Method for producing d-talitol, d-tagatose, and d-psicose

Abstract

The invention relates to a method for producing an aqueous solution containing d-talitol, wherein d-psicose is formed in vitro from d-fructose, which is provided in a dissolved form in an aqueous solution, by treating the d-fructose with an epimerase. The d-psicose is then reduced in order to form d-talitol in vitro by treating the d-psicose with a NAD(P)H-based oxidoreductase. The D-talitol contained in the aqueous solution can be oxidized in a simple manner by treating the d-talitol with an oxidoreductase in order to form d-tagatose or d-psicose.

Description

[0001] Process for the preparation of D-talitol, D-tagatose and D-psicose

[0002] The present invention relates to the preparation of D-talitol. The invention further relates to the preparation of an aqueous solution containing D-tagatose or D-psicose.

[0003] Background of the invention

[0004] The hexavalent sugar alcohol D-talitol (also known as D-altritol) is one of the so-called rare sugars due to its low natural occurrence (e.g., in brown algae such as Himanthalia elongata (Reed et al., 1995)). Due to the lack of a simple production method for this sugar alcohol, the potential applications of D-talitol have not been well studied (Li et al., 2013).

[0005] In the so-called Izumoring strategy, D-talitol plays an important role, since its oxidation leads to the ketohexoses D-tagatose and D-psicose as well as to the aldohexoses D-altrose and D-talose, which are also considered rare sugars (Izumori, 2006).

[0006] D-Tagatose is found, among other substances, in the gum of the tropical tree Sterculia setigera (Hirst et al., 1949), in the lichen Roccella (Lindberg, 1955), and in highly heated milk (Adachi, 1958). D-Psicose has been detected in the leaves of rosemary willows (Itea sp.) (Hough & Stacey, 1963) but is also found in processed foods such as confectionery and spice sauces, where it is formed from D-fructose, its C3 epimer, under the influence of heat (Oshima et al., 2006).

[0007] Both D-tagatose and D-psicose are characterized by their sweet taste with a significantly lower energy content (relative to sucrose) (Jiang et al., 2020; Oh, 2007; Roy et al., 2018), which makes both sugars particularly interesting as sweeteners for the food industry.

[0008] D-tagatose and D-psicose are approved as Generally Recognized as Safe (GRAS) sweeteners by the US Food and Drug Administration (FDA), but in the European Union only D-tagatose is on the so-called Novel Foods list and may therefore be used as a sweetener in the EU (Levin, 2002; Ahmed et al., 2022).

[0009] D-tagatose can also be used as a fungicide, inhibiting the D-mannose metabolism of fungi and oomycetes and thus effective against downy mildew (Mochizuki et al., 2020). US 10638752 B2 and Chahed et al. (2021) describe the use of D-tagatose against various pathogens of plant diseases. D-psicose has a positive effect on lipid and carbohydrate metabolism (e.g., antidiabetic) and exhibits anti-inflammatory and antioxidant effects (Zhang et al., 2016; Jiang et al., 2020; Chen et al., 2022).

[0010] There are several chemical or biotechnological routes for the production of D-tagatose, such as the base-induced isomerization of D-galactose obtained from lactose (EP 0518874 B1; US ​​9150938 B2; US 8802843 B2), the enzymatic isomerization of D-galactose using L-arabinose isomerase (Heath et al., 1958; Oh, 2007; Roy et al., 2018), the production starting from mono-, di-, oligo- or polysaccharides via phosphorylated intermediates with the C4-epimerization of D-fructose-6-phosphate to D-tagatose-6-phosphate as the main step (EP 3322803 B1; US ​​10138506 B2; US 11034988 B2; Dai et al., 2020; Lee et al., 2020). al., 2017) or the oxidation of the sugar alcohol galactitol with galactitol 2-dehydrogenase (EC 1.1.1.16) (Roy et al., 2018; Shaw, 1956).

[0011] D-tagatose can be produced directly from D-fructose by C4 epimerization. Through targeted optimization of the enzyme tagaturonate-3-epimerase from Thermotoga petrophila, a tagatose-4-epimerase (T4E) was developed, which produces 213 g / l D-tagatose from 700 g / l D-fructose in 2 h at 80 °C in the presence of 1.5 mM Ni. 2+ produced (Shin et al., 2020). Other epimerases derived from thermophilic microorganisms are also known (e.g., EP 3006568 B1; EP 3211078 B1; US ​​10196626 B2; US 11180785 B2; US 11408017 B2; EP 3677675 A1), but these also require elevated reaction temperatures (> 50 °C) and the addition of Zn, Ni, or Mn ions for optimal activity.

