Method and system for the enzymatic reduction of an organic keto compound

Abstract

The invention relates to a method for the enzymatic reduction of an organic keto compound in order to form an organic hydroxy compound, wherein NAD(P)H is used as a cofactor of the enzymatic reduction, and the oxidized cofactor NAD(P)+ produced during the reduction is enzymatically reduced using 2-propanol as a cosubstrate in order to form NAD(P)H, thereby forming acetone which is separated by evaporation. The method is characterized in that the evaporation is carried out in a thin-film treatment device.

Description

[0001] Method and apparatus for the enzymatic reduction of an organic keto compound

[0002] The invention relates to a process for the enzymatic reduction of an organic keto compound to an organic hydroxy compound, wherein NAD(P)H is used as a cofactor in the reduction and the oxidized cofactor NAD(P) produced in the reduction + The substance is enzymatically reduced with 2-propanol as a co-substrate to NAD(P)H, forming acetone, and the acetone is separated by evaporation. The invention further relates to an apparatus for carrying out the process.

[0003] Background of the invention

[0004] In contrast to classical chemical-catalytic reactions, biocatalytic reactions can generally be carried out either in vivo (fermentations, whole-cell processes) or in vitro (enzymatically in cell-free systems) under mild reaction conditions (aqueous environment, ambient temperature, ambient pressure). Furthermore, the biocatalysts used (such as cells or enzymes) are non-toxic and biodegradable.

[0005] An important class of enzymes are oxidoreductases, which catalyze reductions and oxidations. Approximately 50% of all oxidoreductases utilize nicotinamide adenine dinucleotide cofactors (NAD₂). + / NADH or NADP + / NADPH; hereinafter generally summarized as NAD(P)) as redox cofactors (Selles Vidal et al., 2018).

[0006] Due to the high cost of NAD(P), redox cofactors in enzymatically catalyzed reactions (in vitro) are not used in stoichiometric amounts, but in catalytic amounts. To enable the highest possible conversion of substrate to product, the cofactors required by the reaction enzymes (NAD(P)) must be present in sufficient quantities. + or NAD(P)H) are regenerated.

[0007] The regeneration of cofactors can be carried out chemically (heterogeneous catalytic, electrochemical, photocatalytic), but of course also enzymatically (Chenault et al., 1988; Wang et al., 2017).

[0008] To convert NAD(P)H from NAD(P) + For enzymatic regeneration, for example formate dehydrogenase (formate CO2) or glucose dehydrogenase (D-glucose D-gluconate) can be used (Chenault et al., 1988; Wang et al., 2017).

[0009] Another method involves using alcohol dehydrogenase (ADH) and an oxidizable alcohol such as 2-propanol (isopropanol) as a hydride donor, from which acetone is formed by oxidation. Acetone's lower boiling point facilitates removal; however, large quantities of 2-propanol are required to shift the equilibrium towards the product side (Wang et al., 2017). Alternatively, the equilibrium can also be shifted by evaporating the volatile acetone (US 10113192 B2).

[0010] The interconversion of keto and hydroxy groups via redox reactions plays a crucial role in organic (synthetic) chemistry. Both chemical and biocatalytic methods for this are described in the literature. For the enzymatic reduction of ketones to alcohols, alcohol dehydrogenases are the biocatalysts of choice, but other enzyme classes such as ene reductases or Baeyer-Villiger monooxygenases are also suitable (Hollmann et al., 2021). Furthermore, depending on the keto compound, aldo-keto reductases such as aldose reductases or various hydroxysteroid dehydrogenases (Penning, 2015), as well as xylose reductases, polyol dehydrogenases, or glucose-fructose oxidoreductase (Intasian et al., 2021) can also be used for the reduction, to name just a few examples. The state of the art thus describes a wide range of enzymes for the reduction of keto groups.

[0011] The reduction of the keto group in D-fructose leads to two diastereomers - D-sorbitol and D-mannitol.

[0012] D-Mannitol has applications as a non-cariogenic and low-calorie sweetener (for diabetic food), a pharmaceutical (diuretic, treatment of kidney failure or as an excipient in tablets and inhalers) for the production of specialty chemicals (Saha et al., 2011; Bhatt et al., 2013; Chen et al., 2020).

[0013] Since natural sources, for example in fruits and vegetables, but also in seaweed, cannot meet the increasing demand for D-mannitol (Saha et al., 2011), methods for the synthetic production of D-mannitol have been and are being developed.

[0014] More than 50,000 tons of D-mannitol per year are produced by hydrogenating a syrup consisting of 50% D-fructose and 50% D-glucose at high pressures and temperatures using a Raney nickel catalyst. The resulting product is a mixture of D-mannitol and D-sorbitol (25:75), as only half of the D-fructose is converted to D-mannitol. Separating the two sugar alcohols requires complex and expensive processes (Bhatt et al., 2013).

[0015] Biocatalytic processes for the production of D-mannitol have also been described. For example, mannitol dehydrogenases catalyze the reduction of D-fructose to D-mannitol (US 7867740 B2; Kavanagh et al., 2002; Krahulec et al., 2011), but fermentative processes are also known.

[0016] US 7358072 B2 describes a continuous fed-batch fermentation using Lactobacillus intermedius NRRL B-3693, which produced 166 g / L of D-mannitol (295 g total) and 140 g D-glucose (as a secondary carbon source) in 22 hours (resulting in 232 g of D-mannitol from a calculated volume of 1.4 liters). Overall, only about 53% of the added sugars (D-fructose / D-glucose) were converted to D-mannitol.

[0017] Enzymatic reductions of keto compounds using NAD(P)H-dependent enzymes in the presence of cofactor regeneration systems are also known. Common regeneration systems include hydrogenases (cosubstrate Hz), formate dehydrogenases (cosubstrate formate), glucose dehydrogenases (cosubstrate glucose), and also alcohol dehydrogenases (cosubstrate e.g. isopropanol) (Hollmann et al., 2021).

[0018] Zucca et al. (2009) described the conversion of cinnamaldehyde to cinnamyl alcohol in a substrate-coupled process using an alcohol dehydrogenase from Saccharomyces cerevisiae, employing ethanol or 2-propanol as the oxidizable co-substrate. An air stream was applied to the reaction mixture.

[0019] Liang et al. (2010) described the asymmetric reduction of the keto group in (Ej-methyl-2-(3-(3-(2-(7-chloroquinolin-2-yl)vinyl)phenyl)-3-oxopropyl)benzoate to the corresponding (S)-alcohol, whereby the reduction and cofactor regeneration (cosubstrate 2-propanol) were carried out using an engineered ketoreductase. A similar system was used by Ju et al. (2014) to reduce a 3-keto piperidine derivative to the corresponding (S)-alcohol. A reaction time of 24 h is reported.

