Method for preparing an aqueous solution containing D-psicose

Abstract

The present invention relates to a method for forming a first D-psicose from D-fructose present in an aqueous solution by treatment with an epimerase in vitro, and then reducing the first D-psicose to allitol by treatment with a respective NAD(P)H-dependent oxidoreductase in vitro, and after inactivation of the epimerase and / or ultrafiltration, reducing the first D-psicose to allitol to form D-psicose. + The present invention relates to a method for preparing an aqueous solution containing D-psicose by adding a D-dependent oxidoreductase and then removing the inactivated epimerase and oxidoreductase.

Description

[Technical Field]

[0001] The present invention relates to a method for preparing an aqueous solution containing D-psicose. [Background technology]

[0002] D-Psicose The monosaccharide D-psicose, also known as D-allulose, is a ketohexose rarely found in nature (Zhang et al., 2016). It has been detected, inter alia, in the leaves of sweet spirea (Itea sp.) (Hough & Stacey, 1966), but is also found in processed foods such as confectionery and spice sauces, where it is formed from its C3 epimer, D-fructose, under the influence of heat (Oshima et al., 2006).

[0003] D-psicose is of interest to the food industry due to its sweet taste. Compared to sucrose, D-psicose has 70% of the relative sweetening power but a lower energy content (0.2 kcal / g), which corresponds to approximately a 95% calorie reduction (compared to sucrose) (Jiang et al., 2020).

[0004] In the United States, D-psicose is recognized as a generally recognized as safe (GRAS) sweetener by the U.S. Food and Drug Administration (FDA), but it is not yet approved in the EU ( Ahmed et al., 2022 ).

[0005] Furthermore, 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).

[0006] Due to its rarity in nature, D-psicose is mainly produced synthetically (chemically or biotechnologically).

[0007] Epimerization of D-fructose to D-psicose can be carried out by boiling in pyridine under reflux followed by yeast fermentation to remove other hexoses, but only 6.8% of the theoretical yield of D-psicose is achieved (Doner, 1979). Another method involves epimerization of D-fructose using molybdate ions as a catalyst, which converts only 0.5% of D-fructose to D-psicose (Bilik & Tihlarik, 1974). Inefficient chemical synthetic routes have now been replaced by more efficient biotechnological processes.

[0008] In 1993, Izumori et al. described ketose-3-epimerase from Pseudomonas cichorii ST-24 for producing D-psicose from D-fructose (Izumori et al., 1993) and patented it (EP 0592202 B1). Ketose-3-epimerases can be divided into three groups based 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).

[0009] However, the conversion of D-fructose to D-psicose via ketose-3-epimerase does not proceed completely, and an equilibrium between the two epimers is formed. Depending on the reaction conditions (temperature between 40 and 70 °C, pH between 6 and 11), the ratio is between 80:20 and 62.5:37.5 (D-fructose:D-psicose). Many epimerases also use divalent metal ions, such as Mn, as cofactors. 2+ or Co 2+ (toxicity) (Zhang et al., 2016; Jiang et al., 2020).

[0010] The equilibrium during epimerization can be affected not only by temperature or pH, but also by the addition of (toxic) borate. The favorable formation of D-psicose-borate complexes shifts the equilibrium toward D-psicose (Kim et al., 2008; Lim et al., 2009). EP 3643786 A2 and US 11028420 B2 describe chromatographic separation of D-psicose-borate complexes using simulated moving bed (SMB) chromatography. EP 3395952 B1 and US 10550414 B2 disclose that the conversion rate during epimerization using DPE can be increased up to 67% by adding sodium aluminate and up to 52% by adding potassium iodate (compared to 25% without the addition of aluminate or iodate).

[0011] Zhu et al. (2020) presented a system consisting of two enzymes (exoinulase from Bacillus velezenis and DAE from Ruminococcus sp.) that could convert inulin from Helianthus tuberosus L. (Jerusalem artichoke) 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 juice) into D-psicose. They were able to enrich the juice with 16–19% D-psicose (relative to the total carbohydrate content).

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

[0013] Patel et al. (2018) used Smt3-DPE (a fusion protein) immobilized on magnetic iron oxide nanoparticles to produce D-psicose from D-fructose in fruit pulp washings. The immobilized epimerase was able to convert 20% of the D-fructose, which was then separated using 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. In this way, 190 g / L of D-psicose was produced from 750 g / L of D-fructose at 55°C in 12 hours. The remaining D-fructose was fermented to ethanol by K. marxianus. Dedania et al. (2020) immobilized DPE from A. tumefaciens on titanium dioxide nanoparticles and were able to convert 36% of D-fructose to D-psicose. The immobilized enzyme could be reused up to nine times.

[0015] The D-fructose / D-psicose mixture resulting from the epimerization of D-fructose can be separated either by chromatographic methods or by "biological methods" (e.g., fermentation of excess D-fructose to 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 probiotic microorganisms (Lactobacillus or Saccharomyces). EP 3423460 B1 describes a process for purifying a D-fructose / D-psicose mixture to obtain highly pure D-psicose. EP 3553069 A1 and Van Duc Long et al. (2009) disclose 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 drawbacks: 1) the position of the equilibrium on the D-fructose side, 2) the 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] One option to avoid thermodynamically unfavorable epimerization is an enzymatic cascade with a phosphorylated intermediate, the final step of which, dephosphorylation, is irreversible and thus drives the cascade ( Li et al., 2021b ).

[0018] The cascade described in nearly identical form by Li et al. (2021b) and in US Pat. No. 11,168,342 B2 and US Pat. No. 10,907,182 B2 shows D-glucose-1-phosphate (G1P) as the central intermediate. G1P is first converted to D-glucose-6-phosphate (G6P) by phosphoglucomutase, which then converts it to D-fructose-6-phosphate (F6P) by glucose-6-phosphate isomerase. F6P is then epimerized to D-psicose-6-phosphate by D-allulose-6-phosphate epimerase, which then dephosphorylates it to D-psicose by D-allulose-6-phosphate phosphatase.

