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

[0001] Process for the preparation of an aqueous solution containing D-psicose

[0002] The present invention relates to a process for preparing an aqueous solution containing D-psicose.

[0003] Background of the invention

[0004] D-Psicosis

[0005] The monosaccharide D-psicose, also known as D-allulose, is a ketohexose rarely occurring in nature (Zhang et al., 2016). It has been detected, among other things, in the leaves of rosemary willow (Itea sp.) (Hough & Stacey, 1966), but is also found in processed foods such as confectionery and spice sauces, where it is formed from D-fructose, its Ca epimer, under the influence of heat (Oshima et al., 2006).

[0006] 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 its energy content is low (0.2 kcal / g), corresponding to a calorie reduction of approximately 95% (relative to sucrose) (Jiang et al., 2020).

[0007] In the USA, D-psicose is recognized by the US Food and Drug Administration (FDA) as a Generally Recognized as Safe (GRAS) sweetener, but is not yet approved in the EU (Ahmed et al., 2022).

[0008] In addition, D-psicose also has positive effects on lipid metabolism and carbohydrate metabolism (e.g., antidiabetic) and has anti-inflammatory and antioxidant effects (Zhang et al., 2016; Jiang et al., 2020; Chen et al., 2022).

[0009] Due to its low natural occurrence, D-psicose is largely produced synthetically (chemically or biotechnologically).

[0010] The epimerization of D-fructose to D-psicose can be achieved by refluxing in pyridine followed by removal of the other hexoses by yeast fermentation, although this method only achieves 6.8% of the theoretical yield of D-psicose (Doner, 1979). Another method involves the epimerization of D-fructose with molybdate ions as a catalyst, whereby only 0.5% of the D-fructose is converted to D-psicose (Bilik & Tihlärik, 1974).

[0011] The inefficient chemical synthesis routes have now been replaced by more efficient biotechnological processes. In 1993, Izumori et al. described a ketosis 3-epimerase from Pseudomonas cichorii ST-24 for the production of D-psicose from D-fructose (Izumori et al., 1993), which was also patented (EP 0592202 Bl). Ketosis 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).

[0012] However, the conversion of D-fructose to D-psicose by ketose 3-epimerases is not complete; rather, an equilibrium ratio is formed between the two epimers. Depending on the reaction conditions (temperature between 40 and 70 °C, pH between 6 and 11), this ratio ranges between 80:20 and 62.5:37.5 (D-fructose:D-psicose). Many of the epimerases also require a divalent metal ion such as Mn. 2+ or Co 2+ (toxic) as a cofactor (Zhang et al., 2016; Jiang et al., 2020).

[0013] The equilibrium during epimerization can be influenced not only by temperature or pH, but also by the addition of (toxic) borate. Due to the preferential formation of a D-psicose-borate complex, the equilibrium is shifted toward D-psicose (Kim et al., 2008; Lim et al., 2009). EP 3643786 A2 and US 11028420 B2 describe the chromatographic separation of the D-psicose-borate complex using simulated moving bed (SMB) chromatography. EP 3395952 B1 and US 10550414 B2 disclose that the conversion during epimerization using DPE can be increased to up to 67% with the addition of sodium aluminate and up to 52% with potassium iodate (comparison: 25% without the addition of aluminate or iodate).

[0014] Zhu et al. (2020) presented a system consisting of two enzymes (exo-inulinase from Bacillus velezenis and DAE from Ruminococcus sp.) that can 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 juices) into D-psicose. They were able to enrich the juices with 16–19% D-psicose (based on the total carbohydrate content).

[0015] Juneja et al. (2019) analyzed the technoeconomic aspects of a modified corn dry grind process, in which D-psicose is produced from ground corn in addition to ethanol using a modified yeast strain (through 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 the 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 by saccharification, enzymatic isomerization, and epimerization. Patel et al. (2018) used an Smt3-DPE (fusion protein) immobilized on magnetic iron oxide nanoparticles to produce D-psicose from D-fructose in fruit pomace washing solutions.The immobilized epimerase was able to convert 20% of the D-fructose and was separated with a magnet after completion of the reaction.

[0016] Yang et al. (2018) transferred the DPE gene from Agrobacterium tumefaciens into the thermotolerant bacterium Kluyveromyces marxianus. This enabled 190 g / l of D-psicose to be produced from 750 g / l D-fructose in 12 h at 55 °C, and the remaining D-fructose was fermented by K. marxianus to ethanol. Dedania et al. (2020) immobilized DPE from A. tumefaciens on titanium dioxide nanoparticles and converted 36% of the D-fructose into D-psicose. Furthermore, the immobilized enzyme could be reused up to nine times.

[0017] The D-fructose / D-psicose mixtures resulting from the epimerization of D-fructose can be separated either by chromatographic methods or by the "biological method" (fermentation of the excess D-fructose to, for example, ethanol) (Jiang et al., 2020). US 2021 / 0189441 A1 describes the separation of a D-fructose / D-psicose mixture, whereby the D-fructose is converted into 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 highly pure D-psicose. EP 3553069 A1 and Van Duc Long et al. (2009) disclose a method for the separation of D-psicose and D-fructose based on SMB chromatography.

[0018] However, the production of D-psicose via the epimerase route has the following disadvantages: 1) position of the equilibrium on the side of D-fructose, 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.

[0019] One way to circumvent the thermodynamically unfavorable epimerization is through enzyme cascades with phosphorylated intermediates. The final step, dephosphorylation, is irreversible and thus drives the cascade (Li et al., 2021b).

[0020] A cascade described in almost identical form by Li et al. (2021b) as well as in US 11168342 B2 and US 10907182 B2 features D-glucose-l-phosphate (G1P) as a central intermediate. G1P is first converted to D-glucose-6-phosphate (G6P) by phosphoglucomutase and then further to D-frucose-6-phosphate (F6P) by glucose-6-phosphate isomerase. F6P is then epimerized to D-psicose-6-phosphate by D-allulose-6-phosphate epimerase, which is subsequently dephosphorylated to D-psicose by D-allulose-6-phosphate phosphatase. G1P can be produced directly by the action of phosphorylases on, for example, maltose and amylodextrins (obtained by the hydrolysis of starch), cellodextrins (obtained by the hydrolysis of cellulose) or sucrose, consuming phosphate.Since the terminal sugar monomers of the oligo- and polysaccharides cannot be phosphorylated by the corresponding phosphorylases, polyphosphate glucokinase (D-glucose) must be used. G6P) or polyphosphate fructokinase (D-fructose F6P) can be used, whereby additional polyphosphates must be added as a phosphate source to increase yields (US 11168342 B2; US 10907182 B2). Starch serves as the substrate for the cascade of Li et al. (2021b), which is converted to D-psicose with yields of 79% (at 50 g / l substrate concentration; reaction time 24 h).

[0021] Wang et al. (2020) also developed an enzymatic cascade for the production of D-psicose starting from starch, which, however, is converted to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate in several steps. Dihydroxyacetone phosphate is converted with D-glyceraldehyde under the action of L-fuculose l-phosphate aldolase (FucA) to D-psicose l-phosphate, which is 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).

[0022] An enzyme cascade starting from glycerol has also been described in the literature. This 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 alditol oxidase), which in turn serve as a substrate for an aldolase (such as FucA). After removal 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, in turn, describes a method for the production of D-psicose from dihydroxyacetone and D-glyceraldehyde using fructose-6-phosphate aldolase and DTE (D-fructose intermediate).

[0023] Xiao et al. coupled the epimerization of D-fructose with the conversion of D-psicose to D-psicose-l-phosphate using L-rhamnulose kinase (consuming adenosine triphosphate (ATP)) to shift the epimerization equilibrium. Subsequent removal of the phosphate group by an acid phosphatase yields D-psicose (99% conversion of 20 mM D-fructose). However, ATP must be regenerated with a polyphosphate kinase in the presence of polyphosphate (Xiao et al., 2019).

[0024] A major disadvantage of routes using phosphorylated intermediates is the use of expensive, energy-rich phosphate compounds such as polyphosphate or ATP in stoichiometric amounts to introduce the phosphate groups. This problem can be partially circumvented by the use of phosphorylases, 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.

[0025] Fermentation processes for the production of D-psicose are also known. Zhang et al. (2021) presented a fermentation process based on the co-cultivation of engineered Bacillus subtilis and Escherichia coli for the co-production of D-psicose (titer 11.7 g / L; conversion: 69.5%) and the enzyme lipase. US 2017 / 0298400 A1 describes the expression of DPE (e.g., from Agrobacterium tumefaciens) in various microorganisms. EP 3088515 B1 and US 9701953 B2 describe a D-psicose-producing Ensifer adhaerens strain. EP 2470668 B1 discloses the immobilization of a GRAS microorganism (Corynebacterium glutamicum KCCM 11046) with expressed DPE on a sodium alginate carrier.

[0026] Allitol as an intermediate

[0027] The unfavorable equilibrium of 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 regeneration enzyme for the cofactor nicotinamide adenine dinucleotide NADH), D-fructose can be converted in vitro to the sugar alcohol allitol (Takeshita et al., 2000).

[0028] Through an oxidation step, allitol can subsequently be converted back to D-psicose. This can be achieved microbially, for example, with Enterobacter aerogenes IK7 (complete oxidation of 100 g / l allitol in 24 h) or Bacillus pallidus Y25 (48% conversion of 50 g / l allitol in 48 h) (Gullapalli et al., 2007; Poonperm et al., 2007).