[0020] A process of the type described above is known from US 10113192 B2. This previously known process describes the production of D-fructose from D-glucose. In the first step, 2.5% (w / v) D-glucose was oxidized to 2-keto-D-glucose (D-glucosone) using a pyranose-2-oxidase, whereby the resulting hydrogen peroxide was degraded to water and oxygen by a catalase. After 24 h, the reaction mixture was heated to deactivate the enzymes of the first step. Subsequently, the reduction step to D-fructose was carried out using a xylose reductase from Candida tropicalis. The required cofactor NADPH was regenerated using an alcohol dehydrogenase from Lactobacillus kefir and 2-propanol as a cosubstrate. The reaction was carried out at 30 °C with continuous shaking (850 rpm). Furthermore, an open system is used to allow the evaporation of acetone and shift the reaction towards D-fructose.Additional 2-propanol was administered after 6, 18, and 24 hours. This resulted in 91% conversion of D-glucose to D-fructose, although this process took 72 hours (step 1: 24 hours, step 2: 48 hours).

[0021] This is where the present invention comes in, aiming to provide a method of the type described above that can be implemented with high throughput and a shorter reaction time. Detailed description of the invention

[0022] This problem is solved in a process for the enzymatic reduction of an organic keto compound to an organic hydroxy compound by using NAD(P)H as a cofactor for the enzymatic reduction and by using the oxidized cofactor NAD(P) produced by the enzymatic reduction. +enzymatically reduced with 2-propanol as a co-substrate to NAD(P)H with the formation of acetone and acetone is separated by evaporation, and is characterized in that the evaporation of acetone is carried out in a thin-film treatment device.

[0023] In a preferred embodiment of the process according to the invention, the separated acetone is reduced to 2-propanol and this is used to reduce further oxidized cofactor NAD(P) + used.

[0024] Surprisingly, it has been shown that in the process according to the invention not only can the equilibrium of the regeneration reaction and thus of the main reaction (reduction of the organic keto compound) be shifted further towards the product (organic hydroxy compound), but also that the reaction takes place in a much shorter time (see examples and figures 8 and 9).

[0025] To evaporate the acetone in the thin-film treatment device, an increased temperature and / or a reduced pressure can advantageously be provided. Preferably, the acetone in the thin-film treatment device can be evaporated at a temperature between 25 and 55 °C, particularly between 30 and 45 °C, with or without applying a reduced pressure. The reduced pressure in the thin-film treatment device is preferably between 40 and 90 mbar.

[0026] For the purposes of this application, an "organic keto compound" is understood to be an organic molecule which bears at least one keto group (aldehyde or ketone, with ketone being preferred), with the exception of acetone. An "organic hydroxy compound" is understood to be a molecule which has at least one hydroxy (alcohol) group.

[0027] Preferred organic keto compounds are D-fructose (reduction products D-mannitol and D-sorbitol), D-glucose (reduction product D-sorbitol, see US 9644227 B2 or US 10253340 B2), 2-keto-D-glucose / D-glucosone (reduction products D-fructose (see US 10113192 B2) and D-mannose), D-xylose (reduction product xylitol, see US 9970038 B2), L-arabinose (reduction product L-arabitol), D-galactose (reduction product D-galactitol), D-mannose (reduction product D-mannitol), D-psicose (reduction products D-talitol and allitol), glycolaldehyde (reduction product ethylene glycol), glyceraldehyde (reduction product Glycerol, see WO 2024 / 100202 Al), Pyruvate (reduction product lactate, see WO 2024 / 100202 Al), (3a,5β)-3-Hydroxy-7-oxocholan-24-ic acid / 7-Ketolithocholic acid (reduction products (3a,5β,7a)-3,7-Dihydroxycholan-24-ic acid / Chenodeoxycholic acid and (3a,5β,7β)-3,7-Dihydroxycholan-24-ic acid / Ursodeoxycholic acid, see US 9644227 B2), (3a,5β,7a)-3,7-Dihydroxy-12-oxocholan-24-ic acid / 12-ketochenodeoxycholic acid (reduction products (3a,5β,7a,12a)-3,7,12-trihydroxycholan-24-ic acid / cholic acid and (3a,5β,7a,12β)-3,7,12-trihydroxycholan-24-ic acid, see US 9644227 B2), (3a,5β,7β)-3,7-dihydroxy-12-oxocholan-24-ic acid / 12-ketochenodeoxycholic acid,

[0028] (Reduction products (3a,5β,7β,12a)-3,7,12-trihydroxycholan-24-acid / ursocholic acid and (3a,5β,7β,12β)-3,7,12-trihydroxycholan-24-acid), (3a,5β,12a)-3,12-dihydroxy-7-oxocholan-24-acid / 7-ketodeoxycholic acid (Reduction products (3a,5β,7a,12a)-3,7,12-trihydroxycholan-24-acid / cholic acid and (3a,5β,7β,12a)-3,7,12-trihydroxycholan-24-acid / ursocholic acid), (3a,5β,12β)-3,12-dihydroxy-7-oxocholan-24-acid (Reduction products (3a,5β,7a,12β)-3,7,12-Trihydroxycholan-24-ic acid and (3a,5β,7β,12β)-3,7,12-Trihydroxycholan-24-ic acid), (3a,5β)-3-Hydroxy-7,12-dioxocholan-24-ic acid (reduction products (3a,5β,12a)-3,12-Dihydroxy-7-oxocholan-24-ic acid / 7-ketodeoxycholic acid, (3a,5β,12β)-3,12-Dihydroxy-7-oxocholan-24-ic acid, (3a,5β,7a)-3,7-Dihydroxy-12-oxocholan-24-ic acid / 12-ketochenodeoxycholic acid and (3a,5β,7β)-3,7-Dihydroxy-12- oxocholan-24-acid / 12-ketoursodeoxycholic acid, see US 9644227 B2).

[0029] In a particularly preferred embodiment of the process according to the invention, the organic keto compound is D-fructose and the organic hydroxy compound is D-mannitol.

[0030] In a further particularly preferred embodiment of the process according to the invention, the organic keto compound D-psicose and the organic hydroxy compound D-talitol or allitol are used.

[0031] In a further particularly preferred embodiment of the process according to the invention, the organic keto compound D-glucosone (2-keto-D-glucose) and the organic hydroxy compound D-mannose are used.

[0032] In a further particularly preferred embodiment of the process according to the invention, the organic keto compound is D-mannose and the organic hydroxy compound is D-mannitol.

[0033] In a further particularly preferred embodiment of the process according to the invention, the organic keto compound (3a,5β)-3-hydroxy-7,12-dioxocholan-24-ic acid and the organic hydroxy compound (3a,5β,7β)-3,7-dihydroxy-12-oxocholan-24-ic acid are used.

