e-cholanic acid (12oxo-KLC) of the formula

    [0079] and reduction to the stereoisomeric hydroxy compound of 3α,7β-dihydroxy-12-oxo-5β-conic acid (12-keto- or ursodeoxycholanic acid) of the formula

    [0080] B) via oxidation to obtain 3a,12a-dihydroxy-7-oxo-5β-cholanic acid of the formula

    [0081] followed by enzymatic oxidation to obtain 3a-hydroxy-7,12-dioxo-5e-cholanic acid(12oxo-KLC) of formula XI, and subsequent reduction to obtain 3a,7e-dihydroxy-stereoisomeric hydroxy compound 12-oxo-5e-cholanic (12-keto-ursodeoxycholanic) of formula XII, or
    [0082] C) via oxidation to obtain 3a,12a-dihydroxy-7-oxo-5e-cholanic acid of formula XIII, followed by enzymatic reduction to obtain 3α,7β,12α-trihydroxy-5β-conic acid from formula

    [0083] and subsequent oxidation to obtain 3α,7β-dihydroxy-12-oxo-5β-conic acid stereoisomeric hydroxy compound (12-keto-ursodeoxycholanic acid) of formula XII; or
    [0084] in any combination of A), B) and/or C)
    [0085] for example, using 3 stereospecific hydroxysteroid dehydrogenases, 2 of which have the opposite stereospecificity.
    [0086] In a preferred embodiment of the present invention, a process according to the present invention is characterized by the fact that a C5- or C6 sugar is used as a substrate, for example, that process is used for the isomerization of C5- or C6 sugars.
    [0087] In a preferred embodiment of the present invention, a process according to the present invention is characterized by the fact that a glucose isomerization occurs via reduction to sorbitol and oxidation to give fructose, for example, which process is used for isomerization of glucose via reduction to sorbitol and subsequent oxidation to give fructose.
    [0088] A process according to the present invention is preferably carried out in an aqueous system, in which it is possible that the substrate for the oxidation and reduction reaction is partially provided in an undissolved state in the form of a suspension and /or as a second liquid phase.
    [0089] In a particular embodiment, a process according to the present invention is characterized by the fact that the substrate(s) for the oxidation reaction(s) involved in the formation of a product is /are provided in the reaction charge at a concentration of at least 5% (w/v) and more, preferably 7% (w/v) and more, particularly preferably 9% (w/v) and more, for example, from 5% (w/v) to 20% (w/v), such as from 5% (w/v) to 15% (w/v), for example, from 5% (w/v) v) to 12% (w/v), such as from 5% (w/v) to 10% (w/v).
    [0090] In a particular embodiment, a process according to the present invention is characterized by the fact that, on the whole, a change of >70%, in particular >90%, is achieved in the product formation reactions.
    [0091] In a process according to the present invention, a buffer may 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. In addition, or alternatively, ions for stabilizing 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 of NAD(P)+ and NAD(P)H is usually between 0.001 mM and 10 mM, preferably between 0.01 mM and 1 mM.
    [0092] Depending on the enzymes used, the process according to the present invention can be carried out at a temperature ranging from 10°C to 70°C, preferably from 20°C to 45°C.
    [0093] Hydroxysteroid dehydrogenases (HSDH) are understood to be enzymes that catalyze the oxidation of the hydroxy groups to give the corresponding keto groups or, conversely, the reduction of keto groups to the corresponding hydroxy groups in the steroid skeleton.
    Appropriate hydroxysteroid dehydrogenases that can be used for the 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 (Clostridium absonum, Clostridium sordelii), Escherichia coli or Bacteroides fragilis.
    [0096] Appropriate enzymes with 7β-HSDH activity can be obtained, for example, from Ruminococcus sp. or Clostridium absonum.
    Appropriate lactate dehydrogenases can be obtained, for example, from Oryctolagus cuniculus.
    [0098] Appropriate alcohol dehydrogenases can be obtained, for example, from Lactobacillus kefir.
    An appropriate xylose reductase can be obtained, for example, from Candida tropicalis.
    Appropriate sorbitol dehydrogenases can be obtained, for example, from sheep liver, Bacillus subtilis or Malus domestica.
    Suitable NADH oxidases can be obtained, for example, from Leuconostoc mesenteroides, Streptococcus mutans, Clostridium aminovalericum.