[0019] G6P can be produced directly by phosphorylase action on maltose and amylodextrin (obtained by starch hydrolysis), cellodextrin (obtained by cellulose hydrolysis), or sucrose, for example, with the consumption of phosphate. Because the terminal sugar monomers of oligosaccharides and polysaccharides cannot be phosphorylated by the corresponding phosphorylases, polyphosphate glucokinase (D-glucose → G6P) or polyphosphate fructokinase (D-fructose → F6P) must be used, and polyphosphate must be added as a phosphate source to increase the yield (US 11168342 B2; US 10907182 B2). The substrate used in the cascade by Li et al. (2021b) is starch, which is converted to D-psicose in 79% yield (at a substrate concentration of 50 g / L; reaction time of 24 h).

[0020] Wang et al. (2020) also developed an enzyme cascade for the production of D-psicose starting from starch, which is converted in several steps to glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. Dihydroxyacetone phosphate is converted to D-psicose-1-phosphate using D-glyceraldehyde under the activity of L-fuculose-1-phosphate aldolase (FucA), which is then dephosphorylated to D-psicose 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] Enzyme cascades starting from glycerol have also been described. This is converted to dihydroxyacetone phosphate (phosphorylated by acid phosphatase and subsequently oxidized by glycerol phosphate oxidase) and D-glyceraldehyde (oxidized by allitol oxidase), which then serves as a substrate for an aldolase (e.g., 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). Meanwhile, WO 2016 / 201110 A1 describes a method for producing D-psicose from dihydroxyacetone and D-glyceraldehyde using fructose-6-phosphate aldolase and DTE (the 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-rhamnulose kinase (which consumes adenosine triphosphate (ATP)) to shift the epimerization equilibrium. Subsequent cleavage of the phosphate group by acid phosphatase yields D-psicose (converting 99% of 20 mM D-fructose). However, ATP must be regenerated by the addition of polyphosphate using polyphosphate kinase (Xiao et al., 2019).

[0023] The main drawback of pathways involving phosphorylated intermediates is the need for stoichiometric amounts of expensive, energy-rich phosphate compounds, such as polyphosphate or ATP, to introduce the phosphate group. While this problem can be partially circumvented by using phosphorylase, the terminal monosaccharide cannot be phosphorylated without the aid of an energy-rich phosphate compound. 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 are also known. Zhang et al. (2021) presented a fermentation process based on the co-culture of engineered Bacillus subtilis and Escherichia coli for the co-production of D-psicose (titer 11.7 g / L; conversion rate: 69.5%) and lipase enzyme. US 2017 / 0298400 A1 describes the expression of DPE in various microorganisms (e.g., from Agrobacterium tumefaciens). EP 3088515 B1 and US 9701953 B2 describe D-psicose-producing Ensifer adhaerens strains. EP 2470668 B1 discloses the immobilization of a GRAS microorganism (Corynebacterium glutamicum KCCM11046) with an expressed DPE on a sodium alginate carrier.

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

[0026] Allitol can then be converted back to D-psicose via an oxidation process, which can be achieved microbially, for example, by Enterobacter aerogenes IK7 (complete oxidation of 100 g / L of allitol in 24 hours) or Bacillus pallidus Y25 (48% conversion of 50 g / L of allitol in 48 hours) (Gullapalli et al., 2007; Poonperm et al., 2007).

[0027] Due to its symmetry, the achiral allitol sugar alcohol forms the 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.

[0028] Theoretical papers by Hold et al. (2009) and Siedentop et al. (2021) address the optimization of enzyme cascades. They note that all components and multiple parameters must be taken into account, particularly those related to the cascade design, the enzymes themselves, the reaction conditions and environment, and process design, and that it is impossible to predict which will be successful. The synthesis of D-psicose is not specifically mentioned.

[0029] Chen et al. (2022) focused on a fermentation route via whole-cell biocatalysis ("in vivo") for the production of D-psicose and concluded that this is the only route with extensive optimization and has the potential for economical and industrial-scale D-psicose production in the future. The advantages of whole-cell biocatalysis are clear: (1) Cells containing enzymes are more readily available than the enzymes themselves, which are often laborious to purify; (2) The interior of the cell provides a suitable microenvironment for the enzyme and also allows for the regeneration of cofactors (NAD(P) + / NAD(P)H) is also possible, (3) Cell walls and membranes protect enzymes from the environment of the reaction medium; (4) Colocalization of multiple enzymes within cells favors local enzyme concentrations and reduces the diffusion of intermediates in cascade reactions.

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

[0031] A team led by Wang et al. (2022, 2023) is also working on the enzymatic biotransformation of sugars, investigating in vitro and in vivo biotransformations. Their goal is to develop 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) described an in vivo method consisting of two whole-cell biocatalysts: a DPE from Clostridiales, a RDH from Providencia alcalifaciens, a FDH from Starkeya, and an additional DPE from Rhizobium straminoryzae. In this way, D-fructose (500 mM = 90 g / L) was converted to 452 mM allitol (90.4% conversion) within 12 hours at 37 °C and pH 6 using 1000 mM sodium formate (2 equivalents relative to D-fructose). Approximately 30 mM D-sorbitol was formed as a by-product. The cells were separated by centrifugation, and proteins leaked into the allitol-containing supernatant were inactivated by heat. E. coli cells containing RDH from Rubrivivax sp. and NADH oxidase from Streptococcus pyogenes were then added to the allitol solution. Allitol (452 ​​mM) was converted to D-psicose (450 mM) at pH 7 within 24 hours.

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

[0034] Accordingly, the object of the present invention is to provide a method for preparing an aqueous solution containing D-psicose, which has set itself the goal of further improving the two-step process for forming D-psicose from D-fructose, and in particular can be carried out in a one-pot manner. [Brief explanation of the drawings]

[0035] [Figure 1] FIG. 1 is a schematic diagram illustrating the method of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0036] According to the present invention, the object is to form a first D-psicose from D-fructose present in an aqueous solution by treatment with an epimerase in vitro, and then reduce the first D-psicose to allitol by treatment with the respective NAD(P)H-dependent oxidoreductase in vitro, and after inactivation of the epimerase and / or ultrafiltration, reduce the first D-psicose to allitol ... + This is achieved by adding a dependent oxidoreductase and then removing the inactivated epimerase and oxidoreductase. Instead of inactivating the epimerase, it can be immobilized on or in a carrier material and filtered from the aqueous solution together with the carrier.

