Method for producing an aqueous solution containing l-psicose

Abstract

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

Description

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

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

[0003] Background of the invention p-Psicose

[0004] 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).

[0005] 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).

[0006] 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).

[0007] 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).

[0008] Due to its low natural occurrence, D-psicose is largely produced synthetically using biotechnology.

[0009] 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).

[0010] 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).

[0011] 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.

[0012] The unfavorable equilibrium of epimerization of D-fructose to D-psicose can, for example, be favorably influenced by downstream redox reactions. By combining a DTE with a 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).

[0013] 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.

[0014] The theoretical papers by Hold et al. (2009) and Siedentop et al. (2021) address the optimization of enzyme cascades. They describe how 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, although success cannot be predicted. A specific synthesis of L-psicose is not mentioned.

[0015] 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:

[0016] (1) Cells that contain the enzymes inside them are more easily accessible than the enzymes themselves, the purification of which is often laborious,

[0017] (2) the interior of the cells provides a suitable microenvironment for the enzymes and also allows cofactor regeneration (NAD(P) + / NAD(P)H),

[0018] (3) cell walls and membranes protect the enzymes against the environment of the reaction medium, and (4) the co-localization of multiple enzymes within the cell favors local enzyme concentrations and reduces the diffusion of intermediates in cascade reactions.

[0019] 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.

[0020] 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.

[0021] 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 oxidized to D-psicose (450 mM) within 24 h at pH 7.

[0022] L-Psicose

[0023] Unlike its enantiomer D-psicose, L-psicose does not occur naturally. L-psicose serves as a starting material for the production of L-fructose (Itoh & Izumori, 1996), L-tagatose (Rao et al., 2008), and L-talitol (Sasahara & Izumori, 2005). A study with mice demonstrated the antiviral activity of L-psicose against herpes simplex virus 1, which causes keratitis (corneal inflammation) (Muniruzzaman et al., 2016).

[0024] Essentially, three different biotechnological routes are known for the production of L-psicose. The aldol reaction of L-glyceraldehyde and dihydroxyacetone phosphate (DHAP), catalyzed by a fructose-l,6-diphosphate aldolase or a tagatose-l,6-diphosphate aldolase, followed by removal of the terminal phosphate group, yields a mixture of L-psicose and L-sorbose. The reaction cascade can be carried out both in vitro and fermentatively in an engineered Corynebacterium glutamicum strain (Yang et al., 2015). Yang et al. (2016) further developed the reaction cascade so that glycerol can also be used as a starting material for the fermentative production of L-psicose. The core of the cascade is also the aldol reaction of L-glyceraldehyde and DHAP.

[0025] The route by Wen et al. (2015) also starts with L-glyceraldehyde and dihydroxyacetone phosphate (DHAP). These are first converted with a rhamnulose bisphosphate aldolase to L-fructose-l-phosphate, which, after dephosphorylation, yields L-fructose. This is then isomerized with a DTE to L-psicose, with the equilibrium shifting toward the product side through phosphorylation using fructokinase (requires adenosine triphosphate (ATP) as a cofactor). After removal of the phosphate group, L-psicose is obtained.

[0026] The second route is based on the use of transketolase (EC 2.2.1.1), a thiamine-dependent enzyme that promotes the reaction between a donor with an a-hydroxy carbonyl group and a C n -aldehyde acceptor, whereby COj elimination leads to a C n+2-Ketose is formed. For example, L-psicose can be produced by reacting L-erythrose with hydroxypyruvate using a thermostable transketolase mutant from Geobacillus stearothermophilus, but thiamine diphosphate is required as a cofactor (Lorilliere et al., 2019).

[0027] The third route involves the microbial oxidation of allitol to L-psicose. Using Gluconobacter frateurii IFO 3254, 100 g / l of allitol can be oxidized to 98% L-psicose (Takeshita et al., 1996). Oxidation by the same organism is also described in JP4761424B2 and JP3711296B2.

[0028] This is where the object of the present invention comes in. The invention aims to provide an efficient and environmentally friendly process for the production of aqueous solutions containing L-psicose with high conversion, which can be carried out in particular in a one-pot process.

[0029] Detailed description of the invention

[0030] 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, to form L-psicose with a corresponding NAD(P) + -dependent oxidoreductase, after which the deactivated epimerase and the oxidoreductases are removed.

[0031] The method according to the invention is shown schematically in the attached figure.

[0032] Surprisingly, it has been shown that the objectives set out in the invention can be achieved if the reaction is not carried out fermentatively, but the enzymes are contained as such in the aqueous solution, i.e. if the reaction is carried out in vitro.

[0033] A preferred variant of the method according to the invention is characterized in that the oxidoreductase for the formation of L-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. 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.

[0034] SEQ ID No. 1:

[0035] ATGTCTCAAGCTATCTCAGCAATCAACGAAAGAGTTGGCCCATTGCCCACTAAAGCACCCCAACTTTCAAA

[0036] AAACGTATTAGACCTCTTTTCCTTGAAAGGCAAGGTAGCATCGGTGACAGGTTCATCGAAGGGAATAGG

[0037] CTTTGCTGTCGCAGAAGCATACGCTCAAGCAGGGGCTGATGTTGCTTTGTGGTACAATTCAAGTCCTGTC GAGGAT AAGGTAAAATACTTGAAAGAGACCTACGGCGTGAAAGTCAAAGCCTACAAGTGTAACGTGGG AAACAGCGAGGAGGTTGAGAAAATAGTAGACCAAATTGCAGAGGACTTCGGCACAATTGACGTTTTTGT TGCTAATGCGGGCATTGCTTGGAGCGAAGGTAGATCTCTCGAGGTTAAGGGTTACGACGCTTGGCAAAA AATTGTCGACTACGACTTGAGCAGTGTATACTACTGTGCTAAGGCAGTGGGTAAGATCTTCCAAGAAAAG GGCTCCGGTTCGTTAATCCTCACCGCATCCATGTCCGGACACATTGTGAATGTTCCTCAAATGCAGGCACC ATACAATGCGGCCAAGGCGGCTGTGATCCATCTAGGAAAATCTTTGGCTGTCGAATGGGCTCCATTTGCC AGGGTCAACACCGTTTCTCCTGGCTATATTGCCACTGATATTGGTGAATTTGTGCTGCCCGATGAGAAGA AAAAGTGGTGGTCATTGACACCCTTGGGTAGAGAAGGTCTTCCTCAAGAACTCACTGGTGCATACTTGTA TTTTGCTTCTAACGCATCGACCTACACTACTGGGGCTGACTTGGTCGTAGACGGTGGCTACACTGTACCAT AA