    [00102] In a process according to the present invention, enzymes are preferably used as recombinantly ultra-expressed proteins in E. coli, wherein the corresponding cell lysates preferably remain without any further purification. Thus, the 1 U enzyme unit corresponds to the amount of enzyme that is needed for the reaction of 1 µmol of substrate per min. Description of Figures
    [00103] Fig. 1 shows the reaction scheme of the epimerization of chenodeoxy cholic acid in ursodeoxy cholic acid via the intermediate 3a-hydroxy-7oxo-5e-cholanic acid, with cofactor regeneration using 2-propanol and pyruvate.
    [00104] Fig. 2 shows the reaction scheme of the epimerization of chenodeoxy cholic acid to ursodeoxy cholic acid via the intermediate 3α-hydroxy-7oxo-5e-cholanic acid, with cofactor regeneration using malate and pyruvate.
    [00105] Fig. 3 shows the reaction scheme of the epimerization of chenodeoxy cholic acid to ursodeoxy cholic acid via the intermediate 3α-hydroxy-7oxo-5e-cholanic acid, with cofactor regeneration using 2-propanol and oxygen.
    [00106] Fig. 4 shows the reaction scheme of the isomerization of glucose to fructose, with cofactor regeneration using 2-propanol and pyruvate.
    [00107] Fig. 5 shows the reaction scheme of the isomerization of glucose to fructose, with cofactor regeneration using 2-propanol and oxygen.
    [00108] Fig. 6 shows the reaction scheme of cholanic epimerization to give 3α,7β-dihydroxy-12-oxo-5β-conic acid via 3a,7a-dihydroxy-12-oxo-5e— cholanic and 3α-hydroxy-7,12-dioxo-5-e-cholanic acid intermediates with cofactor regeneration using 2-propanol and pyruvate.
    [00109] Fig. 7 shows the reaction scheme of the epimerization of cholanic acid to give 3a,7e-dihydroxy-12-oxo-5e-cholanic acid via 3a,7a-dihydroxy-12-oxo-5e-cholanic acid and cholanic acid 3a-hydroxy-7,12-dioxo-5e-cholanic intermediates with cofactor regeneration from the epimerization of cholanic acid to give 3a,7e-dihydroxy-12-oxo-5e-cholanic acid via the 3a,7a-dihydroxy- 12-oxo-5e-cholanic and 3a-hydroxy-7,12-dioxo-5e-cholanic acid intermediates with cofactor regeneration using using 2-propanol and oxygen.
    [00110] Fig. 8 and Fig. 9 show the reaction schemes of the epimerization of cholanic acid to give 3α,7β-dihydr0xy-12-oxo-5β-cholanic acid via the intermediates of 3α,7α-dihydr0xy-12 -oxo-5β- cholanic and 3α-hydroxy-7,12-dioxo-5β- cholanic acid cofactor regeneration using 2-propanol, pyruvate and oxygen.
    [00111] Fig. 10 shows possible reaction schemes of the epimerization of cholanic acid to give 3α,7β-dihydr0xy-12-oxo-5β-cholanic acid via different intermediates and regeneration of cofactor systems. For the regeneration of NAD+ alternatively lactate dehydrogenase (pyruvate as a substrate) and NADH oxidase (oxygen as a substrate) were used. For the regeneration of alcohol dehydrogenase from NADPH (isopropanol as a substrate) was used.
    [00112] Fig. 11 shows the reaction scheme of the epimerization of chenodeoxycholanic acid to give ursodeoxycholanic acid via the 3a-hydroxy-7oxo-5e-cholanic acid intermediate (7-ketolitocholanic acid = 7K-LCA = KLC) with cofactor regeneration using 2-propanol and 2-pentanol (in each case alcohol dehydrogenase) as well as pyruvate (lactate dehydrogenase) and oxagen (NADH oxidase).