[0037] Surprisingly, it has been shown that the method according to the present invention does not substantially form undesirable D-sorbitol and the yield of D-psicose can even be easily increased towards 100%.

[0038] Therefore, the process according to the invention is not carried out fermentatively, but the enzyme is contained intact in aqueous solution. Thus, according to the invention, the process is carried out in vitro.

[0039] A preferred variant of the method of the present invention is to use NAD(P) for the formation of D-psicose from allitol. + Dependent oxidoreductases i) an amino acid sequence having 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) an amino acid sequence encoded by a nucleic acid having 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 binds under stringent conditions 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, or a functional fragment thereof The present invention is characterized in that it comprises or consists of an amino acid sequence selected from the group consisting of:

[0040] This NAD(P) + A "functional fragment" of a dependent oxidoreductase includes an N-terminally and / or C-terminally truncated variant of an oxidoreductase having the amino acid sequence of 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 the enzymatic activity compared to the untruncated oxidoreductase.

[0041] The method according to the invention is illustrated diagrammatically in the accompanying drawing.

[0042] A preferred variant of the method of the invention is the production of oxidized cofactor NAD(P) formed by the reduction of D-psicose to allitol. +The process involves the reduction of NAD(P)H by alcohol dehydrogenase (ADH) and a secondary alcohol, preferably D-glucose or 2-propanol (isopropanol), resulting in the formation of a ketone. 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). Acetone recovered from exhaust gas streams, present either in the gas phase or in solution as a 2-propanol / acetone / water or acetone / water mixture, can be heterogeneously catalytically rehydrogenated to 2-propanol (Al-Rabiah et al., 2022). Hydrogen from sustainable sources ("green hydrogen") may be increasingly used in the future.

[0043] Cofactor regeneration using ADH is known, for example, from EP 2812439 B1 or described in Xu et al. (2021).

[0044] In a further preferred embodiment of the present invention, the oxidized cofactor NAD(P) formed by reduction + is reduced by glucose dehydrogenase and D-glucose with the formation of D-gluconic acid. The use of glucose dehydrogenase to regenerate NAD(P)H is +This is particularly advantageous because during the reduction of D-glucose, D-gluconic acid is formed from D-glucose, which can be obtained from the reaction mixture and used in several fields (e.g., in metal pickling detergents, pharmaceuticals, and as a stabilizer in foods). Furthermore, the use of glucose dehydrogenase allows the use of a mixture containing D-fructose and D-glucose as a substrate for the production of allitol or D-psicose without adding additional D-glucose to the reaction mixture and without prior isomerization of D-glucose to D-fructose. A mixture of D-fructose and D-glucose can be produced, for example, by hydrolysis of sucrose. Particularly preferably, a glucose dehydrogenase derived from Priestia megaterium and comprising the amino acid sequence available under NCBI accession number MDQ0804260.1 is used.

[0045] A further preferred variant of the method according to the invention is the production of the oxidized cofactor NAD(P) by reduction with formate dehydrogenase and formic acid (e.g. sodium formate) with the formation of CO2. + This involves the use of formate dehydrogenase (FDH) for the regeneration of ATP.

[0046] A further preferred variant of the process according to the invention is characterized in that it is carried out as a one-pot reaction without isolation of the intermediate products.

[0047] In the method of the present invention, the enzymes are preferably used as lysates of the corresponding cells that produce them. In contrast to the method described by Wang et al. (2023), which is based on a whole-cell biocatalysis of E. coli with co-expressed recombinant enzymes, the enzymes are expressed individually in a suitable E. coli production strain.

[0048] This allows for optimization of enzyme ratios and is therefore independent of expression levels across the construct, compared to Wang et al. (2023).

[0049] Before carrying out the final step (oxidation), the enzymes (epimerase and reductase and / or dehydrogenase) of the first step (D-fructose → allitol) are inactivated by heat and / or removed by ultrafiltration to prevent the formation of enzymatic by-products. Without proper treatment, most of the D-psicose formed by oxidation may revert to D-fructose.

[0050] Regeneration of the nicotinamide-based cofactor (NAD or NADP) occurs in the reduction of D-psicose to allitol using NAD(P)-dependent alcohol dehydrogenase, glucose dehydrogenase, or formate dehydrogenase, and in the oxidation reaction (second step) using HO-forming NAD(P)H oxidase.

[0051] A particularly preferred concentration of D-fructose is 50 to 250 g / l.

[0052] Particularly preferred temperature ranges for the first step (epimerization and reduction) are between 25 and 45°C, and for the second step (oxidation) between 20 and 30°C.

[0053] A particularly preferred pH range for both steps is between 7 and 8.5.

[0054] In a further preferred embodiment of the method of the invention, the enzymes are present in suspensions and / or homogenates and / or lysates of the respective cells forming them, with lysates being particularly preferred.

[0055] In this context, a suspension refers to a suspension of resting cells. These are harvested (separated from the growth medium) after cultivation and used as a paste or suspended in an appropriate buffer system. In contrast to fermentation methods, in which whole cells are also used, resting cells can no longer grow due to the lack of a carbon source and nutrients and are only useful for substrate conversion (Lin & Tao, 2017). In this context, a homogenate refers to a suspension that has been physically and / or chemically treated (e.g., with pressure, lysozyme, or ultrasound) so that cellular components are released from the cells. A lysate is obtained when the insoluble cellular components of the homogenate are removed, for example, by filtration or centrifugation (see Enzyme Production & Lysate Preparation for details).

[0056] In another variation, the enzyme may also be N-terminally modified with a water-soluble polymer such as polyethylene glycol, may be immobilized in or on a solid matrix, or may be part of a fusion protein.

[0057] In another variation, the enzyme may be present in powdered, freeze-dried or spray-dried form.

[0058] After separation of the enzyme, D-psicose is most preferably present in an aqueous solution, from which solid D-psicose can be obtained, for example, by spray drying (US 2019 / 0315790 A1; Kawakami et al., 2013; Kawakami et al., 2014).

[0059] Because the obtained solution is highly pure, the filtrate can be concentrated to obtain D-psicose in the form of crystals or syrup.

[0060] In a further preferred variant, D-psicose is present in a syrup, which is prepared by concentrating the above-mentioned filtrate or by dissolving crystalline D-psicose, which can be prepared by the method according to the present invention, in water. The syrup according to the present invention preferably has a total solids content of approximately 50% to approximately 90% by weight. The D-psicose content in the syrup according to the present invention is approximately 80% to approximately 99% by weight based on dry matter.