[0029] Due to its symmetry, the achiral sugar alcohol allitol forms an interface between the D- and L-hexoses in the so-called Izumoring strategy for the bioproduction of rare sugars (Izumori, 2006; Hassanin et al., 2017). Thus, it can also serve as a precursor for the production of other rare monosaccharides.

[0030] The theoretical papers by Hold et al. (2009) and Siedentop et al. (2021) address the optimization of enzyme cascades. They describe that all components and a multitude of parameters must be considered, especially parameters of the cascade design, the enzymes themselves, the reaction conditions and environment, and also the process design, whereby success cannot be predicted. A specific synthesis of D-psicose is not mentioned. Chen et al. (2022) turn to the fermentative route via whole-cell biocatalysts ("in vivo") for the production of D-psicose and conclude that only this route has the potential to produce D-psicose economically and on an industrial scale in the future through various optimizations. The advantages of whole-cell biocatalysts are obvious:

[0031] (1) Cells that contain the enzymes inside them are more easily accessible than the enzymes themselves, the purification of which is often laborious,

[0032] (2) the interior of the cells provides a suitable microenvironment for the enzymes and also allows cofactor regeneration (NAD(P) + / NAD(P)H),

[0033] (3) the cell walls and membranes protect the enzymes against the environment of the reaction medium, and

[0034] (4) the co-localization of multiple enzymes within the cell favors local enzyme concentrations and reduces the diffusion of intermediates in cascade reactions.

[0035] The authors therefore see "microbial cell factories" as the best opportunity to produce D-psicose on a large scale, so that the average consumer will also be able to enjoy this rare sugar in the near future.

[0036] A research group led by Wang et al. (2022, 2023) is also investigating the enzymatic biotransformation of sugars, investigating the biotransformations in vitro and in vivo. The goal is to develop an economical production method that can be carried out on an industrial scale.

[0037] For the production of D-psicose from D-fructose, Wang et al. (2023) describe an in vivo process consisting of two E. coli whole-cell biocatalysts. In the first step (conversion of D-fructose to allitol), E. coli cells containing a DPE from Clostridiales, an RDH from Providentia alcalifaciens, an FDH from Starkeya, and another DPE from Rhizobium straminoryzae are used. In this way, D-fructose (500 mM = 90 g / L) was converted to 452 mM allitol within 12 h at 37 °C and pH 6 using 1000 mM sodium formate (two equivalents based on D-fructose) (conversion 90.4%). Approximately 30 mM D-sorbitol was formed as a byproduct. The cells were separated by centrifugation, and any released proteins in the allitol-containing supernatant were inactivated by heat. E. coli cells, which contained an RDH from Rubrivivax sp. and an NADH oxidase from Streptococcus pyogenes, were then added to the allitol solution.Allitol (452 ​​mM) was converted to D-psicose (450 mM) within 24 h at pH 7. Wang et al. (2023) further describe that, to their knowledge, the conversion rate of 90% is the highest ever achieved for the production of D-psicose from D-fructose and announce that they intend to further optimize the in vivo route, as the theoretical conversion rate of the proposed two-step process is 100%. A disadvantage of the Wang et al. process is the formation of the byproduct D-sorbitol, which is formed by the reduction of D-fructose.

[0038] This is where the object of the present invention comes in and aims to further improve the two-stage process for the formation of D-psicose from D-fructose and to provide a process for the production of aqueous solutions containing D-psicose, which can in particular also be carried out in a one-pot process.

[0039] Detailed description of the invention

[0040] The object is achieved according to the invention by forming a first D-psicose from D-fructose, which is dissolved 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 corresponding NAD(P)H-dependent oxidoreductase in vitro and, after deactivation and / or ultrafiltration of the epimerase, is reacted with a corresponding NAD(P) + -dependent oxidoreductase, after which the deactivated epimerase and the oxidoreductases are removed. Instead of deactivating the epimerase, it is also possible to immobilize it on or in a support material and filter it out of the aqueous solution together with the support.

[0041] It has surprisingly been found that in the process according to the invention practically no undesired D-sorbitol is formed and that even the yield of D-psicose can be increased further towards 100% in a simple manner.

[0042] The process according to the invention is therefore not carried out by fermentation; rather, the enzymes are contained as such in the aqueous solution. The process according to the invention is therefore carried out in vitro.

[0043] A preferred variant of the process according to the invention is characterized in that the NAD(P) +-dependent oxidoreductase for the formation of D-psicose from allitol comprises an amino acid sequence which is selected from the group consisting of: i) an amino acid sequence which has an identity to SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10 or SEQ ID No. 12 of at least 80%, ii) an amino acid sequence which is encoded by a nucleic acid which has an identity to SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9 or SEQ ID No. 11 of at least 80%, and iii) an amino acid sequence which is encoded by a nucleic acid which binds under stringent conditions to a nucleic acid molecule having 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, or a functional fragment thereof.

[0044] A "functional fragment" of this NAD(P) +-dependent oxidoreductase comprises an N-terminally and / or C-terminally truncated variant of the oxidoreductase having 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%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, enzyme activity compared to the non-truncated oxidoreductase.

[0045] The method according to the invention is shown schematically in the attached figure.

[0046] A preferred variant of the process according to the invention consists in that the oxidized cofactor NAD(P) formed by the reduction of D-psicose to allitol +by means of an alcohol dehydrogenase (ADH) and a secondary alcohol to form a ketone, with the secondary alcohol preferably being 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 separated due to its volatility (Xu et al., 2021). Acetone recovered from the exhaust stream can be hydrogenated back to 2-propanol using heterogeneous catalysis (Al-Rabiah et al., 2022), either in the gas phase or in solution, as 2-propanol / acetone / water mixtures or as acetone / water mixtures. In the future, hydrogen from sustainable sources ("green hydrogen") could be increasingly used.

[0047] The regeneration of the cofactor by means of ADH is previously known, for example, from EP 2812439 Bl or described by Xu et al. (2021).

[0048] In a further preferred embodiment of the present invention, the oxidized cofactor NAD(P) produced by the reduction + by means of glucose dehydrogenase and D-glucose to form D-gluconate. The use of glucose dehydrogenase to regenerate 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 a wide variety of areas (e.g., in metal mordants, in medicines, and as a stabilizer in food, etc.). Furthermore, the use of glucose dehydrogenase makes it possible to use a mixture comprising D-fructose and D-glucose as a substrate for producing allitol or D-psicose, without adding additional D-glucose to the reaction mixture and without D-glucose being isomerized beforehand to D-fructose. Mixtures of D-fructose and D-glucose can be produced, for example, by hydrolysis of sucrose. Particular preference is given to using a glucose dehydrogenase derived from Priestia megaterium, which comprises an amino acid sequence available under the NCBI accession number MDQ0804260.1.

[0049] A further preferred variant of the process according to the invention comprises the use of a formate dehydrogenase (FDH) for the regeneration of the oxidized cofactor NAD(P) produced by the reduction + using a formate dehydrogenase and formate (e.g. sodium formate) to form CO2.

[0050] 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 intermediate products.

[0051] In the method according to the invention, the enzymes are preferably used as lysate of the corresponding cells producing them. In contrast to the method described by Wang et al. (2023), which is based on E. coli whole-cell biocatalysts with co-expressed recombinant enzymes, the enzymes are expressed individually in suitable E. coli production strains. This allows for optimization of the enzyme ratios to each other, which is thus independent of the expression level in the overall construct, compared to Wang et al. (2023).

[0052] Before the final step (oxidation), the enzymes (epimerase and reductase and / or dehydrogenase) of the first step (D-fructose allitol) are deactivated by heat and / or removed by ultrafiltration to prevent the formation of byproducts by the enzymes. Without appropriate treatment, much of the D-psicose produced by oxidation would be converted back to D-fructose by the epimerase.

[0053] The regeneration of the nicotinamide-based cofactors (NAD or NADP) occurs in the case of the reduction of D-psicose to allitol with an NAD(P)-dependent alcohol dehydrogenase, glucose dehydrogenase or formate dehydrogenase and in the case of the oxidation reactions (second step) with a HjO-forming NAD(P)H oxidase.

[0054] The most preferred concentration of D-fructose is 50 - 250 g / l.

[0055] The particularly preferred temperature range for the first step (epimerization and reduction) is between 25 and 45 °C, for the second step (oxidation) between 20 and 30 °C.

[0056] The particularly preferred pH range for both steps is between 7 and 8.5. In a further preferred variant of the method according to the invention, the enzymes are present in a suspension and / or in the homogenate and / or in the lysate of the corresponding cells producing them, with lysates being particularly preferred.

[0057] In this context, suspension means a suspension of resting cells. These are harvested after cultivation (separated from the nutrient medium) and used as a paste or suspended in a suitable buffer system. In contrast to fermentative processes, which also work with whole cells, the resting cells can no longer grow due to the lack of carbon sources and nutrients; instead, they only serve to convert substrates (Lin & Tao, 2017). In this context, homogenate refers to a physically and / or chemically treated suspension (e.g., treated by pressure, lysozyme, or ultrasound), whereby the 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).

[0058] 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.

[0059] In another variant, the enzymes can be in powder form, in lyophilized or spray-dried form.

[0060] After separation of the enzymes, D-psicose is particularly preferably present in an aqueous solution, from which solid D-psicose can be obtained, for example, by spray drying (US 2019 / 0315790 Al; Kawakami et al., 2013; Kawakami et al., 2014).

[0061] Due to the high purity of the obtained solution, it is possible to concentrate the filtrate and obtain D-psicose in crystalline form or in the form of a syrup.

[0062] In a further preferred variant, the D-psicose is present in a syrup, which is prepared by concentrating the above-described filtrate or by dissolving crystalline D-psicose, which can be produced by the process according to the invention, in water. The syrup according to the invention preferably has a total solids content of about 50% to about 90% by weight. The D-psicose content in the syrup according to the invention is about 80% to about 99% by weight, based on the dry matter.