    [00113] In the figures the following abbreviations when used: Bacillus subtilis sorbitol dehydrogenase BsSDH CA = 3a,7a,12a-trihydroxy-5e-cholanic acid 7β-CA = 3α,7β,12α, -trihydroxy-5e-cholanic acid Caoxo NADH oxidase from Clostridium aminovalericum CDC, CDCA 3a,7a-dihydroxy-5e-cholanic acid CtXR Candida tropicalis xylose reductase 7α-HSDH dehydrogenase 7a-hydroxysteroid 7β-HSDH dehydrogenase 7e-hydroxysteroid 12α-hydroxysteroid 12α-hydroxysteroid 12α-hydroxysteroid = 12asteroid KLC 3a-hydroxy-7-oxo-5e-cholanic acid 7K-LCA = 3a-hydroxy-7-oxo-5e-cholanic acid LacDH lactate dehydrogenase NAD(H)-dependent LkADH Lactobacillus kefir NADP(H)-dependent alcohol dehydrogenase Lmoxid = NADH oxidase from Leuconostoc mesenteroides MalDH E. coli malate dehydrogenase NADP(H)-dependent 7oxo-CA = 3α,12a-dihydroxy-7-oxo-5e-cholanic acid 12oxo-CDC = 3a,7a-dihydroxy acid -12-oxo-5e-cholanic 12oxo-KLC = 3a-hydroxy-7,12-dioxo-5e-cholanic 12oxo-UDC = 3a,7e-dihydroxy-12-oxo-5e-cholanic acid SISDH of Sheep liver sorbitol hydrogenase SmOxo NADH oxidase from Streptococcus mutans UDC. UDCA 3a,7p-dihydroxy-5p-cholanic acid
    [00114] In the following examples, all temperature data is given in degrees Celsius (°C). The following abbreviations are used: EtOAc ethyl acetate h hour(s) IPA (2-propanol) isopropyl alcohol MeOH methanol Rt room temperature Example 1 Epimerization of chenodeoxy cholic acid to ursodeoxy cholic acid by 7α-hydroxysteroid dehydrogenase and 7β-hydroxysteroid dehydrogenase, using a lactate dehydrogenase and alcohol dehydrogenase-dependent cofactor regeneration system
    [00115] A load of 0.5 mL contains 50 mg of chenodeoxy cholic acid, 12 U of recombinant 7α-hydroxysteroid dehydrogenase from Escherichia coli, 6 U of recombinant 7β-hydroxysteroid dehydrogenase from Ruminococcus torques as well as NAD+a 0.5 mM and 0.3 mM NADPH. For NAD+ regeneration, 6 U of recombinant lactate dehydrogenase and 350 mM sodium pyruvate are used. For NADPH regeneration, 6 U of recombinant alcohol dehydrogenase from Lactobacillus kefir and initially 2.4% IPA (w/v) are used. The reaction is carried out in an aqueous potassium phosphate buffer (100 mM, pH = 7.8) at 25°C, with continuous stirring (850 rpm). An open system continues to be used in order to facilitate the evaporation of acetone and to shift the reaction towards ursodeoxycholic acid. 1.6% (w/v) IPA is additionally dosed in after 6 h, 2.4% (w/v) IPA after 16 h, 3.9% (w/v) IPA after 24 h e0.8% (w/v) IPA after 40 h. Furthermore, 20 µl of 4-methyl-2-pentanol is added after 24 h. 200 µl of 2-pentanol as well as 1.6% (w/v) of IPA are added after 46 h. After 48 h, the ratio of ursodeoxycholic acid to all bile acids in the reaction mixture is >97%. Example 2 Epimerization of chenodeoxy cholic acid to ursodeoxy cholic acid by 7α-hydroxysteroid dehydrogenase and 7β-hydroxysteroid dehydrogenase, using a lactate dehydrogenase and malate dehydrogenase-dependent cofactor regeneration system
    [00116] A load of 0.5 mL contains 50 mg of chenodeoxy cholic acid, 20 U of recombinant 7a-hydroxysteroid dehydrogenase from Escherichia coli, 20 U of recombinant 7β-hydroxysteroid dehydrogenase from Ruminococcus torques as well as NAD+a 1 mM and 1 mM NADPH. For NAD+ regeneration, 10 U of lactate dehydrogenase (Sigma-Aldrich) is used, and for starting the reaction, 16.5 mM sodium pyruvate is used. For NADPH regeneration, 20 U of recombinant malate dehydrogenase from Escherichia coli and 320 mM sodium malate are used. The reaction is carried out in an aqueous potassium phosphate buffer (100 mM, pH = 7.8) at 25°C, with continuous stirring (850 rpm). An open system continues to be used in order to allow nascent CO2 to escape. 20 U of 7α-HSDH as well as 10 U of lactate dehydrogenase were additionally dosed at after 16 h and after 40 h. 10 U of 7β-HSDH was additionally dosed at after 20 h, 24 h, 44 h and 48 h. Furthermore, 10 U of malate dehydrogenase was additionally dosed in after 40 h. After 72 h, the ratio of ursodeoxycholic acid to all bile acids in the reaction mixture is about 90%. Example 3 Epimerization of chenodeoxy cholic acid to ursodeoxy cholic acid by 7α-hydroxysteroid dehydrogenase and 7β-hydroxysteroid dehydrogenase, using a cofactor regeneration system dependent on NADH oxidase and alcohol dehydrogenase