[0038] SEQ ID NO. 2: MSQAISAINERVGPLPTKAPQLSKNVLDLFSLKGKVASVTGSSKGIGFAVAEAYAQAGADVALWYNSSPVEDK VKYLKETYGVKVKAYKCNVGNSEEVEKIVDQIAEDFGTIDVFVANAGIAWSEGRSLEVKGYDAWQKIVDYDLS SVYYCAKAVGKIFQEKGSGSLILTASMSGHIVNVPQMQAPYNAAKAAVIHLGKSLAVEWAPFARVNTVSPGYI ATDIGEFVLPDEKKKWWSLTPLGREGLPQELTGAYLYFASNASTYTTGADLVVDGGYTVP

[0039] 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. The use of such a system allows the recycling of 2-propanol without the emission of climate-damaging CO2 and also avoids large amounts of waste (such as unreacted sodium formate in the FDH regeneration system).

[0040] 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).

[0041] 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.). In addition, the use of glucose dehydrogenase makes it possible to use a mixture comprising D-fructose and D-glucose as a substrate for the production of allitol without additional D-glucose being added to the reaction mixture and without D-glucose being isomerized beforehand into 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 which is derived from Priestia megaterium and which comprises an amino acid sequence which is available under the NCBI accession number M DQ0804260.1.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.

[0042] 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.

[0043] This allows for optimization of the enzyme ratios to each other and is thus independent of the expression level in the overall construct compared to Wang et al. (2023).

[0044] 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. Especially in the case of D-psicose, without appropriate treatment, a large portion of the D-psicose produced by oxidation would be converted back to D-fructose by the epimerase.

[0045] 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 or glucose dehydrogenase and in the case of the oxidation reactions (second step) with a HjO-forming NAD(P)H oxidase.

[0046] The most preferred concentration of D-fructose is 50 - 250 g / l.

[0047] 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.

[0048] The particularly preferred pH range for both steps is between 7 and 8.5.

[0049] 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 forming them, with lysates being particularly preferred.

[0050] 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).

[0051] 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.

[0052] In another variant, the enzymes can be in powder form, in lyophilized or spray-dried form.

[0053] After separation of the enzymes, L-psicose is preferably present in an aqueous solution, from which solid L-psicose can be obtained, for example, by spray drying (see for D-psicose: US 2019 / 0315790 Al; Kawakami et al., 2013; Kawakami et al., 2014).

[0054] Due to the high purity of the obtained solution, it is possible to concentrate the filtrate and obtain L-psicose in crystalline form or in the form of a syrup.

[0055] In a further preferred variant, the L-psicose is present in a syrup, wherein the syrup is prepared by concentrating the above-described filtrate or by dissolving crystalline L-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 L-psicose content in the syrup according to the invention is about 80% to about 99% by weight, based on the dry matter.

[0056] Accordingly, a further aspect of the present invention relates to a syrup comprising L-psicose, which can be produced by the process according to the invention.

[0057] In a particularly preferred embodiment of the process, only enzymes from the enzyme groups epimerases and oxidoreductases are used for the conversion of the starting material, with one or more of these enzymes being selected from each of these groups.

[0058] The epimerase used in the process can 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), with the former being particularly preferred. 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).

[0059] 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).

[0060] 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. 6 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 5 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. 5.

[0061] Particularly suitable for cofactor regeneration in general is an alcohol dehydrogenase whose amino acid sequence is at least 80% identical to SEQ ID No. 6 or which is encoded by a nucleic acid which has an identity to SEQ ID No. 5 of at least 80% or which binds under stringent conditions to a nucleic acid molecule with the nucleic acid sequence SEQ ID No. 5.

[0062] SEQ ID Nr. 5:

[0063] ATGAAAGCTGCAGTTGTGGAACAATTTAAAAAGCCGTTACAAGTGAAAGAAGTGGAAAAACCTAAGAT CTCATACGGGGAAGTATTAGTGCGCATCAAAGCGTGTGGGGTATGCCATACAGACTTGCATGCCGCAC ATGGCGACTGGCCTGTAAAGCCTAAACTGCCTCTCATTCCTGGCCATGAAGGCGTCGGTGTAATTGAAG AAGTAGGTCCTGGGGTAACACATTTAAAAGTTGGAGATCGCGTAGGTATCCCTTGGCTTTATTCGGCGT GCGGTCATTGTGACTATTGCTTAAGCGGACAAGAAACATTATGCGAACGTCAACAAAACGCTGGCTATT CCGTCGATGGTGGTTATGCTGAATATTGCCGTGCTGCAGCCGATTATGTCGTAAAAATTCCTGATAACTT ATCGTTTGAAGAAGCCGCTCCAATCTTTTGCGCTGGTGTAACAACATATAAAGCGCTCAAAGTAACAGG CGCAAAACCAGGTGAATGGGTAGCCATTTACGGTATCGGCGGGCTTGGACATGTCGCAGTCCAATACG CAAAGGCGATGGGGTTAAACGTCGTTGCTGTCGATTTAGGTGATGAAAAACTTGAGCTTGCTAAACAA CTTGGTGCAGATCTTGTCGTCAATCCGAAACATGATGATGCAGCACAATGGATAAAAGAAAAAGTGGG CGGTGTGCATGCGACTGTCGTCACAGCTGTTTCAAAAGCCGCGTTCGAATCAGCCTACAAATCCATTCG TCGCGGTGGTGCTTGCGTACTCGTCGGATTACCGCCGGAAGAAATACCTATTCCAATTTTCGATACAGT ATTAAATGGAGTAAAAATTATTGGTTCTATCGTTGGTACGCGCAAAGACTTACAAGAGGCACTTCAATT TGCAGCAGAAGGAAAAGTAAAAACAATTGTCGAAGTGCAACCGCTTGAAAACATTAACGACGTATTCGATCGTATGTTAAAAGGGCAAATTAACGGCCGCGTCGTGTTAAAAGTAGATTAA

[0064] SEQ ID No. 6:

[0065] M KAAVVEQFKKPLQVKEVEKPKISYGEVLVRIKACGVCHTDLHAAHGDWPVKPKLPLIPGHEGVGVIEEV GPGVTHLKVGDRVGIPWLYSACGHCDYCLSGQETLCERQQNAGYSVDGGYAEYCRAAADYVVKIPDNLS FEEAAPIFCAGVTTYKALKVTGAKPGEWVAIYGIGGLGHVAVQYAKAMGLNVVAVDLGDEKLELAKQLG ADLVVNPKHDDAAQWIKEKVGGVHATVVTAVSKAAFESAYKSIRRGGACVLVGLPPEEIPIPIFDTVLNGV KIIGSIVGTRKDLQEALQFAAEGKVKTIVEVQPLENINDVFDRM LKGQINGRVVLKVD

[0066] The alcohol dehydrogenase for cofactor regeneration mentioned here preferably comprises an amino acid sequence that has an identity to SEQ ID No. 6 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. 6.