[0034] Figures 1 to 5 shown in the accompanying illustrations depict the reaction schemes of preferred embodiments of the processes according to the invention. The designation A stands for D-fructose, B for D-mannitol, C for 2-propanol, D for acetone, E for (3a,5β)-3-hydroxy-7,12-dioxocholan-24-ic acid, F for (3a,5β,7β)-3,7-dihydroxy-12-oxocholan-24-ic acid / 12-ketoursodeoxycholic acid, G for D-psicose, H for D-talitol, I for allitol, J for 2-keto-D-glucose (D-glucosone), K for D-mannose, a for reduction, b for cofactor regeneration, c for hydrogenation, and d for epimerization.

[0035] The process is preferably carried out in an aqueous medium.

[0036] The oxidoreductase for the enzymatic reduction of D-fructose to D-mannitol is preferably a mannitol dehydrogenase (NADH-dependent: EC 1.1.1.67; NADPH-dependent: EC 1.1.1.138) or a sorbose reductase (EC 1.1.1.289).

[0037] The oxidoreductase for the reduction of D-fructose to D-mannitol preferably comprises an amino acid sequence selected from the group consisting of: i) an amino acid sequence having at least 80% identity with SEQ ID No. 2, ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity with SEQ ID No. 1, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a complementary strand of a nucleic acid molecule or to a nucleic acid molecule with the nucleic acid sequence SEQ ID No. 1, wherein stringent conditions preferably include one or more washing steps at 65 °C and a salt concentration of 0.1x to 2x SSC.

[0038] SEQ ID No. 1:

[0039] ATGTCACACCCAACATCAGTTATTAACGAGCAGGTAGGCCCATTACCAACTAAGGCCCCACAACTTTCTA AGAATGTGAATGATTTGTTCTCATTAAAGGGTAAGGTAGCTTCTGTCACCGGTTCCTCAGGAGGGATTG GTTGGGCTGTAGCTGAAGCTTATGCCCAAGCAGGAGCAGACGTTGCTGTTTGGTACAATTCTAAAAAT GCAGATGCTAAGGCTGAATACTTAACTAAGACTTACGGTGTGAAGTCGAAGGCATACAAGTGTAACAT TTCTGATCCAGAAGACGTCGAAAAGGTAATTGGACAAATTGAAAAGGATTTCGGTACCATTGACGTTTT CGTTGCCAATGCCGGTGTTCCATGGACAGAAGGTAGAAGTATTGAAGTTGAAGGATATGATTCGTGGA AGAAGGTTATAGATTTGGACTTGAGTGGTGTCTACTATTGCGCTAAGGCTGTCGGAAAGATTTTCAAGA AGAATGGTAAGGGTTCGCTTGTGTTCACAGCATCAATGTCTGGCCACATTGTGAATGTTCCACAATTGC AAGCTCCATACAACGCCGCAAAGGCCGGTGTTTTGCACTTGAGTAAGTCATTAGCTGTCGAATGGGCCC CATTCGCTAGAGTTAACACCATTTCACCTGGTTATATTGCCACAGAAATTTCCGACTTCGTTCCGGACGA TGTCAAGGCTAAGTGGTGGCAATTAATTCCATTAGGAAGAGAAGCTCTTCCACAAGAATTAGTTGGTGC TTACTTATATTTTGCATCCGATGCGTCTACTTACACTACAGGATCGGACTTATTAGTCGATGGTGGTTAC TCTGCTCCATAA SEQ. ID No. 2:

[0040] MSHPTSVINEQVGPLPTKAPQLSKNVNDLFSLKGKVASVTGSSGGIGWAVAEAYAQAGADVAVWYNSKN ADAKAEYLTKTYGVKSKAYKCNISDPEDVEKVIGQIEKDFGTIDVFVANAGVPWTEGRSIEVEGYDSWKKVID LDLSGVYYCAKAVGKIFKKNGKGSLVFTASMSGHIVNVPQLQAPYNAAKAGVLHLSKSLAVEWAPFARVNTI SPGYIATEISDFVPDDVKAKWWQLIPLGREALPQELVGAYLYFASDASTYTTGSDLLVDGGYSAP

[0041] The oxidoreductase for the reduction of D-fructose to D-mannitol preferably comprises or consists of an amino acid sequence having at least 80% identity with SEQ ID No. 2, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%, and particularly preferably 100%. The oxidoreductase according to the invention for the reduction of D-fructose to D-mannitol particularly preferably comprises or consists of the amino acid sequence SEQ ID No. 2.

[0042] Alternatively, the oxidoreductase for the reduction of D-fructose to D-mannitol from D-fructose preferably comprises or consists of an amino acid sequence encoded by a nucleic acid exhibiting at least 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%, and particularly 100% identity with SEQ ID No. 1. Particularly preferably, the nucleic acid encoding the oxidoreductase according to the invention for the reduction of D-fructose to D-mannitol comprises or consists of the nucleic acid sequence SEQ ID No. 1.

[0043] The term "identity," as used here, refers to the percentage of identical nucleotide or amino acid matches between at least two nucleotide or amino acid sequences aligned using a standardized algorithm. Such an algorithm can, in a standardized and reproducible manner, introduce gaps into the compared sequences to optimize the alignment between two sequences and thus achieve a more meaningful comparison of the two sequences.

[0044] The percentage identity between sequences can be determined using one or more computer algorithms or programs known in the prior art or described herein. According to the invention, the Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990), provided by the National Center for Biotechnology Information (NCBI), is used to determine the identity. The BLAST software suite includes various programs, including a tool called "BLAST 2 Sequences," which is used for the direct pairwise comparison of two nucleotide or amino acid sequences. "BLAST 2 Sequences" can also be accessed and used interactively via the NCBI World Wide Web. The blastn program (for nucleotide sequences) uses as parameters a word length (W) of 28, an expectation (E) of 0.05, M = 1, N = -2, and a comparison of both strands.For amino acid sequences, the blastp program uses as specifications a word length of 3 and an expectation (E) of 0.05 and the BLOSUM62 scoring matrix (Henikoff & Henikoff, 1989), alignments (B) of 50, expectation (E) of 0.05, M = 1, N = - 2.

[0045] Alternatively, the oxidoreductase for the reduction of D-fructose to D-mannitol preferably comprises or consists of an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a complementary strand of a nucleic acid molecule or to a nucleic acid molecule with the nucleic acid sequence SEQ. ID No. 1. As used herein, the stringent conditions refer to conditions under which so-called specific hybrids, but no non-specific hybrids, are formed.

[0046] For example, hybridization can be carried out using conventionally known methods, such as those described by J. Sambrook et al. in Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory (1989). Stringent conditions refer to conditions in which washing is performed at 65 °C and a salt concentration of 0.1x to 2x SSC (where 1xSSC is understood to be a mixture of 0.15 M sodium chloride / 0.015 M sodium citrate).