[0063] Accordingly, a further aspect of the present invention relates to a syrup comprising D-psicose, which can be produced by the process according to the invention. In a particularly preferred embodiment of the process, 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.

[0064] The epimerase used in the process may originate 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.

[0065] The enzymes used for the reduction of D-psicose and the oxidation of allitol belong to the group of oxidoreductases (see Table 1 for details).

[0066] The alcohol dehydrogenase (ADH) used for cofactor regeneration can originate from one of the groups EC 1.1.1.1 (NAD-dependent ADH) and EC 1.1.1.2 (NADP-dependent ADH).

[0067] The NAD(P)-dependent alcohol dehydrogenase for cofactor regeneration preferably comprises or consists of an amino acid sequence selected from the group consisting of: i) an amino acid sequence having an identity to SEQ ID No. 18 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 17 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 17.

[0068] Particularly suitable for cofactor regeneration in general is an alcohol dehydrogenase whose amino acid sequence is at least 80% identical to SEQ ID No. 18 or which is encoded by a nucleic acid that has at least 80% identity to SEQ ID No. 17 or binds under stringent conditions to a nucleic acid molecule with the nucleic acid sequence SEQ ID No. 17, or a functional fragment of this alcohol dehydrogenase. A "functional fragment" of the alcohol dehydrogenase comprises an N-terminally and / or C-terminally truncated variant of the alcohol dehydrogenase with the amino acid sequence SEQ ID No. 18, which has at least 50%, preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, enzyme activity compared to the non-truncated alcohol dehydrogenase.

[0069] SEQ ID No. 17: ATGAAAGCTGCAGTTGTGGAACAATTTAAAAAGCCGTTACAAGTGAAAGAAGTGGAAAAACCTAAGAT CTCATACGGGGAAGTATTAGTGCGCATCAAAGCGTGTGGGGTATGCCATACAGACTTGCATGCCGCAC ATGGCGACTGGCCTGTAAAGCCTAAACTGCCTCTCATTCCTGGCCATGAAGGCGTCGGTGTAATTGAAG AAGTAGGTCCTGGGGTAACACATTTAAAAGTTGGAGATCGCGTAGGTATCCCTTGGCTTTATTCGGCGT GCGGTCATTGTGACTATTGCTTAAGCGGACAAGAAACATTATGCGAACGTCAACAAAACGCTGGCTATT CCGTCGATGGTGGTTATGCTGAATATTGCCGTGCTGCAGCCGATTATGTCGTAAAAATTCCTGATAACTT ATCGTTTGAAGAAGCCGCTCCAATCTTTTGCGCTGGTGTAACAACATATAAAGCGCTCAAAGTAACAGG CGCAAAACCAGGTGAATGGGTAGCCATTTACGGTATCGGCGGGCTTGGACATGTCGCAGTCCAATACG CAAAGGCGATGGGGTTAAACGTCGTTGCTGTCGATTTAGGTGATGAAAAACTTGAGCTTGCTAAACAA CTTGGTGCAGATCTTGTCGTCAATCCGAAACATGATGATGCAGCACAATGGATAAAAGAAAGGTGGG CGGTGTGCATGCGACTGTCGTCACAGCTGTTTCAAAAGCCGCGTTCGAATCAGCCTACAAATCCATTCG TCGCGGTGGTGCTTGCGTACTCGTCGGATTACCGCCGGAAGAAATACCTATTCCAATTTTCGATACAGT ATTAAATGGAGTAAAAATTATTGGTTCTATCGTTGGTACGCGCAAAGACTTACAAGAGGCACTTCAATT TGCAGCAGAAGGAAAAGTAAAAACAATTGTCGAAGTGCAACCGCTTGAAAACATTAACGACGTATTCGATCGTATGTTAAAAGGGCAAATTAACGGCCGCGTCGTGTTAAAAGTAGATTAA

[0070] SEQ ID No. 18:

[0071] M KAAVVEQFKKPLQVKEVEKPKISYGEVLVRIKACGVCHTDLHAAHGDWPVKPKLPLIPGHEGVGVIEEV GPGVTHLKVGDRVGIPWLYSACGHCDYCLSGQETLCERQQNAGYSVDGGYAEYCRAAADYVVKIPDNLS FEEAAPIFCAGVTTYKALKVTGAKPGEWVAIYGIGGLGHVAVQYAKAMGLNVVAVDLGDEKLELAKQLG ADLVVNPKHDDAAQWIKEKVGGVHATVVTAVSKAAFESAYKSIRRGGACVLVGLPPEEIPIPIFDTVLNGV KIIGSIVGTRKDLQEALQFAAEGKVKTIVEVQPLENINDVFDRM LKGQINGRVVLKVD

[0072] The alcohol dehydrogenase for cofactor regeneration mentioned here preferably comprises an amino acid sequence that has an identity to SEQ ID No. 18 of at least 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%, and in particular 100%. The alcohol dehydrogenase for cofactor regeneration according to the invention particularly preferably comprises or consists of the amino acid sequence SEQ ID No. 18.

[0073] Alternatively, the alcohol dehydrogenase for cofactor regeneration preferably comprises an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 17 of at least 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%, and 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 SEQ ID No. 17.

[0074] 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 having an identity to SEQ ID No. 18 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 17 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 17, or a functional fragment thereof.

[0075] The alcohol dehydrogenases disclosed here can be used to regenerate NAD(P) + or NAD(P)H, ie for the reduction of NAD(P) +or for the oxidation of NAD(P)H, in a wide variety of enzymatic reactions. The alcohol dehydrogenases according to the invention are particularly preferred for the cofactor regeneration of NAD(P) + , which is formed during the reduction of D-psicose to allitol by an NAD(P)H-dependent oxidoreductase.

[0076] The cofactor NAD(P) produced during the reduction of D-psicose to allitol + is reduced to NAD(P)H using formate and a formate dehydrogenase with the formation of CO2 (cofactor regeneration).

[0077] Particularly preferably, a formate dehydrogenase is used which comprises or consists of the amino acid sequence SEQ ID No. 2, or a functional fragment of this formate dehydrogenase. The preferably used formate dehydrogenase is preferably encoded by the nucleic acid sequence SEQ ID No. 1. A "functional fragment" of the formate dehydrogenase comprises an N-terminally and / or C-terminally truncated variant of the formate dehydrogenase with the amino acid sequence SEQ ID No. 2, which has at least 50%, preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, enzyme activity compared to the non-truncated formate dehydrogenase.

[0078] SEQ ID NO. 1: ATGGCGAAAATACTTTGCGTTCTCTATGACGATCCGGTCGACGGCTACCCGAAGACCTATGCGCGCG

[0079] ACGACCTGCCGAAGATCGACCACTATCCGGGCGGGCAGACGCTGCCCACGCCCAAGGCGATCGACTT CACGCCGGGCGCGCTGCTCGGCTCGGTCTCCGGCGAGCTCGGCCTGCGCAAATACCTGGAAGCCAAC GGCCATACCTTCGTCGTCACCTCCGATAAGGACGGCCCGGATTCGGTGTTCGAGAGGGAACTCGTCG ACGCCGACGTGGTGATCTCGCAGCCCTTCTGGCCGGCCTATCTGACGCCCGAGCGCATCGCCAAGGC GAAGAACCTGAAGCTCGCGCTCACCGCCGGCATCGGCTCCGATCATGTCGATCTTCAGTCAGCTATCG ACCGTGGCATCACTGTGGCCGAAGTCACATATTGCAACTCGATCAGCGTCGCCGAGCACGTGGTGAT GATGATCCTCGGCCTGGTACGAAACTACATTCCCTCGCATGACTGGGCGCGCAAGGGCGGCTGGAAC ATAGCCGACTGCGTAGAGCACTCCTACGACCTCGAGGGCATGACCGTCGGCTCGGTGGCCGCCGGCC GCATCGGCCTCGCCGTGCTGCGCCGCCTCGCGCCGTTCGACGTGAAGCTGCACTATACCGACCGCCA CCGTCTGCCAGAAGCGGTCGAGAAGGAGCTGGGCCTCGTCTGGCACGATACCCGCGAGGACATGTA CCCGCATTGCGACGTGGTCACGCTCAACGTGCCGCTGCACCCCGAAACCGAGCACATGATCAATGAC GAGACGCTGAAGCTGTTCAAGCGCGGCGCCTATATCGTCAACACCGCCCGCGGCAAGCTCGCCGACC GCGACGCCATCGTCCGCGCGATCGAGAGCGGGCAGCTCGCGGGCTATGCCGGCGACGTGTGGTTCC CGCAGCCGGCTCCGAAGGACCACCCCTGGCGCACCATGAAGTGGGAAGGCATGACGCCGCACATCTCCGGCACCTCGCTCTCTGCCCAGGCGCGCTACGCGGCGGGCACGCGCGAGATCCTCGAATGCTTCTT CGAGGGCCGGCCGATCCGCGACGAGTACCTGATCGTGCAGGGCGGCGCGCTCGCGGCACCGGCGC GCATTCCTACTCGAAGGGCAATGCGACCGGCGGTTCGGAAGAGGCCGCGAAGTTCAAGAAGGCTGG CTGA

[0080] SEQ. ID Nr. 2:

[0081] MAKILCVLYDDPVDGYPKTYARDDLPKIDHYPGGQTLPTPKAIDFTPGALLGSVSGELGLRKYLEANGHTFV VTSDKDGPDSVFERELVDADVVISQPFWPAYLTPERIAKAKNLKLALTAGIGSDHVDLQSAIDRGITVAEVT YCNSISVAEHVVMM ILGLVRNYIPSHDWARKGGWNIADCVEHSYDLEGMTVGSVAAGRIGLAVLRRLAP FDVKLHYTDRHRLPEAVEKELGLVWHDTREDMYPHCDVVTLNVPLHPETEHM INDETLKLFKRGAYIVNT ARGKLADRDAIVRAIESGQLAGYAGDVWFPQPAPKDHPWRTMKWEGMTPHISGTSLSAQARYAAGTR EILECFFEGRPIRDEYLIVQGGALAGTGAHSYSKGNATGGSEEAAKFKKAG

[0082] According to a further 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 having an identity to SEQ ID No. 2 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 1 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 1, or a functional fragment thereof.