[0061] Therefore, a further aspect of the present invention relates to a syrup containing D-psicose that can be produced by the method according to the present invention.

[0062] In a particularly preferred embodiment of the method, only enzymes from the group of enzymes epimerases and oxidoreductases are used for the conversion of the starting material, one or more of these enzymes being selected from each of these groups.

[0063] The epimerase used in the present method may be from one of the 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.

[0064] The enzymes used for the reduction of D-psicose and the oxidation of allitol are from the group of oxidoreductases (see Table 1 for details).

[0065] 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).

[0066] The NAD(P)-dependent alcohol dehydrogenase for cofactor regeneration is preferably i) an amino acid sequence having at least 80% identity to SEQ ID NO: 18; ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity to SEQ ID NO: 17, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 17 The amino acid sequence comprises or consists of an amino acid sequence selected from the group consisting of:

[0067] Generally, particularly suitable for cofactor regeneration are alcohol dehydrogenases whose amino acid sequence has at least 80% identity to SEQ ID NO: 18, or is encoded by a nucleic acid having at least 80% identity to SEQ ID NO: 17, or which bind under stringent conditions to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 17, or functional fragments of such alcohol dehydrogenases. A "functional fragment" of an alcohol dehydrogenase includes N-terminally and / or C-terminally truncated variants of an alcohol dehydrogenase having the amino acid sequence of SEQ ID NO: 18, which have 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 the enzymatic activity of the uncleaved alcohol dehydrogenase. SEQ ID NO:17:

number

number

[0068] The alcohol dehydrogenase for cofactor regeneration referred to herein preferably comprises an amino acid sequence which has at least 80%, more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, and especially 100% identity to SEQ ID NO: 18. 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.

[0069] Alternatively, the alcohol dehydrogenase for cofactor regeneration preferably comprises an amino acid sequence encoded by a nucleic acid having at least 80%, more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, and especially 100% identity to SEQ ID NO: 17. 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.

[0070] A further aspect of the invention relates to the use of an alcohol dehydrogenase for cofactor regeneration, the alcohol dehydrogenase comprising: i) an amino acid sequence having at least 80% identity to SEQ ID NO: 18; ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity to SEQ ID NO: 17, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 17, or a functional fragment thereof The amino acid sequence comprises or consists of an amino acid sequence selected from the group consisting of:

[0071] The alcohol dehydrogenases disclosed herein are capable of converting NAD(P) into NAD(P) in various enzymatic reactions. + or regeneration of NAD(P)H, i.e., NAD(P) +or the oxidation of NAD(P)H. Particularly preferred is the reduction of NAD(P)H formed during the reduction of D-psicose to allitol by NAD(P)H-dependent oxidoreductases. + and the use of the alcohol dehydrogenase of the present invention in cofactor regeneration.

[0072] NAD(P) produced during the reduction of D-psicose to allitol + The cofactor is reduced to NAD(P)H by formate and formate dehydrogenase (cofactor regeneration), with the formation of CO2.

[0073] Particularly preferred is 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. The preferred formate dehydrogenase used is preferably encoded by the nucleic acid sequence of SEQ ID NO: 1. A "functional fragment" of formate dehydrogenase includes N-terminally and / or C-terminally truncated variants of formate dehydrogenase having the amino acid sequence of SEQ ID NO: 2, which have 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 the enzymatic activity of the untruncated formate dehydrogenase. SEQ ID NO:1:

number

number

[0074] According to a further preferred embodiment of the present invention, the formate dehydrogenase used for cofactor regeneration is i) an amino acid sequence having at least 80% identity to SEQ ID NO: 2; ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity to SEQ ID NO: 1; and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 1, or a functional fragment thereof The amino acid sequence comprises an amino acid sequence selected from the group consisting of:

[0075] The formate dehydrogenase preferably comprises an amino acid sequence having at least 80%, more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, and especially 100% identity to SEQ ID NO:2.

[0076] Alternatively, the formate dehydrogenase preferably comprises an amino acid sequence encoded by a nucleic acid having at least 80%, more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, and especially 100% identity to SEQ ID NO:1.

[0077] Another aspect of the present invention relates to the use of formate dehydrogenase or a functional fragment thereof for cofactor regeneration, the formate dehydrogenase comprising: i) an amino acid sequence having at least 80% identity to SEQ ID NO: 2; ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity to SEQ ID NO: 1; and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 1, or a functional fragment thereof The amino acid sequence comprises or consists of an amino acid sequence selected from the group consisting of:

[0078] Glucose dehydrogenases (GDHs) used for cofactor regeneration are in the groups EC1.1.1.47 (glucose-1-dehydrogenase), EC1.1.1.118 (glucose-1-dehydrogenase (NAD + )), EC1.1.1.119 (glucose-1-dehydrogenase (NADP +)) or EC 1.1.1.360 (glucose / galactose-1-dehydrogenase).

[0079] The NAD(P)-dependent glucose dehydrogenase for cofactor regeneration is preferably i) an amino acid sequence having at least 80% identity to SEQ ID NO: 20; ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity to SEQ ID NO: 19, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 19 The amino acid sequence comprises or consists of an amino acid sequence selected from the group consisting of:

[0080] Generally, particularly suitable for cofactor regeneration are glucose dehydrogenases whose amino acid sequence has at least 80% identity to SEQ ID NO:20, or encoded by a nucleic acid having at least 80% identity to SEQ ID NO:99, or which bind under stringent conditions to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO:19, or functional fragments of such glucose dehydrogenases. A "functional fragment" of glucose dehydrogenase includes N-terminally and / or C-terminally truncated variants of glucose dehydrogenase having the amino acid sequence of SEQ ID NO:18, which have 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 the enzymatic activity compared to the uncleaved alcohol dehydrogenase. SEQ ID NO: 19:

number

number

[0081] The glucose dehydrogenase for cofactor regeneration referred to herein preferably comprises an amino acid sequence having at least 80%, more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, and especially 100% identity to SEQ ID NO: 20. Particularly preferably, the glucose dehydrogenase of the invention for cofactor regeneration comprises or consists of the amino acid sequence of SEQ ID NO: 20.