[0047] The invention also relates to a plant for carrying out the process, which comprises a reactor and a thin-film treatment device connected to the reactor via a line, which is connected to the reactor via a further line.

[0048] The invention further relates to a plant for carrying out the process, comprising a reactor, a thin-film treatment device connected to the reactor via a line, which is connected to the reactor via a further line and to a hydrogenation reactor via a third line, wherein the hydrogenation reactor is connected to the reactor via a fourth line.

[0049] For the purposes of this application, a thin-film treatment device is understood to be a device that enables the evaporation of acetone from a thin liquid layer or film on a heated surface (optionally under reduced pressure). This could be, for example, a thin-film evaporator, a falling-film evaporator, or a rotary evaporator.

[0050] Thin-film or falling-film evaporators have already proven their worth in the downstream processing of biotechnological processes (e.g. fermentations) for concentrating diluted product solutions, although high temperatures often have to be avoided in order not to impair the fermentation products.

[0051] In a thin-film evaporator, the liquid to be evaporated is spread as a thin film on the heated surface of the vessel by means of a rotor with wiper blades, where evaporation takes place. In a falling-film evaporator, on the other hand, the liquid to be evaporated is passed through a bundle of heated tubes, where evaporation occurs in a thin layer of liquid. In both cases, a reduced pressure can be applied depending on the application (Dechow, 1989).

[0052] The acetone separated in the thin-film treatment apparatus is subsequently preferably catalytically reduced (hydrogenated) to 2-propanol with hydrogen. This hydrogenation can be carried out, for example, in the gas phase using copper-aluminum mixed oxide catalysts (Basu & Pradhan, 2020), ruthenium nanoparticles on activated carbon and nano-zinc oxide (Al-Rabiah et al., 2022), Raney nickel catalysts (US 7041857 Bl), or platinum catalysts (Demir et al., 2021). Hydrogenation in the presence of water is also known (Lemcoff, 1977).

[0053] A preferred embodiment of the apparatus for carrying out the process is schematically illustrated in the accompanying Figure 6. Reactor 1 is equipped with a stirrer Ml, an acetone sensor 2, and other sensors 3 such as a pH electrode, pOj probe, manometer, etc. At the start of the reaction, reactor 1 contains an aqueous reaction solution containing a substrate, additives such as buffers, the enzymes necessary for the conversion of the substrate and any intermediates, as well as an oxidoreductase and 2-propanol. In the reaction shown in Figure 1, D-fructose is reduced to D-mannitol with NAD(P)H, and the resulting NAD(P) +The 2-propanol is regenerated using 2-propanol, forming acetone. During the reaction, acetone is formed from 2-propanol via cofactor regeneration by oxidoreductase. An increase in the acetone content in the reaction solution is detected in the gas phase by acetone sensor 2. As soon as a threshold value, determined according to the enzymes used (e.g., 2% by volume in solution), is detected, the reaction solution is pumped via line 5 into the thin-film evaporator 6 by a pump 4, where acetone (and a portion of the 2-propanol and water) is evaporated under vacuum. Reference numeral 8 indicates a line for extracting the vaporous mixture (acetone with small amounts of 2-propanol and water), which can be condensed and processed by distillation. The reaction solution, now depleted of acetone, is returned to reactor 1 via line 7.

[0054] Another preferred variant of the system for carrying out the procedure is shown in the attached document.

[0055] Figure 7 shows a schematic representation. Reactor 1 is equipped with a stirrer Ml, an acetone sensor 2, and other sensors 3 such as a pH electrode, pOz probe, manometer, etc. At the start of the reaction, reactor 1 contains an aqueous reaction solution containing a substrate, additives such as buffers, the enzymes necessary for the conversion of the substrate and any intermediates, as well as an oxidoreductase and 2-propanol. In the reaction shown in Figure 1, D-fructose is reduced to D-mannitol with NAD(P)H, and the resulting NAD(P) +The 2-propanol is regenerated using 2-propanol, forming acetone. During the reaction, acetone is formed from 2-propanol via cofactor regeneration by oxidoreductase. An increase in the acetone content in the reaction solution is detected in the gas phase by acetone sensor 2. As soon as a threshold value, determined according to the enzymes used (e.g., 2% by volume in solution), is detected, the reaction solution is pumped via line 5 into the thin-film evaporator 6 by a pump 4, where acetone (and a portion of the 2-propanol and water) is evaporated under vacuum. The reaction solution, now depleted of acetone, is returned to reactor 1 via line 7. The acetone / 2-propanol / water-containing vapor is fed via line 8 to a hydrogenation reactor 9 (with agitator M2), where hydrogen (Hz) is introduced via line 10. There, the catalytic reduction of acetone to 2-propanol takes place at Hz (see above).The resulting 2-propanol / water mixture is returned to reactor 1 via line 11, where the 2-propanol is again available for cofactor regeneration.

[0056] A further advantage of the removal of acetone according to the invention is that the enzyme activity is not impaired. For example, alcohol dehydrogenase (ADH) s The fruit fly Drosophila melanogaster is susceptible to non-competitive product inhibition by acetone (Winberg and McKinley-McKee, 1994). By removing acetone from the reaction mixture according to the invention, a reduction or inhibition of enzyme activity can be prevented and the reaction rate increased.

[0057] However, a decisive advantage of the process according to the invention is that larger quantities of co-substrate can be dispensed with, since this is continuously regenerated, which brings environmental and safety advantages (e.g. less waste, reduced storage capacities required for 2-propanol and acetone, lower co-substrate concentrations in the reactor and associated concentrations of 2-propanol and acetone in the headspace).

[0058] Since acetone can also be hydrogenated in the presence of water, the distillative separation of the acetone / 2-propanol / water-containing mixture formed in the thin-film treatment device is not necessary.

[0059] Preferably, the hydrogen used for the reduction of acetone is of sustainable origin ("green hydrogen"). Solid D-mannitol can be obtained from the aqueous solution, for example, by crystallization or spray drying. Chromatographic separation of byproducts or the substrate is not necessary.

[0060] materials

[0061] D-Fructose, NAD + , NADH disodium salt, NADP +-Disodium salt, NADPH tetrasodium salt, acetone, 2-propanol, and acetonitrile were obtained from PanReac AppliChem (ITW Reagents); D-glucose, D-mannitol, IPTG (isopropyl β-D-thiogalactopyranoside), and phosphoric acid were obtained from Sigma-Aldrich; Allitol from TCI; D-psicose from Hunan Garden Naturals Inc. (China); D-talitol from Biosynth; D-mannose, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and sodium dodecyl sulfate (SDS) were obtained from Carl Roth; and triethanolamine (TEA) was obtained from Chem-Lab NV. (3a,5β)-3-hydroxy-7,12-dioxocholano-24-ic acid was prepared according to US 9644227 B2. D-Glucoson (2-Keto-D-Glucose) was produced by enzymatic oxidation of D-glucose (using commercial pyranose-2-oxidase from Cariolus sp.).

