METHOD FOR PRODUCING AN AQUEOUS SOLUTION CONTAINING D-PSICOSE

Abstract

The present invention relates to a method for producing an aqueous solution containing D-psicose, by a first D-psicose being formed from D-fructose, which is dissolved in an aqeuous solution, by treatment with an epimerase in vitro, after which the first D-psicose is reduced to form allitol by treatment with a corresponding NAD(P)H-dependent oxidoreductase in vitro, and after deactivation and / or ultrafiltration of the epimerase in order to form D-psicose is treated with a corresponding NAD(P)+-dependent oxidoreductase, after which the deactiavted epimerase and the oxidoreductases are removed.

Description

1 / 56 METHOD FOR PRODUCING AN AQUEOUS SOLUTION CONTAINING D-PSICOSE

[001] The present invention relates to a method for preparing an aqueous solution containing D-psicose. BACKGROUND OF THE INVENTION D-Psychosis

[002] The polysaccharide D-psicose, also known as Dalulose, is a ketohexose that is rarely found in nature (Zhang et al., 2016). It has been detected, among others, in the leaves of the sweetspires shrub species (Itea sp.) (Hough & Stacey, 1996), but is also found in processed foods such as candies and hot sauces, where it is formed from D-fructose, its C3 epimer, under the influence of heat (Oshima et al., 2006).

[003] D-psicose is of interest to the food industry due to its sweet taste. Compared to sucrose, D-psicose has a relative sweetening power of 70%, but a low energy content (0.2 kcal / g), which corresponds to a caloric reduction of approximately 95% (compared to sucrose) (Jiang et al., 2020).

[004] In the United States, D-psicose has been recognized by the Food and Drug Administration (FDA) as a Generally Recognized as Safe (GRAS) sweetener, but in Europe it has not yet been approved (Ahmed et al., 2022).

[005] In addition, D-psicose has positive effects on lipid and carbohydrate metabolism (e.g., antidiabetic) and is anti-inflammatory and antioxidant (Zhang et al., 2016; Jiang et al., 2020; Chen et al., 2022).

[006] Due to its scarcity in nature, D-psicose is produced primarily synthetically (chemically or biotechnologically).

[007] The epimerization of D-fructose into D-psicose can be realized Petition 870250081800, dated 11 / 09 / 2025, p. 8 / 88 2 / 56 zada via boiling in pyridine under reflux, followed by the removal of other hexoses through yeast fermentation, although only 6.8% of the theoretical yield of D-psicose is obtained (Doner, 1979). Another method involves the epimerization of D-fructose with molybdate ions as a catalyst, in which only 0.5% of the D-fructose is converted to D-psicose (Bílik & Tihlárik, 1974). Inefficient chemical synthesis routes have, however, been replaced by more efficient biotechnological processes.

[008] In 1993, Izumori et al. described a Pseudomonas cichorii ST-24 ketose-3-epimerase for the production of D-psicose from D-fructose (Izumori et al., 1993), which was also patented (EP 0592202 B1). Ketose-3-epimerases can be divided into three groups depending on their substrate specificity: 1) D-tagatose-3-epimerase (DTE), 2) D-psicose-3-epimerase (DPE) or D-allulose-3-epimerase (DAE), and 3) L-ribulose-3-epimerase (LRE) (Zhang et al., 2016; Jiang et al., 2020).

[009] However, the conversion of D-fructose to D-psicose via ketose-3-epimerases does not occur completely, but an equilibrium ratio between the two epimers is formed. Depending on the reaction conditions (temperature between 40 and 70 °C, pH between 6 and 11), it is between 80:20 and 62.5:37.5 (D-fructose:D-psicose). Many epimerases also require a divalent metal ion such as Mn2+ or Co2+ (toxic) as a cofactor (Zhang et al., 2016; Jiang et al., 2020).

[0010] The equilibrium during epimerization can be influenced not only by temperature or pH, but also by the addition of borate (toxic). Due to the preferential formation of a D-psicose-borate complex, the equilibrium shifts towards D-psicose (Kim et al., 2008; Lim et al., 2009). Documents EP 3643786 A2 and US 11028420 B2 describe the chromatographic separation of the D-psicose complex. Petition 870250081800, dated 11 / 09 / 2025, p. 9 / 88 3 / 56 psychose-borate using simulated moving bed (SMB) chromatography. EP 3395952 B1 and US 10550414 B2 reveal that conversion during epimerization using DPE can be increased by up to 67% with the addition of sodium aluminate and by up to 52% with potassium iodate (comparison: 25% without the addition of aluminate or iodate).

[0011] Zhu et al., (2020) presented a system composed of two enzymes (exo-inulase from Bacillus velezensis and DAE from Ruminococcus sp.) by which inulin from Helianthus tuberosus L. (Jerusalem artichoke) can be converted into a syrup consisting of D-glucose, D-fructose, and D-psicose (1:3:1). Li et al., (2021a) used a system consisting of invertase, D-glucose isomerase, and immobilized DAE from Pirellula sp. SH-Sr6A to convert sucrose, D-glucose, and D-fructose (from fruit juices) into D-psicose. They were able to enrich the juices with 16-19% D-psicose (based on total carbohydrate content).

[0012] In a study, Juneja et al. (2019) analyzed the techno-economic aspects of a modified dry milling process for corn, in which, in addition to ethanol, D-psicose is also produced from milled corn using a modified yeast strain (through the expression of a DPE). The authors calculated that 390.4 l of ethanol and 75.3 kg of D-psicose can be obtained from one ton of corn and that the minimum selling price for D-psicose produced by the described process is US$ 1.29 / kg (compared to the market price of US$ 10-20 / kg in 2018). WO 2020 / 057560 A1 and WO 2020 / 057561 A1 describe the production of D-psicose from starch through saccharification, enzymatic isomerization, and epimerization.

[0013] Patel et al. (2018) used Smt3-DPE (fusion protein) immobilized on magnetic iron oxide nanoparticles to produce D-psicose from D-fructose from berry washing solutions Petition 870250081800, dated 11 / 09 / 2025, p. 10 / 88 4 / 56 fruit extract. The immobilized epimerase was able to convert 20% of the D-fructose and was separated with a magnet after the reaction was complete.

[0014] Yang et al., (2018) transferred the DPE gene from Agrobacterium tumefaciens to the thermotolerant bacterium Kluyveromyces marxianus. Thus, 190 g / l of D-psicose could be produced from 750 g / l of D-fructose in 12 ha at 55 °C, with the remaining D-fructose being fermented into ethanol by K. marxianus. Dedania et al. (2020) immobilized DPE from A. tumefaciens on titanium dioxide nanoparticles and were able to convert 36% of the D-fructose into D-psicose. Furthermore, the immobilized enzyme could be reused up to nine times.

[0015] D-fructose / D-psicose mixtures produced during the epimerization of D-fructose can be separated either by chromatographic methods or by biological methods (fermentation of excess D-fructose in, for example, ethanol) (Jiang et al., 2020). US 2021 / 0189441 A1 describes the separation of a D-fructose / D-psicose mixture in which D-fructose is converted to L-lactic acid by a probiotic microorganism (Lactobacillus or Saccharomyces). EP 3423460 B1 describes a process for purifying a D-fructose / D-psicose mixture and obtaining high-purity D-psicose. EP 3553069 A1 and Van Duc Long et al. (2009) describe a method for separating D-psicose and D-fructose based on SMB chromatography.

[0016] However, the production of D-psicose via the epimerase pathway has the following disadvantages: 1) equilibrium position on the D-fructose side, 2) addition of (partially toxic) metal ions as cofactors for many epimerases, 3) low activity and long-term stability of the epimerases, and 4) complex separation of the product mixture.

[0017] An option to circumvent thermodynamic epimerization Petition 870250081800, dated 11 / 09 / 2025, page 11 / 88 5 / 56 Most unfavorable are enzyme cascades with phosphorylated intermediates. The final step, dephosphorylation, is irreversible and therefore drives the cascade (Li et al., 2021b).

[0018] A cascade described in an almost identical manner by Li et al., (2021b), as well as in US 11168342 B2 and US 10907182 B2, shows D-glucose-1-phosphate (G1P) as the central intermediate. G1P is first converted to D-glucose-6-phosphate (G6P) by phosphoglucomutase and then converted to D-fructose-6-phosphate (F6P) by glucose-6-phosphate isomerase. F6P is then epimerized by D-allulose-6-phosphate epimerase to D-psicose-6-phosphate, which is subsequently dephosphorylated by D-allulose-6-phosphate phosphatase to D-psicose.

[0019] G1P can be produced directly, for example, by the action of phosphorylases on, for example, maltose and amylodextrins (obtained by hydrolysis of starch), cellodextrins (obtained by hydrolysis of cellulose), or sucrose, with the consumption of phosphate. As the terminal sugar monomers of oligo- and polysaccharides cannot be phosphorylated by the corresponding phosphorylases, polyphosphate glucokinase (D-glucose^G6P) or polyphosphate fructokinase (D-fructose^F6P) has to be used, where polyphosphates must be further added as a phosphate source in order to increase yields (US 11168342 B2; US 10907182 B2). The substrate used for the cascade by Li et al. (2021b) is starch, which is converted to D-psicose with yields of 79% (at a substrate concentration of 50 g / l; reaction time of 24 h).

[0020] Wang et al. (2020) also developed an enzymatic cascade for the production of D-psicose from starch, which, however, is converted to glyceraldehyde-3-phosphate and dihydroxyacetone phosphate in several steps. Dihydroxyacetone phosphate is converted to D-psicose-1-phosphate with D-glyceraldehyde under the activity of L-fuculose-1-phosphate aldolase (FucA), which is dephosphorylated to D Petition 870250081800, dated 11 / 09 / 2025, page 12 / 88 6 / 56 psychosis by phosphatase (95% yield at a titer of 15.2 mM). Glyceraldehyde-3-phosphate is further converted to 2-deoxy-D-ribose (Wang et al., 2020).

[0021] An enzyme cascade from glycerol is also described in the literature. It is converted into dihydroxyacetone phosphate (phosphorylation of glycerol with an acid phosphatase and subsequent oxidation with a glycerol phosphate oxidase) and D-glyceraldehyde (oxidation of glycerol with allitol oxidase), which in turn serve as substrates for an aldolase (such as FucA). After cleavage of the phosphate group, a mixture of D-sorbose and D-psicose is obtained, which can be separated chromatographically (Li et al., 2020). WO 2016 / 201110 A1, on the other hand, describes a method for producing D-psicose from dihydroxyacetone and D-glyceraldehyde using fructose-6-phosphate aldolase and DTE (intermediate product D-fructose).

[0022] Xiao et al. coupled the epimerization of D-fructose with the conversion of D-psicose to D-psicose-1-phosphate using L-ramulose kinase (consuming adenosine triphosphate (ATP)) to shift the epimerization equilibrium. Subsequent cleavage of the phosphate group by an acid phosphatase yields D-psicose (99% conversion of 20 mM D-fructose). However, ATP must be regenerated with the addition of polyphosphate using a polyphosphate kinase (Xiao et al., 2019).

[0023] A major disadvantage of routes with phosphorylated intermediates is the use of expensive, energy-rich phosphate compounds, such as polyphosphate or ATP, in stoichiometric amounts to introduce the phosphate groups. Using phosphorylases, this problem can be partially avoided, but terminal monosaccharides cannot be phosphorylated without the aid of energy-rich phosphate compounds. Furthermore, the remaining phosphate compounds and phosphate ions must be removed after the reaction is complete.