[0082] Alternatively, the glucose dehydrogenase for cofactor regeneration preferably comprises an amino acid sequence encoded by a nucleic acid having at least 80%, more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, and especially 100% identity to SEQ ID NO: 19. Particularly preferably, the nucleic acid encoding the glucose dehydrogenase of the invention for cofactor regeneration comprises or consists of the nucleic acid sequence of SEQ ID NO: 19.

[0083] A further aspect of the present invention relates to the use of glucose dehydrogenase for cofactor regeneration, the glucose dehydrogenase comprising: i) an amino acid sequence having at least 80% identity to SEQ ID NO: 20; ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity to SEQ ID NO: 19, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 19, or a functional fragment thereof The amino acid sequence comprises or consists of an amino acid sequence selected from the group consisting of:

[0084] The NAD(P)H oxidase used for cofactor regeneration (see FIG. 1) may be from one of the groups EC 1.6.3.1 (NAD(P)H oxidase (HO forming)), EC 1.6.3.2 (NAD(P)H oxidase (HO forming)), EC 1.6.3.3 (NADH oxidase (HO forming)) and EC 1.6.3.4 (NADH oxidase (HO forming)), where the HO forming class is particularly preferred.

[0085] Particularly preferred HO-forming NAD(P)H oxidases preferably i) an amino acid sequence having at least 80% identity to SEQ ID NO: 14 or SEQ ID NO: 16; ii) an amino acid sequence encoded by a nucleic acid having 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 binds under stringent conditions to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 13 or SEQ ID NO: 15; or Its functional fragments The amino acid sequence comprises or consists of an amino acid sequence selected from the group consisting of:

[0086] A "functional fragment" of an NAD(P)H oxidase includes an N-terminally and / or C-terminally truncated variant of an NAD(P)H oxidase having the amino acid sequence of 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 the enzymatic activity of the uncleaved NAD(P)H oxidase. SEQ ID NO: 13:

number

number

number

number

[0087] Preferably used HO-forming NAD(P)H oxidases preferably comprise or consist of an amino acid sequence having at least 80%, more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, and especially 100% identity to SEQ ID NO: 16 or SEQ ID NO: 14. Particularly preferably, the HO-forming NAD(P)H oxidase comprises or consists of the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 14.

[0088] Alternatively, the HO-forming NAD(P)H oxidase preferably has an amino acid sequence encoded by a nucleic acid having at least 80%, more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, and especially 100% identity to SEQ ID NO: 15 or SEQ ID NO: 13. Particularly preferably, the nucleic acid encoding the HO-forming NAD(P)H oxidase comprises or consists of the nucleic acid sequence of SEQ ID NO: 15 or SEQ ID NO: 13.

[0089] A further aspect of the present invention is i) an amino acid sequence having at least 80% identity to SEQ ID NO: 16 or SEQ ID NO: 14; ii) an amino acid sequence encoded by a nucleic acid having 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 binds under stringent conditions to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 15 or SEQ ID NO: 13; or Its functional fragments Cofactor regeneration (NAD(P)H to NAD(P)) comprising or consisting of an amino acid sequence selected from the group consisting of: + The present invention relates to the use of HO-forming NAD(P)H oxidase for the treatment of inflammatory bowel diseases.

[0090] Combined with cofactor regeneration, the enzymatic strategy presented here enables a biocatalytic, environmentally friendly, and highly efficient production method for D-psicose.

[0091] The first NAD(P)H-dependent oxidoreductase for reducing D-psicose to allitol is preferably i) an amino acid sequence having 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 having 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 binds under stringent conditions to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 3, SEQ ID NO: 9, or SEQ ID NO: 11; The amino acid sequence comprises or consists of an amino acid sequence selected from the group consisting of:

[0092] Generally particularly suitable for reducing a first D-psicose to allitol or D-psicose to allitol are oxidoreductases whose amino acid sequence has at least 80% identity to SEQ ID NO:4, SEQ ID NO:10, or SEQ ID NO:12, or is encoded by a nucleic acid having at least 80% identity to SEQ ID NO:3, SEQ ID NO:9, or SEQ ID NO:1, or which bind under stringent conditions to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO:3, SEQ ID NO:9, or SEQ ID NO:11. Moreover, surprisingly, this oxidoreductase can be used to oxidize allitol to D-psicose.

[0093] NAD(P) for the formation of allitol from D-psicose +The dependent oxidoreductase is preferably i) an amino acid sequence having 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) an amino acid sequence encoded by a nucleic acid having 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 binds under stringent conditions 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 The amino acid sequence comprises or consists of an amino acid sequence selected from the group consisting of: SEQ ID NO:3:

number

number

number

number

number

number

number

number

number

number

[0094] The oxidoreductase referred to herein for the reduction of D-psicose to allitol and / or the oxidation of allitol to D-psicose preferably comprises an amino acid sequence having at least 80%, more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, and especially 100% identity to SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12. Particularly preferably, the oxidoreductase of the present invention for the reduction of D-psicose to allitol and / or the oxidation of allitol to D-psicose comprises or consists of the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 10, or SEQ ID NO: 12, and the oxidoreductase for the oxidation of allitol to D-psicose comprises or consists of the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 8.

[0095] Alternatively, the oxidoreductase for the reduction of D-psicose to allitol and / or the oxidation of allitol to D-psicose preferably comprises an amino acid sequence encoded by a nucleic acid having at least 80%, more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, and particularly 100% identity to SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 9, or SEQ ID NO: 11. Particularly preferably, the nucleic acid encoding the oxidoreductase of the present 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.

[0096] As used herein, the term "identity" refers to the percentage of identical nucleotides or amino acids between at least two nucleotide or amino acid sequences that have been aligned ("aligned") using a standardized algorithm. Such algorithms can insert gaps in the sequences being compared in a standardized and reproducible manner to optimize the alignment between the two sequences and thus achieve a more meaningful comparison of the two sequences.

[0097] The percentage identity between sequences can be determined using one or more computer algorithms or programs known in the state of the art or described herein. According to the present invention, identity is determined using the Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990) provided by the National Center for Biotechnology Information (NCBI). The BLAST software suite includes several programs, including a tool called "BLAST2 sequences," which is used for direct pairwise comparison of two nucleotide or amino acid sequences. "BLAST2 sequences" can also be searched and used interactively on the Internet via the NCBI World Wide Web page. The blastn program (for nucleotide sequences) uses as default 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 defaults a word length of 3 and 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.