    [00117] A load of 0.5 mL contains 50 mg of chenodeoxy cholic acid, 12 U of recombinant 7α-hydroxysteroid dehydrogenase from Escherichia coli, 7.5 U of recombinant 7β-hydroxysteroid dehydrogenase from Ruminococcus torques as well as NAD + at 1 mM and NADPH at 1 mM. For NAD+ regeneration, 20 U of recombinant NADH oxidase from Clostridium aminovalericum are used. For NADPH regeneration, 5 U of recombinant alcohol dehydrogenase from Lactobacillus kefir and initially 2% IPA (w/v) are used. The reaction is carried out in an aqueous potassium phosphate buffer (100 mM, pH 6) at 25°C, with continuous stirring (850 rpm). An open system continues to be used in order to facilitate the evaporation of acetone and to shift the reaction towards ursodeoxycholic acid. 2% IPA is additionally dosed after 18 h, 22 h, 26 h and 41 h as well as 5% IPA after 41 h and after 48 h. 20 U of NADH oxidase are additionally administered in after 24 h, and 7.5 U of 7β-hydroxysteroid dehydrogenase as well as 5 U of alcohol dehydrogenase are additionally administered in after 41 h. After 48 h, the proportion of ursodeoxycholic acid to all bile acids in the reaction mixture is about 95-98%. Example 4 Bile acid reprocessing and analytics
    [00118] On completion of the reactions as described in Examples 1 to 3, the reaction mixture is extracted with EtOAc. Subsequently, the solvent is removed by evaporation. The evaporation residue is dissolved in a mixture of MeOH:acetonitrile:sodium phosphate buffer pH = 3, 0.78 g/L (40:30:37) and the conversion of chenodeoxy cholic acid to ursodeoxy cholic acid is monitored by HPLC. Thus, a reverse phase separation column (ZORBAX®Eclipse® XDB C18, flow 0.8 mL/min) and a fraction of light detector (RID), Agilent 1260 Infinity®, both from Agilent Technologies Inc., they're used. Example 5 Conversion of glucose to fructose via a xylose reductase and a sorbitol dehydrogenase, using an alcohol dehydrogenase to recycle NADPH and a lactate dehydrogenase to recycle NAD+
    [00119] A load of 0.5 ml contains 50 mg/ml glucose and 6 U/ml recombinant xylose reductase from Candida tropicalis (ultra-expressed in E. coli BL21 (DE3)) and NADP+ to 0.1 mM. For cofactor regeneration, 7% IPA and recombinant alcohol dehydrogenase from Lactobacillus kefir (ultra-expressed in E. coli BL21 (DE3)) are added. Enzymes are used in the form of cell lysates. The reaction takes place for 24 h at 40°C and pH = 9 (50 mM tris HCl buffer) in an open system with continuous stirring (900 rpm). The open system leads to the removal of acetone, which directs the reaction towards the formation of sorbitol. In the open system, water and IPA evaporate too, so that they are additionally administered at after 6 h and after 21 h. In this way every hour a total volume of 0.5 ml as well as an IPA concentration of 7% (v/v) is adjusted. After 24 h, the reaction vessel is incubated at 60°C under vacuum in order to inactivate the enzymes and evaporate the organic solvents. After cooling to room temperature, recombinant sorbitol dehydrogenase from Bacillus subtilis (ultra-expressed in E. coli BL21 (DE3)) is added at a concentration of 5 U/ml, ZnCl2 at a final concentration of 1 mM and NAD+ at a final concentration of 0.1 mM. For cofactor regeneration, 5 U/mL (final concentration) of lactate dehydrogenase from rabbit muscles (Sigma Aldrich) and 300 mM pyruvate are used. The charge is covered up to 0.5 mL with water. The reaction takes place for another 24 h at 40°C in a closed system with continuous stirring (900 rpm). Conversion of D-glucose to D-fructose of >90% is achieved. Example 6 Conversion of glucose to fructose via a xylose reductase and a sorbitol dehydrogenase, using an alcohol dehydrogenase for NADPH recycling and an NADH oxidase for NAD+ recycling
    A load of 0.5 ml contains 50 mg/ml glucose, 6 U/ml recombinant xylose reductase from Candida tropicalis (ultra-expressed in E. coli BL21 (DE3)) and NADP+a 0, 1 mM. For cofactor regeneration, 7% (v/v) of IPA and recombinant alcohol dehydrogenase from Lactobacillus kefir (ultra-expressed in E. coli BL21 (DE3)) are added. Enzymes are used in the form of cell lysates. The reaction takes place for 24 h at 40°C and pH = 8 (50 mM Tris HCl buffer) in an open system with continuous stirring (900 rpm). The open system leads to the removal of acetone, which directs the reaction towards the formation of sorbitol. In the open system, water and IPA evaporate too, so they are additionally administered at after 6 h and after 21 h. In this way every hour a total volume of 0.5 ml as well as an IPA concentration of 