[0067] 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. 5 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. 5.

[0068] 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. 6 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 5 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. 5. The alcohol dehydrogenases disclosed herein can be used for the regeneration of 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.

[0069] 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).

[0070] 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. 10 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 9 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. 9.

[0071] Particularly suitable for cofactor regeneration in general is a glucose dehydrogenase whose amino acid sequence is at least 80% identical to SEQ ID No. 10 or which is encoded by a nucleic acid which has an identity to SEQ ID No. 9 of at least 80% or which binds under stringent conditions to a nucleic acid molecule with the nucleic acid sequence SEQ ID No. 9.

[0072] SEQ ID No. 9:

[0073] ATGTATACAGATTTAAAAGATAAAGTAGTTGTAATTACAGGTGGATCAACAGGTTTAGGACGCGCCAA TGGCTGTTCGTTTCGGTCAAAGAAGCAAAAGTTTTTACTATTACAACATGAAAGAAGCT TTAGATGCGAAAAAAGAAGTAGAAGAGGCAGGACAAGCAATTCGATCGATCGATCCAGTCAGTCAGCT AAAGAAGAAGATGTTGTAAACCTTGTTCAAACAGCTATTAAAGAATTCGGTACATTAGACGTTATGAT TAATAACGCTGGTGTTGAAAACCCAGTTCCTTCATGAGTTATCTTTAGACAACTGGAATAAAGTTAT TGATACAAACTTAACAGGTGCATTCTTAGGAAGCCGTGAGTAACTTCATTCATTCATTGATTGAGGAATAAGTTAT TTAAAGGAAACGTTTAACATGTCTAGTGTTCATGAAATGATTCCTTGGCCATTATTTGTTCATTACG CAGCAAGTAAAGGCGGTATGAAACTAATGACGGAAACATTGGCTCTTGAATATGCGCCAAAAGGTAT CCGCGTAAATAACATTGGACCAGGTGCGATGAACACCAATTAGCCAGCATTGATTGAATTTGGCCAAAAGGTAT GTACAACGTGCAGACGTAGAAAGCATGATTCCAATGGGTTACATCGGTAAACCAGAAGAAGTAGCA GCAGTTGCAGCATTCTTAGCATCATCAAGCAAGCTATGTAACAGGTTACATTATTTGCTGATGG TGGTATGACGAAATACCCTTCTTTCCAAGCAGGAAGAGGCTAA

[0074] SEQ ID No. 10:

[0075] MYTDLKDKVVVITGGSTGLGRAMAVRFGQEEAKVVINYYNNEEEALDAKKEVEEAGGQAIIVQGDVTKEED VVNLVQTAIKEFGTLDVMINNAGVENPVPSHELSLDNWNKVIDTNLTGAFLGSREAIKYFVENDIKGNVIN MSSVHEMIPWPLFVHYAASKGGMKLMTETLALEYAPKGIRVNNIGPGAMNTPINAEKFADPVQRADVES MIPMGYIGKPEEVAAVAAFLASSQASYVTGITLFADGGMTKYPSFQAGRG

[0076] The glucose dehydrogenase for cofactor regeneration mentioned here preferably comprises an amino acid sequence that has an identity to SEQ ID No. 10 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. 10.

[0077] 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. 9 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. 9.

[0078] 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. 10 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 9 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. 9. The NAD(P)H oxidase used for cofactor regeneration (see Figure 1) can be selected 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.

[0079] 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. 8 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 7 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. 7.

[0080] SEQ ID No. 7:

[0081] ATGAAAGTAGTAGTAGTAGGCTGTACACATGCAGGAACAGCGGCAGTTAAGACGATTTTAAATGAACATC CAGATGCATCAGTATCAGTATATGAGCGTAATGACAATGTCTCATTTCTATCTTGTGGGATTGCGTTGTATG TTGGTGGAGTTGTGAAAGATCCTGCAGGTTTGTTTTATTCAAGTCCAGAAGAACTTGCATCAATGGGCGCG AAAATTAACATGGAACACAATGTGAAAAATATAGATAATGAGAATAAGGTCGTAGTAATTGAGAATTTAAA AACAGGCGAAACATTTGAAGAAAGCTATGATAAGTTGGTAATGACAACTGGATCATGGCCAATTATTCCTC CAATTGATGGAATCAATAGTGAAAATATTCTTTTGTGTAAAAACTATAACCAAGCAAATGAAATTATTAAAG AATCAAAAAATGCTAAAAAGATTGTCATTGTTGGTGGTGGCTATATTGCGATTGAATTAGTTGAGGCATTT GCAGAATCTGGCAAGCAAGTGACGCTAGTTGCGCGTAGCGATCGTATTTTACGTAAATATTTAGATGCTGA ATTCACTTCTGTTTTAGAGCATGATTTACAAGAAAGAGGCGTTACGCTAGCTTTAAACCAAACCGTCGAGAA ATTTGTTGCCAATGAATCAGGTGCTGTGACAGCTGTGAAAACACCAGTTGGAGAATATGAGGCTGATTTAG TTATTTTATGTGTTGGATTTAAACCAAATACTGATTTGTTGAAGGATAAAGTAGAGATGTTGCCAAATGGTG CCATCGTAGTGGATGAATATATGAGAACAAGCGATGAAGCGATTTTTGCTGCTGGCGATAGTTGCGCGGTT CATTATAATCCAACTGGAGGGCTCTGCGTATATTCCGTTAGCTACAAATGCAGTTAGAATGGGAGCTTTAGTTGGGAAAAAATATTGTTTCTCCAACAGTTAAATATCGTGGCACGCAAGCAACTTCTGGTTTATATTTTTGGT TTTAATATAGGTTCAACCGGATTGACTGAAAATAGCTCCTCATTTTGGCGTAGGGTTCGTTCAGTAGTT GTAGAAGATAATTATCGTCCAGAGTTTTCGACAGAGAGAGAGTAGTGATTGATT AGGAACGAATCGGATTGTTGGAGGTCAAATCATGTCAAAATATGATGTGACACAATCTGCCAATACGTTAT CTTTATGTGTTCAAATAAAATGACGATTGAGGATTTGGCTTATGTAGATTTCTTTCCAACCTCACTTTGA TCGTCCTTGGAACTATTTAATATTTAGCGCAAGCTGACTGAGCAGACTAGCAGCAGACTATTTTCCAACCTCACTTTGA ID No. 8:

[0082] MKVVVVGCTHAGTAAVKTILNEHPDASVVYERNDNVSFLSCGIALYVGGVVKDPAGLFYSSPEELASMGAKIN MECHANICS IVGGGYIAIELVEAFAESGKQVTLVARSDRILRKYLDAEFTSVLEHDLQERGVTLALNQTVEKFVANESGAVTAVK TPVGEYEADLVILCVGFKPNTDLLKDKVEM LPNGAIVVDEYMRTSDEAIFAGDSCAVHYNPTGGSAYIPLATN AVRMGALVGKNIVSPTVKYRGTQATSGLYLFGFNIGSTGLTENSAPHFGVEVRSVVVEDNYRPEFMPTTEKVT MKLVYEVGTNRIVGGQIMSKYDVTQSANTLSLCVQNKMTIEDLAYVDFFFQPHFDRPWNYLNILAQAQER KLAAV

[0083] The preferably used HjO-forming NAD(P)H oxidase preferably comprises or consists of an amino acid sequence having an identity to SEQ ID No. 8 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 HjO-forming NAD(P)H oxidase comprises or consists of the amino acid sequence SEQ ID No. 8.

[0084] Alternatively, the HjO-forming NAD(P)H oxidase preferably comprises an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 7 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 HjO-forming NAD(P)H oxidase comprises or consists of the nucleic acid sequence SEQ ID No. 7.

[0085] A further aspect of the present invention relates to the use of a HjO-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. 8 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 7 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. 7.

[0086] The enzymatic strategy presented here, in combination with cofactor regeneration, enables a biocatalytic, environmentally friendly, and highly efficient production process for the manufacture of L-psicose. The oxidoreductase used according to the invention to form L-psicose from allitol is preferably a mannitol dehydrogenase-like short-chain dehydrogenase / reductase. Surprisingly, it has been shown that mannitol dehydrogenase-like short-chain dehydrogenases / reductases are capable of converting allitol into psicose in the presence of the cofactor NAD(P). + to L-psicose. Accordingly, a further aspect of the present invention relates to a process for producing L-psicose comprising the step of treating allitol with an oxidoreductase, preferably a mannitol dehydrogenase-like short-chain dehydrogenase / reductase, in the presence of the cofactor NAD(P) + .

[0087] The oxidoreductase for the formation of L-psicose from allitol 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.

[0088] An NAD(P)H-dependent oxidoreductase for reducing 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. 12 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. 3, SEQ ID No. 11 or SEQ ID No. 13 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. 11 or SEQ ID No. 13.

[0089] 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. 12 or SEQ ID No. 14 or which is encoded by a nucleic acid which has an identity to SEQ ID No. 3, SEQ ID No. 11 or SEQ ID No. 13 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. 11 or SEQ ID No. 13. These oxidoreductases can also surprisingly be used for the oxidation of allitol to D-psicose.

[0090] SEQ ID No. 3:

[0091] ATGACACAGTCTCTTCAGGGCAAGATCGTCGCCATTACTGGCGCGGCTTCGGGCATTGGCCTCGAATGC

[0092] GCCCGCTATCTCATCGAAGCTGGCGCGGTGGTCTATCTTCTGGACCGTGACGCCAAAACTCTCGAAGAC

[0093] AAAACGGCAGAACTCGGCAGCCAGGCCCATGCGATCATCGTCGATCTCTTCGACTACAAAACCGTAGAT

[0094] GCTGCGGTCGCGCAGATCGTTGAGGAGCAGGGCCGGATCGATGTTTTCCACGCCAACGCCGGCGCGTA

[0095] TGTGGGCGGCAATGTCTGGGAAGGGGATCCCGATAGCTGGGATGCGATGCTGCACCTGAACATCAAT

[0096] GCAGCTTTCCGCTCGGTCCGTGCCGTGCTGCCGCAGATGATGAAGCAGGAAAGTGGCGATATCGTCAT

[0097] GACCAGCTCGATCGCGGGCATGATCCCCATCATGGCCGAGCCGATCTACACGGCCTCGAAACATGCCG

[0098] TGCAGGCATTCGTACATACGGTGCGCCGACAGGTCGCGAAGTATGGAATTCGTGTGGGTGCAATCCAG

[0099] CCTGGTCCTGTAGTCACGCCTTTGCTGAAAGACTGGGATCAGGCCCGTCTTGAAGCCAACATCAAAGCG

[0100] GGTGCCCTGATGGAAGCCAAGGAAGTCGCCGAAGCCCTGATCTTCATCCTGACACGTTCAAAGGGTGT

[0101] CATGGTGCGGGATCTGGTCGTTCTGCCACATAACTTCGACGCCTAAGCTTAAGCGGCCGCACTCGAGCA CCACCACCACCACCACTGAGATCCGGCTGCTAA

[0102] SEQ ID Nr. 4:

[0103] MTQSLQGKIVAITGAASGIGLECARYLIEAGAVVYLLDRDAKTLEDKTAELGSQAHAIIVDLFDYKTVDAAVA QIVEEQGRIDVFHANAGAYVGGNVWEGDPDSWDAMLHLNINAAFRSVRAVLPQMMKQESGDIVMTSSI AGMIPIMAEPIYTASKHAVQAFVHTVRRQVAKYGIRVGAIQPGPVVTPLLKDWDQARLEANIKAGALM EA KEVAEALIFILTRSKGVMVRDLVVLPHNFDA

[0104] SEQ ID Nr. 11:

[0105] ATGACCTCTCCTCTCCAGGGTAAGATAGCCGCCATCACGGGCGGGGCTTCGGGCATCGGCCTCGAATG

[0106] TGTCCGCCAGATCGCCGCAAGTGGTGCCACGGTTTATATTCTCGACCGCGACCATCAGGCGCTCGACAA

[0107] GGCGCGCGAAGAATTGGGCGAGCGCGTTCATACCATCGAGGTCGATCTCTTCCGTTACGAAACGGTCG

[0108] ATCGCGCCATCGAAACCATCGTGTCCGAACAAGGACGCATCGACATTCTCCATGTCAATGCGGGCGCGT

[0109] ATATCGGCGGCAATGTCTGGGAAGGCGATCCCGATAAATGGGACAAGATGCTGAATCTCAACATCAAC

[0110] GCCGCCTTCCGTTCCGCCCGCGCCGTCATGCCCGCCATGATGAAGCAGAAAAGCGGCGATATCATCATG

[0111] ACAAGCTCGATCGCAGGCATCGTCCCGATCCCGGCGGAGCCGATCTACACGGCTTCCAAACATGCGGT

[0112] GCAAGCCTTCGCTCACACCATACGCCGCCAGTTGGCCCCGTTCGGCATCCGCGTCGGCGCCATCCAGCC

[0113] CGGCCCGGTCGTCACGCCCTTGCTCAATGATTGGGACCCCGAGCGCCTTAAAGCCAATATCGAGGCTG

[0114] GCGCCATGATGCAGCCTTCTGACGTCGCCGAAGCCGTGGTTTTCATGCTGTCCCGCCGCAAGGGAACG

[0115] GTAATCCGCGACTTGGTTCTGTTACCCCATTCTTTCGACGTCTAA SEQ. ID Nr. 12:

[0116] MTSPLQGKIAAITGGASGIGLECVRQIAASGATVYILDRDHQALDKAREELGERVHTIEVDLFRYETVDRAIET IVSEQGRIDILHVNAGAYIGGNVWEGDPDKWDKM LNLNINAAFRSARAVM PAMMKQKSGDIIMTSSIAGI VPIPAEPIYTASKHAVQAFAHTIRRQLAPFGIRVGAIQPGPVVTPLLNDWDPERLKANIEAGAM MQPSDVA EAVVFMLSRRKGTVIRDLVLLPHSFDV

[0117] SEQ ID Nr. 13:

[0118] ATGGCTATATCTCGAAAACAACGTAGCTGCAATTACAGGTGCCGCTTCAGGTATCGGTCTCGAATGT GCACGCACACTGATCAAAGCAGGCGCTAAAGTTGTCCTCATTGACCGAGCAGAAGATAGACTAAATCA ATTGGTCGCAGAATTAGGTGAAAATGCAATTCCATTAGTTATCGATTTAATGAAACCAGAACAAGTCGA TGGCATGTTAGCGCGTATTATCGAAAAGGCAGGCAGATTAGATATCTTTCATGCTAATGCTGGAGCTTA CATTGGTGGGCCCGTAGCCGAAGGCGATCCCGATGTTTGGGATAAAGTCCTAAATTTAAATGTTAATGC CGCATTCCGCTGTGTTCGCGCAGTTCTACCACACTTTATCGCACAAAAGTCAGGCGATATTCTATTCACC AGCTCTATCGCTGGTATGGTTCCCGTAATTTGGGAGCCTATTTACACGGCATCAAAATTTGCGGTTCAA GCATTCGTTCATTCTACTCGCCGTCAGGTTTCTGAACACGGTGTCCGTGTTGGTGCTGTATTACCTGGTC CTGTTGTTACTGCGTTATTAGATGATTGGCCAAAAGAAAAACTTGAAGAAGCTTTAGCTAACGGTAGTT TAATGCAACCCATTGAAGTTGCTGAGGCTGTTCTATTCATGCTGACGCGTCCAAGAATATTACAATTCG CGATTTAGTTATTTTACCCAATAGTGTTGACCTCTAA

[0119] SEQ ID No. 14:

[0120] MAISLENNVAAITGAAASGIGLECARTLIKAGAKVVLIDRAEDRLNQLVAELGENAIPLVIDLMKPEQVDGMLA RIIEKAGRLDIFHANAGAYIGGPVAEGDPDVWDKVLNLNVNAAFRCVRAVLPHFIAQKSGDILFTSSIAGMV PVIWEPIYTASKFAVQAFVHSTRRQVSEHGVRVGAVLPGPPVVTALLDDWPKEKLEEALANGSLMQPIEVAE AVLFMLTRPRNITIRDLVILPNSVDL

[0121] The oxidoreductases mentioned here for the reduction of D-psicose to allitol and / or for the formation of L-psicose from allitol preferably comprise an amino acid sequence which has an identity to SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 12 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 oxidoreductase according to the invention for the reduction of D-psicose to allitol comprises or consists of the amino acid sequence SEQ ID No. 4, SEQ ID No. 12 or SEQ ID No. 14 and the oxidoreductase for the formation of L-psicose from allitol comprises or consists of the amino acid sequence SEQ ID No. 2.Alternatively, the oxidoreductases for reducing D-psicose to allitol and / or for forming L-psicose from allitol preferably comprise an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 11 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 oxidoreductase according to the invention for reducing D-psicose to allitol comprises or consists of the nucleic acid sequence SEQ ID No. 3, SEQ ID No. 11 or SEQ ID No. 13, and the nucleic acid encoding the oxidoreductase for forming L-psicose from allitol comprises or consists of the nucleic acid sequence SEQ ID No. 1.

[0122] 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.

[0123] 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 BLOSUIVI62 scoring matrix (Henikoff & Henikoff, 1989), alignments (B) of 50, expectation (E) of 10, M = 5, N = - 4.

[0124] Alternatively, the oxidoreductases for reducing D-psicose to allitol and / or for forming L-psicose from allitol 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: 1, SEQ ID NO: 3, SEQ ID NO: 11, or SEQ ID NO: 13. As used herein, stringent conditions refer to conditions under which so-called specific hybrids, but not nonspecific 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).

[0125] One aspect of the present invention relates to the use of an oxidoreductase for the formation of L-psicose from allitol, wherein the oxidoreductase 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.