[0062] Production of enzymes & production of lysates

[0063] General information on the expression of recombinant enzymes in E. coli

[0064] For recombinant enzyme production in an Escherichia co / / strain, the gene to be expressed was first amplified in PCR using genomic DNA or its synthetically adapted equivalent (adapted to the codon usage of E. coli) as a template, along with specific oligonucleotides that additionally carry recognition sequences for restriction endonucleases. The resulting DNA was then isolated from the reaction mixture. After nucleic acid digestion with the restriction enzymes Sphl and Hind II I, the gene fragment encoding the target enzyme was ligated into the Sphl-Hind II I backbone of the expression vector pQE70-Kan. The ligation product was transformed into chemically competent E. co / / cells (ToplOF'), and the resulting colonies were used for plasmid isolation and restriction analysis.

[0065] The result of the cloning step was verified by restriction enzyme digestion and DNA sequencing. The resulting construct carries the target gene under the IPTG-inducible T5 promoter.

[0066] For the overexpression of the enzyme in E. coli, the resulting expression plasmid was transformed into competent RB791 expression cells. After 24 h incubation at 37 °C, the resulting colonies were inoculated into LB medium for expression assays. The following day, expression cultures with an optical density (OD550) of 0.02 were inoculated and shaken at 37 °C until an OD550 of 0.3 was reached. Subsequently, the temperature was lowered to 25 °C, and the cultures were induced with 0.1 mM IPTG upon reaching an OD550 of 0.5. After 22 h, the cultures were harvested (separated from the medium by centrifugation as a cell pellet) and analyzed for the expression of the recombinant enzyme using SDS-gel electrophoresis and activity determination (for use in a use test or optical zymatic assay).

[0067] Production of cell lysates using Sonifier digestion

[0068] To prepare a cell suspension, the cell pellet produced according to the above procedure was weighed into a suitable container and mixed with buffer and lysozyme (final concentration 0.5 mg / ml) (e.g., triethanolamine (TEA)-HCl) and dissolved with stirring. The biomass fraction is typically 20% by mass, the remainder being the buffer.

[0069] A Branson Sonifier 450 was used for cell disruption. The suspension was treated three times with 15 ultrasonic pulses each (device settings: Timer = 15; Duty Cycle = 50; Output Control = 3-5).

[0070] The resulting homogenate was centrifuged for 10 min at 4 °C and 16000 rpm (Eppendorf centrifuge 5417R) to separate the insoluble cell fragments and obtain the lysate.

[0071] Table 1. Enzyme classes and donor organisms for the enzymes used in the examples.

[0072] * Note: In the NCBI database entry, mannitol dehydrogenase is classified as a sorbose reductase; however, region 27 to 276 is also described in the entry as "mannitol dehydrogenase (MDH)-Iike, classical (c) SDRs; cd05352". The MDH activity of sorbose reductases is also known from the literature (e.g., Sugisawa et al., 1991).

[0073] ** Note: In the NCBI database entry, the enzyme is classified as "glucose-l-dehydrogenase", however, EP 4464785 Al describes the reduction of D-psicose to D-talitol with this enzyme.

[0074] Analytical methods

[0075] High Performance Liquid Chromatography (HPLC)

[0076] An Agilent HPLC 1260 Infinity II Series system was used to quantify sugars and sugar alcohols by HPLC (High Performance Liquid Chromatography). Detection was performed using a refractive index detector (RI detection). A Phenomenex Rezex RPM monosaccharide Ca2+ (8%) or Phenomenex Rezex RPM monosaccharide Pb2+ (8%) column with a corresponding guard column was used for measurement and isocratically eluted with 3.5 v% 2-propanol or pure water (flow rate 0.5 or 0.6 ml / min).

[0077] HPLC was also used to quantify (3a,5β)-3-hydroxy-7,12-dioxocholano-24-ic acid and (3a,5β,7β)-3,7-dihydroxy-12-oxocholano-24-ic acid. Detection was performed using a UV detector at 200 nm. A Merck Purosphere® STAR RP-18 endcapped (5 pm) column was used for the measurements, with elution performed using a gradient method from water (pH 2.6 with phosphoric acid) and acetonitrile. High-performance anion exchange chromatography (HPAEC) was also employed.

[0078] HPAEC was additionally used to quantify the sugars and sugar alcohols. The analytes were detected by pulsed amperometric detection (PAD). For this purpose, a Dionex™ CarboPac™ PA210-fast-4pm column with a suitable guard column was used and eluted with a NaOH gradient.

[0079] Head Space Gas Chromatography (HS-GC)

[0080] For the quantification of 2-propanol and acetone, a Trace 1300 gas chromatograph (Thermo Scientific) with a TriPlus 500 headspace autosampler and a Supelco WATERCOL 1910 column (length 30 m, diameter 0.25 mm, film thickness 0.2 pm) was used.

[0081] Determination of enzyme activities (optical-enzymatic assay)

[0082] Enzyme activities in the lysates were determined using a Shimadzu UV-1900 spectrophotometer. The formation and consumption of NAD(P)H at a wavelength of 340 nm were monitored by measuring changes in absorption. The measurements were performed with 0.2 mM cofactor (NAD(P)H). + or NAD(P)H). For this purpose, 20 µl of a 10 mM stock solution of the cofactor were placed in a cuvette (Greiner bio-one semi-micro cuvette made of polystyrene), and the desired pH was adjusted with 100 mM TEA-HCl buffer (870 µl). 10 µl of lysate (diluted or undiluted) and 100 µl of substrate solution were added to the cuvette, and the measurement was started immediately thereafter. The measurements were carried out at 25 °C as standard. The extinction coefficient of NADH / NADPH at 340 nm (E = 6220 L mol) was used. 1 cm 1The enzyme activity of the lysate can be determined in U / ml (based on the volume of the lysate) or U / g (based on the biomass used for production). 1 U represents 1 pmol of substrate conversion per minute (1 U = 1 pmol / min = l.67-10 _8 cat).

[0083] The following examples describe preferred variants of the process according to the invention in more detail. The lysates used in these examples were produced according to the processes described above. Example 1

[0084] Reduction of D-fructose to D-mannitol in the reactor (Figure 1)

[0085] The reaction was carried out in a reactor, with the evaporation of acetone being performed in a conventional manner (applying an air stream).

[0086] The reactor setup was carried out in a Labfors 5 benchtop bioreactor (Infors AG). A glass reactor (volume 3.4 L) with an attached stirrer and pH electrode was used. pH control was achieved by adding either NaOH or H₂SO₄.