[0024] Fermentation processes for the production of D-psicose Petition 870250081800, dated 11 / 09 / 2025, p. 13 / 88 7 / 56 are also known. Zhang et al. (2021) presented a fermentation process based on the co-cultivation of Bacillus subtilis and Escherichia coli engineered for the joint production of D-psicose (titer 11.7 g / l; conversion: 69.5%) and the lipase enzyme. US 2017 / 0298400 A1 describes the expression of DPE (e.g., from Agrobacterium tumefaciens) in several microorganisms. EP 3088515 B1 and US 9701953 B2 describe a strain of Ensifer adhaerens producing D-psicose. EP 2470668 B1 describes the immobilization of a GRAS microorganism (Corynebacterium glutamicum KCCM 11046) with DPE expressed on a sodium alginate carrier. Allitol as an intermediate

[0025] The unfavorable equilibrium position of D-fructose epimerization in D-psicose can also be favorably influenced by downstream redox reactions. By combining a DTE with a ribitol dehydrogenase (RDH; EC 1.1.1.56) and a formate dehydrogenase (FDH; as a regenerating enzyme for the nicotinamide adenine dinucleotide cofactor NADH), D-fructose can be converted in vitro into the sugar alcohol allitol (Takeshita et al., 2000).

[0026] Alitol can then be converted back into D-psicose via an oxidation step. This can be achieved, for example, microbially with Enterobacter aerogenes IK7 (complete oxidation of 100 g / l of allytol in 24 h) or Bacillus pallidus Y25 (48% conversion of 50 g / l of allytol in 48 h) (Gullapalli et al., 2007; Poonperm et al., 2007).

[0027] Due to its symmetry, the achiral allitol sugar alcohol forms an interface between D and L hexoses in the so-called Izumoring strategy for the bioproduction of rare sugars (Izumori, 2006; Hassanin et al., 2017). Therefore, it can also serve as a precursor for the production of other rare monosaccharides. Petition 870250081800, dated 11 / 09 / 2025, page 14 / 88 8 / 56

[0028] The theoretical articles by Hold et al. (2009) and Siedentop et al. (2021) address the optimization of enzyme cascades. It is described that all components and a plurality of parameters must be taken into consideration, especially parameters relating to the cascade design, the enzymes themselves, the reaction conditions and environment, and also the process design, and that it is not possible to predict which ones will be successful. The synthesis of D-psicose is not specifically mentioned.

[0029] Chen et al. (2022) address the fermentation route via whole-cell biocatalysts (in vivo) for the production of D-psicose and conclude that this is the only route, with different optimizations, that has the potential for the economical and industrial-scale production of D-psicose in the future. The advantages of whole-cell biocatalysts are obvious: (1) cells containing enzymes inside are more easily accessible than the enzymes themselves, whose purification is usually laborious; (2) the interior of cells provides a suitable microenvironment for enzymes and also allows cofactor regeneration (NAD(P)+ / NAD(P)H); (3) cell walls and membranes protect enzymes from the reaction medium environment; and (4) colocalization of multiple enzymes within cells favors local enzyme concentrations and reduces diffusion of intermediates in cascade reactions.

[0030] Consequently, the authors see microbial cell factories as the best opportunity to produce D-psicose on a large scale, so that ordinary consumers can also enjoy this rare sugar in the near future.

[0031] A team led by Wang and others (2022, 2023) tam Petition 870250081800, dated 11 / 09 / 2025, page 15 / 88 9 / 56 is also working on the enzymatic biotransformation of sugars, investigating biotransformations in vitro and in vivo. Their goal is the development of an economical production method that can be carried out on an industrial scale.

[0032] For the production of D-psicose from D-fructose, Wang et al. (2023) describe an in vivo method consisting of two whole-cell E. coli biocatalysts, in which, in the first step (conversion of D-fructose to allitol), E. coli cells are used, which include DPE from Clostridiales, an RDH from Providencia alcalifaciens, an FDH from Starkeya, as well as an additional DPE from Rhizobium straminoryzae. In this way, D-fructose (500 mM = 90 g / l) was converted to 452 mM of allitol within 12 hours at 37 °C and pH 6 using 1000 mM of sodium formate (two equivalents based on D-fructose) (conversion of 90.4%). Approximately 30 mM of D-sorbitol were formed as a byproduct. The cells were separated by centrifugation, and the proteins that leaked into the supernatant containing allitol were deactivated by heat. Then, E. coli cells containing an RDH from Rubrivivax sp. and an NADH oxidase from Streptococcus pyogenes were added to the allitol solution.Alitol (452 ​​mM) transformed into D-psicose (450 mM) in 24 hours at pH 7.

[0033] Wang et al. (2023) further describe that, to their knowledge, the 90% conversion rate is the highest conversion rate ever achieved for the production of D-psicose from D-fructose and announce their intention to further optimize the pathway in vivo, since the theoretical conversion rate of the proposed two-step process is 100%. A disadvantage of the process according to Wang et al. is the formation of the byproduct D-sorbitol, which is formed by the reduction of D-fructose.

[0034] This is where the objective of the present invention comes in, which aims to further improve the two-step process for the formation of Petition 870250081800, dated 11 / 09 / 2025, p. 16 / 88 10 / 56 To obtain D-psicose from D-fructose and to provide a method for preparing aqueous solutions containing D-psicose, which can, in particular, also be carried out in a one-container method. DETAILED DESCRIPTION OF THE INVENTION

[0035] According to the invention, this objective is achieved by forming a first D-psicose from D-fructose, which is present in aqueous solution, through treatment with an epimerase in vitro, after which the first D-psicose is reduced to allitol through treatment with a respective NAD(P)H-dependent oxidoreductase in vitro and, after deactivation and / or ultrafiltration of the epimerase, a respective NAD(P)+-dependent oxidoreductase is added for the formation of D-psicose, then the deactivated epimerase and the oxidoreductases are removed. Instead of deactivating the epimerase, it is also possible to immobilize it on or in a carrier material and filter it from the aqueous solution along with the carrier.

[0036] Surprisingly, it has been shown that virtually no unwanted D-sorbitol is formed in the method according to the invention and that the yield of D-psicose can be easily increased to up to 100%.

[0037] The process according to the invention is therefore not carried out in a fermentative manner, but the enzymes are contained as such in the aqueous solution. According to the invention, the process is therefore carried out in vitro.

[0038] A preferred variant of the invention's method features the NAD(P)+-dependent oxidoreductase for the formation of D-psicose from allitol comprising an amino acid sequence selected from the group consisting of: i) an amino acid sequence with at least 80% identity to SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 12, Petition 870250081800, dated 11 / 09 / 2025, p. 17 / 88 11 / 56 ii) an amino acid sequence encoded by a nucleic acid with at least 80% identity to SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 11, and iii) an amino acid sequence encoded by a nucleic acid that, under strict conditions, binds to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 9 or SEQ ID NO: 11 or a functional fragment thereof.

[0039] A functional fragment of this NAD(P)+-dependent oxidoreductase comprises an N-terminal and / or C-terminal truncated variant of the oxidoreductase with the amino acid sequence SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 12, which has at least 50%, preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90% and even more preferably at least 95%, of enzymatic activity compared with the non-truncated oxidoreductase.

[0040] The method according to the invention is shown schematically in the attached figure.

[0041] A preferred variant of the invention's method consists in which the oxidized NAD(P)+ cofactor formed by the reduction of D-psicose to allitol is reduced by means of an alcohol dehydrogenase (ADH) and a secondary alcohol to form a ketone, where the secondary alcohol is preferably D-glucose or 2-propanol (isopropanol). 2-propanol is a very inexpensive hydrogen donor for the regeneration of NAD(P)H, and the oxidation product, acetone, is easily separable due to its volatility (Xu et al., 2021). The acetone recovered from the exhaust gas stream can be catalytically re-hydrogenated heterogeneously to 2-propanol (Al-Rabiah et al., 2022), either in the gas phase or in solution, present as mixtures of 2 Petition 870250081800, dated 11 / 09 / 2025, page 18 / 88 12 / 56 propanol / acetone / water or acetone / water mixtures, while hydrogen from sustainable sources (green hydrogen) may be increasingly used in the future.

[0042] Cofactor regeneration using ADH is, for example, known from EP 2812439 B1 or was described in Xu et al. (2021).

[0043] In a further preferred embodiment of the present invention, the oxidized cofactor NAD(P)+ formed by the reduction is reduced by means of a glucose dehydrogenase and D-glucose, with the formation of D-gluconate. The use of a glucose dehydrogenase for the regeneration of NAD(P)H is particularly advantageous because, during the reduction of NAD(P)+, D-gluconate is formed from D-glucose, which can be obtained from the reaction mixture and used in various areas (e.g., in metal pickling agents, in pharmaceuticals and as stabilizers in food, etc.). Furthermore, the use of glucose dehydrogenase allows the use of a mixture comprising D-fructose and D-glucose as a substrate for the production of allitol or D-psicose without the addition of additional D-glucose to the reaction mixture and without the D-glucose being isomerized to D-fructose beforehand. Mixtures of D-fructose and D-glucose can, for example, be produced through the hydrolysis of sucrose.Particularly preferred is a glucose dehydrogenase originating from Priestia megaterium and comprising an amino acid sequence available under NCBI accession number MDQ0804260.1.

[0044] Another preferred variant of the method according to the invention comprises the use of a formate dehydrogenase (FDH) for the regeneration of the oxidized cofactor NAD(P)+ produced by reduction via a formate dehydrogenase and formate (e.g., sodium formate) with the formation of CO2.

[0045] A preferred additional variant of the invention method Petition 870250081800, dated 11 / 09 / 2025, page 19 / 88 13 / 56 is characterized by being carried out as a single-vessel reaction, without isolating any intermediate products.

[0046] In the invention method, the enzymes are preferably used as a lysate of the corresponding cells that produce them. In contrast to the method described by Wang et al. (2023), which is based on whole-cell E. coli biocatalysts with co-expressed recombinant enzymes, the enzymes are individually expressed in suitable E. coli production strains.

[0047] This allows for an optimization of enzyme ratios and is therefore independent of the expression level in the overall construct compared to Wang et al., (2023).

[0048] Before performing the final step (oxidation), the enzymes (epimerase and reductase and / or dehydrogenase) from the first step (D-fructose ^ allyl) are deactivated by heat and / or removed through ultrafiltration in order to avoid the formation of byproducts by the enzymes. Without proper treatment, much of the D-psicose formed by oxidation would be converted back into D-fructose.

[0049] The regeneration of nicotinamide-based cofactors (NAD or NADP) occurs, in the case of a reduction of D-psicose to allitol, with an NAD(P)-dependent alcohol dehydrogenase, glucose dehydrogenase or formate dehydrogenase, and in the case of oxidation reactions (second step), with an H2O-forming NAD(P)H oxidase.

[0050] The particularly preferred concentration of D-fructose is 50 to 250 g / l.

[0051] The particularly preferred temperature range for the first stage (epimerization and reduction) is between 25 and 45 °C and, for the second stage (oxidation), between 20 and 30 °C.

[0052] The pH range particularly preferred for both stages is between 7 and 8.5. Petition 870250081800, dated 11 / 09 / 2025, page 20 / 88 14 / 56

[0053] In a further preferred embodiment of the method of the invention, the enzymes are present in a suspension and / or in the homogenate and / or lysate of the respective cells that form them, a lysate being particularly preferred.

[0054] In this context, suspension refers to a suspension of resting cells. These are harvested after cultivation (separated from the growth medium) and used as a paste or suspended in a suitable buffer system. Unlike fermentative methods, where whole cells are also used, resting cells can no longer grow due to a lack of carbon and nutrient sources, but serve only for substrate conversion (Lin & Tao, 2017). In this context, homogenate refers to a suspension treated physically and / or chemically (e.g., by pressure, lysozyme, or ultrasound) so that the cell components are released from the cells. A lysate is obtained when the insoluble cell components of the homogenate are removed, for example, by filtration or centrifugation (see Production of the enzymes & preparation of the lysates for more details).

[0055] In another variant, the enzymes can also be modified at the N-terminus with a water-soluble polymer such as polyethylene glycol, immobilized in or on a solid matrix, or be part of a fusion protein.

[0056] In another variant, the enzymes may be present in powder form, freeze-dried or spray-dried.

[0057] After separation of the enzymes, D-psicose is preferably present in an aqueous solution, from which solid D-psicose can, for example, be obtained by spray drying (US 2019 / 0315790 A1; Kawakami et al., 2013; Kawakami et al., 2014).