[0012] In a recent study, tagatose 4-epimerases from Thermotoga neapolitana and Kosmotoga olearia were tested. They were expressed in Corynebacterium glutamicum and used as whole-cell biocatalysts. Conversions of 13.8% D-tagatose (T4E from T. neapolitana) and 14.4% D-tagatose (T4E from K. olearia; 21.7% after codon optimization) were achieved (30 g / L D-fructose as substrate, 60 °C for 2.5 h). However, heavy metal salts (1 mM NiSCU for T. neapolitana and 3 mM COSO4 for K. olearia) were also required (Jeon et al., 2023).

[0013] Since D-tagatose is a sweetener, heavy metal ions are undesirable in the finished product. The higher reaction temperatures and incomplete conversion are further disadvantages of C4 epimerization.

[0014] D-psicose, in turn, can be obtained by epimerization of D-fructose with a ketosis 3-epimerase (Izumori et al., 1993). 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).

[0015] 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).

[0016] The D-fructose / D-psicose mixtures resulting from the epimerization of D-fructose must 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). In US 2021 / 0189441 A1, the excess D-fructose is converted into L-lactic acid by a probiotic microorganism (Lactobacillus or Saccharomyces). EP 3423460 B1 describes a process using ion exchange and continuous chromatography for the purification of a D-fructose / D-psicose mixture and the production of 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 simulated moving bed chromatography.

[0017] There are several other biotechnological routes for the production of D-psicose, such as the production starting from mono-, di-, oligo- or polysaccharides via phosphorylated intermediates with the C3 epimerization of D-fructose 6-phosphate to D-psicose 6-phosphate as the main step (Li et al., 2021; US ​​11168342 B2; US 10907182 B2) or the aldol reaction of dihydroxyacetone phosphate and D-glyceraldehyde with subsequent dephosphorylation of D-psicose l-phosphate (Wang et al., 2020; Li et al., 2020).

[0018] The unfavorable equilibrium of epimerization from D-fructose to D-psicose can also be favorably influenced by downstream reactions. One possibility is the targeted phosphorylation of D-psicose to D-psicose-l-phosphate with L-rhamnulose kinase and subsequent dephosphorylation with an acid phosphatase (Xiao et al., 2019). The disadvantage of this process is the stoichiometric consumption of the expensive phosphate donor adenosine triphosphate (ATP).

[0019] An alternative is the combination of epimerase with ribitol dehydrogenase (RDH) and formate dehydrogenase (FDH; oxidizes formate to CO2) as a regeneration enzyme for the cofactor nicotinamide adenine dinucleotide (NADH), which converts D-fructose via D-psicose to allitol (Takeshita et al., 2000). Through a subsequent oxidation step, allitol can be converted back to D-psicose.

[0020] Wang et al. (2023) describe a system consisting of two E. coli whole-cell biocatalysts for the conversion of D-fructose (90 g / L) to D-psicose with a conversion of 90%. 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 were used. In this way, D-fructose (500 mM = 90 g / L) was converted to 452 mM allitol (conversion 90.4%) within 12 h at 37 °C and pH 6 using 1000 mM sodium formate (two equivalents based on D-fructose). Approximately 30 mM D-sorbitol (the reduction product of D-fructose) was formed as a byproduct. The cells were separated by centrifugation, and exuded proteins in the allitol-containing supernatant were deactivated by heat. Then, E. coli cells expressing an RDH from Rubrivivax sp.and an NADH oxidase from Streptococcus pyogenes were added to the allitol solution. Allitol (452 ​​mM) was oxidized to D-psicose (450 mM) within 24 h at pH 7, and the byproduct D-sorbitol was reoxidized to D-fructose. The overall conversion of the process was 90%.

[0021] In an article, Izumori (2002) describes a toolbox consisting of an epimerase (D-tagatose 3-epimerase) and oxidoreductases for the bioproduction of ketohexoses with hexitols as intermediates (known as the Izumoring strategy). The examples cited in the article for the production of hexitols from ketohexoses and vice versa use exclusively microbial cells as catalysts (Izumori, 2002).

[0022] Heterogeneous catalytic hydrogenation of D-psicose over Raney nickel or platinum group metals yields a mixture of the epimers allitol and D-talitol, which requires chromatographic separation (US 2004 / 0143024 Al). In contrast, the use of microorganisms allows the stereoselective reduction of ketoses to sugar alcohols (preferential formation of one of the two epimers).