[0098] Alternatively, the oxidoreductase for reducing D-psicose to allitol and / or oxidizing allitol to D-psicose preferably comprises an amino acid sequence encoded by a nucleic acid that 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 under stringent conditions. As used herein, "stringent conditions" refer to conditions under which so-called specific hybrids are formed but nonspecific hybrids are not. For example, stringent conditions include hybridization in 6xSSC (sodium chloride / sodium citrate) at 45°C, followed by washing with 0.2-1xSSC, 0.1% SDS at 50-65°C, or such conditions may include hybridization in 1xSSC at 65-70°C, followed by washing with 0.3xSSC at 65-70°C. Hybridization can be carried out by conventional methods such as those described in J. Sambrook et al. in Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory (1989).

[0099] One aspect of the present invention relates to the use of an oxidoreductase for reducing D-psicose to allitol and / or oxidizing allitol to D-psicose, the oxidoreductase comprising: i) an amino acid sequence having 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 having 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 binds under stringent conditions to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 3, SEQ ID NO: 9, or SEQ ID NO: 11; The amino acid sequence comprises an amino acid sequence selected from the group consisting of:

[0100] A further aspect of the present invention relates to the use of an oxidoreductase for the oxidation of allitol to D-psicose, the oxidoreductase comprising: i) an amino acid sequence having 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) an amino acid sequence encoded by a nucleic acid having 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 binds under stringent conditions 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 The amino acid sequence comprises an amino acid sequence selected from the group consisting of:

[0101] Depending on the reaction (reduction or oxidation), the oxidoreductase enzymes of the invention require the corresponding cofactor, as described above.

[0102] material D-psicose was purchased from TCI and Hunan Garden Naturals Inc. (China), allitol was purchased from TCI, D-fructose, NADPH tetrasodium salt, and methanol were purchased from PanReac AppliChem (ITW Reagents), D-glucose, sodium gluconate, and IPTG (isopropyl-β-D-thiogalactopyranoside) were purchased from Sigma-Aldrich, and potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and NAD + , NADH disodium salt, NADP + Disodium salt and sodium dodecyl sulfate (SDS) were purchased from Carl Roth, and triethanolamine (TEA) was purchased from Chem-Lab NV.

[0103] Enzyme production and lysate preparation General information regarding the expression of recombinant enzymes in E. coli For recombinant enzyme production in Escherichia coli strains, the gene to be expressed was first amplified in PCR using genomic DNA or its synthetic equivalent, adapted to E. coli codon usage, as a template, along with specific oligonucleotides containing additional restriction endonuclease recognition sequences, and isolated from the reaction mixture. After digestion of the nucleic acid with the restriction enzymes SphI and HindIII, the gene fragment encoding the target enzyme was ligated into the SphI- and HindIII-cleaved backbone of the expression vector pQE70-Kan. The ligation product was transformed into chemically competent Top10F E. coli cells, and the resulting colonies were used for plasmid isolation and restriction analysis.

[0104] The results of the cloning process were verified by restriction enzyme digestion and DNA sequencing. The resulting construct harbors the target gene under an IPTG-inducible T5 promoter.

[0105] For overexpression of the enzyme in E. coli, the resulting expression plasmid was transformed into competent expression cells RB791. After 24 hours of incubation at 37°C, the resulting colonies were inoculated into LB medium for expression assays.

[0106] The next day, grow the expression culture to an optical density OD of 0.02. 550 and inoculated at an OD of 0.3. 550 The mixture was shaken at 37°C until an OD of 0.5 was reached. The temperature was then lowered to 25°C. 550 Once the ATP concentration reached 0.1 mM, the cultures were induced with 0.1 mM IPTG. After 22 hours, the cultures were harvested (separated from the medium by centrifugation in the form of a cell pellet) and analyzed for expression of the recombinant enzyme using SDS gel electrophoresis and activity determination (using a test or optical enzyme assay).

[0107] Preparation of cell lysates using sonifier disruption To prepare the cell suspension, the cell pellet prepared according to the method described above was weighed into an appropriate container, mixed with buffer and lysozyme (final concentration 0.5 mg / ml) (e.g., triethanolamine (TEA)-HCl), and dissolved under stirring. The mass fraction of biomass was usually 20%, with the remainder being buffer.

[0108] 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).

[0109] The resulting homogenate was centrifuged at 16,000 rpm for 10 minutes at 4°C (Eppendorf centrifuge 5417R) to separate insoluble cell fragments and obtain a lysate.

[0110] [Table 1]

[0111] Analysis method High-performance liquid chromatography D-psicose, D-fructose, D-glucose, and allitol were quantified by HPLC (high-performance liquid chromatography) using an Agilent HPLC 1260 Infinity II Series system. Detection was performed using a refractive index detector (RI detection). Measurements were performed using a Phenomenex Rezex RPM-Monosaccharide Pb+2 (8%) column with an appropriate precolumn, and eluted isocratically with ultrapure water.

[0112] High-Performance Anion-Exchange Chromatography D-gluconic acid / D-gluconate was quantified using HPAEC (high-performance anion exchange chromatography) on a Dionex ICS6000 system equipped with an AS-AP autosampler. Measurements were performed using conductivity detection (CD) coupled to a Dionex AERS500 electrolytically regenerated suppressor in external water mode. Analytes were separated using a Dionex lonPac AS11-HC-4 μm column with an appropriate precolumn and NaOH gradient. The mobile phase was further pretreated with a Dionex ATC anion trap column.

[0113] Determination of enzyme activity (optical-enzyme assay) The enzyme activity in the lysates was determined using a Shimadzu UV-1900 spectrophotometer. For this purpose, the formation or consumption of NAD(P)H was monitored via the change in absorbance at a wavelength of 340 nm. Measurements were performed in the presence of 0.2 mM of the cofactor (NAD(P) + or NAD(P)H). For this purpose, a cuvette (Greiner Bio-One Semi-Micro Cuvette made of polystyrene) was filled with 20 μl of a 10 mM stock solution of the cofactor and adjusted to the desired pH 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 carried out at 25°C by default. The enzyme activity of the lysate was determined by the extinction coefficient of NADH / NADPH at 340 nm (E = 6220 Lmol -1 cm -1 ) can be used to calculate the conversion rate in U / ml (based on the volume of lysate) or U / g (based on the biomass used for production), where 1 U represents 1 pmol of substrate converted per minute (1 U = 1 pmol / min = 1 μmol / min = 1.67 10 -8 kat).