[0087] Initially, 207.4 ml of a D-fructose solution (482 g / l), 219.2 ml of deionized water and 45.5 ml of a 200 mM TEA-HCl buffer (pH 8) were placed in the reactor and brought to 30 °C while stirring.

[0088] To start the reaction, 14.5 kL of mannitol dehydrogenase I lysate, 12 kL of alcohol dehydrogenase I lysate, and 5 ml of a 5 mM NADP⁻ solution were used. + -solution and 55 ml of 2-propanol were added. The concentration of D-fructose at the start of the reaction was 200 g / l.

[0089] An airflow of 0.15 l / min was applied to the reactor to remove acetone formed during cofactor regeneration. After 12 hours, 20 ml of 2-propanol was added.

[0090] During operation, samples were continuously taken from the reaction mixtures and analyzed as follows: 50 µl of a mixture were mixed with 200 µl of acetonitrile and incubated in an Eppendorf Thermomixer at 85 °C and 1200 rpm for 15 min. The sample was briefly centrifuged, mixed with 750 µl of deionized water, vortexed, and then centrifuged for 5 min at max. g. 200 µl of the supernatant were transferred to an HPLC vial with an insert and analyzed by HPLC (Rl detection). To determine the acetone and 2-propanol content, 250 µl of the mixture were diluted with 4.75 mL of KjCOa solution (0.2 g / mL) in an HS-GC vial and analyzed by HS-GC.

[0091] The concentration profile is shown in Figure 8 (for discussion, see Example 2). Example 2

[0092] Reduction of D-fructose to D-mannitol in a rotary evaporator (Figure 1)

[0093] The reaction was carried out in a thin-film evaporator (in this example a rotary evaporator), whereby the evaporation of acetone was performed by applying a reduced pressure to a thin film.

[0094] A 1-liter round-bottom flask was filled with 207.4 ml of a D-fructose solution (482 g / l), 219.2 ml of deionized water, 45.5 ml of a 200 mM TEA-HCl buffer (pH 8), 14.5 kL of mannitol dehydrogenase I lysate, 12 kL of alcohol dehydrogenase I lysate, and 5 ml of a 5 mM NADP + The solution was filled with 55 ml of 2-propanol. The concentration of D-fructose at the start of the reaction was 200 g / l.

[0095] The round-bottom flask was heated in a water bath to 30 °C at a pressure of 50 mbar and rotated at 210 rpm. The acetone and 2-propanol content of the condensate collected in the receiving flask was determined by HS-GC.

[0096] The acetone-containing distillate obtained in the rotary evaporator was transferred to an autoclave and treated with hydrogen (20 bar) and Raney nickel (63 mg) as a catalyst for 2 h. The resulting 2-propanol-containing solution was returned to the reaction mixture in the round-bottom flask, thus eliminating the need to add any additional 2-propanol, as is required in the reactor (see Example 1).

[0097] The process according to the invention can therefore be carried out in a plant according to Figure 7, wherein reference numeral 1 represents the reactor, reference numeral 6 the thin-film treatment device, reference numeral 9 the hydrogenation reactor and reference numeral 11 the return line for the 2-propanol / water mixture.

[0098] During operation, samples were continuously taken from the reaction mixtures and analyzed as follows: 50 µl of a mixture were mixed with 200 µl of acetonitrile and incubated in an Eppendorf Thermomixer at 85 °C and 1200 rpm for 15 min. The sample was briefly centrifuged, mixed with 750 µl of deionized water, vortexed, and then centrifuged for 5 min at max. g. 200 µl of the supernatant were transferred to an HPLC vial with an insert and measured by HPLC (Rl detection). To determine the acetone and 2-propanol content, 250 µl of the mixture were diluted with 4.75 mL of KjCOa solution (0.2 g / mL) in an HS-GC vial and measured by HS-GC. The concentration profile is shown in Figure 8.It shows that the conversion in the rotary evaporator (solid black line, Example 2) not only proceeds significantly faster than in the reactor (light gray dotted line, Example 1), but also that the process according to the invention leads to a complete conversion in only 8 h, whereas in the reactor itself only 90% of the D-fructose could be converted after 53 h.

[0099] Example 3

[0100] Conversion of invert sugar to D-mannitol

[0101] The reactor setup was carried out in a Labfors 5 benchtop bioreactor (Infors AG). A glass reactor (volume 3.4 L) with an attached stirrer, pC sensor, and pH electrode was used. pH control was achieved by adding either NaOH or H₂SO₄.

[0102] Initially, 197.4 ml of an invert sugar solution (D-glucose / D-fructose 1 / 1; 507 g / l), 201.0 ml of deionized water and 42.7 ml of a 500 mM TEA-HCl buffer (pH 8) were placed in the reactor and brought to 30 °C while stirring.

[0103] To start the reaction, 10 ml of a lysate containing pyranose-2-oxidase and catalase, 6 kU xylose reductase lysate, 16.1 kU mannitol dehydrogenase I lysate, 34 kU alcohol dehydrogenase I lysate, and 55 ml of 2-propanol were added. The invert sugar concentration at the start of the reaction was 200 g / l.

[0104] An airflow of 0.05 l / min and an overpressure of 320 mbar were applied.

[0105] During operation, samples were continuously taken from the reactor solution and analyzed as follows: 50 µl of the mixture were mixed with 200 µl of acetonitrile and incubated in an Eppendorf Thermomixer at 85 °C and 1200 rpm for 15 min. The sample was briefly centrifuged, mixed with 750 µl of deionized water, vortexed, and then centrifuged for 5 min at max. g. 200 µl of the supernatant were transferred to an HPLC vial with an insert and analyzed by HPLC (Rl detection).

[0106] After 2 hours of operation, the entire reactor contents were transferred to a 1-liter round-bottom flask. Acetone (and 2-propanol) were evaporated for 60 minutes using a rotary evaporator at 30 °C, 50 mbar pressure, and a rotation speed of 210 rpm. The loss was then replenished with deionized water up to a volume of 500 ml, and 30 ml of 2-propanol was added. This procedure was repeated after 5 hours and 22 hours. After 26 hours, 93% of the invert sugar had been converted to D-mannitol.

[0107] The example shows that the inventive method can also be used in combination with a C-dependent oxidation reaction (D-glucose). D-glucoson with P2O) can be performed.

[0108] Example 4

[0109] Reduction of (3a,5β)-3-hydroxy-7,12-dioxocholano-24-ic acid to (3a,5β,7β)-3,7-dihydroxy-12-oxocholano-24-ic acid in the reactor (Figure 2)

[0110] The reaction was carried out in a reactor, with the evaporation of acetone being performed in a conventional manner (applying an air stream).

[0111] The reactor setup was carried out in a Labfors 5 benchtop bioreactor (Infors AG). A glass reactor (volume 3.4 L) with an attached stirrer and pH electrode was used. pH control was achieved by adding either NaOH or H₂SO₄.