[0126] Disclosed is the use of an oxidoreductase for reducing D-psicose to allitol, 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. 12 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. 3, SEQ ID No. 11 or SEQ ID No. 13 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. 11 or SEQ ID No. 13.

[0127] Depending on the reaction (reduction or oxidation), the oxidoreductases according to the invention require corresponding cofactors, as mentioned above.

[0128] Materials

[0129] D-Psicose was purchased from TCI and Hunan Garden Naturals Inc. (China), allitol and L-psicose were 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.

[0130] Production of enzymes & preparation of lysates

[0131] General information on the expression of recombinant enzymes in E. coli

[0132] 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.

[0133] 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.

[0134] 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.

[0135] 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).

[0136] Preparation of cell lysates using sonifier disruption

[0137] 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.

[0138] 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).

[0139] 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.

[0140] Table 1. Enzyme classes and donor organisms for the enzymes used in the examples (SDR = oxidoreductase from the short-chain dehydrogenase / reductase family).

[0141] Analytical methods

[0142] High Performance Liquid Chromatography

[0143] An Agilent HPLC 1260 Infinity II Series system was used to quantify D-psicose / L-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 and eluted isocratically with ultrapure water.

[0144] High Performance Anion Exchange Chromatography

[0145] 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.

[0146] Determination of enzyme activities (optical-enzymatic assay)

[0147] Enzyme activities in the lysates were determined using a Shimadzu UV-1900 spectrophotometer. 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 _8kat). 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.

[0148] Example 1

[0149] Conversion of D-fructose to L-psicose (cofactor regeneration with ADH during reduction step)

[0150] The following components were mixed in a glass vial: 70.6 μl of deionized water, 250 μl of a 200 mM TEA-HCl buffer (pH 8), 50 μl of a D-fructose solution (500 g / L), and 25 μl of D-psicose-3-epimerase lysate. Subsequently, 35 μl of SDR I lysate, 8 U of alcohol dehydrogenase lysate, and 5 μl of a 10 mM NAD + solution and 50 μl of 2-propanol were added. The mixture was incubated for a total of 20 h with continuous shaking (Eppendorf Thermomixer; 35 °C, 800 rpm).

[0151] The mixture was heated to 70 °C for 60 min. After cooling, 25 μl of oxidoreductase lysate (SEQ. ID No. 2), 10 U of NAD(P)H oxidase lysate and 5 μl of 5 mM NADP + solution was added. The mixture was incubated for a further 20 h with continuous shaking (Eppendorf Thermomixer; 24 °C, 800 rpm).

[0152] For analysis, 100 μl of the mixture 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).

[0153] In this way, 97.7% of the D-fructose (50 g / l) was converted to L-psicose (found concentration: 45.8 g / l).

[0154] D-psicose and L-psicose could not be distinguished by the chromatographic method used (elution at the same retention time), so 25 μl of D-psicose 3-epimerase lysate was added to the vial. The mixture was incubated for 2 h at 35 °C and analyzed again by HPLC.

[0155] No change in the amount of psicose was observed after the addition of epimerase, which supports L-psicose as the product. Example 2

[0156] Conversion of D-fructose to L-psicose (cofactor regeneration with GDH during reduction step)

[0157] The following components were mixed in a glass vial: 134 μl of deionized water, 50 μl of a 500 mM TEA-HCl buffer (pH 7.5), 250 μl of a solution containing D-fructose and D-glucose (300 g / L each), and 25 μl of D-psicose-3-epimerase lysate. Subsequently, 35 μl of SDR I lysate, 2 U of glucose dehydrogenase lysate, and 5 μl of a 10 mM NAD +solution was added. The mixture was incubated with continuous shaking (Eppendorf Thermomixer; 35 °C, 800 rpm) for a total of 30 h.

[0158] After cooling the mixture to 24 °C, 25 μl of oxidoreductase lysate (SEQ ID No. 2), 10 U of NAD(P)H oxidase lysate and 5 μl of a 5 mM NADP + solution was added. The mixture was incubated for a further 20 h with continuous shaking (Eppendorf Thermomixer; 24 °C, 800 rpm).

[0159] For analysis, 100 μl of the mixture 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 measured by HPLC (RI detection). For HPAEC measurements (conductivity detection), the clear supernatant was diluted 1:250.

[0160] In this way, 25.6% of the D-fructose (150 g / l) was converted to L-psicose (found concentration: 29.9 g / l) and the D-glucose was completely converted to D-gluconate (found concentration: 158 g / l).

[0161] Example 3

[0162] Oxidation of allitol to L-psicose

[0163] The following components were mixed in a glass vial: 30 μl of deionized water, 250 μl of a 200 mM TEA-HCl buffer (pH 8), 125 μl of an allitol solution (200 g / l), 50 μl of oxidoreductase lysate (SEQ ID No. 2), 40 μl of NAD(P)H oxidase lysate and 5 μl of a 10 mM NADP + -solution. The mixture was incubated with continuous shaking (Eppendorf Thermomixer; 35 °C, 800 rpm) for a total of 20 h.

[0164] For analysis, 100 μl of the mixture 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). In this way, 98.2% of the allitol (50 g / l) was oxidized to L-psicose (found concentration: 50.1 g / l).

[0165] literature

[0166] Zhang, W., Yu, S., Zhang, T., Jiang, B., & Mu, W. (2016). Recent advances in D-allulose: Physiological functionalities, applications, and biological production. Trends in Food Science and Technology, 54, 127-137. https: / / doi.Org / 10.1016 / j.tifs.2016.06.004

[0167] Hough, L., & Stacey, B. E. (1996). Variation in the allitol content of Itea plants during photosynthesis. Phytochemistry, 5(1), 171-175. https: / / doi.org / 10.1016 / S0031-9422(00)85095-5

[0168] Oshima, H., Kimura, I., & Izumori, K. (2006). Psicose Contents in Various Food Products and its Origin. Food Science and Technology Research, 12(2), 137-143. https: / / doi.org / 10.3136 / fstr.12.137

[0169] Jiang, S., Xiao, W., Zhu, X., Yang, P., Zheng, Z., Lu, S., Jiang, S., Zhang, G., & Liu, J. (2020). Review on D-Allulose: In vivo Metabolism, Catalytic Mechanism, Engineering Strain Construction, Bio-Production Technology. Frontiers in Bioengineering and Biotechnology, 8, 26. https: / / doi.org / 10.3389 / fbioe.2020.00026