7% (v/v) was adjusted. After 24 h, the reaction vessel is incubated at 60°C under vacuum in order to inactivate the IPA evaporating enzymes as well as any acetone that has formed. After cooling to room temperature, recombinant D-sorbitol dehydrogenase from Bacillus subtilis (ultra-expressed in BL21 to E. coli (DE3)) is added to a final concentration of 5 U/ml, CaCl2 to a final concentration of 1 mM and a mixture of NAD+ and NADH at a final concentration of 0.1 mM. For cofactor regeneration, 10 U/ml (final concentration) of NADH oxidase from Leuconostoc mesenteroids (ultra-expressed in E. coli BL21 (DE3)) are used. Enzymes are used in the form of cell lysates. The charge is covered to 0.5 mL with water. The reaction takes place for 24 h at 40°C in an open system, with continuous agitation (900 rpm), in order to guarantee sufficient oxygen supply for the NADH oxidase from the air. Due to the fact that open system at 40°C water evaporates. Thus, after 6 h and after 21 h it is completely filled with water to a volume of 0.5 ml. The conversion of D-glucose to D-fructose of about 98% is achieved. Example 7 Sugar reprocessing and analytics
    [00121] The load is incubated at 65°C for 10 min for enzyme inactivation and is subsequently centrifuged. The supernatant is then filtered over a 0.2 µM PVDF filter and analyzed by linker exchange HPLC (Agilent Technologies Inc.). In doing so, sugars and polyols are separated via a lead column from Showa Denko K.K. (Shodex®Sugar SP0810) with a flow of 0.5 ml/min of water (VWR International GmbH, Grade HPLC) at 80°C. Detection takes place with the aid of a light refraction detector (RID, Agilent 1260 Infinity®, Agilent Technologies Inc.). An inline filter from Agilent Technologies Inc. and, as a pre-column, an anion exchange column (Shodex®Axpak-WAG), a reversed phase column (Shodex®Asahipak®ODP-50 6E) and a pre- sugar column (SUGAR SPG) from Showa Denko KK are used. Example 8 Bioconversion of cholanic acid to 3α,7β-dihydroxy-12-oxo-5e—cholanic acid by 12α-hydroxysteroid dehydrogenase, 7a-hydroxysteroid dehydrogenase, and 7β-hydroxysteroid dehydrogenase using a lactate dehydrogenase and a alcohol dehydrogenase-dependent cofactor regeneration system
    [00122] A load of 0.5 ml contains 25 mg of cholanic acid 12.5 U of recombinant 12a-hydroxysteroid dehydrogenase from Eggertella lente or Lysinibacillus sphaericus, 16 U of recombinant 7a-hydroxysteroid dehydrogenase from Escherichia coli , 6 U of recombinant 7e-hydroxysteroid dehydrogenase from Ruminococcus torques, as well as 1 mM NAD+ and 1 mM NADPH. For the regeneration of NAD+12.5 U of recombinant lactate dehydrogenase from Oryctolagus cuniculus (muscle isoform) and 200 mM sodium pyruvate are used. For the regeneration of NADPH 5 U of recombinant alcohol dehydrogenase from Lactobacillus kefir and initially 2% IPA (w/v) are used. The reaction is carried out in an aqueous potassium phosphate buffer (100 mM, pH 7.8) at 25°C under continuous stirring (850 rpm). An open system is further used in order to allow evaporation of acetone and to shift the reaction towards 3α,7β-dihydr0xy-12-oxo-5β-cholanic acid. After 18 h and 24 h 2% IPA (w/v) is dosed in additionally. After 48 h 61% of the cholanic acid used is reacted with 3a,7a-dihydroxy-12-oxo-5e-cholanic acid. Example 9 Bioconversion of cholanic acid to 3α,7β-dihydroxy-12-oxo-5e—cholanic acid by 12a-hydroxysteroid dehydrogenase, 7a-hydroxysteroid dehydrogenase and 7β-hydroxysteroid dehydrogenase using a lactate dehydrogenase, NADH-oxidase and alcohol dehydrogenase-dependent cofactor regeneration system
    [00123] In a load of 0.5 ml it contains 25 mg of cholanic acid, 12.5 U of recombinant 12a-hydroxysteroid dehydrogenase from Eggertella lente or Lysinibacillus sphaericus, 16 U of recombinant 7a-hydroxysteroid dehydrogenase from Escherichia coli, 6 U of recombinant 7e-hydroxysteroid dehydrogenase from Ruminococcus torques, as well as 1 mM NAD+ and 1 mM NADPH. For the regeneration of NAD+5 U of recombinant NADH oxidase from Leuconostoc mesenteroides and 12.5 U of recombinant lactate dehydrogenase from Oryctolagus cuniculus (muscle isoform) and 200 mM of sodium pyruvate are used. For the regeneration of NADPH 5 U of recombinant alcohol dehydrogenase from Lactobacillus kefir and initially 2% IPA (w/v) are used. The reaction is carried out in an aqueous potassium phosphate buffer (100 mM, pH 7.8) at 25°C under continuous stirring (850 rpm).