[0112] Initially, 500 ml of a solution of (3a,5ß)-3-hydroxy-7,12-dioxocholan-24-acid (50 g / l) in 50 mM TEA-HCl buffer (pH 8.4) was heated to 25 °C under stirring.

[0113] To start the reaction, 3.25 kU of 7β-hydroxysteroid dehydrogenase lysate, 2.5 kU of alcohol dehydrogenase I lysate, and 10 mg of NADP were used. + and 40 ml of 2-propanol was added.

[0114] An airflow of 2 l / min was applied to the reactor to remove acetone formed during cofactor regeneration.

[0115] During operation, samples were continuously taken from the reaction mixtures and analyzed as follows: 150 µl of the mixture were mixed with 750 µl of solvent mixture (acetonitrile / H₂O / 50% H₃PO₄ (90 + 9 + 1)) and incubated in an Eppendorf Thermomixer at 55 °C and 1200 rpm for 15 min. The sample was centrifuged for 5 min at max. g. 100 µl of the supernatant were diluted with 900 µl of solvent mixture in an HPLC vial and analyzed by HPLC (UV detection). To determine the acetone and 2-propanol content, 250 µl of the mixture were diluted with 4.75 mL of KzCOa solution (0.2 g / mL) in an HS-GC vial and analyzed by HS-GC.

[0116] The concentration profile is shown in Figure 9 (for discussion, see Example 5). Example 5

[0117] Reduction of (3a,5β)-3-hydroxy-7,12-dioxocholano-24-ic acid to (3a,5β,7β)-3,7-dihydroxy-12-oxocholano-24-ic acid in a rotary evaporator (Figure 2)

[0118] The reaction was carried out in a thin-film evaporator (in this example a rotary evaporator), whereby the evaporation of acetone was performed by applying a reduced pressure to a thin film.

[0119] A 1 l round-bottom flask was filled with 500 ml of a solution of (3a,5ß)-3-hydroxy-7,12-dioxocholan-24-ic acid (50 g / l), 3.25 kll 7ß-hydroxysteroid dehydrogenase lysate, 2.5 kll alcohol dehydrogenase I lysate, 10 mg NADP + and filled with 40 ml of 2-propanol.

[0120] The round-bottom flask was heated in a water bath to 25 °C at a pressure of 50 mbar and rotated at 210 rpm. The acetone and 2-propanol content of the condensate collected in the receiving flask was determined by HS-GC.

[0121] The acetone-containing distillate obtained in the rotary evaporator was transferred to an autoclave and treated with hydrogen (20 bar) and Raney nickel (63 mg) as a catalyst for 2 h. The resulting 2-propanol-containing solution was returned to the reaction mixture in the round-bottom flask, thus eliminating the need to add any additional 2-propanol, as is required in the reactor (see Example 4).

[0122] The process according to the invention can therefore be carried out in a plant according to Figure 7, wherein reference numeral 1 represents the reactor, reference numeral 6 the thin-film treatment device, reference numeral 9 the hydrogenation reactor and reference numeral 11 the return line for the 2-propanol / water mixture.

[0123] During operation, samples were continuously taken from the reaction mixtures and analyzed as follows: 150 µl of the mixture were mixed with 750 µl of solvent mixture (acetonitrile / H₂O / 50% H₃PO₄ (90 + 9 + 1)) and incubated in an Eppendorf Thermomixer at 55 °C and 1200 rpm for 15 min. The sample was centrifuged for 5 min at max. g. 100 µl of the supernatant were diluted with 900 µl of solvent mixture in an HPLC vial and measured by HPLC (UV detection). To determine the acetone and 2-propanol content, 250 µl of the mixture were diluted with 4.75 mL of KjCO₃ solution (0.2 g / mL) in an HS-GC vial and measured by HS-GC. The concentration profiles are shown in Figure 9. It shows that the conversion in the rotary evaporator (Example 5; solid black line; 81% conversion in 5 h) is significantly faster than in the reactor (Example 4; light gray dotted line; 66% conversion in 5 h).

[0124] Example 6

[0125] Conversion of D-fructose to D-talitol in a rotary evaporator (Figure 3)

[0126] A 1-liter round-bottom flask was filled with 92.2 ml of a D-fructose solution (542 g / l), 267.2 ml of deionized water, 29.9 ml of a 500 mM TEA-HCl buffer (pH 8), 25 ml of epimerase lysate, 35 ml of glucose dehydrogenase lysate (see Table 1), 12 kU alcohol dehydrogenase I lysate, and 10 ml of a 10 mM NAD+ solution. + The container was filled with a solution and 35 ml of 2-propanol. The concentration of D-fructose at the start of the reaction was 100 g / l.

[0127] The round-bottom flask was heated in a water bath to 40 °C at a pressure of 80 mbar and rotated at 210 rpm. The acetone and 2-propanol content of the condensate collected in the receiving flask was determined by HS-GC, and the evaporated 2-propanol was continuously replenished (20 ml of 2-propanol and 20 ml of deionized water were added every 60 min).

[0128] Five hours after the start of the reaction, the round-bottom flask was removed from the rotary evaporator and stirred using a magnetic stir bar at 300 rpm and 40 °C. After 15 hours, the flask was reconnected to the rotary evaporator and incubated for another 5 hours at 40 °C, 80 mbar, and 210 rpm. During this time, the evaporated 2-propanol was continuously replenished.

[0129] During operation, samples were continuously taken from the reaction mixtures and analyzed as follows: 50 µl of a mixture were mixed with 200 µl of methanol and incubated in an Eppendorf Thermomixer at 60 °C and 1200 rpm for 15 min. The sample was briefly centrifuged, mixed with 750 µl of deionized water, vortexed, and then centrifuged for 5 min at max. g. 200 µl of the supernatant were transferred to an HPLC vial with an insert and measured by HPLC (Rl detection). To determine the acetone and 2-propanol content, 250 µl of the mixture were diluted with 4.75 ml of KjCOa solution (0.2 g / ml) in an HS-GC vial and measured by HS-GC.

[0130] In this way, 95% of the D-fructose could be converted to D-talitol in 25 hours.

[0131] This example shows that the inventive process can also be carried out in combination with an epimerase (D-fructose D-psicose). Example 7

[0132] Conversion of D-fructose to allitol in a rotary evaporator (Figure 3)

[0133] A 1-liter round-bottom flask was filled with 217 ml of a D-fructose solution (461 g / l), 116 ml of deionized water, 23.3 ml of a 500 mM TEA-HCl buffer (pH 8), 25 ml of epimerase lysate, 70 ml of short-chain reductase lysate, 12 kU alcohol dehydrogenase II lysate, and 10 ml of a 10 mM NAD + The solution was filled with 35 ml of 2-propanol. The concentration of D-fructose at the start of the reaction was 200 g / l.