[0058] Due to the high purity of the solution obtained, it is possible to concentrate Petition 870250081800, dated 11 / 09 / 2025, p. 21 / 88 15 / 56 extract the filtrate and obtain D-psicose in crystalline form or in the form of a syrup.

[0059] In a further preferred variant, D-psicose is present in a syrup, where the syrup is prepared by concentrating the filtrate described above or by dissolving crystalline D-psicose, which can be prepared using the method according to the invention, in water. The syrup according to the invention preferably has a total solids content of approximately 50% by weight to approximately 90% by weight. The D-psicose content in the syrup according to the invention is approximately 80% by weight to approximately 99% by weight, based on the dry matter.

[0060] Consequently, a further aspect of the present invention relates to a syrup comprising D-psicose, which can be produced by the method according to the invention.

[0061] In a particularly preferred embodiment of the method, only enzymes from the epimerase and oxidoreductase enzyme groups are used for the conversion of the starting material, with one or more of these enzymes being selected from each of these groups.

[0062] The epimerase used in the method may be from one of the following groups EC 5.1.3.30 (D-psicose-3-epimerase) or EC 5.1.3.31 (D-tagatose-3-epimerase / L-ribulose-3-epimerase), the former being particularly preferred.

[0063] The enzymes used for the reduction of D-psicose and for the oxidation of allitol belong to the group of oxidoreductases (see Table 1 for details).

[0064] The alcohol dehydrogenase (ADH) used for cofactor regeneration may belong to one of the groups EC 1.1.1.1 (NAD-dependent ADH) and EC 1.1.1.2 (NADP-dependent ADH).

[0065] A NAD(P)-dependent alcohol dehydrogenase for cofactor regeneration preferably comprises or consists of a Petition 870250081800, dated 11 / 09 / 2025, p. 22 / 88 16 / 56 amino acid sequence selected from the group consisting of: i) an amino acid sequence with at least 80% identity to SEQ ID NO: 18, ii) a nucleic acid-encoded amino acid sequence with at least 80% identity to SEQ ID NO: 17, and iii) a nucleic acid-encoded amino acid sequence that, under stringent conditions, binds to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 17.

[0066] Particularly suitable for cofactor regeneration in general is an alcohol dehydrogenase whose amino acid sequence has at least 80% identity with SEQ ID NO: 18, or which is encoded by a nucleic acid having at least 80% identity with SEQ ID NO: 17, or which binds, under stringent conditions, to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 17, or to a functional fragment of that alcohol dehydrogenase. A functional fragment of alcohol dehydrogenase comprises an N-terminal and / or C-terminal truncated variant of alcohol dehydrogenase with the amino acid sequence SEQ ID NO: 18, which has at least 50%, preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, and even more preferably at least 95% enzymatic activity compared to the non-truncated alcohol dehydrogenase. SEQ ID NO: 17: ATGAAAGCTGCAGTTGTGGAACAATTTAAAGCCGTTACAAGTGA AAGAAGTGGAAAAACCTAAGATCTCATACGGGGAAGTATTAGTGC GCATCAAAGCGTGGGGTATGCCATACAGACTTGCATGCCGCAC ATGGCGACTGGCCTGTAAGCCTAAACTGCCTCATTCTCCTGTCCTGGGGAAGTATTAGTGC Petition 870250081800, of 11 / 09 / 2025, p. 23 / 88 17 / 56 ATGAAGGCGTCGGTGTAATTGAAGAAGTAGGTCCTGGGGTAACAC ATTTAAAAGTTGGAGATCGCGTAGGTATCCCTTGGCTTTATTCGGC GTGCGGTCATTGTGACTATTGCTTAAGCGGACAAGAAACATTATG CGAACGTCAACAAAACGCTGGCTATTCCGTCGATGGTGGTTATGC TGAATATTGCCGTGCTGCAGCCGATTATGTCGTAAAAATTCCTGAT AACTTATCGTTTGAAGAAGCCGCTCCAATCTTTTGCGCTGGTGTAA CAACATATAAAGCGCTCAAAGTAACAGGCGCAAAACCAGGTGAAT GGGTAGCCATTTACGGTATCGGCGGGCTTGGACATGTCGCAGTC CAATACGCAAAGGCGATGGGGTTAAACGTCGTTGCTGTCGATTTA GGTGATGAAAAACTTGAGCTTGCTAAACAACTTGGTGCAGATCTT GTCGTCAATCCGAAACATGATGATGCAGCACAATGGATAAAAGAA AAAGTGGGCGGTGTGCATGCGACTGTCGTCACAGCTGTTTCAAAA GCCGCGTTCGAATCAGCCTACAAATCCATTCGTCGCGGTGGTGCT TGCGTACTCGTCGGATTACCGCCGGAAGAAATACCTATTCCAATTT TCGATACAGTATTAAATGGAGTAAAAATTATTGGTTCTATCGTTGG TACGCGCAAAGACTTACAAGAGGCACTTCAATTTGCAGCAGAAGG AAAAGTAAAAACAATTGTCGAAGTGCAACCGCTTGAAAACATTAAC GACGTATTCGATCGTATGTTAAAAGGGCAAATTAACGGCCGCGTC GTGTTAAAAGTAGATTAA SEQ ID NO: 18: MKAAVVEQFKKPLQVKEVEKPKISYGEVLVRIKACGVCHTDLHAAHG DWPVKPKLPLIPGHEGVGVIEEVGPGVTHLKVGDRVGIPWLYSACGH CDYCLSGQETLCERQQNAGYSVDGGYAEYCRAAADYVVKIPDNLSF EEAAPIFCAGVTTYKALKVTGAKPGEWVAIYGIGGLGHVAVQYAKAM GLNVVAVDLGDEKLELAKQLGADLVVNPKHDDAAQWIKEKVGGVHA TVVTAVSKAAFESAYKSIRRGGACVLVGLPPEEIPIPIFDTVLNGVKIIG SIVGTRKDLQEALQFAAEGKVKTIVEVQPLENIDVFDRMLKGQINGR VVLKVD

[0067] Alcohol dehydrogenase for regeneration of menthol cofactor. Petition 870250081800, dated 11 / 09 / 2025, page 24 / 88 18 / 56 described herein preferably comprises an amino acid sequence that has at least 80% identity with SEQ ID NO: 18, more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, in particular 100%. Particularly preferably, the alcohol dehydrogenase according to the invention for cofactor regeneration comprises or consists of the amino acid sequence of SEQ ID NO: 18.

[0068] Alternatively, the alcohol dehydrogenase for cofactor regeneration preferably comprises an amino acid sequence encoded by a nucleic acid with at least 80% identity to SEQ ID NO: 17, more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, in particular 100%. Particularly preferably, the nucleic acid encoding the alcohol dehydrogenase for cofactor regeneration according to the invention comprises or consists of the nucleic acid sequence of SEQ ID NO: 17.

[0069] A further aspect of the present invention relates to the use of an alcohol dehydrogenase for cofactor regeneration, wherein the alcohol dehydrogenase comprises or consists of an amino acid sequence selected from the group consisting of: i) an amino acid sequence with at least 80% identity to SEQ ID NO: 18, ii) a nucleic acid-encoded amino acid sequence with at least 80% identity to SEQ ID NO: 17, and iii) a nucleic acid-encoded amino acid sequence that, under stringent conditions, binds to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 17, or a functional fragment thereof. Petition 870250081800, dated 11 / 09 / 2025, p. 25 / 88 19 / 56

[0070] The alcohol dehydrogenases disclosed herein can be used for the regeneration of NAD(P)+ or NAD(P)H, that is, for the reduction of NAD(P)+ or the oxidation of NAD(P)H, in a variety of enzymatic reactions. Particularly preferred is the use of the alcohol dehydrogenases of the invention in the regeneration of NAD(P)+ cofactor, which is formed during the reduction of D-psicose to allyl by means of an NAD(P)H-dependent oxidoreductase.

[0071] The cofactor NAD(P)+ produced during the reduction of Dpsicose to allytol is reduced to NAD(P)H by formate and a formate dehydrogenase with the formation of CO2 (cofactor regeneration).

[0072] The use of a formate dehydrogenase comprising or consisting of the amino acid sequence of SEQ ID NO: 2 or a functional fragment of this formate dehydrogenase is particularly preferred. The preferred formate dehydrogenase used is preferably encoded by the nucleic acid sequence of SEQ ID NO: 1. A functional fragment of the formate dehydrogenase comprises an N-terminal and / or C-terminal truncated variant of the formate dehydrogenase with the amino acid sequence of SEQ ID NO: 2, which has at least 50%, preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, and even more preferably at least 95%, enzymatic activity compared to the non-truncated formate dehydrogenase. SEQ ID NO: 1: ATGGCGAAAATACTTTGCGTTCTCTATGACGATCCGGTCGACGGC TACCCGAAGACCTATGCGCGCGACGACCTGCCGAAGATCGACCA CTATCCGGGCGGGCAGACGCTGCCCACGCCCAAGGCGATCGACT TCACGCCGGGCGCGCTGCTCGGCTCGGCTCCGGCGAGCTCGG Petition 870250081800, of 11 / 09 / 2025, p. 26 / 88 20 / 56 CCTGCGCAAATACCTGGAAGCCAACGGCCATACCTTCGTCGTCAC CTCCGATAAGGACGGCCCGGATTCGGTGTTCGAGAGGGAACTCG TCGACGCCGACGTGGTGATCTCGCAGCCCTTCTGGCCGGCCTAT CTGACGCCCGAGCGCATCGCCAAGGCGAAGAACCTGAAGCTCGC GCTCACCGCCGGCATCGGCTCCGATCATGTCGATCTTCAGTCAGC TATCGACCGTGGCATCACTGTGGCCGAAGTCACATATTGCAACTC GATCAGCGTCGCCGAGCACGTGGTGATGATGATCCTCGGCCTGG TACGAAACTACATTCCCTCGCATGACTGGGCGCGCAAGGGCGGC TGGAACATAGCCGACTGCGTAGAGCACTCCTACGACCTCGAGGG CATGACCGTCGGCTCGGTGGCCGCCGGCCGCATCGGCCTCGCC GTGCTGCGCCGCCTCGCGCCGTTCGACGTGAAGCTGCACTATAC CGACCGCCACCGTCTGCCAGAAGCGGTCGAGAAGGAGCTGGGC CTCGTCTGGCACGATACCCGCGAGGACATGTACCCGCATTGCGA CGTGGTCACGCTCAACGTGCCGCTGCACCCCGAAACCGAGCACA TGATCAATGACGAGACGCTGAAGCTGTTCAAGCGCGGCGCCTATA TCGTCAACACCGCCCGCGGCAAGCTCGCCGACCGCGACGCCATC GTCCGCGCGATCGAGAGCGGGCAGCTCGCGGGCTATGCCGGCG ACGTGTGGTTCCCGCAGCCGGCTCCGAAGGACCACCCCTGGCGC ACCATGAAGTGGGAAGGCATGACGCCGCACATCTCCGGCACCTC GCTCTCTGCCCAGGCGCGCTACGCGGCGGGCACGCGCGAGATC CTCGAATGCTTCTTCGAGGGCCGGCCGATCCGCGACGAGTACCT GATCGTGCAGGGCGGCGCGCTCGCCGGCACCGGCGCGCATTCCTACTCGAAGGGCAATGCGACCGGCGGTTCGGAAGAGGCCGCGAA GTTCAAGAAGGCTGGCTGA SEQ ID NO: 2: MAKILCVLYDDPVDGYPKTYARDDLPKIDHYPGGQTLPTPKAIDFTPG ALLGSVSGELGLRKYLEANGHTFVVTSDKDGPDSVFERELVDADVVI SQPFWPAYLTPERIAKAKNLKLALTAGIGSDHVDLQSAIDRGITVAEVT YCNSISVAEHVVMMILGLVRNYIPSHDWARKGGWNIADCVEHSYDLE Petition 870250081800, dated 11 / 09 / 2025, p. 27 / 88 21 / 56 GMTVGSVAAGRIGLAVLRRLAPFDVKLHYTDRHRLPEAVEKELGLVW HDTREDMYPHCDVVTLNVPLHPETEHMINDETLKLFKRGAYIVNTAR GKLADRDAIVRAIESGQLAGYAGDVWFPQPAPKDHPWRTMKWEGM TPHISGTSLSAQARYAAGTREILECFFEGRPIRDEYLIVQGGALAGTG AHSYSKGNATGGSEEAAKFKKAG

[0073] According to another preferred embodiment of the present invention, the formate dehydrogenase used for cofactor regeneration comprises an amino acid sequence selected from the group consisting of: i) an amino acid sequence with at least 80% identity to SEQ ID NO: 2, ii) a nucleic acid-encoded amino acid sequence with at least 80% identity to SEQ ID NO: 1, and iii) a nucleic acid-encoded amino acid sequence that, under stringent conditions, binds to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 1, or a functional fragment thereof.