    [00124] An open system is further used in order to allow evaporation of acetone and to shift the reaction towards 3a,7e-dihydroxy-12-oxo-5e-cholanic acid. After 18 h and 24 h 2% IPA (w/v) is dosed in additionally. After 48 h 70% of the used cholanic acid is reacted to 3α,7α-dihydroxy-12-oxo-5e-cholanic acid. Example 10 Epimerization of chenodeoxy cholanic acid to ursodeoxy cholanic acid using 7α-hydroxysteroid dehydrogenase and 7β-hydroxysteroid dehydrogenase under use of an alcohol dehydrogenase and lactate dehydrogenase-dependent cofactor regeneration system. Advantage of adding manganese chloride (MnCl2)
    [00125] A load of 0.5 mL contains 50 mg of chenodeoxy cholanic acid, 12 U of recombinant 7α-hydroxysteroid dehydrogenase from Escherichia coli, 6 U of recombinant 7β-hydroxysteroid dehydrogenase from Ruminococcus torques, as well as NAD+ at 0.5 mM and NADPH at 0.3 mM. For the regeneration of NAD+6 U of recombinant lactate dehydrogenase and 350 mM of sodium pyruvate are used. For the regeneration of NADPH 6 U of recombinant alcohol dehydrogenase from Lactobacillus kefir and initially 2.4% of IPA (w/v) are used. The reaction is carried out in an aqueous potassium phosphate buffer (100 mM, pH = 7.8) with 5 mM MnCl2 at 25°C and under continuous stirring (850 rpm). An open system is further used in order to allow evaporation of acetone and to shift the reaction towards ursodeoxy cholanic acid. 1.6% (w/v) of IPA after 6 h, 2.4% (w/v) of IPA after 16 h and 3.9% (w/v) of IPA after 24 h are dosed in additionally . After 36 h 200 μl of 2-pentanol as well as 3% (w/v) of IPA are added and after 48 h 100 μl of 2-pentanol and 4% (w/v) of IPA are dosed in additionally. After 64 h the ursodeoxy cholanic acid portion of all bile acids in a reaction mixture is >99%. in particular, the part of chenodeoxy cholanic acid is about 0.3%. In a control load without the addition of MnCl2 the chenodeoxy cholanic acid part is about 2% and the ursodeoxy cholanic acid part about 97.5% (average value of 5 experiments each). 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 cofactor regeneration system as well as a NADH-dependent cofactor regeneration system combined oxidase and lactate dehydrogenase.