[0134] The round-bottom flask was heated in a water bath to 35 °C at a pressure of 60 mbar and rotated at 210 rpm. The acetone and 2-propanol content of the condensate collected in the receiving flask was determined by HS-GC, and the evaporated 2-propanol was continuously replenished (20 ml of 2-propanol and 20 ml of deionized water were added every 60 min).

[0135] Six and a half hours after the start of the reaction, the round-bottom flask was removed from the rotary evaporator and stirred using a magnetic stir bar at 300 rpm and 35 °C. After 15 hours, the flask was reconnected to the rotary evaporator and incubated at 35 °C, 60 mbar, and 210 rpm.

[0136] During operation, samples were continuously taken from the reaction mixtures and analyzed as follows: 50 µl of a mixture were mixed with 200 µl of methanol and incubated in an Eppendorf Thermomixer at 60 °C and 1200 rpm for 15 min. The sample was briefly centrifuged, mixed with 750 µl of deionized water, vortexed, and then centrifuged for 5 min at max. g. 200 µl of the supernatant were transferred to an HPLC vial with an insert and measured by HPLC (Rl detection). To determine the acetone and 2-propanol content, 250 µl of the mixture were diluted with 4.75 ml of KjCOa solution (0.2 g / ml) in an HS-GC vial and measured by HS-GC.

[0137] In this way, 77% of the D-fructose could be converted to allitol in 21.5 h.

[0138] The example shows that the inventive method can also be carried out in combination with an epimerase (D-fructose D-psicose).

[0139] Example 8

[0140] Reduction of 2-keto-D-glucose (D-glucosone) to D-mannose in a rotary evaporator (Figure 4)

[0141] A 1 L round-bottom flask was filled with 251 ml of a D-glucosone solution (199 g / l), 133 ml of deionized water, 36.1 ml of a 500 mM TEA-HCl buffer (pH 8), 50 ml of mannitol dehydrogenase I-lysate, 12 kU of alcohol dehydrogenase I-lysate, and 10 ml of a 10 mM NAD + The container was filled with a solution and 30 ml of 2-propanol. The concentration of D-glucosone at the start of the reaction was 100 g / l.

[0142] The round-bottom flask was heated in a water bath to 35 °C at a pressure of 50 mbar and rotated at 210 rpm. The acetone and 2-propanol content of the condensate collected in the receiving flask was determined by HS-GC, and the evaporated 2-propanol was continuously replenished (10 ml of 2-propanol and 20 ml of deionized water added every 60 min).

[0143] Six hours after the start of the reaction, the round flask was removed from the rotary evaporator and stirred using a magnetic stir bar at 300 rpm and 35 °C for 15 hours.

[0144] During operation, samples were continuously taken from the reaction mixtures and analyzed as follows: 100 µl of a mixture were mixed with 200 µl of methanol and incubated in an Eppendorf Thermomixer at 60 °C and 1200 rpm for 15 min. The sample was briefly centrifuged, mixed with 700 µl of deionized water, vortexed, and then centrifuged for 5 min at max. g. 200 µl of the supernatant were transferred to an HPLC vial with an insert and measured by HPLC (Rl detection). The supernatant was diluted 1:1000 and measured by HPAEC (PAD). To determine the acetone and 2-propanol content, 250 µl of the mixture were diluted with 4.75 mL of KjCOa solution (0.2 g / mL) in an HS-GC vial and measured by HS-GC.

[0145] In this way, 99% of the D-glucoson could be converted to D-mannose in 21 hours.

[0146] Example 9

[0147] Reduction of D-mannose to D-mannitol in a rotary evaporator (Figure 5)

[0148] A 1-liter round-bottom flask was filled with 46.8 ml of a D-mannose solution (534 g / l), 151 ml of deionized water, 19.4 ml of a 500 mM TEA-HCl buffer (pH 8), 25 ml of xylose reductase lysate, 6 kU of alcohol dehydrogenase I lysate, and 5 ml of a 5 mM NADP + The container was filled with a solution and 15 ml of 2-propanol. The concentration of D-mannose at the start of the reaction was 100 g / l.

[0149] The round-bottom flask was heated in a water bath to 30 °C at a pressure of 50 mbar and the rotary evaporator was set to 210 rpm. The acetone and 2-propanol content of the condensate collected in the receiving flask was determined by HS-GC, and the evaporated 2-propanol was continuously replaced (10 mL of 2-propanol and 20 mL of deionized water added every 60 min). Five hours after the start of the reaction, the round-bottom flask was removed from the rotary evaporator and stirred with a magnetic stir bar at 300 rpm and 30 °C. After 16 hours, the flask was reconnected to the rotary evaporator and incubated for another 6 hours at 30 °C, 50 mbar, and 210 rpm. During this time, the evaporated 2-propanol was again continuously replaced (5 mL of 2-propanol added every 60 min).

[0150] During operation, samples were continuously taken from the reaction mixtures and analyzed as follows: 100 µl of a mixture were mixed with 200 µl of methanol and incubated in an Eppendorf Thermomixer at 60 °C and 1200 rpm for 15 min. The sample was briefly centrifuged, mixed with 700 µl of deionized water, vortexed, and then centrifuged for 5 min at max. g. 200 µl of the supernatant were transferred to an HPLC vial with an insert and measured by HPLC (Rl detection). The supernatant was diluted 1:1000 and measured by HPAEC (PAD). To determine the acetone and 2-propanol content, 250 µl of the mixture were diluted with 4.75 mL of KjCOa solution (0.2 g / mL) in an HS-GC vial and measured by HS-GC.

[0151] In this way, 72% of the D-mannose could be converted to D-mannitol in 27 hours.

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Claims

Patent claims 1. A process for the enzymatic reduction of an organic keto compound to an organic hydroxy compound, wherein NAD(P)H is used as a cofactor for the enzymatic reduction and the oxidized cofactor NAD(P) produced during the reduction + enzymatically reduced with 2-propanol as a co-substrate to form acetone to NAD(P)H and acetone is separated by evaporation, characterized in that the evaporation is carried out in a thin-film treatment device.

2. The method according to claim 1, characterized in that the acetone separated by evaporation is reduced to 2-propanol and this is used to reduce further oxidized cofactor NAD(P) + is used.

3. Apparatus for carrying out the method according to claim 1, comprising a reactor (1), a thin-film treatment device (6) connected to the reactor (1) via a line (5) and connected to the reactor (1) via a line (7).

4. Plant according to claim 3, comprising a reactor (1), a thin-film treatment device (6) connected to the reactor (1) via a line (5), which is connected to the reactor (1) via a line (7) and to a hydrogenation reactor (9) via a line (8), wherein the hydrogenation reactor (9) is connected to the reactor (1) via a line (11).

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