[0170] Ahmed, A., Khan, T. A., Ramdath, D. D., Kendall, C. W. C., & Sievenpiper, J. L. (2022). Rare sugars and their health effects in humans: a systematic review and narrative synthesis of the evidence from human trials, Nutrition Reviews, 80(2), 255-270. https: / / doi.org / 10.1093 / nutrit / nuab012

[0171] Chen, Z., Gao, X.-D., & Li, Z. (2022). Recent Advances Regarding the Physiological Functions and Biosynthesis of D-Allulose. Frontiers in Microbiology, 13, 881037. https: / / doi.org / 10.3389 / fmicb.2022.881037

[0172] Izumori, K., Khan, A. R., Okaya, H., & Tsumura, T. (1993). A New Enzyme, D-Ketohexose 3-Epimerase, from Pseudomonas sp. ST-24. Bioscience, Biotechnology, and Biochemistry, 57(6), 1037-1039. https: / / doi.org / 10.1271 / bbb.57.1037

[0173] Takeshita, K., Ishida, Y., Takada, G., & Izumori, K. (2000). Direct Production of Allitol from D-Fructose by a Coupling Reaction Using D-Tagatose 3-Epimerase, Ribitol Dehydrogenase and Formate Dehydrogenase. Journal of Bioscience and Bioengineering, 90(5), 545-548. https: / / doi.org / 10.1016 / 51389-1723(01)80038-4

[0174] Izumori, K. (2006). Izumoring: a strategy for bioproduction of all hexoses. Journal of Biotechnology, 124(4), 717-722. https: / / doi.org / 10.1016 / jjbiotec.2006.04.016

[0175] Hassanin, H. A. M., Mu, W., Koko, M. Y. F., Zhang, T., Masamba, K., Jiang, B. (2017). Allitol: production, properties and applications. International Journal of Food Science and Technology, 52(1), 91-97. https: / / doi.org / 10.llll / ijfs.13290 Hold, C., & Panke, S. (2009). Towards the engineering of in vitro systems. Journal of the Royal Society Interface, 6(4), S507-S521. https: / / doi.org / 10.1098 / rsif.2009.0110.focus

[0176] Siedentop, R., Claaßen, C., Rother, D., Lütz, S., & Rosenthal, K. (2021). Getting the Most Out of Enzyme Cascades: Strategies to Optimize In Vitro Multi-Enzymatic Reactions. Catalysts, 11(10), 1183. https: / / doi.org / 10.3390 / catallll01183

[0177] Wang, L., Hu, M., & Tao, Y. (2022). Efficient production of D-glucosamine by diacetylchitobiose deacetylase catalyzed deacetylation of N-acetyl-D-glucosamine. Biotechnology Letters, 44, 473-483. https: / / doi.org / 10.1007 / sl0529-022-03225-2

[0178] Wang, L., Chen, K., Zheng, P., Huo, X., Liao, F., Zhu, L., Hu, M., & Tao, Y. (2023). Enhanced production of D-psicose from D-fructose by a redox-driven multi-enzyme cascade system. Enzyme and Microbial Technology, 163, 110172. https: / / doi.Org / 10.1016 / j.enzmictec.2022.110172

[0179] Itoh , H. & Izumori , K. (1996). Enzymatic production of L-tagatose and L-fructose from L-sorbose and L-psychose, respectively. Journal of Fermentation and Bioengineering, 81(4), 351-353. https: / / doi.org / 10.1016 / 0922-338X(96)80590-3

[0180] Rao , D. , Gullapalli , P. , Yoshihara , A. , Jenkinson , SF , Morimoto , K. , Takata , G. , Akimitsu , K. , Tajima , S. , Fleet , GWJ , & Izumori , K. (2008). Direct Production of L-Tagatose from L-Psychoses by Enterobacter aerogenes 230S. Journal of Bioscience and Bioengineering, 106(5), 473-480. https: / / doi.org / 10.1263 / jbb.106.473

[0181] Sasahara , H. , & Izumori , K. (2005). Production of L-thalitol from L-psicose by Metschnikowia koreensis LAI isolated from soy sauce mash. Journal of Bioscience and Bioengineering, 100(3), 335-338. https: / / doi.org / 10.1263 / jbb.100.335

[0182] Muniruzzaman, S., McIntosh, M., Hossain, A., Izumori, K., & Bhattacharjee, P. S. (2016). A novel rare sugar inhibitor of murine herpes simplex keratitis. Journal of Pharmacological Sciences, 131(2), 126- 130. https: / / doi.org / 10.1016 / jjphs.2016.05.004

[0183] Yang, J., Li, J., Men, Y., Zhu, Y., Zhang, Y., Sun, Y., & Ma, Y. (2015). Biosynthesis of L-Sorbose and L- Psicose Based on C-C Bond Formation Catalyzed by Aldolases in an Engineered Corynebacterium glutamicum Strain. Applied and Environmental Microbiology, 81(13), 4284-4294. https: / / doi.org / 10.1128 / AEM.00208-15 Yang, J., Zhu, Y., Men, Y., Sun, S., Zeng, Y., Zhang, Y., Sun, Y. &, Ma, Y. (2016). Pathway Construction in Corynebacterium glutamicum and Strain Engineering To Produce Rare Sugars from Glycerol. Journal of Agricultural and Food Chemistry, 64(50), 9497-9505. https: / / doi.org / 10.1021 / acs.jafc.6b03423

[0184] Wen, L, Huang, K., Wei, M., Meisner, J., Liu, Y., Garner, K., Zang, L., Wang, X., Li, X., Fang, J., Zhang, H. & Wang, P. G. (2015). Facile Enzymatic Synthesis of Ketoses. Angewandte Chemie International Edition, 54(43), 12654-12658. https: / / doi.org / 10.1002 / anie.201505714

[0185] Lorilliere, M., Dumoulin, R., L'enfant, M., Rambourdin, A., Thery, V., Nauton, L., Fessner, W.-D., Charmantray, F., & Hecquet, L. (2019). Evolved Thermostable Transketolase for Stereoselective Two- Carbon Elongation of Non-Phosphorylated Aldoses to Naturally Rare Ketoses. ACS Catalysis, 9(6), 4754-4763. https: / / doi.org / 10.1021 / acscatal.9b01339