    [00126] A loadq of 0.5 mL contains 50 mg of chenodeoxy cholanic acid, 12 U of recombinant 7β-hydroxysteroid dehydrogenase from Escherichia coli, 6 U of recombinant 7β-hydroxysteroid dehydrogenase from Ruminococcus torques, as well as 0.5 mM NAD+ and 0.3 mM NADPH. For the regeneration of NAD+6 U of recombinant lactate dehydrogenase and 350 mM of sodium pyruvate are used. For the regeneration of NAD+ in addition to 9 U of recombinant NADH oxidase from Leuconostoc mesenteroids, as well as 6 U of recombinant NADH oxidase from Clostridium aminovalericum are used. For the regeneration of NADPH 6 U of recombinant alcohol dehydrogenase from Lactobacillus kefir and initially 2.4% (w/v) of IPA are used. The reaction is carried out in an aqueous potassium phosphate buffer (100 mM, pH = 7.8) at 25°C under continuous stirring (850 rpm). An open system is further used in order to allow evaporation of acetone and to shift the reaction towards ursodeoxy cholanic acid. After 6 h 1.6% (w/v) of IPA, after 16 h 2.4% (w/v) of IPA and after 24 h 3.9% (w/v) of IPA are dosed in additionally. After 36 h 200 µl of 2-pentanol as well as 3% (w/v) of IPA are added and after 48 h 100 µl of 2-pentanol and 4% (w/v) of IPA are additionally administered in. After 64 h the ursodeoxy cholanic acid share of all bile acids in a reaction mixture is >99%. in particular the part of chenodeoxy cholanic acid is about 0.2%. In a control load without addition of NADH oxidase the chenodeoxy cholanic acid part is about 2% and the ursodeoxy cholanic acid part is about 97.5% (same control load as in example 11; mean values of 5 experiences each). Example 12 Epimerization of chenodeoxy cholanic acid to ursodeoxy acid 7α-hydroxysteroid dehydrogenase and 7β-hydroxysteroid dehydrogenase under use of an alcohol dehydrogenase-dependent cofactor regeneration system as well as a NADH-dependent cofactor regeneration system combined oxidase and lactate dehydrogenase. Additive effect of 2-pentanol and 2-propanol
    [00127] A load of 50 ml contains 5 g of chenodeoxy cholanic acid, 24 U/ml of recombinant 7β-hydroxysteroid dehydrogenase from Escherichia coli, 12 U/ml of recombinant 7β-hydroxysteroid dehydrogenase from Ruminococcus torques as well as 055 mM NAD+ and 0.3 mM NADPH. For the regeneration of NAD+12 U/ml recombinant lactate dehydrogenase and 350 mM sodium pyruvate are used. For the regeneration of NAD+ additionally 18 U/ml of recombinant NADH oxidase from Leuconostoc mesenteroides as well as 12 U/ml of recombinant NADH oxidase from Clostridium aminovalericum are used. For the regeneration of NADPH 12 U/ml of recombinant alcohol dehydrogenase from Lactobacillus kefir and initially 1.5% (w/v) of IPA are used. The reaction is carried out in an aqueous potassium phosphate buffer (100 mM, pH = 7.8) with 5 mM MnCl2 at 25°C. On a three-neck piston it is agitated with an agitator at about 100 rpm. Removal of acetone originating from the reaction is affected by a stream of air (about 400-600 mL/min) through the reaction vessel. Since at the same time 2-propanol is also evaporated, additional dosed administration is necessary, for example, in an amount of 75 mL (1.5 h), 0.75 mL (3 h), 0.5 mL (4 h), 0.75 ml (6 h), 0.75 ml (8 h), 0.5 ml (11 h), 0.5 ml (14 h), 0.5 ml (17 h), 0, 5ml (21h), 1ml (23h), 2.5ml (25h), 4ml (29h). After about 30 h 20 ml of 2-Pentanol as well as 2 ml of 2-Propanol are added. After 46 h of the total reaction time the 7-ketolite cholanic acid part is about 1% (related to the sum of chenodeoxy cholanic acid, ursodeoxy cholanic acid and 7-ketolite cholanic acid. In addition 2-propanol is added: 3 mL (46 h), 4 mL (52 h), 4 mL (54 h) as well as 10 mL of 2-pentanol. be decreased to less than 0.2%.The portion of ursodeoxycholanic acid is >99%. Example 13 Bile Acid Processing and Analyticals
    [00128] After completion of the reaction as described in examples 8 to 12, the bile acids that are present in the experiments can be analyzed via a method described in example 4.
    权利要求:
    Claims (12)
    [0001]
    1. Process for enzymatic regeneration of the re-dox cofactors NAD+/NADH and NADP+/NADPH in a one-pot reaction, in which, as a result of at least two more enzymatically catalyzed redox reactions occurring in the same reaction batch (formation reactions of product ), the redox cofactor NAD+/NADH accumulates in its reduced form as NADH and the redox cofactor NADP+/NADPH accumulates in its oxidized form as NADP+, characterized by the fact that: (a) in the regeneration reaction that reconverts NADH in NAD+, oxygen is reduced through an NADH oxidase or pyruvate through a lactate dehydrogenase, and (b) in the regeneration reaction that reconverts NADP+ to NADPH, 2-propanol is oxidized through a dehydrogenase of alcohol or malate by means of a malate dehydrogenase.