[0074] The formate dehydrogenase preferably comprises an amino acid sequence with at least 80% identity with SEQ ID NO: 2, more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, in particular 100%.

[0075] Alternatively, the formate dehydrogenase preferably comprises an amino acid sequence encoded by a nucleic acid with at least 80% identity with SEQ ID NO: 1, more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, in particular 100%.

[0076] Another aspect of the present invention relates to the use of a formate dehydrogenase for cofactor regeneration or a fragment. Petition 870250081800, dated 11 / 09 / 2025, p. 28 / 88 22 / 56 functional element thereof, wherein formate dehydrogenase comprises or consists of an amino acid sequence selected from the group consisting of: i) an amino acid sequence with at least 80% identity to SEQ ID NO: 2, ii) an amino acid sequence encoded by a nucleic acid with at least 80% identity to SEQ ID NO: 1, and iii) an amino acid sequence encoded by a nucleic acid that, under strict conditions, binds to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 1, or a functional fragment thereof.

[0077] The glucose dehydrogenase (GDH) used for cofactor regeneration can be from one of the following groups: EC 1.1.1.47 (glucose-1-dehydrogenase), EC 1.1.1.118 (glucose-1-dehydrogenase (NAD+)), EC 1.1.1.119 (glucose-1-dehydrogenase (NADP+)), or EC 1.1.1.360 (glucose / galactose-1-dehydrogenase).

[0078] NAD(P)-dependent glucose dehydrogenase for cofactor regeneration preferably comprises or consists of an amino acid sequence selected from the group consisting of: i) an amino acid sequence with at least 80% identity to SEQ ID NO: 20, ii) a nucleic acid-encoded amino acid sequence with at least 80% identity to SEQ ID NO: 19, and iii) a nucleic acid-encoded amino acid sequence that, under stringent conditions, binds to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 19.

[0079] Particularly suitable for cofactor regeneration in general is a glucose dehydrogenase whose amino acid sequence has at least 80% identity with SEQ ID NO: 20 or that is encoded by a nucleic acid with at least 80% identity. Petition 870250081800, dated 11 / 09 / 2025, p. 29 / 88 23 / 56 with SEQ ID NO: 99, or that binds, under stringent conditions, to a nucleic acid molecule with the nucleic acid sequence of SEQ ID NO: 19 or to a functional fragment of this glucose dehydrogenase. A functional fragment of glucose dehydrogenase comprises an N-terminal and / or C-terminal truncated variant of glucose dehydrogenase with the amino acid sequence of SEQ ID NO: 18, which has at least 50%, preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, and even more preferably at least 95% enzymatic activity compared to the non-truncated alcohol dehydrogenase. SEQ ID NO: 19: ATGTATACAGATTTAAAAGATAAAGTAGTTGTAATTACAGGTGGAT CAACAGGTTTAGGACGCGCAATGGCTGTTCGTTTCGGTCAAGAAG AAGCAAAAGTTGTTATTATTACAACAATGAAGAAGAAGCTTT AGATGCGAAAAAAGAAGTAGAAGAGGCAGGCGGACAAGCAATCAT CGTTCAAGGCGACGTAACAAAAGAAGAAGATGTTGTAAACCTTGT TCAAACAGCTATTAAAATTCGGTACATTAGACGTTATTAATA ACGCTGGTGTTGAAAACCCAGTTCCTTCATGAGTTATCTTTAGA CAACTGGAATAAAGTTATTGATACAAACTTAACTAGGTGCATTCTTA GGAAGCCGTGAAGCAATCAAATATTGTTGAAAACGACATTAAAG GAAACGTTTAACATGTCTAGTGTTCATGAAATGATTCCTTGGCC ATTATTTGTTCATTACGCAGCAAGTAAAGGCGGTATGAAACTAATG ACGGAAACATTGGCTCTTGAATATGCGCCAAAAGGTATCCGCGTA AATAACATTGGACCAGGTGCGATGAACACACCAATTAACGCAGAG AAATTTGCAGATCCTGTACAACGTGCAGACGTAGAAAGCATGATT CCAATGGGTTACATCGGTAAACCAGAAGAAGTAGCAGCAGTTGCA GCATTCTTAGCATCATCACAAGCAAGCTATGTAACAGGTTATCAT TATTTGCTGATGGTGGTATGACGAAATACCCTTCTTTCCAAGCAGG Petition 870250081800, of 11 / 09 / 2025, p. 30 / 88 24 / 56 AAGAGGCTAA SEQ ID NO: 20: MYTDLKDKVVVITGGSTGLGRAMAVRFGQEEAKVVINYYNNEEEALD AKKEVEEAGGQAIIVQGDVTKEEDVVNLVQTAIKEFGTLDVMINNAGV ENPVPSHELSLDNWNKVIDTNLTGAFLGSREAIKYFVENDIKGNVINM SSVHEMIPWPLFVHYAASKGGMKLMTETLALEYAPKGIRVNNIGPGA MNTPINAEKFADPVQRADVESMIPMGYIGKPEEVAAVAAFLASSQAS YVTGITLFADGGMTKYPSFQAGRG

[0080] The glucose dehydrogenase for cofactor regeneration mentioned herein preferably comprises an amino acid sequence with at least 80% identity to SEQ ID NO: 20, more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, in particular 100%. Particularly preferably, the glucose dehydrogenase for cofactor regeneration of the invention comprises or consists of the amino acid sequence of SEQ ID NO: 20.

[0081] Alternatively, the glucose dehydrogenase for cofactor regeneration preferably comprises an amino acid sequence encoded by a nucleic acid with at least 80% identity to SEQ ID NO: 19, more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, in particular 100%. Particularly preferably, the nucleic acid encoding the inventive glucose dehydrogenase for cofactor regeneration comprises or consists of the nucleic acid sequence of SEQ ID NO: 19.

[0082] A further aspect of the present invention relates to the use of a glucose dehydrogenase for cofactor regeneration, wherein the glucose dehydrogenase comprises or consists of an amino acid sequence selected from the group consisting of: Petition 870250081800, dated 11 / 09 / 2025, page 31 / 88 25 / 56 i) an amino acid sequence with at least 80% identity to SEQ ID NO: 20, ii) a nucleic acid-encoded amino acid sequence with at least 80% identity to SEQ ID NO: 19, and iii) a nucleic acid-encoded amino acid sequence that, under stringent conditions, binds to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 19 or a functional fragment thereof.

[0083] The NAD(P)H oxidase used for cofactor regeneration (see Figure 1) can be from one of the following groups: EC 1.6.3.1 (NAD(P)H oxidase (H2O2 forming)), EC 1.6.3.2 (NAD(P)H oxidase (H2O forming)), EC 1.6.3.3 (NADH oxidase (H2O2 forming)) and EC 1.6.3.4 (NADH oxidase (H2O forming)), with the H2O-forming classes being particularly preferred.

[0084] A particularly preferred H2O-forming NAD(P)H oxidase comprises or preferably consists of an amino acid sequence selected from the group consisting of: i) an amino acid sequence with at least 80% identity to SEQ ID NO: 14 or SEQ ID NO: 16, ii) a nucleic acid-encoded amino acid sequence with at least 80% identity to SEQ ID NO: 13 or SEQ ID NO: 15, and iii) a nucleic acid-encoded amino acid sequence that, under stringent conditions, binds to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 13 or SEQ ID NO: 15, or a functional fragment thereof.