[0114] The following examples explain in more detail preferred variants of the method according to the invention: The lysates used in these examples were prepared according to the procedure described above.

[0115] Example 1 Production of D-psicose from D-fructose - Cofactor regeneration with ADH and 2-propanol The reaction was carried out in a Labfors5 benchtop bioreactor (Infors AG). A glass reactor (volume 3.4 L) equipped with a stirrer and a pH electrode was used as the vessel. The pH was controlled by adding 1 M NaOH or 1 M H2SO4.

[0116] At the start, 50 ml of D-fructose solution (500 g / l), 246.1 ml of deionized water and 96 ml of 200 mM TEA-HCl buffer (pH 8) were placed in the reactor and brought to 35° C. with stirring.

[0117] To start the reaction, 25 ml of D-psicose 3-epimerase lysate was added, followed by 25 ml of SDR I lysate, 4 kU of alcohol dehydrogenase lysate, and 10 ml of 10 mM NAD. + solution, and 40 ml of 2-propanol was added.

[0118] During the run, samples were continuously taken from the reactor solution and analyzed as follows: 100 μl of reactor solution was mixed with 200 μl of methanol and incubated for 15 min at 60 °C and 1200 rpm in an Eppendorf Thermomixer. The sample was briefly centrifuged in a centrifuge, mixed with 700 μl of deionized water, vortexed, and then centrifuged at maximum g for 5 min. 200 μl of the supernatant was transferred to an HPLC vial with an insert and measured by HPLC (RI detection).

[0119] After 23 hours of operation, 15 ml of 2-propanol was added, after 50 hours 20 ml was added, and after 77 hours 15 ml was added.

[0120] After 74 hours, 5 ml of D-psicose 3-epimerase lysate was added, and after 77 hours, 15 ml of SDR I lysate and 2.4 kU of alcohol dehydrogenase lysate were added.

[0121] After 97 hours, 94% of the D-fructose (50 g / l) was converted to allitol.

[0122] The entire contents of the reactor were then heated to 70°C for 60 minutes (to inactivate D-psicose 3-epimerase), cooled to 24°C, and then added to 25 ml of xylitol dehydrogenase lysate, 10 kU of NADH oxidase lysate, and 10 ml of 10 mM NAD + The solution was added.

[0123] Allitol was completely oxidized to D-psicose within 3 hours. The reactor contents were heated to 70°C, the pH was adjusted to 4, and the mixture was stirred at 70°C for 30 minutes. The enzyme was filtered using a glass frit (P3).

[0124] In this way, 94% of the D-fructose was converted to D-psicose, and no D-sorbitol was detectable.

[0125] The filtrate was concentrated using a rotary evaporator to a syrup having a D-psicose concentration of 520 g / l, and the remaining acetone and 2-propanol were also separated.

[0126] Example 1 shows that the epimerase can be denatured by heat (in a one-pot process) and the resulting precipitate does not interfere with further reactions.

[0127] Example 2 Production of D-psicose from D-fructose - Cofactor regeneration by GDH and D-glucose The reaction was carried out in a Multifors benchtop bioreactor (Infors AG). A glass reactor (volume 1 L) equipped with a stirrer and a pH electrode was used as the vessel. The pH was controlled by adding 5 M NaOH or 1 M H2SO4.

[0128] At the start, 17.5 g of D-fructose and 17.5 g of D-glucose (final concentration of each 50 g / l), 217.8 ml of deionized water, and 26.5 ml of 500 mM potassium phosphate buffer (pH 7.5) were placed in a reactor and brought to 35°C with stirring.

[0129] To initiate the reaction, 17.5 ml of D-psicose 3-epimerase lysate was added, followed by 24.5 ml of SDR I lysate, 0.4 kU of glucose dehydrogenase lysate, and 3.5 ml of 10 mM NAD+ solution.

[0130] During the run, samples were continuously taken from the reactor solution and analyzed as follows: 100 μl of reactor solution was mixed with 200 μl of methanol and incubated for 15 min at 60 °C and 1200 rpm in an Eppendorf Thermomixer. The sample was briefly centrifuged, mixed with 700 μl of deionized water, vortexed, and then centrifuged at maximum g for 5 min. 200 μl of the supernatant was transferred to an HPLC vial with an insert and measured by HPLC (RI detection). For HPAEC measurement (conductivity detection), the clear supernatant was diluted 1:250.

[0131] After 16 hours, only allitol and D-gluconic acid could be detected in the reactor.

[0132] The entire contents of the reactor were then heated to 70°C for 60 minutes (to inactivate D-psicose 3-epimerase), cooled to 30°C, and then 17.5 ml of xylitol dehydrogenase lysate, 7 kU of NADH oxidase lysate, and 3.5 ml of 10 mM NAD+ solution were added.

[0133] Allitol was completely oxidized to D-psicose within 27 hours.

[0134] In this way, 17.5 g of D-fructose was oxidized to 100% D-psicose (17.8 g in solution). Neither D-sorbitol nor D-fructose was detectable in the resulting solution.

[0135] 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 enzyme was filtered using a glass frit (P3).

[0136] Example 3 Production of D-psicose from D-fructose - Cofactor regeneration using FDH and sodium formate The reaction was carried out in a Multifors benchtop bioreactor (Infors AG). A glass reactor (volume 1 L) equipped with a stirrer and a pH electrode was used as the vessel. The pH was controlled by adding 5 M NaOH or 6 M H2SO4.

[0137] At the start, 70 ml of D-fructose solution (500 g / l), 26.3 ml of deionized water, and 43.8 ml of 8 M sodium formate solution were placed in the reactor and heated to 37° C. with stirring.

[0138] To start the reaction, 25 ml of D-psicose 3-epimerase lysate was added, followed by 35 ml of SDR I lysate, 7 kU of formate dehydrogenase lysate, and 17.5 ml of 10 mM NAD. + The solution was added.