[0083] The formate dehydrogenase preferably comprises an amino acid sequence having an identity to SEQ ID NO: 2 of at least 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%, in particular 100%.

[0084] Alternatively, the formate dehydrogenase preferably comprises an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 1 of at least 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%, in particular 100%.

[0085] A further aspect of the present invention relates to the use of a formate dehydrogenase for cofactor regeneration 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 having an identity to SEQ ID No. 2 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 1 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 1.

[0086] The glucose dehydrogenase (GDH) used for cofactor regeneration can be selected from one of the groups EC 1.1.1.47 (glucose-l-dehydrogenase), EC 1.1.1.118 (glucose-l-dehydrogenase (NAD + )), EC 1.1.1.119 (glucose-l-dehydrogenase (NADP +)) or EC 1.1.1.360 (glucose / galactose-l-dehydrogenase).

[0087] The 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 having an identity to SEQ ID No. 20 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 19 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 19.

[0088] Particularly suitable for cofactor regeneration in general is a glucose dehydrogenase whose amino acid sequence is at least 80% identical to SEQ ID No. 20 or which is encoded by a nucleic acid that has at least 80% identity to SEQ ID No. 19 or binds under stringent conditions to a nucleic acid molecule with the nucleic acid sequence SEQ ID No. 19, or a functional fragment of this glucose dehydrogenase. A "functional fragment" of the glucose dehydrogenase comprises an N-terminally and / or C-terminally truncated variant of the glucose dehydrogenase with the amino acid sequence SEQ ID No. 18, which has at least 50%, preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, enzyme activity compared to the non-truncated glucose dehydrogenase.

[0089] SEQ ID No. 19:

[0090] ATGTATACAGATTTAAAAGATAAAGTAGTTGTAATTACAGGTGGATCAACAGGTTTAGGACGCGCCAA TGGCTGTTCGTTTCGGTCAAAGAAGCAAAAGTTTTTACTATTACAACATGAAAGAAGCT TTAGATGCGAAAAAAGAAGTAGAAGAGGCAGGACAAGCAATTCGATCGATCGATCCAGTCAGTCAGCT AAAGAAGAAGATGTTGTAAACCTTGTTCAAACAGCTATTAAAGAATTCGGTACATTAGACGTTATGAT TAATAACGCTGGTGTTGAAAACCCAGTTCCTTCATGAGTTATCTTTAGACAACTGGAATAAAGTTAT TGATACAAACTTAACAGGTGCATTCTTAGGAAGCCGTGAGTAACTTCATTCATTCATTGATTGAGGAATAAGTTAT TTAAAGGAAACGTTTAACATGTCTAGTGTTCATGAAATGATTCCTTGGCCATTATTTGTTCATTACG CAGCAAGTAAAGGCGGTATGAAACTAATGACGGAAACATTGGCTCTTGAATATGCGCCAAAAGGTAT CCGCGTAAATAACATTGGACCAGGTGCGATGAACACCAATTAGCCAGCATTGATTGAATTTGGCCAAAAGGTAT GTACAACGTGCAGACGTAGAAAGCATGATTCCAATGGGTTACATCGGTAAACCAGAAGAAGTAGCA GCAGTTGCAGCATTCTTAGCATCATCAAGCAAGCTATGTAACAGGTTACATTATTTGCTGATGG TGGTATGACGAAATACCCTTCTTTCCAAGCAGGAAGAGGCTAA

[0091] SEQ ID No. 20:

[0092] MYTDLKDKVVVITGGSTGLGRAMAVRFGQEEAKVVINYYNNEEEALDAKKEVEEAGGQAIIVQGDVTKEED VVNLVQTAIKEFGTLDVMINNAGVENPVPSHELSLDNWNKVIDTNLTGAFLGSREAIKYFVENDIKGNVIN MSSVHEMIPWPLFVHYAASKGGMKLMTETLALEYAPKGIRVNNIGPGAMNTPINAEKFADPVQRADVES MIPMGYIGKPEEVAAVAAFLASSQASYVTGITLFADGGMTKYPSFQAGRG

[0093] The glucose dehydrogenase for cofactor regeneration mentioned here preferably comprises an amino acid sequence that has an identity to SEQ ID No. 20 of at least 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%, and in particular 100%. The glucose dehydrogenase for cofactor regeneration according to the invention particularly preferably comprises or consists of the amino acid sequence SEQ ID No. 20.

[0094] Alternatively, the glucose dehydrogenase for cofactor regeneration preferably comprises an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 19 of at least 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%, and in particular 100%. Particularly preferably, the nucleic acid encoding the glucose dehydrogenase for cofactor regeneration according to the invention comprises or consists of the nucleic acid sequence SEQ ID No. 19.

[0095] 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: i) an amino acid sequence having an identity to SEQ ID No. 20 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 19 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 19, or a functional fragment thereof.

[0096] The NAD(P)H oxidase used for cofactor regeneration (see Figure 1) can originate from one of the 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.

[0097] A particularly preferably used H2O-forming NAD(P)H oxidase preferably comprises or consists of an amino acid sequence selected from the group consisting of: i) an amino acid sequence having an identity to SEQ ID No. 14 or SEQ ID No. 16 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 13 or SEQ ID No. 15 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 13 or SEQ ID No. 15, or a functional fragment thereof.

[0098] A "functional fragment" of this NAD(P)H oxidase comprises an N-terminally and / or C-terminally truncated variant of the NAD(P)H oxidase having 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%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, enzyme activity compared to the non-truncated NAD(P)H oxidase.

[0099] SEQ ID No. 13:

[0100] ATGAGCAAAATTGTTATCGTGGGTGCAAATCATGCAGGCACCGCAGCAATTAATACCATTCTGGATAATTA TGGCAGCGAAAATGAAGTGGTTGTGTTTGATCAGAATAGCAACATTAGCTTTCTGGGTTGTGGTATGGCAC TGTGGATTGGTAAACAAATTAGCGGTCCGCAGGGTCTGTTTTATGCAGATAAAGAAAGCCTGGAAGCAAA AGGTGCCAAAATCTATATGGAAAGTCCGGTTACCGCCATTGATTATGATGCAAAACGTGTTACCGCACTGG TTAATGGTCAAGAACATGTTGAAAGCTACGAGAAACTGATTCTGGCAACCGGTAGCACCCCGATTCTGCCT CCGATTAAAGGTGCAGCCATTAAAGAAGGTAGTCGCGATTTTGAAGCAACCCTGAAAAATCTGCAGTTCGT GAAACTGTATCAGAATGCCGAAGATGTGATTAACAAACTGCAGGATAAAAGCCAGAATCTGAATCGTATT GCAGTTGTTGGTGCAGGTTATATTGGTGTTGAACTGGCAGAAGCATTTAAACGTCTGGGTAAAGAAGTGA TTCTGATTGACGTTGTTGATACCTGTCTGGCAGGTTATTATGATCAGGATCTGAGCGAAATGATGCGTCAG AATCTGGAAGATCATGGTATCGAACTGGCATTTGGTGAAACCGTTAAAGCAATTGAAGGTGATGGTAAAG TGGAACGTATTGTTACCGATAAAGCAAGCCATGATGTGGATATGGTTATTCTGGCAGTTGGTTTTCGTCCG AATACAGCACTGGGTAATGCAAAACTGAAAACCTTTCGTAATGGTGCCTTTCTGGTGGATAAAAAACAAGA AACCAGCATCCCGGATGTTTATGCAATTGGTGATTGTGCAACCGTGTATGATAATGCCATTAACGACACCAACTATATTGCACTGGCAAGCAATGCACTGCGTAGCGGTATTGTTGCAGGTCATAATGCAGCCGGTCATAAA CTGGAAAGTCTGGGTGTTCAGGGTAGCAATGGTATTTCAATTTTTGGCCTGAATATGGTTAGCACCGGTCT GACCCAAGAAAAAGCCAAACGTTTTGGTTATAATCCGGAAGTTACCGCCTTTACCGATTTTCAGAAAGCCA GCTTTATCGAGCATGATAACTATCCGGTTACGCTGAAAATTGTGTATGACAAAGATAGCCGTCTGGTTCTG GGTGCACAGATGGCCAGCAAAGAAGATATGAGCATGGGTATTCACATGTTTAGCCTGGCCATTCAAGAGA AAGTTACCATTGAACGTCTGGCCCTGCTGGATTATTTCTTTCTGCCGCATTTTAATCAGCCGTACAACTATAT GACCAAAGCAGCACTGAAAGCCAAATAA

[0101] SEQ. ID Nr. 14:

[0102] MSKIVIVGANHAGTAAINTILDNYGSENEVVVFDQNSNISFLGCGMALWIGKQISGPQGLFYADKESLEAKGAKI

[0103] YM ESPVTAIDYDAKRVTALVNGQEHVESYEKLILATGSTPILPPIKGAAIKEGSRDFEATLKNLQFVKLYQNAEDVI

[0104] NKLQDKSQNLNRIAVVGAGYIGVELAEAFKRLGKEVILIDVVDTCLAGYYDQDLSEMM RQNLEDHGIELAFGET

[0105] VKAIEGDGKVERIVTDKASHDVDMVILAVGFRPNTALGNAKLKTFRNGAFLVDKKQETSIPDVYAIGDCATVYD

[0106] NAINDTNYIALASNALRSGIVAGHNAAGHKLESLGVQGSNGISIFGLNMVSTGLTQEKAKRFGYNPEVTAFTDF

[0107] QKASFIEHDNYPVTLKIVYDKDSRLVLGAQMASKEDMSMGIHM FSLAIQEKVTIERLALLDYFFLPHFNQPYNY MTKAALKAK

[0108] SEQ. ID No. 15:

[0109] ATGAAAGTAGTAGTAGTAGGCTGTACACATGCAGGAACAGCGGCAGTTAAGACGATTTTAAATGAACATC

[0110] CAGATGCATCAGTATCAGTATATGAGCGTAATGACAATGTCTCATTTCTATCTTGTGGGATTGCGTTGTATG

[0111] TTGGTGGAGTTGTGAAAGATCCTGCAGGTTTGTTTTATTCAAGTCCAGAAGAACTTGCATCAATGGGCGCG

[0112] AAAATTAACATGGAACACAATGTGAAAAATATAGATAATGAGAATAAGGTCGTAGTAATTGAGAATTTAAA

[0113] AACAGGCGAAACATTTGAAGAAAGCTATGATAAGTTGGTAATGACAACTGGATCATGGCCAATTATTCCTC

[0114] CAATTGATGGAATCAATAGTGAAAATATTCTTTTGTGTAAAAACTATAACCAAGCAAATGAAATTATTAAAG

[0115] AATCAAAAAATGCTAAAAAGATTGTCATTGTTGGTGGTGGCTATATTGGAATTGAATTAGTTGAGGCATTT

[0116] GCAGAATCTGGCAAGCAAGTGACGCTAGTTGATGGATTAGATCTGTATTTAAACAAATATTTAGTGCTGA

[0117] ATTCACTTCTGTTTTAGAGCATGATTTACAAGAAAGAGGCGTTACGCTAGCTTTAAACCAAACCGTCGAGAA

[0118] ATTTGTTGCCAATGAATCAGGTGCTGTGACAGCTGTGAAAACACCAGTTGGAGAATATGAGGCTGATTTAG

[0119] TTATTTTATGTGTTGGATTTAAACCAAATACTGATTTGTTGAAGGATAAAGTAGAGATGTTGCCAAATGGTG

[0120] CCATCGTAGTGGATGAATATATGAGAACAAGCGATGAAGCGATTTTTGCTGCTGGCGATAGTTGCGCGGTT

[0121] CATTATAATCCAACTGGAGGCTCTGCGTAATTCCGTTAGCTACAAATGCAGTTTAGATGGGAGCTTTAGTT

[0122] GGGAAAAAATTGTTTCTCCCAACAGTTAAATATCGTGGCACGCAAGCAACTTCTGGTTTATATTTTATTGGT

[0123] TTTAATATAGGTTCAACCGGATTGACTGAAAATAGCGCTCCTCATTTTGGCGTAGAGGTTCGTTCAGTAGTT

[0124] GTAGAAGATAATTATCGTCCAGAGTTTATGCCGCAACAGAGAAAGTAACGATGAAATTAGTTTATGAAGT

[0125] AGGAACGAATCGGATTGTTGGAGGTCAAATCATGTCAAAATATGATGTGACACAATCTGCCAATACGTTAT

[0126] CTTTATGTGTTCAAAAATAAAATGACGATTGAGGATTTGGCTTATGTAGATTTCTTCTCAACCTCACTTTGA TCGTCCTTGGAACTATTTAATATTTTAGCGCAAGCTGTTGAGCAAGAGCGTAAACTAGCAAAATAA

[0127] SEQ ID No. 16: MKVVVVGCTHAGTAAVKTILNEHPDASVVYERNDNVSFLSCGIALYVGGVVKDPAGLFYSSPEELASMGAKIN MECHANICS IVGGGYIGIELVEAFAESGKQVTLVDGLDRLNKYLDAEFTSVLEHDLQERGVTLALNQTVEKFVANESGAVTAVK TPVGEYEADLVILCVGFKPNTDLLKDKVEM LPNGAIVVDEYMRTSDEAIFAAGDSCAVHYNPTGGSAYIPLATN AVRMGALVGKNIVSPTVKYRGTQATSGLYLFGFNIGSTGLTENSAPHFGVEVRSVVVEDNYRPEFMPTTEKVT MKLVYEVGTNRIVGGQIMSKYDVTQSANTLSLCVQNKMTIEDLAYVDFFFQPHFDRPWNYLNILAQAQER KLAAV

[0128] The preferably used F O-forming NAD(P)H oxidase preferably comprises or consists of an amino acid sequence having an identity to SEQ ID No. 16 or SEQ ID No. 14 of at least 80%, even 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 F O-forming NAD(P)H oxidase comprises or consists of the amino acid sequence SEQ ID No. 16 or SEQ ID No. 14.

[0129] Alternatively, the F O-forming NAD(P)H oxidase preferably comprises an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 15 or SEQ ID No. 13 of at least 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%, in particular 100%. Particularly preferably, the nucleic acid encoding the F O-forming NAD(P)H oxidase comprises or consists of the nucleic acid sequence SEQ ID No. 15 or SEQ ID No. 13.

[0130] A further aspect of the present invention relates to the use of an F O-forming NAD(P)H oxidase for cofactor regeneration (NAD(P)H to NAD(P) +) which comprises or consists of an amino acid sequence selected from the group consisting of: i) an amino acid sequence having an identity to SEQ ID No. 16 or SEQ ID No. 14 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 15 or SEQ ID No. 13 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid which binds under stringent conditions to a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 15 or SEQ ID No. 13, or a functional fragment thereof.

[0131] The enzymatic strategy presented here in combination with cofactor regeneration enables a biocatalytic, environmentally friendly and highly efficient production process for the manufacture of D-psicose. The NAD(P)H-dependent oxidoreductase for the reduction of the first D-psicose to allitol preferably comprises or consists of an amino acid sequence selected from the group consisting of: i) an amino acid sequence having an identity to SEQ ID No. 4, SEQ ID No. 10 or SEQ ID No. 12 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ. ID No. 3, SEQ ID No. 9 or SEQ ID No. 11 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 3, SEQ ID No. 9 or SEQ ID No. 11.

[0132] Particularly suitable for reducing the first D-psicose to allitol or D-psicose to allitol in general is an oxidoreductase whose amino acid sequence is at least 80% identical to SEQ ID No. 4, SEQ ID No. 10 or SEQ ID No. 12 or which is encoded by a nucleic acid which has an identity to SEQ ID No. 3, SEQ ID No. 9 or SEQ ID No. 11 of at least 80% or which binds under stringent conditions to a nucleic acid molecule with the nucleic acid sequence SEQ ID No. 3, SEQ ID No. 9 or SEQ ID No. 11. This oxidoreductase can also surprisingly be used for the oxidation of allitol to D-psicose.

[0133] The NAD(P) +-dependent oxidoreductase for the formation of D-psicose from allitol preferably comprises or consists of an amino acid sequence which is selected from the group consisting of: i) an amino acid sequence which has an identity to SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10 or SEQ ID No. 12 of at least 80%, ii) an amino acid sequence which is encoded by a nucleic acid which has an identity to SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9 or SEQ ID No. 11 of at least 80%, and iii) an amino acid sequence which is encoded by a nucleic acid which binds under stringent conditions to a nucleic acid molecule having 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 binds.

[0134] SEQ ID No. 3:

[0135] ATGACACAGTCTCTTCAGGGCAAGATCGTCGCCATTACTGGCGCGGCTTCGGGCATTGGCCTCGAATGC GCCCGCTATCTCATCGAAGCTGGCGCGGTGGTCTATCTTCTGGACCGTGACGCCAAAACTCTCGAAGAC AAAACGGCAGAACTCGGCAGCCAGGCCCATGCGATCATCGTCGATCTCTTCGACTACAAAACCGTAGAT GCTGCGGTCGCGCAGATCGTTGAGGAGCAGGGCCGGATCGATGTTTTCCACGCCAACGCCGGCGCGTA TGTGGGCGGCAATGTCTGGGAAGGGGATCCCGATAGCTGGGATGCGATGCTGCACCTGAACATCAAT GCAGCTTTCCGCTCGGTCCGTGCCGTGCTGCCGCAGATGATGAAGCAGGAAAGTGGCGATATCGTCAT

[0136] GACCAGCTCGATCGCGGGCATGATCCCCATCATGGCCGAGCCGATCTACACGGCCTCGAAACATGCCG

[0137] TGCAGGCATTCGTACATACGGTGCGCCGACAGGTCGCGAAGTATGGAATTCGTGTGGGTGCAATCCAG

[0138] CCTGGTCCTGTAGTCACGCCTTTGCTGAAAGACTGGGATCAGGCCCGTCTTGAAGCCAACATCAAAGCG

[0139] GGTGCCCTGATGGAAGCCAAGGAAGTCGCCGAAGCCCTGATCTTCATCCTGACACGTTCAAAGGGTGT

[0140] CATGGTGCGGGATCTGGTCGTTCTGCCACATAACTTCGACGCCTAAGCTTAAGCGGCCGCACTCGAGCA

[0141] CCACCACCACCACCACTGAGATCCGGCTGCTAA

[0142] SEQ. ID Nr. 4:

[0143] MTQSLQGKIVAITGAASGIGLECARYUEAGAVVYLLDRDAKTLEDKTAELGSQAHAIIVDLFDYKTVDAAVA

[0144] QIVEEQGRIDVFHANAGAYVGGNVWEGDPDSWDAMLHLNINAAFRSVRAVLPQMMKQESGDIVMTSSI

[0145] AGMIPIMAEPIYTASKHAVQAFVHTVRRQVAKYGIRVGAIQPGPVVTPLLKDWDQARLEANIKAGALM EA

[0146] KEVAEALIFILTRSKGVMVRDLVVLPHNFDA

[0147] SEQ. ID Nr. 5:

[0148] ATGAGCACACCGGAAAATCTGAGCTTTGTGCTGCAGAAACCGTTTGATGTGAAATTTGAAGATCGTCCG

[0149] ATTCCGAAACTGAGCGATCCGTATAGCGTTAAAATTCAGGTGAAAAAAACCGGCATTTGCGGTAGTGA

[0150] TGTTCACTATTTCACCCATGGTGCAATTGGTGATTTTGTTGTTAAAGCACCGATGGTTCTGGGTCATGAA

[0151] AGCAGCGGTGTTGTTCTGGAAGTTGGTAGCGAAGTTAAAAGCCTGAAAGTTGGTGATCGTGTTGCAAT

[0152] GGAACCGGGTGTTCCGAGCCGTCATAGTGATGAGTATAAAAGCGGTCGTTATAATCTGTGTCCGCACA

[0153] TGGCATTTGCAGCAACCCCTCCGTATGATGGCACCCTGTGTAAATACTATATTCTGCCGGAAGATTTCTG

[0154] CGTTAAACTGCCGGAACATGTTAGCCTGGAAGAAGGTGCACTGGTTGAACCGCTGAGCGTTGCAGTTC

[0155] ATAGCAGCAAACTGGGTAACATTAAACCGGGTAGCCATGTTGCAATTTATGGTGCAGGTCCGGTTGGT

[0156] CTGCTGGTTGCAGCAGTTGCAAGCGCATTTGGTGCAGAAAGCGTTACCATTATTGATCTGGTTGAAAGC

[0157] CGTCTGAATCTGGCAAAAGAACTGGGTGCAACCGCAACCGTTCAGGTTGATTTTAAAGATACCCCGAA

[0158] AGAAAGCGCAGCAAAAGTTGTTGCAGCAAATAATGGCATTGCACCGGATGTTGTTATTGATGCAAGCG

[0159] GTGCAGAAGCAAGCATTAATTCAGCCATTAATGCAATTCGTCCGGGTGGCACCTATGTTCAGGTGGGTA

[0160] TGGGTAAACCGGATGTGAGCTTTCCGATTGCAACCCTGATTGGTAAAGAACTGACCGTTAAAGGTAGC

[0161] TTTCGTTATGGTTATGGTGATTATCCGCTGGCAGTTAGCCTGCTGGCAAGCGGTAAAGTTAATGTGAAA

[0162] AAACTGATCACCCATGAAGTGAAATTCGAGGATGCAGCAGAAGCATTTCAGCTGGTTCGTGATGGTAA

[0163] AGCCATTAAATGTATTATCAACGGTCCGGAATAA

[0164] SEQ ID Nr. 6:

[0165] MSTPENLSFVLQKPFDVKFEDRPIPKLSDPYSVKIQVKKTGICGSDVHYFTHGAIGDFVVKAPMVLGHESSGV

[0166] VLEVGSEVKSLKVGDRVAMEPGVPSRHSDEYKSGRYNLCPHMAFAATPPPYDGTLCKYYILPEDFCVKLPEHV SLEEGALVEPLSVAVHSSKLGNIKPGSHVAIYGAGPVGLLVAAVASAFGAESVTIIDLVESRLNLAKELGATAT

[0167] VQVDFKDTPKESAAKVVAANNGIAPDVVIDASGAEASINSAINAIRPGGGTYVQVGMGKPDVSFPIATLIGKE

[0168] LTVKGSFRYGYGDYPLAVSLLASGKVNVKKLITHEVKFEDAAEAFQLVRDGKAIKCIINGPE

[0169] SEQ ID No. 7:

[0170] ATGAATAACAACCTGCCGAAAACCATGAAAGCAGCAGTTATGCATGGCACCCGTGAAATTAGCATTGA

[0171] AACCCTGCCGATTCCGCAGATTGATGAAAATGAAGTTCTGATCAAAGTTATGGCCGTTGGTATTTGTGG

[0172] TAGCGATCTGCACTATTACACCCAGGGTCGTATTGGTAAATACAAAGTGGAAAAACCGTTTATCCTGGG

[0173] TCATGAATGTAGCGGTGAAGTTGTTGCAATTGGTAGCGCAGTTGAACGTTTTCGTGTTGGTGATCGTGT

[0174] TGCCGTTGAACCGGGTGTTACCTGTGGTCATTGTGAAGCATGTAAAGAGGGTCGTTATAATCTGTGTCC

[0175] GGATGTTCAGTTTCTGGCAACCCCTCCGGTTGATGGTGCATTTGTTCAGTATATCAAAATGCGCCAGGA

[0176] TTTCGTTTTTCTGATTCCGAATAGCCTGAGCTATGAAGATGCAGCACTGATTGAACCGTTTAGCGTGGG

[0177] TATTCATGCAGCAACCCGTACCAAACTGCAGCCTGGTAGCACCATTGCAATTATGGGTATGGGTCCGGT

[0178] TGGTCTGATGGCAGTTGCAGCAGCAAAAGCATTTGGTGCAAGCACCATTATTGCAACCGATCTGGAAC

[0179] CGCTGCGTCTGGAAGCAGCCAAACGTATGGGTGCAACCCATGTTATTAACATTCGTGAACAGGATCCG

[0180] CTGAACGAGATTAAAAACATTACCGAAAATGTGGGTGTTGATGTTGCATGGGAAACCGCAGGTAATCC

[0181] GAAAGCACTGCAGAGCAGCCTGAGCAGCATTCGTCGTGGTGGTAAACTGGCAATTGTTGGTCTGCCGA

[0182] GCCAGAGCGATATTCCGCTGGATGTTCCGTTTATTGCCGATAATGAAATCGATATCTATGGCATCTTTCG

[0183] CTATGCAAACACCTATCCGAAAGGCATCAAATTTCTGACCAGCGGTGCAATTGATACCAAAAATCTGGT

[0184] TACCGATCGTTATCCGCTGGCAGGTACACGTGAAGCAATGGAACGTGCACTGAATTTCAAAAACGAAT

[0185] GCCTGAAAATCATCGTGTATCCGAACGAATAA

[0186] SEQ. ID Nr. 8:

[0187] MNNNLPKTMKAAVM HGTREISIETLPIPQIDENEVLIKVMAVGICGSDLHYYTQGRIGKYKVEKPFILGHECS

[0188] GEVVAIGSAVERFRVGDRVAVEPGVTCGHCEACKEGRYNLCPDVQFLATPPVDGAFVQYIKM RQDFVFLIP

[0189] NSLSYEDAALIEPFSVGIHAATRTKLQPGSTIAIMGMGPVGLMAVAAAKAFGASTIIATDLEPLRLEAAKRMG

[0190] ATHVINIREQDPLNEIKNITENVGVDVAWETAGNPKALQSSLSSIRRGGKLAIVGLPSQSDIPLDVPFIADNEI

[0191] DIYGIFRYANTYPKGIKFLTSGAIDTKNLVTDRYPLAGTREAM ERALNFKNECLKIIVYPNE

[0192] SEQ ID Nr. 9:

[0193] ATGACCTCTCCTCTCCAGGGTAAGATAGCCGCCATCACGGGCGGGGCTTCGGGCATCGGCCTCGAATG

[0194] TGTCCGCCAGATCGCCGCAAGTGGTGCCACGGTTTATATTCTCGACCGCGACCATCAGGCGCTCGACAA

[0195] GGCGCGCGAAGAATTGGGCGAGCGCGTTCATACCATCGAGGTCGATCTCTTCCGTTACGAAACGGTCG

[0196] ATCGCGCCATCGAAACCATCGTGTCCGAACAAGGACGCATCGACATTCTCCATGTCAATGCGGGCGCGT

[0197] ATATCGGCGGCAATGTCTGGGAAGGCGATCCCGATAAATGGGACAAGATGCTGAATCTCAACATCAAC GCCGCCTTCCGTTCCGCCCGCGCCGTCATGCCCGCCATGATGAAGCAGAAAAGCGGCGATATCATCATG

[0198] ACAAGCTCGATCGCAGGCATCGTCCCGATCCCGGCGGAGCCGATCTACACGGCTTCCAAACATGCGGT GCAAGCCTTCGCTCACACCATACGCCGCCAGTTGGCCCCGTTCGGCATCCGCGTCGGCGCCATCCAGCC CGGCCCGGTCGTCACGCCCTTGCTCAATGATTGGGACCCCGAGCGCCTTAAAGCCAATATCGAGGCTG GCGCCATGATGCAGCCTTCTGACGTCGCCGAAGCCGTGGTTTTCATGCTGTCCCGCCGCAAGGGAACG GTAATCCGCGACTTGGTTCTGTTACCCCATTCTTTCGACGTCTAA

[0199] SEQ. ID Nr. 10:

[0200] MTSPLQGKIAAITGGASGIGLECVRQIAASGATVYILDRDHQALDKAREELGERVHTIEVDLFRYETVDRAIET IVSEQGRIDILHVNAGAYIGGNVWEGDPDKWDKM LNLNINAAFRSARAVM PAMIVIKQKSGDIIIVITSSIAGI VPIPAEPIYTASKHAVQAFAHTIRRQLAPFGIRVGAIQPGPVVTPLLNDWDPERLKANIEAGAM MQPSDVA

[0201] EAVVFMLSRRKGTVIRDLVLLPHSFDV

[0202] SEQ. ID Nr. 11:

[0203] ATGGCTATATCTCGAAAACAACGTAGCTGCAATTACAGGTGCCGCTTCAGGTATCGGTCTCGAATGT GCACGCACACTGATCAAAGCAGGCGCTAAAGTTGTCCTCATTGACCGAGCAGAAGATAGACTAAATCA ATTGGTCGCAGAATTAGGTGAAAATGCAATTCCATTAGTTATCGATTTAATGAAACCAGAACAAGTCGA

[0204] TGGCATGTTAGCGCGTATTATCGAAAAGGCAGGCAGATTAGATATCTTTCATGCTAATGCTGGAGCTTA CATTGGTGGGCCCGTAGCCGAAGGCGATCCCGATGTTTGGGATAAAGTCCTAAATTTAAATGTTAATGC CGCATTCCGCTGTGTTCGCGCAGTTCTACCACACTTTATCGCACAAAAGTCAGGCGATATTCTATTCACC AGCTCTATCGCTGGTATGGTTCCCGTAATTTGGGAGCCTATTTACACGGCATCAAAATTTGCGGTTCAA GCATTCGTTCATTCTACTCGCCGTCAGGTTTCTGAACACGGTTGTCCGTGTTGGTGCTGTATTACCTGGTC CTGTTGTTACTGCGTTATTAGATGATTGGCCAAAAGAAAAACTTGAAGAAGCTTTAGCTAACGGTAGTT

[0205] TAATGCAACCCATTGAAGTTGCTGAGGCTGTTCTATTCATGCTGACGCGTCCAAGAAATATTACAATTCG CGATTTAGTTATTTTACCCAATAGTGTTGACCTCTAA

[0206] SEQ ID No. 12:

[0207] MAISLENNVAAITGAAASGIGLECARTLIKAGAKVVLIDRAEDRLNQLVAELGENAIPLVIDLMKPEQVDGMLA RIIEKAGRLDIFHANAGAYIGGPVAEGDPDVWDKVLNLNVNAAFRCVRAVLPHFIAQKSGDILFTSSIAGMV PVIWEPIYTASKFAVQAFVHSTRRQVSEHGVRVGAVLPGPPVVTALLDDWPKEKLEEALANGSLMQPIEVAE

[0208] AVLFMLTRPRNITIRDLVILPNSVDL

[0209] The oxidoreductases mentioned here for the reduction of D-psicose to allitol and / or for the oxidation of allitol to D-psicose preferably comprise an amino acid sequence which has an identity to SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10 or SEQ ID No. 12 of at least 80%, even more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, in particular 100%. The oxidoreductase according to the invention for the reduction of D-psicose to allitol and / or for the oxidation of allitol to D-psicose particularly preferably comprises the amino acid sequence SEQ ID No. 4, SEQ ID No. 10 or SEQ. ID No. 12 or consists of this, the oxidoreductase for the oxidation of allitol to D-psicose comprises or consists of one of the amino acid sequences SEQ ID No. 6 or SEQ ID No. 8.

[0210] Alternatively, the oxidoreductases for reducing D-psicose to allitol and / or for oxidizing allitol to D-psicose preferably comprise an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9 or SEQ ID No. 11 of at least 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%, in particular 100%. Particularly preferably, the nucleic acid which encodes the oxidoreductase according to the invention for the reduction of D-psicose to allitol and / or for the oxidation of allitol to D-psicose comprises or consists of the nucleic acid sequence SEQ ID No. 3, SEQ ID No. 9 or SEQ ID No. 11, and the oxidoreductase which encodes the oxidoreductase for the oxidation of allitol to D-psicose comprises or consists of the nucleic acid sequence SEQ ID No. 5 or SEQ ID No. 7.

[0211] The term "identity," as used herein, refers to the percentage of identical nucleotide or amino acid matches between at least two nucleotide or amino acid sequences aligned using a standardized algorithm. Such an algorithm can, in a standardized and reproducible manner, insert gaps into the compared sequences to optimize the alignment between two sequences, thus achieving a more meaningful comparison of the two sequences.

[0212] The percentage identity between sequences can be determined using one or more computer algorithms or programs known in the 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 various programs, including a tool called "BLAST 2 Sequences," which is used for the direct pairwise comparison of two nucleotide or amino acid sequences. "BLAST 2 Sequences" can also be accessed and used interactively via the NCBI World Wide Web. The blastn program (for nucleotide sequences) uses 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.

[0213] Alternatively, the oxidoreductases for reducing D-psicose to allitol and / or for oxidizing allitol to D-psicose preferably comprise an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule having 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 not non-specific hybrids, are formed. For example, the 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 may be performed by conventionally known methods, such as those described by J. Sambrook et al.in Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory (1989).

[0214] One aspect of the present invention relates to the use of an oxidoreductase for the reduction of D-psicose to allitol and / or 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 having an identity to SEQ ID No. 4, SEQ ID No. 10 or SEQ ID No. 12 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 3, SEQ ID No. 9 or SEQ ID No. 11 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 3, SEQ ID No. 9 or SEQ ID No. 11.

[0215] 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 having an identity to SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10 or SEQ ID No. 12 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9 or SEQ ID No. 11 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid which binds under stringent conditions to a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 3, SEQ ID No. ID No. 5, SEQ ID No. 7, SEQ ID No. 9 or SEQ ID No. 11.

[0216] Depending on the reaction (reduction or oxidation), the oxidoreductases according to the invention require corresponding cofactors, as mentioned above.

[0217] Materials

[0218] 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, IPTG (isopropyl-ß-D-thiogalactopyranoside) were purchased from Sigma-Aldrich, potassium dihydrogen phosphate, di-potassium hydrogen phosphate, 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.

[0219] Production of enzymes & preparation of lysates

[0220] General information on the expression of recombinant enzymes in E. coli

[0221] 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 codon usage of E. coli, as a template, together with specific oligonucleotides additionally carrying recognition sequences for restriction endonucleases. The gene fragment encoding the target enzyme was isolated from the reaction mixture. After nucleic acid digestion 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 E. coli cells (ToplOF), and the resulting colonies were used for plasmid isolation and restriction analysis.

[0222] The result of the cloning step was verified by restriction enzyme digestion and DNA sequencing. The resulting construct carries the target gene under the IPTG-inducible T5 promoter.

[0223] For overexpression of the enzyme in E. coli, the resulting expression plasmid was transformed into the competent expression cells RB791. After 24 h of incubation at 37 °C, the resulting colonies were inoculated into LB medium for expression assays.

[0224] The next day, expression cultures with an optical density (OD550) of 0.02 were inoculated and shaken at 37°C until an OD550 of 0.3 was reached. The temperature was then lowered to 25°C, and the cultures were induced with 0.1 mM IPTG upon reaching an OD550 of 0.5. After 22 h, the cultures were harvested (separated from the medium by centrifugation in the form of a cell pellet) and analyzed for expression of the recombinant enzyme using SDS gel electrophoresis and an activity determination (use in a USE test or optical enzymatic assay).

[0225] Preparation of cell lysates using sonifier disruption

[0226] To prepare a cell suspension, the cell pellet prepared according to the above procedure was weighed into a suitable container and mixed with buffer and lysozyme (final concentration 0.5 mg / ml) (e.g., triethanolamine (TEA) - HCl) and dissolved with stirring. The mass fraction of biomass is typically 20%, with the remainder being buffer.

[0227] 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).

[0228] The resulting homogenate was centrifuged for 10 min at 4 °C and 16000 rpm (Eppendorf Centrifuge 5417R) to separate the insoluble cell fragments and obtain the lysate.

[0229] Table 1. Enzyme classes and donor organisms for the enzymes used in the examples (SDR = oxidoreductase from the short-chain dehydrogenase / reductase family).

[0230] Analytical methods

[0231] High Performance Liquid Chromatography

[0232] An Agilent HPLC 1260 Infinity II Series system was used to quantify D-psicose, D-fructose, D-glucose, and allitol using HPLC (high-performance liquid chromatography). Detection was performed using a refractive index detector (RI detection). A Phenomenex Rezex RPM monosaccharide Pb+2 (8%) column with a corresponding precolumn was used for the measurement, eluted isocratically with ultrapure water.

[0233] High Performance Anion Exchange Chromatography

[0234] 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 a Dionex AERS 500 electrolytically regenerated suppressor in external water mode. A Dionex IonPac AS11-HC-4pm column with a corresponding precolumn and a NaOH gradient was used to separate the analytes. The mobile phase was additionally pretreated with a Dionex ATC Anion Trap Column.

[0235] Determination of enzyme activities (optical-enzymatic assay) Enzyme activities in the lysates were determined 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 via the change in absorbance. The 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 was placed in a cuvette (Greiner bio-one semi-micro cuvette made of polystyrene), and the desired pH value 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. The measurements were carried out at 25 °C as standard. The extinction coefficient of NADH / NADPH at 340 nm (E = 6220 L mol 1 cm 1 ), 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). 1 U represents 1 pmol of substrate turnover per minute (1 U = 1 pmol / min = l.67-10 _8 cat).