[0085] A functional fragment of this NAD(P)H oxidase comprises an N-terminal and / or C-terminal truncated variant of NAD(P)H oxidase with the amino acid sequence SEQ ID NO: 4, SEQ ID Petition 870250081800, dated 11 / 09 / 2025, p. 32 / 88 26 / 56 NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 12, which has at least 50%, preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90% and even more preferably at least 95% enzymatic activity compared to non-truncated NAD(P)H oxidase. SEQ ID NO: 13: ATGAGCAAAATTGTTATCGTGGGTGCAAATCATGCAGGCACCGCA GCAATTAATACCATTCTGGATAATTATGGCAGCGAAAATGAAGTGG TTGTGTTTGATCAGAATAGCAACATTAGCTTTCTGGGTTGTGGTAT GGCACTGTGGATTGGTAAACAAATTAGCGGTCCGCAGGGTCTGTT TTATGCAGATAAAGAAAGCCTGGAAGCAAAAGGTGCCAAAATCTA TATGGAAAGTCCGGTTACCGCCATTGATTATGATGCAAAACGTGTT ACCGCACTGGTTAATGGTCAAGAACATGTTGAAAGCTACGAGAAA CTGATTCTGGCAACCGGTAGCACCCCGATTCTGCCTCCGATTAAA GGTGCAGCCATTAAAGAAGGTAGTCGCGATTTTGAAGCAACCCTG AAAAATCTGCAGTTCGTGAAACTGTATCAGAATGCCGAAGATGTG ATTAACAAACTGCAGGATAAAAGCCAGAATCTGAATCGTATTGCAG TTGTTGGTGCAGGTTATATTGGTGTTGAACTGGCAGAAGCATTTAA ACGTCTGGGTAAAGAAGTGATTCTGATTGACGTTGTTGATACCTGT CTGGCAGGTTATTATGATCAGGATCTGAGCGAAATGATGCGTCAG AATCTGGAAGATCATGGTATCGAACTGGCATTTGGTGAAACCGTT AAAGCAATTGAAGGTGATGGTAAAGTGGAACGTATTGTTACCGAT AAAGCAAGCCATGATGTGGATATGGTTATTCTGGCAGTTGGTTTTC GTCCGAATACAGCACTGGGTAATGCAAAACTGAAAACCTTTCGTA ATGGTGCCTTTCTGGTGGATAAAAAACAAGAAACCAGCATCCCGG ATGTTTATGCAATTGGTGATTGTGCAACCGTGTATGATAATGCCAT TAACGACACCAACTATATTGCACTGGCAAGCAATGCACTGCGTAGCGGTATTGTTGCAGGTCATAATGCAGCCGGTCATAAACTGGAAAG Petition 870250081800, of 11 / 09 / 2025, p. 33 / 88 27 / 56 TCTGGGTGTTCAGGGTAGCAATGGTATTTCAATTTTTGGCCTGAAT ATGGTTAGCACCGGTCTGACCCAAGAAAAAGCCAAACGTTTTGGT TATAATCCGGAAGTTACCGCCTTTACCGATTTCGAAAGCCAGCT TTATCGAGCATGATAACTATCCGGTTACGCTGAAATTGTGTGAAT CAAAGATAGCCGTCTGGTTCTGGGTGCACAGATGGCCAGCAAAG AAGATATGAGCATGGGTATTCACATGTTTAGCCTGGCCATTCAAGA GAAAGTTACCATTGAACGTCTGGCCCTGCTGGATTATTTCTTTCTG CCGCATTTTAATCAGCCGTACAACTATATGACCAAAGCAGACCAACTGAACATGA SEQ ID NO: 14: MSKIVIVGANHAGTAAINTILDNYGSENEVVVFDQNSNISFLGCGMAL WIGKQISGPQGLFYADKESLEAKGAKIYMESPVTAIDYDAKRVTALVN GQEHVESYEKLILATGSTPILPPIKGAAIKEGSRDFEATLKNLQFVKLY QNAEDVINKLQDKSQNLNRIAVVGAGYIGVELAEAFKRLGKEVILIDVV DTCLAGYYDQDLSEMMRQNLEDHGIELAFGETVKAIEGDGKVERIVT DKASHDVDMVILAVGFRPNTALGNAKLKTFRNGAFLVDKKQETSIPD VYAIGDCATVYDNAINDTNYIALASNALRSGIVAGHNAAGHKLESLGV QGSNGISIFGLNMVSTGLTQEKAKRFGYNPEVTAFTDFQKASFIEHDN YPVTLKIVYDKDSRLVLGAQMASKEDMSMGIHMFSLAIQEKVTIERLA LLDYFFLPHFKKKNYMTKKNYM SEQ ID NO: 15: ATGAAAGTAGTAGTAGTAGGCTGTACACATGCAGGAACAGCGGCA GTTAAGACGATTTTAAATGAACATCCAGATGCATCAGTATCAGTAT ATGAGCGTAATGACAATGTCTCATTTCTATCTTGTGGGATTGCGTT GTATGTTGGTGGAGTTGTGAAAGATCCTGTCAGTTTCTTTT AGTCCAGAAGAACTTGCATCAATGGGCGCGAAAATTAACATGGAA CACAATGTGAAAAATAGATAATGAATAAGGTCGTAGTAATTG AGAATTTAAAAACAGGCGAAACATTTGAAGAAAGCTATGATAAGTT Petition 870250081800, of 11 / 09 / 2025, p. 34 / 88 28 / 56 GGTAATGACAACTGGATCATGGCCAATTATTCCTCCAATTGATGGA ATCAATAGTGAAAATATTCTTTTGTGTAAAAAACTATAACCAAGCAAA TGAAATTATTAAAGAATCAAAAAAATGCTAAAAGATTGTCATTGTTG GTGGTGGCTATATTGGAATTGAATTAGTTGAGGCATTTGCAGAATC TGGCAAGCAAGTGACGCTAGTTGAATGGATTAGATCTTTTAAAC AAATATTTAGATGCTGAATTCACTTCTGTTTTAGCATGATTTACA AGAAAGAGGCGTTACGCTAGCTTTAAACCAAACCGTCGAGAAATT TGTTGCCAATGAATCAGGTGCTGTGACAGCTGTGAAACACCAGT TGGAGAATATGAGGCTGATTTAGTTATTTATGTGTTGGATTTAAA CCAAATACTGATTTGTTGAAGGATAAAGTAGAGATGTTGCCAAATG GTGCCATCGTAGTGGATGAATATATGAGAACAAGCGATGAAGCGA TTTTTGCTGCTGGCGATAGTTGCGCGGTTCATTATAATCCAACTGG AGGCTCTGCGTATATTCCGTTAGCTACAAATGCAGTTTAGAATGGG AGCTTTTAGTTGGGAAAAAATTGTTTCTCCAACAGTTAAATATCGT GGCACGCAAGCAACTTCTGGTTTATTTTATTTGGTTTTAATATAG GTTCAACCGGATTGACTGAAAATAGCGCTCCTCATTTTGGCGTAG AGGTTCGTTCAGTAGTTGTAGAAGATAATTATCGTCCAGAGTTTAT GCCGACAACAGAGAAAGTAACGAATGAAATTAGTTTATGAAGTAG AACGAATCGGATTGGAGGTCAAATCATGTCAAAAATAGATGTG ACACAATCTGCCAATACGTTATCTTTATGTTCAAAATAAAATGAC GATTGAGGATTTGGCTTATGTAGATTTCTTCTTCCAACCTCACTTTGATCGTCCTTGGAACTATTTAAATATTTTAGCGCAAGCAGCTGTTG AGCAAGAGCGTAAACTAGCAAAATAA SEQ ID NO: 16: MKVVVVGCTHAGTAAVKTILNEHPDASVSVYERNDNVSFLSCGIALY VGGVVKDPAGLFYSSPEELASMGAKINMEHNVKNIDNENKVVVIENL KTGETFEESYDKLVMTTGSWPIIPPIDGINSENILLCKNYNQANEIKES KNAKKIVIVGGGYIGIELVEAFAESGKQVTLVDGLDRLNKYLDAEFTS VLEHDLQERGVTLALNQTVEKFVANESGAVTAVKTPVGEYEADLVIL Petition 870250081800, of 11 / 09 / 2025, p. 35 / 88 29 / 56 CVGFKPNTDLLKDKVEMLPNGAIVVDEYMRTSDEAIFAAGDSCAVHY NPTGGSAYIPLATNAVRMGALVGKNIVSPTVKYRGTQATSGLYLFGF NIGSTGLTENSAPHFGVEVRSVVVEDNYRPEFMPTTEKVTMKLVYEV GTNRIVGGQIMSKYDVTQSANTLSLCVQNKMTIEDLAYVDFFFQPHF DRPWNYLNILAQAEQERKLAK

[0086] The preferably used H2O-forming NAD(P)H oxidase comprises or consists of an amino acid sequence with at least 80% identity to SEQ ID NO: 16 or SEQ ID NO: 14, more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, in particular 100%. Particularly preferably, the H2O-forming NAD(P)H oxidase comprises or consists of the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 14.

[0087] Alternatively, the NAD(P)H H2O-forming oxidase preferably has an amino acid sequence encoded by nucleic acid with at least 80% identity to SEQ ID NO: 15 or SEQ ID NO: 13, more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, in particular 100%. Particularly preferably, the nucleic acid encoding the NAD(P)H H2O-forming oxidase comprises or consists of the nucleic acid sequence of SEQ ID NO: 15 or SEQ ID NO: 13.

[0088] A further aspect of the present invention relates to the use of a H2O-forming NAD(P)H oxidase for cofactor regeneration (NAD(P)H to NAD(P)+) comprising or consisting of an amino acid sequence selected from the group consisting of: i) an amino acid sequence with at least 80% identity to SEQ ID NO: 16 or SEQ ID NO: 14, Petition 870250081800, dated 11 / 09 / 2025, p. 36 / 88 30 / 56 ii) an amino acid sequence encoded by a nucleic acid with at least 80% identity to SEQ ID NO: 15 or SEQ ID NO: 13, and iii) an amino acid sequence encoded by a nucleic acid that, under stringent conditions, binds to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 15 or SEQ ID NO: 13, or a functional fragment thereof.

[0089] In combination with cofactor regeneration, the enzymatic strategy presented here enables a biocatalytic, environmentally friendly, and highly efficient production method for D-psicose production.

[0090] The NAD(P)H-dependent oxidoreductase for reducing first D-psicose to allytol preferably comprises or consists of an amino acid sequence selected from the group consisting of: i) an amino acid sequence with at least 80% identity to SEQ ID NO: 4, SEQ ID NO: 10, or SEQ ID NO: 12; ii) a nucleic acid-encoded amino acid sequence with at least 80% identity to SEQ ID NO: 3, SEQ ID NO: or SEQ ID NO: 11, and iii) an amino acid sequence encoded by a nucleic acid that, under strict conditions, binds to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 3, SEQ ID NO: 9 or SEQ ID NO: 11.

[0091] Particularly suitable for reducing first Dpsicose in allytol or D-psicose in allytol in general is an oxidoreductase whose amino acid sequence has at least 80% identity with SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12, or that is Petition 870250081800, dated 11 / 09 / 2025, p. 37 / 88 31 / 56 encoded by a nucleic acid with at least 80% identity to SEQ ID NO: 3, SEQ ID NO: 9, or SEQ ID NO: 1, or which, under stringent conditions, binds to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 3, SEQ ID NO: 9, or SEQ ID NO: 11. Furthermore, this oxidoreductase can, surprisingly, be used to oxidize allitol in D-psicose.