[0139] During the run, samples were continuously taken from the reactor solution and analyzed as follows: 100 μl of reactor solution was mixed with 200 μl of methanol and incubated for 15 min at 60 °C and 1200 rpm in an Eppendorf Thermomixer. The sample was briefly centrifuged, mixed with 700 μl of deionized water, vortexed, and then centrifuged at maximum g for 5 min. 200 μl of the supernatant was transferred to an HPLC vial with an insert and measured by HPLC (RI detection).

[0140] After 40 hours, 95% of the D-fructose (100 g / l) was converted to allitol.

[0141] The entire contents of the reactor were then heated to 70°C for 60 minutes (to inactivate D-psicose 3-epimerase), cooled to 24°C, and then added to 25 ml of xylitol dehydrogenase lysate, 10 kU of NADH oxidase lysate, and 10 ml of 10 mM NAD + The solution was added.

[0142] Allitol was completely oxidized to D-psicose within 4 hours. The reactor contents were heated to 70°C, the pH was adjusted to 4, and the mixture was stirred at 70°C for 30 minutes. The enzyme was filtered using a glass frit (P3).

[0143] In this way, 97% of the D-fructose was converted to D-psicose. Trace amounts of D-fructose were still detected in the reaction solution, but no D-sorbitol was detected.

[0144] literature [Table L1] [Table L2] [Table L3] [Table L4] [Table L5] [Table L6]

Claims

1. A first D-psicose is formed from D-fructose present in an aqueous solution by treatment with an epimerase in vitro, and then the first D-psicose is reduced to allitol by treatment with a respective NAD(P)H-dependent oxidoreductase in vitro, and after inactivation of the epimerase and / or ultrafiltration, the respective NAD(P)H-dependent oxidoreductase is removed to form D-psicose. ＋ and then removing the inactivated epimerase and the oxidoreductase.

2. The NAD(P) for the formation of D-psicose from allitol ＋ Dependent oxidoreductases i) an amino acid sequence having 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) an amino acid sequence encoded by a nucleic acid having 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 binds under stringent conditions 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 2. The method of claim 1, wherein the amino acid sequence comprises an amino acid sequence selected from the group consisting of:

3. The oxidized cofactor NAD(P) formed by the reduction ＋ 3. The method according to claim 1, wherein is reduced by an alcohol dehydrogenase and a secondary alcohol with the formation of a ketone.

4. 4. The method according to claim 3, wherein the secondary alcohol is D-glucose or 2-propanol.

5. 5. The process according to any one of claims 1 to 4, characterized in that it is carried out as a one-pot reaction without isolation of the intermediate products.

6. 6. The method according to claim 1, wherein the enzymes are present as lysates of the corresponding cells that produce them.

7. the NAD(P)H-dependent oxidoreductase for the reduction of the first D-psicose to allitol is i) an amino acid sequence having 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 having 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 binds under stringent conditions to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 3, SEQ ID NO: 9, or SEQ ID NO: 11; The method according to any one of claims 1 to 6, characterized in that the amino acid sequence is selected from the group consisting of:

8. The alcohol dehydrogenase i) an amino acid sequence having at least 80% identity to SEQ ID NO: 18; ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity to SEQ ID NO: 17; and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 17 9. The method according to any one of claims 3 to 8, characterized in that it comprises or consists of an amino acid sequence selected from the group consisting of:

9. the oxidized cofactor NAD(P) formed by the reduction ＋ 9. The method according to claim 1, wherein is reduced by glucose dehydrogenase and D-glucose with the formation of D-gluconic acid.

10. Glucose dehydrogenase is i) an amino acid sequence having at least 80% identity to SEQ ID NO: 20; ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity to SEQ ID NO: 19; and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 19 10. The method of claim 9, comprising or consisting of an amino acid sequence selected from the group consisting of:

11. The oxidized cofactor NAD(P) produced by the reaction ＋ But CO ２ 11. The method according to claim 1, wherein the hydroxybenzoate is reduced by formate dehydrogenase and formate with the formation of

12. The formate dehydrogenase i) an amino acid sequence having at least 80% identity to SEQ ID NO:2; ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity to SEQ ID NO: 1; and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 1; 12. The method of claim 11, comprising or consisting of an amino acid sequence selected from the group consisting of:

13. Use of an aqueous solution preparable by the method according to any one of claims 1 to 12 for preparing a syrup containing D-psicose.

14. Use of an oxidoreductase for forming D-psicose from allitol, the oxidoreductase comprising: i) an amino acid sequence having 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) an amino acid sequence encoded by a nucleic acid having 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 binds under stringent conditions 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 Use of an oxidoreductase, characterized in that it comprises an amino acid sequence selected from the group consisting of:

15. Use of an alcohol dehydrogenase for cofactor regeneration, the alcohol dehydrogenase comprising: i) an amino acid sequence having at least 80% identity to SEQ ID NO: 18; ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity to SEQ ID NO: 17; and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 17 10. Use of an alcohol dehydrogenase comprising or consisting of an amino acid sequence selected from the group consisting of:

16. Use of glucose dehydrogenase for cofactor regeneration, said glucose dehydrogenase comprising: i) an amino acid sequence having at least 80% identity to SEQ ID NO: 20; ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity to SEQ ID NO: 19; and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 19 10. Use of a glucose dehydrogenase comprising or consisting of an amino acid sequence selected from the group consisting of:

17. 1. Use of formate dehydrogenase or a functional fragment thereof for cofactor regeneration, the formate dehydrogenase comprising: i) an amino acid sequence having at least 80% identity to SEQ ID NO:2; ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity to SEQ ID NO: 1; and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 1; 10. Use of a formate dehydrogenase or a functional fragment thereof, comprising or consisting of an amino acid sequence selected from the group consisting of:

18. i) an amino acid sequence having at least 80% identity to SEQ ID NO: 14 or SEQ ID NO: 16; ii) an amino acid sequence encoded by a nucleic acid having 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 binds under stringent conditions to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 13 or SEQ ID NO: 15 H for cofactor regeneration comprising or consisting of an amino acid sequence selected from the group consisting of: ２ Use of O-forming NAD(P)H oxidase.

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