[0236] 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 methods described above.

[0237] Example 1

[0238] Production of D-psicose from D-fructose - cofactor regeneration with ADH and 2-propanol

[0239] The reaction was carried out in a Labfors 5 benchtop bioreactor (Infors AG). A glass reactor (volume 3.4 l) with a stirrer and pH electrode was used as the vessel. pH control was achieved by adding 1M NaOH or 1M H2SO4.

[0240] 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 brought to 35 °C while stirring.

[0241] To start the reaction, 25 ml of D-psicose-3-epimerase lysate was added. Then, 25 ml of SDR I lysate, 4 kU of alcohol dehydrogenase lysate, 10 ml of 10 mM NAD + -solution and 40 ml of 2-propanol.

[0242] During operation, samples were continuously taken from the reactor solution and analyzed as follows: 100 μl of the reactor solution was mixed with 200 μl of methanol and incubated in an Eppendorf Thermomixer at 60 °C and 1200 rpm for 15 min. The sample was briefly centrifuged, mixed with 700 μl of deionized water, vortexed, and then centrifuged for 5 min at max. 1 g. 200 μl of the supernatant was transferred to an HPLC vial with an insert and analyzed by HPLC (RI detection).

[0243] After 23 h of running time, 15 ml of 2-propanol was added, after 50 h 20 ml and after 77 h 15 ml.

[0244] After 74 h, 5 ml of D-psicose-3-epimerase lysate, after 77 h, 15 ml of SDR I lysate and 2.4 kJ of alcohol dehydrogenase lysate were added.

[0245] After 97 h, 94% of the D-fructose (50 g / l) was converted to allitol.

[0246] The entire reactor contents were then heated to 70 °C for 60 min (deactivation of D-psicose-3-epimerase) and after cooling to 24 °C, 25 ml of xylitol dehydrogenase lysate, 10 kU NADH oxidase lysate and 10 ml of a 10 mM NAD + -solution added.

[0247] Allitol was completely oxidized to D-psicose within 3 h. The reactor contents were heated to 70 °C, the pH was adjusted to 4, and stirred for 30 min at 70 °C. The enzymes were filtered off through a glass frit (P3).

[0248] In this way, 94% of the D-fructose was converted to D-psicose. D-sorbitol could not be detected.

[0249] The filtrate was concentrated to a syrup with a D-psicose concentration of 520 g / l using a rotary evaporator, with any remaining acetone and 2-propanol also being removed.

[0250] Example 1 shows that epimerase can be denatured by heat (in a one-pot process) and that the resulting precipitate does not interfere with the further reaction.

[0251] Example 2

[0252] Production of D-psicose from D-fructose - cofactor regeneration with GDH and D-glucose

[0253] The reaction was carried out in a Multifors benchtop bioreactor (Infors AG). A glass reactor (volume 1 L) with an attached stirrer and pH electrode was used as the vessel. pH was controlled by adding 1 / 4 NaOH or 1 / 4 H2SO4. Initially, 17.5 g of D-fructose and 17.5 g of D-glucose (final concentration 50 g / L each), 217.8 ml of deionized water, and 26.5 ml of a 500 mM potassium phosphate buffer (pH 7.5) were placed in the reactor and heated to 35 °C while stirring.

[0254] To start the reaction, 17.5 ml of D-psicose-3-epimerase lysate was added. Then, 24.5 ml of SDR I lysate, 0.4 kJ of glucose dehydrogenase lysate and 3.5 ml of 10 mM NAD + -solution introduced.

[0255] During operation, samples were continuously taken from the reactor solution and analyzed as follows: 100 μl of the reactor solution was mixed with 200 μl of methanol and incubated in an Eppendorf Thermomixer at 60 °C and 1200 rpm for 15 min. The sample was briefly centrifuged, mixed with 700 μl of deionized water, vortexed, and then centrifuged for 5 min at max. 1 g. 200 μl of the supernatant was transferred to an HPLC vial with insert and analyzed by HPLC (RI detection). For HPAEC measurements (conductivity detection), the clear supernatant was diluted 1:250.

[0256] After 16 h, only allitol and D-gluconate could be detected in the reactor.

[0257] The entire reactor contents were then heated to 70 °C for 60 min (deactivation of D-psicose-3-epimerase) and after cooling to 30 °C, 17.5 ml of xylitol dehydrogenase lysate, 7 kU NADH oxidase lysate and 3.5 ml of a 10 mM NAD +-solution added.

[0258] Allitol was completely oxidized to D-psicose within 27 h.

[0259] In this way, 17.5 g of D-fructose was 100% oxidized to D-psicose (17.8 g in solution). D-sorbitol and D-fructose could not be detected in the resulting solution.

[0260] The reactor contents were heated to 70 °C, the pH was adjusted to 4, and stirred at 70 °C for 30 min. The enzymes were filtered through a glass frit (P3).

[0261] Example 3

[0262] Production of D-psicose from D-fructose - cofactor regeneration with FDH and sodium formate

[0263] The reaction was carried out in a Multifors benchtop bioreactor (Infors AG). A glass reactor (volume 1 L) with a stirrer and pH electrode was used as the vessel. pH control was achieved by adding 5M NaOH or 6M H2SO4. Initially, 70 ml of a D-fructose solution (500 g / L), 26.3 ml of deionized water, and 43.8 ml of 8 M sodium formate were placed in the reactor and heated to 37 °C while stirring.

[0264] To start the reaction, 25 ml of D-psicose-3-epimerase lysate were added. Then, 35 ml of SDR I lysate, 7 kJ of formate dehydrogenase lysate and 17.5 ml of 10 mM NAD + -solution introduced.

[0265] During operation, samples were continuously taken from the reactor solution and analyzed as follows: 100 μl of the reactor solution was mixed with 200 μl of methanol and incubated in an Eppendorf Thermomixer at 60 °C and 1200 rpm for 15 min. The sample was briefly centrifuged, mixed with 700 μl of deionized water, vortexed, and then centrifuged for 5 min at max. 1 g. 200 μl of the supernatant was transferred to an HPLC vial with an insert and analyzed by HPLC (RI detection).

[0266] After 40 h, 95% of the D-fructose (100 g / l) was converted to allitol.

[0267] The entire reactor contents were then heated to 70 °C for 60 min (deactivation of D-psicose-3-epimerase) and after cooling to 24 °C, 25 ml of xylitol dehydrogenase lysate, 10 kJ of NADH oxidase lysate and 10 ml of a 10 mM NAD + -solution added.

[0268] Allitol was completely oxidized to D-psicose within 4 h. The reactor contents were heated to 70 °C, the pH was adjusted to 4, and stirred for 30 min at 70 °C. The enzymes were filtered off through a glass frit (P3).

[0269] 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.

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Claims

Patent claims 1. A process for the preparation of an aqueous solution containing D-psicose, in which a first D-psicose is formed from D-fructose, which is dissolved 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 corresponding NAD(P)H-dependent oxidoreductase in vitro and, after deactivation and / or ultrafiltration of the epimerase, to form D-psicose with a corresponding NAD(P) + -dependent oxidoreductase, 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 which is selected from the group consisting of: i) an amino acid sequence which has an identity to SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10 or SEQ ID No. 12 of at least 80%, ii) an amino acid sequence which is encoded by a nucleic acid which has an identity to SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9 or SEQ ID No. 11 of at least 80%, and iii) an amino acid sequence which is encoded by a nucleic acid which binds under stringent conditions to a nucleic acid molecule having 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.

3. A process according to claim 1 or 2, characterized in that the oxidized cofactor NAD(P) produced by the reduction +by an alcohol dehydrogenase and a secondary alcohol to form a ketone.

4. Process according to claim 3, characterized in that the secondary alcohol is D-glucose or 2-propanol.

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

6. Method according to one of claims 1 to 5, characterized in that the enzymes are present as a lysate of the corresponding cells producing them.

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

11.

8. The method according to any one of claims 3 to 8, 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 having an identity to SEQ ID No. 18 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 17 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule having the nucleic acid sequence SEQ ID No.

17.

9. Process according to one of claims 1 to 8, characterized in that the oxidized cofactor NAD(P) produced by the reduction + by means of glucose dehydrogenase and D-glucose to form D-gluconate.

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

19.

11. A process according to any one of claims 1 to 10, characterized in that the oxidized cofactor NAD(P) produced by the reduction + by means of a formate dehydrogenase and formate to form CO2.

12. The method according to claim 11, characterized in that the formate dehydrogenase comprises or consists of an amino acid sequence which is selected from the group consisting of: i) an amino acid sequence which has an identity to SEQ ID No. 2 of at least 80%, ii) an amino acid sequence which is encoded by a nucleic acid which has an identity to SEQ ID No. 1 of at least 80%, and iii) an amino acid sequence which is encoded by a nucleic acid which binds under stringent conditions to a nucleic acid molecule having the nucleic acid sequence SEQ ID No.

1.

13. Use of an aqueous solution producible by a process according to any one of claims 1 to 12 for producing a syrup containing D-psicose.

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

15. 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 having an identity to SEQ ID No. 18 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 17 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule having the nucleic acid sequence SEQ ID No.

17.

16. 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: i) an amino acid sequence having an identity to SEQ ID No. 20 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 19 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule having the nucleic acid sequence SEQ ID No.

19.

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

1.

18. Use of an F O-forming NAD(P)H oxidase for cofactor regeneration, which comprises or consists of an amino acid sequence selected from the group consisting of: i) an amino acid sequence having an identity to SEQ ID No. 14 or SEQ ID No. 16 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 13 or SEQ ID No. 15 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 13 or SEQ ID No. 15.

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