[0092] The NAD(P)+-dependent oxidoreductase for the formation of allitol from D-psicose preferably comprises or consists of an amino acid sequence selected from the group consisting of: i) an amino acid sequence with at least 80% identity to SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12, ii) a nucleic acid-encoded amino acid sequence with at least 80% identity to SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 11, and iii) a nucleic acid-encoded amino acid sequence that, under strict conditions, binds to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 11. SEQ ID NO: 3: ATGACACAGTCTCTTCAGGGCAAGATCGTCGCCATTACTGGCGCG GCTTCGGGCATTGGCCTCGAATGCGCCCGCTATCTCATCGAAGCT GGCGCGGTGGTCTATCTTCTGGACCGTGACGCCAAAACTCTCGAA GACAAAACGGCAGAACTCGGCAGCCAGGGCCCATGCGATCATCGT CGATCTCTTCGACTACAAAACCGTAGATGCTGCGGTCGCGCAGAT CGTTGAGGAGCAGGGCCGGATCGATGTTTTCCACGCCAACGCCG GCGCGTATGTGGGCGGCAATGTCTGGGAAGGGGATCCCGATAGC TGGGATGCGATGCTGCACCTGAACATCAATGCAGCTTTCCGCTCG Petition 870250081800, of 11 / 09 / 2025, p. 38 / 88 32 / 56 GTCCGTGCCGTGCTGCCGCAGATGATGAAGCAGGAAAGTGGCGA TATCGTCATGACCAGCTCGATCGCGGGCATGATCCCCATCATGGC CGAGCCGATCTACACGGCCTCGAAACATGCCGTGCAGGCATTCG TACATACGGTGCGCCGACAGGTCGCGAAGTATGGAATTCGTGTG GGTGCAATCCAGCCTGGTCCTGTAGTCACGCCTTTGCTGAAAGAC TGGGATCAGGCCCGTTCTTGAAGCCAACATCAAAGCGGGTGCCCT GATGGAAGCCAAGGAAGTCGCCGAAGCCCTGATCTTCATCCTGAC ACGTTCAAAGGGTGTCATGGTGCGGGATCTGCAGTCCGTCGTC TAACTTCGACGCCTAAGCTTAAGCGGCCGCACTCGAGCACCACCA CCACCACCACTGAGATCCGGCTGCTAA SEQ ID NO: 4: MTQSLQGKIVAITGAASGIGLECARYLIEAGAVVYLLDRDACTLEDKTA ELGSQAHAIIVDLFDYKTVDAAVAQEEQGRIDVFHANAGAYVGGNV WEGDPDSWDAMLHLNINAAFRSVRAVLPQMMKQESGDIVMTSSIAG MIPIMAEPIYTASKHAVQAFVHTVRRQVAKYGIRVGAIQPGPVVTPLLK DWDQARLEANIKAGALMEAKEVAEALIFILTRSKGVMVRDLVVLPHNF DA SEQ ID NO: 5: ATGAGCACACCGGAAAATCTGAGCTTTGTGCTGCAGAAACCGTTT GATGTGAAATTTGAAGATCGTCCGATTCCGAAACTGAGCGATCCG TATAGCGTTAAAATTCAGGTGAAAAAAACCGGCATTTGCGGTAGT GATGTTCACTATTTCACCCATGGTGCAATTGGTGATTTTGTTGTTA AAGCACCGATGGTTCTGGGTCATGAAAGCAGCGGTGTTGTTCTGG AAGTTGGTAGCGAAGTTAAAAGCCTGAAAGTTGGTGATCGTGTTG CAATGGAACCGGGTGTTCCGAGCCGTCATAGTGATGAGTATAAAA GCGGTCGTTATAATCTGTGTCCGCACATGGCATTTGCAGCAACCC CTCCGTATGATGGCACCCTGTGTAAATACTATATTCTGCCGGAAG ATTTCTGCGTTAAACTGCCGGAACATGTTAGCCTGGAAGAAGGTG Petição 870250081800, de 11 / 09 / 2025, pág. 39 / 88 33 / 56 CACTGGTTGAACCGCTGAGCGTTGCAGTTCATAGCAGCAAACTGG GTAACATTAAACCGGGTAGCCATGTTGCAATTTATGGTGCAGGTC CGGTTGGTCTGCTGGTTGCAGCAGTTGCAAGCGCATTTGGTGCA GAAAGCGTTACCATTATTGATCTGGTTGAAAGCCGTCTGAATCTG GCAAAAGAACTGGGTGCAACCGCAACCGTTCAGGTTGATTTTAAA GATACCCCGAAAGAAAGCGCAGCAAAAGTTGTTGCAGCAAATAAT GGCATTGCACCGGATGTTGTTATTGATGCAAGCGGTGCAGAAGCA AGCATTAATTCAGCCATTAATGCAATTCGTCCGGGTGGCACCTAT GTTCAGGTGGGTATGGGTAAACCGGATGTGAGCTTTCCGATTGCA ACCCTGATTGGTAAAGAACTGACCGTTAAAGGTAGCTTTCGTTATG GTTATGGTGATTATCCGCTGGCAGTTAGCCTGCTGGCAAGCGGTA AAGTTAATGTGAAAAAACTGATCACCCATGAAGTGAAATTCGAGGA TGCAGCAGAAGCATTTCAGCTGGTTCGTGATGGTAAAGCCATTAA ATGTATTATCAACGGTCCGGAATAA SEQ ID NO: 6: MSTPENLSFVLQKPFDVKFEDRPIPKLSDPYSVKIQVKKTGICGSDVH YFTHGAIGDFVVKAPMVLGHESCGVVLEVGSEVKSLKVGDRVAMEP GVPSRHSDEYKSGRYNLCPHMAFAATPPYDGTLCYILPEDFCVKL PEHVSLEEGALVEPLSVAVHSSKLGNIKPGSHVAIYGAGPVGLLVAAV ASAFGAESVTIIDLVESRLNLAKELGATATVQVDFKDTPKESAAKVVA ANNGIAPDVVIDASGAEASINSAINAIRPGGTYVQVGMGKPDVSFPIAT LIGKELTVKGSFRYGYGDYPLAVSLLASGKVNVKKLITHEVKFEDAAE AFQLVRDGKAIKCIINGPE SEQ ID NO: 7: ATGAATAACAACCTGCCGAAACATGAAAGCAGCAGTTATGCAT GGCACCCGTGAAATTAGCATTGAAACCCTGCCGATTCCGCAGATT GATGAAAATGAAGTTCTGATCAAAGTTATGGCCGTTGGTATTTGTG GTAGCGATCTGCACTATTACCCAGGGTCGTATTGGTAAATACA Petition 870250081800, of 11 / 09 / 2025, p. 40 / 88 34 / 56 AAGTGGAAAAACCGTTTATCCTGGGTCATGAATGTAGCGGTGAAG TTGTTGCAATTGGTAGCGCAGTTGAACGTTTTCGTGTTGGTGATC GTGTTGCCGTTGAACCGGGTGTTACCTGTGGTCATTGTGAAGCAT GTAAAGAGGGTCGTTATAATCTGTGTCCGGATGTTCAGTTTCTGG CAACCCCTCCGGTTGATGGTGCATTTGTTCAGTATATCAAAATGCG CCAGGATTTCGTTTTTCTGATTCCGAATAGCCTGAGCTATGAAGAT GCAGCACTGATTGAACCGTTTAGCGTGGGTATTCATGCAGCAACC CGTACCAAACTGCAGCCTGGTAGCACCATTGCAATTATGGGTATG GGTCCGGTTGGTCTGATGGCAGTTGCAGCAGCAAAAGCATTTGGT GCAAGCACCATTATTGCAACCGATCTGGAACCGCTGCGTCTGGAA GCAGCCAAACGTATGGGTGCAACCCATGTTATTAACATTCGTGAA CAGGATCCGCTGAACGAGATTAAAAACATTACCGAAAATGTGGGT GTTGATGTTGCATGGGAAACCGCAGGTAATCCGAAAGCACTGCAG AGCAGCCTGAGCAGCATTCGTCGTGGTGGTAAACTGGCAATTGTT GGTCTGCCGAGCCAGAGCGATATTCCGCTGGATGTTCCGTTTATT GCCGATAATGAAATCGATATCTATGGCATCTTTCGCTATGCAAACA CCTATCCGAAAGGCATCAAATTTCTGACCAGCGGTGCAATTGATA CCAAAAATCTGGTTACCGATCGTTATCCGCTGGCAGGTACACGTG AAGCAATGGAACGTGCACTGAATTTCAAAAACGAATGCCTGAAAAT CATCGTGTATCCGAACGAATAA SEQ ID NO: 8: MNNNLPKTMKAAVMHGTREISIETLPIPQIDENEVLIKVMAVGICGSDL HYYTQGRIGKYKVEKPFILGHECSGEVVAIGSAVERFRVGDRVAVEP GVTCGHCEACKEGRYNLCPDVQFLATPPVDGAFVQYIKMRQDFVFLI PNSLSYEDAALIEPFSVGIHAATRTKLQPGSTIAIMGMGPVGLMAVAA AKAFGASTIIATDLEPLRLEAAKRMGATHVINIREQDPLNEIKNITENVG VDVAWETAGNPKALQSSLSSIRRGGKLAIVGLPSQSDIPLDVPFIADN EIDIYGIFRYANTYPKGIKFLTSGAIDTKNLVTDRYPLAGTREAMERAL NFKNECLKIIVYPNE Petition 870250081800, dated 11 / 09 / 2025, page 41 / 88 35 / 56 SEQ ID NO: 9: ATGACCTCTCCTCTCCAGGGTAAGATAGCCGCCATCACGGGCGG GGCTTCGGGCATCGGCCTCGAATGTGTCCGCCAGATCGCCGCAA GTGGTGCCACGGTTTATATTCTCGACCGCGACCATCAGGCGCTCG ACAAGGCGCGCGAAGAATTGGGCGAGCGCGTTCATACCATCGAG GTCGATCTCTTCCGTTACGAAACGGTCGATCGCGCCATCGAAACC ATCGTGTCCGAACAAGGACGCATCGACATTCTCCATGTCAATGCG GGCGCGTATATCGGCGGCAATGTCTGGGAAGGCGATCCCGATAA ATGGGACAAGATGCTGAATCTCAACATCAACGCCGCCTTCCGTTC CGCCCGCGCCGTCATGCCCGCCATGATGAAGCAGAAAAGCGGCG ATATCATCATGACAAGCTCGATCGCAGGCATCGTCCCGATCCCGG CGGAGCCGATCTACACGGCTTCCAAACATGCGGTGCAAGCCTTC GCTCACACCATACGCCGCCAGTTGGCCCCGTTCGGCATCCGCGT CGGCGCCATCCAGCCCGGCCCGGTCGTCACGCCCTTGCTCAATG ATTGGGACCCCGAGCGCCTTAAAGCCAATATCGAGGCTGGCGCC ATGATGCAGCCTTCTGACGTCGCCGAAGCCGTGGTTTTCATGCTG TCCCGCCGCAAGGGAACGGTAATCCGCGACTTGGTTCTGTTACCC CATTCTTTCGACGTCTAA SEQ ID NO: 10: MTSPLQGKIAAITGGASGIGLECVRQIAASGATVYILDRDHQALDKAR EELGERVHTIEVDLFRYETVDRAIETIVSEQGRIDILHVNAGAYIGGNV WEGDPDKWDKMLNLNINAAFRSARAVMPAMMKQKSGDIIMTSSIAGI VPIPAEPIYTASKHAVQAFAHTIRRQLAPFGIRVGAIQPGPVVTPLLND WDPERLKANIEAGAMMQPSDVAEAVVFMLSRRKGTVIRDLVLLPHSF DV SEQ ID NO: 11: ATGGCTATATCTCTCGAAAACAACGTAGCTGCAATTACAGGTGCC GCTTCAGGTATCGGTCTCGAATGTGCACGCCACACTGATCAAAGCA Petition 870250081800, of 11 / 09 / 2025, p. 42 / 88 36 / 56 GGCGCTAAAGTTGTCCTCATTGACCGAGCAGAAGATAGACTAAAT CAATTGGTCGCAGAATTAGGTGAAAATGCAATTCCATTAGTTATCG ATTTAATGAAACCAGAACAAGTCGATGGCATGTTAGCGCGTATTAT CGAAAAGGCAGGCAGATTAGATATCTTTCATGCTAATGCTGGAGC TTACATTGGTGGGCCCGTAGCCGAAGGCGATCCCGATGTTTGGG ATAAAGTCCTAAATTTAAATGTTAATGCCGCATTCCGCTGTGTTCG CGCAGTTCTACCACACTTTATCGCACAAAAGTCAGGCGATATTCTA TTCACCAGCTCTATCGCTGGTATGGTTCCCGTAATTTGGGAGCCT ATTTACACGGCATCAAAATTTGCGGTTCAAGCATTCGTTCATTCTA CTCGCCGTCAGGTTTCTGAACACGGTGTCCGTGTTGGTGCTGTAT TACCTGGTCCTGTTGTTACTGCGTTATTAGATGATTGGCCAAAAGA AAAACTTGAAGAAGCTTTAGCTAACGGTAGTTTAATGCAACCCATT GAAGTTGCTGAGGCTGTTCTATTCATGCTGACGCGTCCAAGAAAT ATTACAATTCGCGATTTAGTTATTTTACCCAATAGTGTTGACCTCTA A SEQ ID NO: 12: MAISLENNVAAITGAASGIGLECARTLIKAGAKVVLIDRAEDRLNQLVA ELGENAIPLVIDLMKPEQVDGMLARIIEKAGRLDIFHANAGAYIGGPVA EGDPDVWDKVLNLNVNAAFRCVRAVLPHFIAQKSGDILFTSSIAGMV PVIWEPIYTASKFAVQAFVHSTRRQVSEHGVRVGAVLPGPVVTALLD DWPKEKLEEALANGSLMQPIEVAEAVLFMLTRPRNITIRDLVILPNSVD L

[0093] The oxidoreductases mentioned herein for the reduction of D-psicose to allitol and / or for the oxidation of allitol to D-psicose preferably comprise an amino acid sequence with at least 80% identity to SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 12, more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, in particular 100%. Particularly preferably, the oxidoreductase of Petition 870250081800, dated 11 / 09 / 2025, p. 43 / 88 37 / 56 invention for the reduction of D-psicose to alitol and / or for the oxidation of alitol to D-psicose comprises or consists of the amino acid sequence SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12, and the oxidoreductase for the oxidation of alitol to D-psicose comprises or consists of one of the amino acid sequences SEQ ID NO: 6 or SEQ ID NO: 8.

[0094] Alternatively, the oxidoreductases for the reduction of D-psicose to allitol and / or for the oxidation of allitol to D-psicose preferably comprise a nucleic acid-encoded amino acid sequence with at least 80% identity to SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 9 or SEQ ID NO: 11, more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, in particular 100%. Particularly preferably, the nucleic acid encoding the oxidoreductase of the invention for the reduction of D-psicose to allitol and / or the oxidation of allitol to D-psicose comprises or consists of the nucleic acid sequence of SEQ ID NO: 3, SEQ ID NO: 9 or SEQ ID NO: 11, and the oxidoreductase for the oxidation of allitol to D-psicose comprises or consists of the nucleic acid sequence of SEQ ID NO: 5 or SEQ ID NO: 7.

[0095] The term identity as used here refers to the percentage of identical nucleotides or amino acids between at least two nucleotide or amino acid sequences aligned using a standardized algorithm (alignment). Such an algorithm can, in a standardized and reproducible manner, insert gaps in the compared sequences to optimize the alignment between two sequences and thus obtain a more meaningful comparison of the two sequences.

[0096] The percentage identity between sequences can be determined using one or more algorithms or computer programs. Petition 870250081800, dated 11 / 09 / 2025, page 44 / 88 38 / 56 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 identity. The BLAST software suite includes several programs, including a tool called BLAST 2 Sequences which is used for direct pairwise comparison of two nucleotide or amino acid sequences. BLAST 2 Sequences can also be retrieved interactively from the internet via the NCBI website and used. The blastn program (for nucleotide sequences) uses as standards a word length (W) of 11, an expectation (E) of 10, M = 5, N = -4 and a comparison of both strands.For amino acid sequences, the blastp program uses as standards a word length of 3, an expectation (E) of 10 and the BLOSUM62 scoring matrix (Henikoff & Henikoff, 1989), alignments (B) of 50, expectation (E) of 10, M = 5, N = -4.