[0023] In general, it can be said that in recent years there has been a trend towards whole-cell processes (microbial and fermentative processes) in bioproduction processes (cf. Wang et al., 2023). For example, according to Boverio et al. (2023), isolated (uronate) dehydrogenases ("in vitro") have "inherent disadvantages," and Vastano et al. (2019) consider their industrial use "impractical" due to the cofactor requirement (NADH).

[0024] For example, D-talitol can be produced by reducing D-tagatose with the fungus Aureobasidium pullulans (strain 113B) (Muniruzzaman et al., 1994). The reverse reaction can be accomplished using altritol 5-dehydrogenase (EC 1.1.1.407), which is part of the D-altritol catabolism in Agrobacterium tumefaciens C58 (Wichelecki et al., 2015).

[0025] For example, D-Talitol can interact with the bacterium Alcaligenes sp. 701B can be oxidized to D-psicose (Izumori et al., 1990).

[0026] Yoshihara et al. (2006) describe the direct conversion of D-psicose to D-tagatose (via D-talitol) using various fungi from the Mucoraceae family, such as Rhizopus oryzae MYA-2483. However, this process is characterized by long reaction times (8 to 14 days) with only low titers of up to 3 g / l D-talitol and 4.2 g / l D-tagatose at a starting D-psicose concentration of 12 g / l. To make the conversion more economical, the authors propose screening for more effective strains, breeding by mutation and genetic engineering, and bioprocess optimization.

[0027] Sasahara et al. (1998) also investigated the production of D-talitol from D-psicose. There (as well as in JP 2020039301 A and JP 2006166718 A) they describe the reduction of D-psicose to D-talitol using the halotolerant yeast strain Candida famata R28. The authors observed that the reduction can be accelerated in the presence of sugar alcohols such as D-sorbitol. However, the reaction rate is very slow, so the process requires 10 days to convert 10% D-psicose (100 g / L) to 95% D-talitol in the presence of 5% D-sorbitol (50 g / L). When the amount of D-sorbitol used was halved, i.e., 25 g / L, a conversion of only about 55% could be achieved after 10 days.

[0028] This is where the object of the present invention comes in and aims to provide a biotechnological process for the production of D-talitol and aqueous solutions of D-tagatose and / or D-psicose, which can be carried out more efficiently and in particular in a one-pot process.

[0029] Detailed description of the invention

[0030] The object of producing D-talitol is achieved according to the invention by first forming D-psicose from D-fructose, which is dissolved in an aqueous solution, by treating it with an epimerase in vitro, after which the D-psicose is reduced to D-talitol by treating it with an NAD(P)H-dependent oxidoreductase in vitro and the epimerase and the oxidoreductase are separated or deactivated.

[0031] D-talitol can be concentrated and crystallized from the resulting solution. Surprisingly, it has been shown that the reduction of D-psicose to D-talitol can be accomplished in vitro much more efficiently than is known in the prior art (Sasahara et al., 1998). The time course of D-talitol concentration is shown in the attached Figure 2.

[0032] The task of producing an aqueous solution of D-tagatose or D-psicose is solved by reacting the aqueous solution containing D-talitol prepared by the process according to the invention with an NAD(P) + -dependent oxidoreductase to form reduced cofactor NAD(P)H to oxidize D-talitol to D-tagatose or D-talitol to D-psicose, respectively, after which the oxidoreductase is separated. These oxidations are advantageously carried out in vitro.

[0033] The reaction scheme of the process according to the invention is shown in the attached Figure 1. The abbreviation "Reg. Enzyme" in Figure 1 stands for regeneration enzyme.

[0034] In the case of the production of D-psicose, before carrying out the last step (oxidation), the enzymes (epimerase and reductase / dehydrogenase) of the first step (D-fructose D-talitol) are advantageously deactivated by heat or separated by ultrafiltration in order to prevent part of the D-psicose produced by the oxidation from being converted back to D-fructose by the epimerase.

[0035] A preferred embodiment of the process according to the invention for preparing an aqueous solution containing D-talitol consists in that the oxidized cofactor NAD(P) formed by the reduction + is reduced by means of an alcohol dehydrogenase and a secondary alcohol to form a ketone, as described, for example, in EP 2812439 B1.

[0036] The secondary alcohol 2-propanol (isopropanol) has also been shown to be particularly suitable. 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, in solution, or in 2-propanol / acetone / water mixtures. In the future, hydrogen from sustainable sources ("green hydrogen") could be increasingly used.