[0186] Takeshita, K., Shimonishi, T., & Izumori, K. (1996). Production of L-psicose from allitol by Gluconobacter frateurii IFO 3254. Journal of Fermentation and Bioengineering, 81(3), 212-215. https: / / doi.org / 10.1016 / 0922-338X(96)82210-0

[0187] Xu, J., Zhou, H., Yu, H., Deng, T., Wang, Z., Zhang, H., Wu, J., & Yang, L. (2021). Computational design of highly stable and soluble alcohol dehydrogenase for NADPH regeneration. Bioresources and Bioprocessing, 8, 12. https: / / doi.org / 10.1186 / s40643-021-00362-w

[0188] Al-Rabiah, A. A., Boz, I., Akhmedov, V. M., Mostafa, M. M. M., & Bagabas, A. A. (2022). Highly Selective Gas-Phase Catalytic Hydrogenation of Acetone to Isopropyl Alcohol. Catalysts, 12(10), 1251. https: / / doi.org / 10.3390 / catall2101251

[0189] Lin, B., & Tao, Y. (2017). Whole-cell biocatalysts by design. Microbial Cell Factories, 16, 106. https: / / doi.org / 10.1186 / sl2934-017-0724-7

[0190] Kawakami, K., Hasegawa, Y., Deguchi, K., Ohki, S., Shimizu, T., Yoshihashi, Y., Yonemochi, E., & Terada, K. (2013). Competition of Thermodynamic and Dynamic Factors During Formation of Multicomponent Particles via Spray Drying. Journal of Pharmaceutical Sciences, 102(2), 518-529. https: / / doi.org / 10.1002 / jps.23378

[0191] Kawakami, K., Hasegawa, Y., Zhang, S., Yoshihashi, Y., Yonemochi, E., & Terada, K. (2014). Low-Density Microparticles with Petaloid Surface Structure for Pulmonary Drug Delivery. Journal of Pharmaceutical Sciences, 103(4), 1309-1313. https: / / doi.org / 10.1002 / jps.23899

[0192] Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D. J. (1990). Basic local alignment search tool. Journal of Molecular Biology, 215(3), 403-410. https: / / doi.org / 10.1016 / S0022-2836(05)80360-2 Henikoff, S., & Henikoff, J. G. (1992). Amino acid substitution matrices from protein blocks. Proceedings of the National Academy of Sciences of the United States of America, 89(22), 10915-10919. https: / / doi.org / 10.1073 / pnas.89.22.10915

[0193] Sambrook, J., Fritsch, E. R., & Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual (2nd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

[0194] Mu, W., Chu, F., Xing, Q„ Yu, S., Zhou, L., & Jiang, B. (2011). Cloning, Expression, and Characterization of a D-Psicose 3-Epimerase from Clostridium cellulolyticum H10. Journal of Agricultural and Food Chemistry, 59(14), 7785-7792. https: / / doi.org / 10.1021 / jf201356q

[0195] Chan, H.-C., Zhu, Y., Hu, Y., Ko, T.-P., Huang, C.-H., Ren, F., Chen, C.-C., Ma, Y., Guo, R.-T., & Sun, Y. (2012). Crystal structures of D-psicose 3-epimerase from Clostridium cellulolyticum H10 and its complex with ketohexose sugars. Protein & Cell, 3(2), 123-131. https: / / doi.org / 10.1007 / sl3238-012- 2026-5

[0196] Protein [Internet], Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information. Accession No. WP_063903495.1, SDR family oxidoreductase [Gluconobacter frateurii]. Verfügbar unter: https: / / www.ncbi.nlm.nih.gOv / protein / WP_063903495.l / (Zugriff am 06.03.2024)

[0197] Protein [Internet], Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information. Accession No. WP_070401870.1, SDR family oxidoreductase [Kozakia baliensis]. Verfügbar unter: https: / / www.ncbi.nlm.nih.gOv / protein / WP_070401870.l / (Zugriff am 07.03.2024)

[0198] Protein [Internet], Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information. Accession No. WP_068907433.1, SDR family oxidoreductase [Providencia heimbachae]. Verfügbar unter: https: / / www.ncbi.nlm.nih.gOv / protein / WP_068907433.l / (Zugriff am 07.03.2024)

[0199] V. L. Louis, L. Despons, A. Friedrich, et al. (2012). Pichia sorbitophila, an Interspecies Yeast Hybrid, Reveals Early Steps of Genome Resolution After Polyploidization. G3 Genes I Genomes I Genetics, 2(2), 299-311. https: / / doi.org / 10.1534 / g3.lll.000745

[0200] Sakoda, H., & Imanaka, T. (1992). Cloning and sequencing of the gene coding for alcohol dehydrogenase of Bacillus stearothermophilus and rational shift of the optimum pH. Journal of Bacteriology, 174(A), 1397-1402. https: / / doi.Org / 10.1128 / jb.174.4.1397-1402.1992 Protein [Internet], Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information. Accession No. MDQ0804260.1, Glucose 1-dehydrogenase [Priestia megaterium]. Verfügbar unter: https: / / www.ncbi.nlm.nih.gOv / protein / MDQ0804260.l (Zugriff am 06.03.2024)

Claims

Patent claims 1. A process for the preparation of an aqueous solution containing L-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 L-psicose with a corresponding NAD(P) + -dependent oxidoreductase, after which the deactivated epimerase and the oxidoreductases are removed.

2. The method according to claim 1, characterized in that the oxidoreductase for forming L-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. 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.

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. 12 or SEQ ID No. 14 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. 11 or SEQ ID No. 13 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. 11 or SEQ ID No.

13.

8. The method according to any one of claims 3 to 7, characterized in that the alcohol dehydrogenase comprises or consists of an amino acid sequence selected from the group consisting of: i) an amino acid sequence having an identity to SEQ ID No. 6 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 5 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.

5.

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. 10 of at least 80%, ii) an amino acid sequence which is encoded by a nucleic acid which has an identity to SEQ ID No. 9 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.

9.

11. Use of an aqueous solution producible by a process according to any one of claims 1 to 10 for producing a syrup containing L-psicose.

12. Use of an oxidoreductase for the formation of L-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. 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 oxidoreductase for the reduction of D-psicose to allitol, 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. 12 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. 3, SEQ ID No. 11 or SEQ ID No. 13 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. 11 or SEQ ID No.

13.

14. 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. 6 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 5 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.

5.

15. 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. 10 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 9 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.

9.

16. Use of a HjO-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. 8 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 7 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. 7.

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