[0097] Alternatively, oxidoreductases for the reduction of D-psicose to allitol and / or oxidation of allitol to D-psicose preferably comprise a nucleic acid-encoded amino acid sequence which, under stringent conditions, binds to a nucleic acid molecule comprising the nucleic acid sequence SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 11. As used herein, stringent conditions refer to conditions under which so-called specific hybrids, but no non-specific hybrids, are formed. For example, stringent conditions include hybridization in 6xSSC (sodium chloride / sodium citrate) at 45 °C and then washing with 0.2 to 1xSSC, 0.1% SDS at 50 to 65 °C; or such conditions may include hybridization in 1xSSC at 65 to 70 °C and then washing with 0.3xSSC at 65 to 70 °C. Hybridization can be carried out using conventionally known methods, such as Petition 870250081800, dated 11 / 09 / 2025, p. 45 / 88 39 / 56 those described by J. Sambrook and others, in Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory (1989).

[0098] One aspect of the present invention relates to the use of an oxidoreductase for the reduction of D-psicose to allytol and / or the oxidation of allytol to D-psicose, wherein the oxidoreductase comprises an amino acid sequence selected from the group consisting of: i) an amino acid sequence with at least 80% identity to SEQ ID NO: 4, SEQ ID NO: 10, or SEQ ID NO: 12, ii) a nucleic acid-encoded amino acid sequence with at least 80% identity to SEQ ID NO: 3, SEQ ID NO: 9, or SEQ ID NO: 11, and iii) a nucleic acid-encoded amino acid sequence that, under stringent conditions, becomes ligand-bound to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 3, SEQ ID NO: 9, or SEQ ID NO: 11.

[0099] A further aspect of the present invention relates to the use of an oxidoreductase for the oxidation of allitol to D-psicose, wherein the oxidoreductase comprises an amino acid sequence selected from the group consisting of: i) an amino acid sequence with at least 80% identity to SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12, ii) a nucleic acid-encoded amino acid sequence with at least 80% identity to SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 11, and iii) a nucleic acid-encoded amino acid sequence that, under strict conditions, binds to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 11.

[00100] Depending on the reaction (reduction or oxidation), the oxidant Petition 870250081800, dated 11 / 09 / 2025, p. 46 / 88 40 / 56 reductases of the invention require corresponding cofactors, as mentioned above. Materials

[00101] D-Psicose was purchased from TCI and Hunan Garden Naturals Inc. (China), allitol was purchased from TCI, D-fructose, tetrasodium salt of NADPH and methanol were purchased from PanReac AppliChem (ITW Reagents), D-glucose, sodium gluconate, IPTG (isopropyl-e-D-thiogalactopyranoside) were purchased from Sigma-Aldrich, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, NAD+, disodium salt of NADH, disodium salt of NADP+ and sodium dodecyl sulfate (SDS) were purchased from Carl Roth and triethanolamine (TEA) was purchased from Chem-Lab NV. Enzyme production and lysate preparation General information on the expression of recombinant enzymes in E. coli.

[00102] For recombinant enzyme production in an Escherichia coli strain, the gene to be expressed was first amplified by PCR using genomic DNA or its synthetic equivalent adapted to the use of E. coli codons as a template along with specific oligonucleotides, which additionally carried recognition sequences for restriction endonucleases, and isolated from the reaction mixture. After nucleic acid digestion with the restriction enzymes Sphl and HindIII, the gene fragment encoding the target enzyme was ligated to the Sphl and HindIII-cleaved main structure of the pQE70-Kan expression vector. The ligation product was transformed into chemically competent E. coli Top10F cells, and the resulting colonies were used for plasmid isolation and restriction analysis.

[00103] The result of the cloning step was verified by restriction enzyme digestion and DNA sequencing. The resulting construct carries the target gene under the inducible T5 promoter. Petition 870250081800, dated 11 / 09 / 2025, page 47 / 88 41 / 56 by IPTG.

[00104] For enzyme overexpression in E. coli, the resulting expression plasmid was transformed into competent RB791 expression cells. After 24 h of incubation at 37 °C, the resulting colonies were inoculated into LB medium for expression assays.

[00105] The following day, expression cultures with an optical density OD550 of 0.02 were inoculated and agitated 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 when an OD550 of 0.5 was reached. After 22 h, the cultures were collected (separated from the medium by centrifugation in the form of a cell pellet) and analyzed for recombinant enzyme expression using SDS gel electrophoresis and an activity determination (use test or optical enzyme assay). Preparation of cell lysates using sonicator disruption.

[00106] To prepare a cell suspension, the cell pellet prepared according to the above method was weighed into a suitable container and mixed with buffer and lysozyme (final concentration of 0.5 mg / ml) (e.g., triethanolamine (TEA) - HCl) and dissolved under stirring. The biomass mass fraction is generally 20%, the remainder being buffer.

[00107] 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).

[00108] The resulting homogenate was centrifuged for 10 min at 4°C and 16,000 rpm (Eppendorf 5417R centrifuge) to separate the insoluble cell fragments and obtain the lysate. Table 1. Enzyme classes and donor organisms for the enzymes used in the examples (SDR = dehydrogen oxidoreductase family) Petition 870250081800, dated 11 / 09 / 2025, page 48 / 88 42 / 56 genase / short-chain reductase). Enzyme type (EC class) Catalyzed reaction Donor organism Literature / SEQ ID NO: D-Psicose-3epimerase (EC 5.1.3.30) D-fructose ^ D-psicose Clostridium cellulolyticum H10 (Mu et al., 2011; Chan et al., 2012) SDR I D-psicose θ alitol Gluconobacter frateurii (DSM 7146) (NCBI Protein Database: WP_063903495.1); SEQ ID NO: 4 Xylitol dehydrogenase (XDH; EC 1.1.1.9) alitol ^ D-psicose Galactocandida mastotermitis (Candida sp. HA167) (Habenicht et al., 1999); SEQ ID NO: 6 NAD(P)-dependent alcohol dehydrogenase θ D-psicose Priestia megaterium (NCBI Protein Database: WP_013084280.1); SEQ ID NO: 8 SDR II θ D-psicose Kozakia baliensis (NCBI Protein Database: WP_070401870.1); SEQ ID NO: 10 SDR III θ D-psicose Providencia heimbachae (NCBI Protein Database: WP_068907433.1); SEQ ID NO: 12 Alcohol dehydrogenase (ADH; EC 1.1.1.1) 2-propanol ^ acetone (Geo-)Bacillus stearothermophilus NCA1503 (Sakoda & Imanaka, 1992); SEQ ID NO: 18 NADH oxidase I (EC 1.6.3.4) NADH ^ NAD+ Streptococcus mutans (Matsumoto et al., 1996); SEQ ID NO: 14 NADH oxidase II (EC 1.6.3.4) NADH ^ NAD+ Carnobacterium divergens SEQ ID NO: 16. Petition 870250081800, dated 11 / 09 / 2025, p. 49 / 88 43 / 56 Glucose dehydrogenase (GDH; EC 1.1.1.47) D-glucose ^ D-gluconate (via D-gluconolactone) Priestia megaterium (NCBI Protein Database: MDQ0804260.1); SEQ ID NO: 20 Formate dehydrogenase (EC 1.17.1.9) formate ^ CO2 Starkeya novella / Ancylobacter novellus (NCBI Protein Database: WP_013168047.1); SEQ ID NO: 2 Analytical methods High-Performance Liquid Chromatography

[00109] An Agilent HPLC 1260 Infinity II Series system was used to quantify D-psicose, D-fructose, D-glucose, and allitol by HPLC (high-performance liquid chromatography). Detection was performed using a refractive index detector (IR detection). For the measurement, a Phenomenex Rezex RPM-Monosaccharide Pb+2 (8%) column with an appropriate pre-column was used and isocratically eluted with ultrapure water. High-Performance Anion Exchange Chromatography

[00110] A Dionex ICS6000 system with an AS-AP autosampler was used to quantify D-gluconic acid / D-gluconate using HPAEC (High Performance Anion Exchange Chromatography). The measurement was performed using conductivity detection (CD) coupled to an electrolytically regenerated Dionex AERS 500 suppressor in external water mode. A Dionex lonPac AS11-HC-4pm column with an appropriate pre-column and a NaOH gradient was used to separate the analytes. The mobile phase was additionally pretreated with a Dionex ATC Anion Trap column. Determination of enzyme activities (enzymatic-optical assay)

[00111] The enzyme activities in the lysates were determined Petition 870250081800, dated 11 / 09 / 2025, page 50 / 88 44 / 56 using a Shimadzu UV-1900 spectrophotometer. For this purpose, the formation or consumption of NAD(P)H was monitored at a wavelength of 340 nm by means of the change in absorption. Measurements were performed with 0.2 mM cofactor (NAD(P)+ 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 SemiMicro 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. Measurements were performed at 25°C as standard. The 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) using the NADH / NADPH extinction coefficient at 340 nm (E = 6220 L mol-1cm-1).Here, 1 U represents 1 pmol of substrate conversion per minute (1 U = 1 pmol / min = 1 μmol / min = 1.67·10⁸ kat).

[00112] The following examples describe preferred variants of the method according to the invention in more detail. The lysates used in these examples were prepared according to the procedures described above. Example 1 Production of D-psicose from D-fructose - cofactor regeneration with ADH and 2-propanol

[00113] The reaction was carried out in a Labfors 5 benchtop bioreactor (Infors AG). A glass reactor (3.4 l volume) with a stirrer and pH electrode was used as the vessel. The pH was controlled by adding 1M NaOH or 1M H2SO4.

[00114] Initially, 50 ml of a D-fructose solution (500 g / l), 246.1 ml of deionized water and 96 ml of a 200 mM TEA-HCl buffer (pH 8) were placed in the reactor and heated to 35 °C under stirring. Petition 870250081800, dated 11 / 09 / 2025, page 51 / 88 45 / 56

[00115] To initiate the reaction, 25 ml of D-psicose 3epimerase lysate were added. Then, 25 ml of SDR I lysate, 4 kU of alcohol dehydrogenase lysate, 10 ml of a 10 mM NAD+ solution, and 40 ml of 2-propanol were added.

[00116] During the operation, samples were continuously withdrawn from the reactor solution and analyzed as follows: 100 µl of the reactor solution 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 rapidly centrifuged in a centrifuge, mixed with 700 µl of deionized water, vortexed, and then centrifuged for 5 min at a maximum temperature of 100 °C. 200 µl of the supernatant were transferred to an HPLC vial with an insert and measured by HPLC (IR detection).

[00117] After 23 h of running time, 15 ml of 2-propanol were added; after 50 h, 20 ml were added; and after 77 h, 15 ml were added.

[00118] After 74 h, 5 ml of D-psicose 3-epimerase lysate were added and, after 77 h, 15 ml of SDR I lysate and 2.4 kU of alcohol dehydrogenase lysate were added.

[00119] After 97 h, 94% of the D-fructose (50 g / l) had been converted to allitol.

[00120] The entire contents of the reactor were then heated to 70°C for 60 min (D-psicose 3-epimerase deactivation) and, after cooling to 24°C, 25 ml of xylitol dehydrogenase lysate, 10 kU of NADH oxidase lysate and 10 ml of a 10 mM NAD+ solution were added.

[00121] Alitol was completely oxidized to D-psicose within 3 h. The reactor contents were heated to 70°C, the pH was adjusted to 4, and the mixture was stirred for 30 minutes at 70°C. The enzymes were filtered using a glass frit (P3). Petition 870250081800, dated 11 / 09 / 2025, page 52 / 88 46 / 56

[00122] Thus, 94% of D-fructose was converted to D-psicose. D-sorbitol could not be detected.

[00123] The filtrate was concentrated into a syrup with a D-psicose concentration of 520 g / l in a rotary evaporator, with the remaining acetone and 2-propanol also being separated.