[0037] A further preferred embodiment of the process according to the invention for preparing an aqueous solution containing D-talitol consists in that the oxidized cofactor NAD(P) formed by the reduction +by means of a formate dehydrogenase and a salt of formic acid (formate) to form carbon dioxide. A preferred embodiment of this process according to the invention consists in converting the reduced cofactor NAD(P)H produced by the oxidation to NAD(P) by means of an oxidase and oxygen. + is oxidized.

[0038] In a further preferred embodiment of this process according to the invention, the reduced cofactor NAD(P)H produced by the oxidation is converted to NAD(P) by means of NAD(P)-dependent alcohol dehydrogenase and a ketone. + is oxidized to form a secondary alcohol.

[0039] It has also been shown that acetone is particularly suitable as a ketone, with 2-propanol being formed as a reduction product.

[0040] For the regeneration of the cofactors required in the redox reactions, an NAD(P)-dependent alcohol dehydrogenase or an NAD(P)-dependent formate dehydrogenase is used in the case of the reduction of D-psicose to D-talitol, and an F O-forming NAD(P)H oxidase or an NAD(P)-dependent alcohol dehydrogenase is used in the case of the oxidation of the sugar alcohol to D-tagatose or D-psicose.

[0041] A further preferred variant of the processes according to the invention is characterized in that they are carried out as a one-pot reaction without isolation of intermediate products.

[0042] The separation of the enzymes can be achieved, for example, by heat deactivation and / or centrifugation or ultrafiltration.

[0043] The most preferred concentration of D-fructose is 50 - 250 g / l.

[0044] The particularly preferred temperature range for the first step (epimerization and reduction) is between 25 and 40 °C, for the second step (oxidation) between 20 and 35 °C.

[0045] The particularly preferred pH range for both steps is between 7.0 and 8.5.

[0046] In a further preferred variant of the process according to the invention, the enzymes are present in a suspension, in the homogenate and / or in the lysate of the corresponding cells producing them, with lysates being particularly preferred. In addition, the enzymes can also be used in lyophilized, spray-dried, or immobilized form in the process according to the invention. The enzymes used according to the invention can also be used in a substantially purified form, so that the enzyme preparations comprise no or substantially no components originating from the cells or culture in which they were expressed. The enzymes produced, for example, by recombinant expression (e.g. in E. coli) can be purified by methods known in the art in order to be used, after ultrafiltration and DNA removal, among other things, in and for the production of food.

[0047] In this context, suspension means a suspension of resting cells. These are harvested after cultivation (separated from the nutrient medium) and 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 removal of carbon sources and nutrients; instead, they only serve to convert substrates (Lin & Tao, 2017). Homogenate in this context 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).

[0048] 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.

[0049] In another variant, the enzymes can be in powder form, in lyophilized or spray-dried form.

[0050] 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.

[0051] 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.

[0052] The NAD(P)H-dependent oxidoreductase for the reduction of D-psicose to D-talitol preferably comprises an amino acid sequence selected from the group consisting of: i) an amino acid sequence having an identity to SEQ ID No. 2, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18, SEQ ID No. 20, SEQ ID No. 22 or SEQ ID No. 24 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 1, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 21 or SEQ ID No. 23 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid which anneals under stringent conditions to a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 1, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No.

[0053] 15, SEQ ID Nr. 17, SEQ ID Nr. 19, SEQ ID Nr. 21 oder SEQ ID Nr. 23 bindet.

[0054] SEQ ID Nr. 1:

[0055] ATGAACGATCCCGTTCTGGAAGCCAGACCGATTTTGGAGAGATTTCGCCTCGACGGCCGCGTGGCGC TGGTGACCGGCGGTGGGCAGGGCATCGGCCGCGCCTTCGCCCATGCGCTGTGCGAGGCAGGCGCCG CAGTGGCCGTCGTCGATCTGCGCCTGGACCTGGCCGAGGAAGTGGCGCATGAGCTGGTAAACAAGC AGATCGACGCCATCGCAATTCAGGCCGATGTGACGAAGCCCGATCAGGTGCAGACGATGGTTGATGC CATTCTGTCCAGGTGGGGGACGTTGACGATCGGTGTCAACAACGCCGGCATCGGCTTGTGGGCGGAT GCCGAAAGCATGGCATACACAGACTGGCTGCGCGTGATCGATATTGATCTGAACTCGGTTTTCCTCTG CGCCCAGGCTGAAGCGAGGGTCATGCTGCCGGCCGGCTACGGCAAGATCATCAACACGGCGTCGAT GTCCGGCCATATCAGCAACACGCCGCAAAATCAGGCTGCTTACAACACGGCCAAGGCTGGCGTGATC CATCTGACGCGCAGCCTTGCCGCGGAATGGGCCAAGCGGGGAGTGCGCGTCAACAGCATCAGCCCC GGCTATACGCGCACCAAATTGGTTGACGACCTGCTCGCCACGCCGATCGGACAGACGATGTTGCCCA CCTGGATGGGCATGACGCCCATGGGACGAATGGCCGAGGTCACCGATCTGCAAGGCGCCGTCGTCT ACCTGGCGTCGTCGGCGTCCGACTATATGACCGGCCATGACATGGTGATTGACGGCGGCTATTGTTG CTGGTAA