    [0002]
    2. Process according to claim 1, characterized in that the oxidation reaction(s) and reduction reaction(s) occur on the same substrate (molecular structure).
    [0003]
    3. Process according to claim 1 or 2, characterized by the fact that the oxidation reaction(s) and the reduction reaction(s) proceed chronologically in parallel.
    [0004]
    4. Process according to any one of claims 1 to 3, characterized in that, in the regeneration reaction that reconverts NADP+ to NADPH, 2-propanol is oxidized to acetone by means of an alcoholic dehydrogenase.
    [0005]
    5. Process according to any one of claims 1 to 3, characterized in that, in the regeneration reaction that reconverts NADH to NAD+, pyruvate is reduced to lactate by means of a lactate dehydrogenase.
    [0006]
    6. Process according to claim 5, characterized by the fact that, in the regeneration reaction that reconverts NADP+ to NADPH, malate is oxidized to pyruvate and CO2 by means of a malate-dehydrogenase.
    [0007]
    7. Process according to any one of claims 1 to 5, characterized in that it is used to carry out at least one oxidation reaction and at least one reduction reaction, respectively, in the same reaction batch in compounds of general formula
    [0008]
    8. Process according to claim 7, characterized in that it is used to convert dehydroepiandrosterone (DHEA) of the formula
    [0009]
    9. Process according to claim 7, characterized in that it is used for enzymatic epimerization of 3a,7a-dihydroxy-5e-cholanic acid (chenodeoxycholic acid) of the formula
    [0010]
    10. Process according to claim 7, characterized in that it is used for the enzymatic epimerization of 3a,7a,12a-trihydroxy-5e-cholanic acid (cholic acid) of the formula
    [0011]
    11. Process according to any of claims 1 to 6, characterized in that it is used for the isomerization of C5- or Ce-sugars, in particular, for the isomerization of glucose through sorbitol reduction and subsequent oxidation to fructose.
    [0012]
    12. Process according to any one of claims 1 to 11, characterized in that the substrate(s) for the oxidation reaction(s) involved in the formation of a product is/are supplied in the reaction batch at a concentration of 5% (w/v) and more, in particular 7% (w/v) and more, in particular 9% (w/v) and more.
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    EP2812439A1|2014-12-17|
    KR102022137B1|2019-09-17|
    RU2014136162A|2016-03-27|
    CN113337568A|2021-09-03|
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    MX358771B|2018-09-04|
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    NZ627477A|2015-12-24|
    US9644227B2|2017-05-09|
    HRP20191514T1|2019-11-29|
    CA2862384C|2021-07-13|
    AU2013218042B2|2016-07-28|
    MX2014009455A|2014-11-12|
    HK1204010A1|2015-11-06|
    TW201343623A|2013-11-01|
    SA113340270B1|2015-09-03|
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    PT2812439T|2019-09-10|
    SG11201404614XA|2014-10-30|
    KR20140127258A|2014-11-03|
    US20190323051A1|2019-10-24|
    AU2013218042A1|2014-08-07|
    PL2812439T3|2019-11-29|
    JP2015506707A|2015-03-05|
    AR089841A1|2014-09-24|
    HUE044690T2|2019-11-28|
    US20170218417A1|2017-08-03|
    MY172493A|2019-11-27|
    JP6329086B2|2018-05-23|
    US20140377798A1|2014-12-25|
    ES2742381T3|2020-02-14|
    PH12014501770B1|2014-11-10|
    WO2013117584A1|2013-08-15|
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    法律状态:
    2018-03-27| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|
    2019-08-20| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|
    2021-05-25| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|
    2021-07-06| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 06/02/2013, OBSERVADAS AS CONDICOES LEGAIS. |
    优先权:
    申请号 | 申请日 | 专利标题
    EP12450007.5|2012-02-07|
    EP12450007|2012-02-07|
    PCT/EP2012/067781|WO2013117251A1|2012-02-07|2012-09-12|Method for enzymatic redox cofactor regeneration|
    EPPCT/EP2012/067781|2012-09-12|
    ATA1284/2012|2012-12-10|
    AT12842012A|AT513721B1|2012-12-10|2012-12-10|Process for the enzymatic regeneration of redox cofactors|
    PCT/EP2013/052313|WO2013117584A1|2012-02-07|2013-02-06|Method for enzymatic redox cofactor regeneration|
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