[00124] Example 1 shows that epimerase can be denatured by heat (in a one-container process) and that the resulting precipitate does not interfere with the subsequent reaction. Example 2 Production of D-psicose from D-fructose - cofactor regeneration with GDH and D-glucose

[00125] The reaction was carried out in a Multifors benchtop bioreactor (Infors AG). A glass reactor (1 L volume) with a stirrer and pH electrode was used as a container. The pH was controlled by adding 5 M NaOH or 1 M H2SO4.

[00126] Initially, 17.5 g of D-fructose and 17.5 g of D-glucose (final concentration of 50 g / L each), 217.8 mL of deionized water and 26.5 mL of 500 mM potassium phosphate buffer (pH 7.5) were placed in the reactor and heated to 35 °C under stirring.

[00127] To initiate the reaction, 17.5 mL of D-psicose-3epimerase lysate were added. Then, 24.5 mL of SDR I lysate, 0.4 kU of glucose dehydrogenase lysate, and 3.5 mL of a 10 mM NAD+ solution were added.

[00128] During the operation, samples were continuously taken from the reactor solution and analyzed as follows: 100 µl of the reactor solution 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 rapidly centrifuged, mixed with 700 µl of deionized water, vortexed, and then centrifuged for a maximum of 5 min. 200 µl of the supernatant were transferred to a flask. Petition 870250081800, dated 11 / 09 / 2025, page 53 / 88 47 / 56 HPLC with an insert and measured by HPLC (IR detection). For HPAEC (conductivity detection) measurements, the clear supernatant was diluted 1:250.

[00129] After 16 h, only allitol and D-gluconate could be detected in the reactor.

[00130] All the contents of the reactor were then heated to 70°C for 60 minutes (deactivation of D-psicose 3-epimerase) and, after cooling to 30°C, 17.5 ml of xylitol dehydrogenase lysate, 7 kU of NADH oxidase lysate and 3.5 ml of a 10 mM NAD+ solution were added.

[00131] Alitol was completely oxidized to D-psicose in 27 h.

[00132] Thus, 17.5 g of D-fructose could be 100% oxidized to D-psicose (17.8 g in solution). D-sorbitol and D-fructose could not be detected in the resulting solution.

[00133] The reactor contents were heated to 70 °C, the pH was adjusted to 4, and the mixture was stirred for 30 min at 70 °C. The enzymes were filtered through a glass frit (P3). Example 3 Production of D-psicose from D-fructose - cofactor regeneration with FDH and sodium formate.

[00134] The reaction was carried out in a Multifors benchtop bioreactor (Infors AG). A glass reactor (1 L volume) with a stirrer and pH electrode was used as a container. The pH was controlled by adding 5 M NaOH or 6 M H2SO4.

[00135] Initially, 70 mL of a D-fructose solution (500 g / L), 26.3 mL of deionized water, and 43.8 mL of an 8 M sodium formate solution were placed in the reactor and heated to 37 °C with stirring.

[00136] To initiate the reaction, 25 mL of D-psicose-3epimerase lysate were added. Then, 35 mL of SDR I lysate, 7 kU of formate dehydrogenase lysate, and 17.5 mL of a solution were added. Petition 870250081800, dated 11 / 09 / 2025, page 54 / 88 48 / 56 of NAD+10 mM were added.

[00137] During the operation, samples were continuously withdrawn from the reactor solution and analyzed as follows: 100 μL of the reactor solution 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 rapidly centrifuged, mixed with 700 μL of deionized water, vortexed, and then centrifuged for a maximum of 5 min. 200 μL of the supernatant were transferred to an HPLC vial with an insert and measured by HPLC (IR detection).

[00138] After 40 h, 95% of D-fructose (100 g / l) was converted to allitol.

[00139] All the contents of the reactor were then heated to 70 °C for 60 min (D-psicose 3-epimerase deactivation) and, after cooling to 24 °C, 25 ml of xylitol dehydrogenase lysate, 10 kU of NADH oxidase lysate and 10 ml of a 10 mM NAD+ solution were added.

[00140] Alitol was completely oxidized to D-psicose within 4 h. The reactor contents were heated to 70 °C, the pH was adjusted to 4, and the mixture was stirred for 30 minutes at 70 °C. The enzymes were filtered using a glass frit (P3).

[00141] In this way, 97% of the D-fructose was converted to D-psicose. Traces of D-fructose were still detected in the reaction solution, but no D-sorbitol. Literature Zhang, W., Yu, S., Zhang, T., Jiang, B., & Mu, W. (2016). Recent advances in D-allulose: Physiological functionalities, applications, and biological production. Trends in Food Science and Technology, 54, 127-137. https: / / doi.org / 10.1016 / j.tifs.2016.06.004 Hough, L., & Stacey, B. E. (1996). Variation in the allitol content of Itea Petição 870250081800, de 11 / 09 / 2025, pág. 55 / 88 49 / 56 plants during photosynthesis. Phytochemistry, 5 (1), 171-175. https: / / doi.org / 10.1016 / S0031-9422(00)85095-5 Oshima, H., Kimura, I., & Izumori, K. (2006). Psicose Contents in Various Food Products and its Origin. Food Science and Technology Research, 12(2), 137-143. https: / / doi.org / 10.3136 / fstr.12.137 Jiang, S., Xiao, W., Zhu, X., Yang, P., Zheng, Z., Lu, S., Jiang, S., Zhang, G., & Liu, J. (2020). 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Claims

1 / 6 CLAIMS 1. Method for preparing an aqueous solution containing D-psicose, characterized in that it is through the formation of a first D-psicose from a D-fructose, which is present in an aqueous solution, by treatment with an epimerase in vitro, after which the first D-psicose is reduced to allitol by treatment with a respective NAD(P)H-dependent oxidoreductase in vitro and, after deactivation and / or ultrafiltration of the epimerase, a respective NAD(P)+-dependent oxidoreductase was added to form D-psicose, after which the deactivated epimerase and the oxidoreductases are removed.

2. Method according to claim 1, characterized in that the NAD(P)+-dependent oxidoreductase for the formation of D-psicose from allitol comprises an amino acid sequence selected from the group consisting of: (i) an amino acid sequence with at least 80% identity to SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 12, (ii) a nucleic acid-encoded amino acid sequence with at least 80% identity to SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 11, and (iii) a nucleic acid-encoded amino acid sequence that, under stringent conditions, binds to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO:

11.

3. Method according to claim 1 or 2, characterized in that the oxidized cofactor NAD(P)+ formed by the reduction is reduced by means of an alcohol dehydrogenase and a secondary alcohol with the formation of a ketone. Petition 870250081800, dated 11 / 09 / 2025, p. 64 / 88 2 / 6 4. Method according to claim 3, characterized in that the secondary alcohol is D-glucose or 2-propanol.

5. A method according to any one of claims 1 to 4, characterized in that it is carried out as a single-vessel reaction without isolation of any intermediate products.

6. A method according to any one of claims 1 to 5, characterized in that the enzymes are present as a lysate of the corresponding cells that produce them.

7. Method according to any one of claims 1 to 6, characterized in that the NAD(P)H-dependent oxidoreductase for the reduction of first D-psicose to alitol comprises an amino acid sequence selected from the group consisting of: (i) an amino acid sequence with at least 80% identity to SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 12, (ii) an amino acid sequence encoded by a nucleic acid with at least 80% identity to SEQ ID NO: 3, SEQ ID NO: 9 or SEQ ID NO: 11, and (iii) an amino acid sequence encoded by a nucleic acid that, under stringent conditions, binds to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 3, SEQ ID NO: 9 or SEQ ID NO:

11.

8. Method, according to any one of claims 3 to 7, characterized in that the alcohol dehydrogenase comprises or consists of an amino acid sequence selected from the group consisting of: (i) an amino acid sequence with at least 80% identity to SEQ ID NO: 18, (ii) an amino acid sequence encoded by a nucleic acid with at least 80% identity to SEQ ID NO: 17, and (iii) an amino acid sequence encoded by a nucleic acid that, under stringent conditions, binds to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO:

17.

9. A method according to any one of claims 1 to 8, characterized in that the oxidized cofactor NAD(P)+ formed by the reduction is reduced by means of a glucose dehydrogenase and D-glucose with the formation of D-gluconate.

10. Method according to claim 9, characterized in that the glucose dehydrogenase comprises or consists of an amino acid sequence selected from the group consisting of: (i) an amino acid sequence with at least 80% identity to SEQ ID NO: 20, (ii) an amino acid sequence encoded by a nucleic acid with at least 80% identity to SEQ ID NO: 19, and (iii) an amino acid sequence encoded by a nucleic acid that, under stringent conditions, binds to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO:

19.

11. A method according to any one of claims 1 to 10, characterized in that the oxidized cofactor NAD(P)+ produced by the reaction is reduced by means of a formate dehydrogenase and formate with the formation of CO2.

12. Method according to claim 11, characterized in that the formate dehydrogenase comprises or consists of an amino acid sequence selected from the group consisting of: (i) an amino acid sequence with at least 80% identity with SEQ ID NO: 2, (ii) a nucleic acid-encoded amino acid sequence with at least 80% identity with SEQ ID NO: 1, and (iii) a nucleic acid-encoded amino acid sequence that, under stringent conditions, binds to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO:

1.

13. Use of an aqueous solution that can be prepared by the method as defined in any one of claims 1 to 12, characterized in that it is for the preparation of a syrup containing D-psicose.

14. Use of an oxidoreductase, characterized in that it is for the formation of D-psicose from allitol, wherein the oxidoreductase comprises an amino acid sequence selected from the group consisting of: (i) an amino acid sequence with at least 80% identity to SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 12, (ii) a nucleic acid-encoded amino acid sequence with at least 80% identity to SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 11, and (iii) a nucleic acid-encoded amino acid sequence that, under stringent conditions, binds to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO:

11.

15. Use of an alcohol dehydrogenase, characterized in that it is for cofactor regeneration, wherein the alcohol dehydrogenase comprises or consists of an amino acid sequence selected from the group consisting of: Petition 870250081800, dated 11 / 09 / 2025, page 67 / 88 5 / 6 (i) an amino acid sequence with at least 80% identity to SEQ ID NO: 18, (ii) an amino acid sequence encoded by a nucleic acid with at least 80% identity to SEQ ID NO: 17, and (iii) an amino acid sequence encoded by a nucleic acid that, under stringent conditions, binds to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO:

17.

16. Use of a glucose dehydrogenase, characterized in that it is for cofactor regeneration, wherein the glucose dehydrogenase comprises or consists of an amino acid sequence selected from the group consisting of: (i) an amino acid sequence with at least 80% identity to SEQ ID NO: 20, (ii) an amino acid sequence encoded by a nucleic acid with at least 80% identity to SEQ ID NO: 19, and (iii) an amino acid sequence encoded by a nucleic acid that, under stringent conditions, binds to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO:

19.

17. Use of a formate dehydrogenase, characterized in that it is for the regeneration of a cofactor or a functional fragment thereof, wherein the formate dehydrogenase comprises or consists of an amino acid sequence selected from the group consisting of: (i) an amino acid sequence with at least 80% identity to SEQ ID NO: 2, (ii) an amino acid sequence encoded by a nucleic acid with at least 80% identity to SEQ ID NO: 1, and (iii) an amino acid sequence encoded by a nucleic acid that, under stringent conditions, binds to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO:

1.

18. Use of a water-forming NAD(P)H oxidase, characterized in that it is for cofactor regeneration comprising or consisting of an amino acid sequence selected from the group consisting of: (i) an amino acid sequence with at least 80% identity to SEQ ID NO: 14 or SEQ ID NO: 16, (ii) an amino acid sequence encoded by a nucleic acid with at least 80% identity to SEQ ID NO: 13 or SEQ ID NO: 15, and (iii) an amino acid sequence encoded by a nucleic acid that, under strict conditions, binds to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 13 or SEQ ID NO:

15. Petition 870250081800, dated 11 / 09 / 2025, p. 69 / 88

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