[0056] SEQ ID Nr. 2:

[0057] M NDPVLEARPILERFRLDGRVALVTGGGQGIGRAFAHALCEAGAAVVDLRLDLAEEVAHELVNKQIDA IAIQADVTKPDQVQTMVDAILSRWGTLTIGVNNAGIGLWADAESMAYTDWLRVIDIDLNSVFLCAQAEA RVMLPAGYGKIINTASMSGHISNTPQNQAAYNTAKAGVIHLTRSLAAEWAKRGVRVNSISPGYTRTKLVD DLLATPIGQTM LPTWMGMTPMGRMAEVTDLQGAVVYLASSASDYMTGHDMVIDGGYCCW

[0058] SEQ ID No. 5:

[0059] ATGTATCCCAGATTTAAAAGGAAAAGTTGTCGCTATTACAGGAGCTGCTTCAGGATTAGGGAAGGCAA TGGCCATTCGCTTCGGCAAGGAGCAGGCAAAAGTGTTATCAACTACTACAGCAATAAGCAGGATCC GAACGAGGTAAAGGAAGAGGTCATCAAGGCGGGGGAGGAGGAGGAGTTGAGGAGGAGTCGAGCAAG CAAAAGAGGAAGATGTAAAAAACATCGTCCAAACAGCGATTAACGAGTTCGGTACACTCGATATTATGCCGGTCTTGAAAATCCCCGTTCCTTCATGAAATGCCGCTGAAGGATTGGGATAAAG TAATCAGCACGAACTTAACGGCGCTTTTTAAGGAAGCCGTGAAGGTTTAGATTGATTGATTGAAT GATATAAAAGGAAATGTCATTAATATGTCGAGCGTACATGAAGTGATTCCGTGGCCATTATTTGTTCA

[0060] CTATGCGGCAAGTAAAGGCGGAATCAAGCTGATGACGGAAACATTGGCGCTGGAATATGCGCCGAA AGGCATTCGTGTCAACAATATCGGGCCAGGCGCGATCAACGCCAATCAATGCTGAAAAATTTGCT

[0061] GATCCTAAGCAGAGAGCAGATGTAGAAAGCATGATTCCGATGGGATATATCGGTGAACCGGAGGAA

[0062] ATTGCGGCAGTAGCAGCCTGGCTTGCTTCGAAGGAAGCCAGCTACGTCACAGGCATCACGTTATTCG CGGACGGCGGTATGACCCAATATCCTTCCTTCCAGGCAGGACGCGGATAA

[0063] SEQ. ID Nr. 6:

[0064] MYPDLKGKVVAITGAASGLGKAMAIRFGKEQAKVVINYYSNKQDPNEVKEEVIKAGGEAVVVQGDVTKE

[0065] EDVKNIVQTAINEFGTLDIMINNAGLENPVPSHEMPLKDWDKVISTNLTGAFLGSREAIKYFVENDIKGNVI

[0066] NMSSVHEVIPWPLFVHYAASKGGIKLMTETLALEYAPKGIRVNNIGPGAINTPINAEKFADPKQRADVES M IPMGYIGEPEEIAAVAAWLASKEASYVTGITLFADGGMTQYPSFQAGRG

[0067] SEQ. ID Nr. 7:

[0068] ATGACTATTCCTAATACTACTGAAATTACCTCGTTTACAAACCCTGCTCTTGGACCATTACCCACTTCAG

[0069] CACCAGAACTAGCTACTAGTGTGCTTGATTTATTTCTGCTTAAGGGCAAAGTAGCATCAGTTACTGGA

[0070] TCCTCTACTGGTATAGGGTACGCTGTGGCAGAAGCTTTTGCACAGGCAGGTGCCGATGTTGCAATTTG

[0071] GTACAACAATCATCCTGCAACCGAAAAGGCTGAAAAATTGGCTAAAAAATATGGCGTTAAATGCAGG

[0072] GCATACAAATGCAACATTTCCAATGCTGACGATGTTGATCTGACGATTAAGCAGATTGAGCAAGATTT

[0073] TGGAACGATTGATATCTTTGTTGCCAATGCTGGTGTGGCTTGGACTTCTGGTTCCATGGTTGATGTGG

[0074] ACGATAATCATGAAAGTTGGCACAAGATTGTCGACTTGGATTTGAATGGAGTATACTATTGTTGTCAT

[0075] TCAATAGGGAAAGTATTCAAGAGGAAAGGAAAGGGTTCCTTGGTTATTACTTCATCAATGTCGGGCC

[0076] TGATTGTCAATGTTCCACAGCTACAGGCTCCTTATAATGCTGCTAAGGCTGCTGTAAAACATTTAGCTA

[0077] AATCGTTAGCAGTTGAATGGGCTCCATTTGCTAGAGTCAATAGTATATCTCCAGGATATATCACTACT

[0078] GATATTTCTGAATTCGCCGATCCTGAAATGAAGGCAAGATGGTGGCAATTAACTCCTTTGGGGAGAG

[0079] AGGGATTGCCACAAGAATTAGTAGGAGCTTACTTATACTTAGCCTCCAATGCCTCTACATTTACTACG GGAGCCGATCTCGTTGTGGACGGTGGATACACTGCTCCATAA

[0080] SEQ ID No. 8:

[0081] MTIPNTTEITSFTNPALGPLPTSAPELATSVLDLFLLKGKVASVTGSSTGIGYAVAEAFAQAGADVAIWYNN

[0082] HPATEKAEKLAKKYGVKCRAYKCNISNADDVDLTIKQIEQDFGTIDIFVANAGVAWTSGSMVDVDDNHES

[0083] WHKIVDLDLNGVYYCCHSIGKVFKRKGKGSLVITSSMSGLIVNVPQLQAPYNAAKAAVKHLAKSLAVEWA

[0084] PFARVNSISPGYITTDISEFADPEMKARWWQLTPLGREGLPQELVGAYLYLASNASTFTTGADLVVDGGYT AP

[0085] SEQ ID Nr. 9: ATGGAAAAACTCCGCCTCGATAACCGCGTCGCCATCGTCACCGGCGGCGCACAGAACATCGGCCTGG

[0086] CCTGCGTCACCGCACTGGCCGAGGCTGGCGCCCGCGTCATCATCGCCGATCTCGATGAAGCCATGGC

[0087] CGCAAAAGCCGTGGAAGATCTGCGCATGGAAGGCCATGATGTCAGCAGCGTCGTCATGGACGTCAC

[0088] GAACACGGAAAGTGTGCAGAACGCTGTCCGCAGCGTTCACGAGCAGGAAGGCCGCGTGGATATCCT

[0089] GGTCGCCTGCGCCGGCATCTGCATTTCCGAAGTCAAGGCCGAGGACATGACGGATGGCCAGTGGCTC

[0090] AAGCAGGTCGATATCAACCTGAACGGCATGTTCCGCTCCTGTCAGGCCGTCGGGCGCATCATGCTCG

[0091] AACAGAAACAGGGCGTCATTGTCGCAATCGGCTCCATGTCCGGCCTGATCGTCAACCGCCCGCAGCA

[0092] GCAGGCGGCCTATAATGCGTCGAAAGCCGGCGTTCACCAGTATATCCGCTGCCTCGCCGCAGAATGG

[0093] GCTCCCCATGGCATTCGTGCCAACGCCGTTGCCCCCACCTTATATCGAAACGACGCTCACCCGCTTCGG

[0094] CATGGAAAAGCCGGAACTCTACGACGCCTGGATCGCCGGAACCCCGATGGGCCGCGTGGGCCAGCC

[0095] CGATGAAGTCGCTTCCGTCGTGCAGTTCCTGGCGTCCGACGCAGCCAGCCTGATGACCGGCGCCATC GTGAATGTGGATGCGGGCTTCACCGTCTGGTAA

[0096] SEQ. ID No. 10:

[0097] I EKLRLDNRVAIVTGGAQNIGLACVTALAEAGARVIIADLDEAMAAKAVEDLRMEGHDVSSVVMDVTN

[0098] TESVQNAVRSVHEQEGRVDILVACAGICISEVKAEDMTDGQWLKQVDINLNGM FRSCQAVGRIM LEQK

[0099] QGVIVAIGSMSGLIVNRPQQQQAAYNASKAGVHQYIRSLAAEWAPHGIRANAVAPTYIETTLTRFGM EKPE

[0100] LYDAWIAGTPMGRVGQPDEVASVVQFLASDAASLMTGAIVNVDAGFTVW

[0101] SEQ. ID No. 11:

[0102] ATGTCTCAAGCTATCTCAGCAATCAACGAAAGAGTTGGCCCATTGCCCACTAAAGCACCCCAACTTTC

[0103] AAAAAACGTATTAGACCTCTTTTCCTTGAAAGGCAAGGTAGCATCGGTGACAGGTTCATCGAAGGGA

[0104] ATAGGCTTTGCTGTCGCAGAAGCATACGCTCAAGCAGGGGCTGATGTTGCTTTGTGGTACAATTCAA

[0105] GTCCTGTCGAGGATAAGGTAAAATACTTGAAAGAGACCTACGGCGTGAAAGTCAAAGCCTACAAGTG

[0106] TAACGTGGGAAACAGCGAGGAGGTTGAGAAAATAGTAGACCAAATTGCAGAGGACTTCGGCACAAT

[0107] TGACGTTTTTGTTGCTAATGCGGGCATTGCTTGGAGCGAAGGTAGATCTCTCGAGGTTAAGGGTTAC

[0108] GACGCTTGGCAAAAAATTGTCGACTACGACTTGAGCAGTGTATACTACTGTGCTAAGGCAGTGGGTA

[0109] AGATCTTCCAAGAAAAGGGCTCCGGTTCGTTAATCCTCACCGCATCCATGTCCGGACACATTGTGAAT

[0110] GTTCCTCAAATGCAGGCACCATACAATGCGGCCAAGGCGGCTGTGATCCATCTAGGAAAATCTTTGG

[0111] CTGTCGAATGGGCTCCATTTGCCAGGGTCAACACCGTTTCTCCTGGCTATATTGCCACTGATATTGGTG

[0112] AATTTGTGCTGCCCGATGAGAAGAAAAAGTGGTGGTCATTGACACCCTTGGGTAGAGAAGGTCTTCC

[0113] TCAAGAACTCACTGGTGCATACTTGTATTTTGCTTCTAACGCATCGACCTACACTACTGGGGCTGACTT GGTCGTAGACGGTGGCTCACTGTACCATAA

[0114] SEQ ID No. 12: MSQAISAINERVGPLPTKAPQLSNKNVLDFSLKGKVASVTGSSKGIGFAVAEAYAQAGADVALWYNSSPVE

[0115] DKVKYLKETYGVKVKAYKCNVGNSEEVEKIVDQIAEDFGTIDVFVANAGIAWSEGRSLEVKGYDAWQKIV

[0116] DYDLSSVYYCAKAVGKIFQEKGSGSLILTASMSGHIVNVPQMQAPYNAAKAAVIHLGKSLAVEWAPFARV

[0117] NTVSPGYIATDIGEFVLPDEKKKWWSLTPLGREGLPQELTGAYLYFASNASTYTTGADLVVDGGYTVP

[0118] SEQ ID No. 13:

[0119] ATGTCACACCCAACATCAGTTATTAACGAGCAGGTAGGCCCATTACCAACTAAGGCCCCACACACTTTC

[0120] TAAGAATGTGAATGATTTGTTCTCATTAAAGGGTAAGGTAGCTTCTGTCACCGGTTCCTCAGGAGGGGA

[0121] TTGGTTGGGCTGTAGCTGAAGCGTATGCCCAAGCAGGAGCAGACGTTGCTGTTTGGTACAATTCTAA

[0122] AAATGCAGATGCTAAGGCTGAATACTTAACTAAGACTTACGGTGTGAAGTCGAAGGCATACAAGTGT

[0123] AACATTTCTGATCCAGAAGACGTCGAAAAGGTAATTGGACAAATTGAAAAGGATTTCGGTACCATTG

[0124] ACGTTTTCGTTGCCAATGCCGGTGTTCCATGGACAGAAGGTAGAAGTATTGAAGTTGAAGGATATGA

[0125] TTCGTGGAAGAAGGTTATAGATTTGGACTTGAGTGGTGTCTACTATTGCGCTAAGGCTGTCGGAAAG

[0126] ATTTTCAAGAAGAATGGTAAGGGTTCGCTTGTGTTCACAGCATCAATGTCTGGCCACATTGTGAATGT

[0127] TCCACAATTGCAAGCTCCATACAACGCCGCAAAGGCCGGTGTTTTGCACTTGAGTAAGTCATTAGCTG

[0128] TCGAATGGGCCCCATTCGCTAGAGTTAACACCATTTCACCTGGTTATATTGCCACAGAAATTTCCGACT

[0129] TCGTTCCGGACGATGTCAAGGCTAAGTGGTGGCAATTAATTCCATTAGGAAGAGAAGCTCTTCCACA AGAATTAGTTGGTGCTTACTTATATTTTGCATCCGATGCGTCTACTTACACTACAGGATCGGACTTATT AGTCGATGGTGGTTACTCTGCTCCATAA

[0130] SEQ. ID Nr. 14:

[0131] MSHPTSVINEQVGPLPTKAPQLSKNVNDLFSLKGKVASVTGSSGGIGWAVAEAYAQAGADVAVWYNSK

[0132] NADAKAEYLTKTYGVKSKAYKCNISDPEDVEKVIGQIEKDFGTIDVFVANAGVPWTEGRSIEVEGYDSWKK

[0133] VIDLDLSGVYYCAKAVGKIFKKNGKGSLVFTASMSGHIVNVPQLQAPYNAAKAGVLHLSKSLAVEWAPFA

[0134] RVNTISPGYIATEISDFVPDDVKAKWWQLIPLGREALPQELVGAYLYFASDASTYTTGSDLLVDGGYSAP

[0135] SEQ ID Nr. 15:

[0136] ATGGGCAAGACTGTAGCAACTGGTATTGAAACCCCCCAACCTTATCCACAATTGCCTGAACATGTTAT

[0137] GGACATGTTTTCATTAAAAGGTAAGGTGGCCTCTGTGACTGGTGCTTCCGGTGGTATTGGTTATGAAG

[0138] TGGCCGTTGCATTTGCACAGGCAGGTGCCAATGTTGCTATGTGGTACAACTCGCATTCTGTCGAGGA

[0139] GGAAGCTGAAAAGCTGTCGAAGAAATACAACGTCACCGTTAAGGCTTATAAATGTTCATTGACTGAT

[0140] ACTAAGGCTGTTGAAGAAACTGTGCAACAGATTAAGAAGGACTTTGGTGGTAGAATTGATATAATGG

[0141] TGGCTAATGCTGGTGTGGCGTGGGATAAGGGTCCCTTGACCGAGCTGGCCGAAAAAGATTCTGAGCT

[0142] TTGTGACAAGGAATGGCAGAAAGTTTTGAGCATCGATATCAATGGTGTCTATAATGCAGCCAAGAGT

[0143] ATTGGTCCAATTTTCAAGAAGCAAGGATCAGGTTCTTTCATTGCAACTGGTTCGATGTCTGGTCACATT GCAAACGTTCCGCAATTACAGGTCGCATACAATACTGCAAAGGCTGCCGTTATTCATATGTGCAAATC

[0144] GCTGGCTGTCGAATGGACAGGTTACGCCCGTGCTAACACTGTATCCCCAGGTTACGTAGCCACTCCAT

[0145] TGAATGCTGGGATGGACGAAGAGATGCTAAAGAAATGGAACACCTTAGTCCCACTAGGTCGTCTCGC

[0146] TTTGCCCAAGGAAATGGTTGGTGCTTATTTGTACTTGGCCTCTAATGCCTCAACATACACGACCGGTA GCGACATACTAGTGGATGGTGGTTACTGTAGTGTTTAA

[0147] SEQ. ID Nr. 16:

[0148] MGKTVATGIETPQPYPQLPEHVMDM FSLKGKVASVTGASGGIGYEVAVAFAQAGANVAMWYNSHSVE

[0149] EEAEKLSKKYNVTVKAYKCSLTDTKAVEETVQQIKKDFGGRIDIMVANAGVAWDKGPLTELAEKDSELCDK

[0150] EWQKVLSIDINGVYNAAKSIGPIFKKQGSGSFIATGSMSGHIANVPQLQVAYNTAKAAVIHMCKSLAVEW

[0151] TGYARANTVSPGYVATPLNAGMDEEMLKKWNTLVPLGRLALPKEMVGAYLYLASNASTYTTGSDILVDG GYCSV

[0152] SEQ. ID Nr. 17:

[0153] ATGGTTGATAAGATTCTGGATAGGTTCAGTTTAAAAGGGAAAACCGCATTGGTGACAGGTGGTGGTC

[0154] AGGGTATTGGACGCGGATATGCTTTTGCGCTGGGAGAAGCCGGGGCCAAGGTAGCCATTGTTGATA

[0155] TCAATGGAGAAACTGCTGAAGCGACAGCCAAGGACCTGCAAGCAGCGGGGATTGCATCAATGTCCT

[0156] ATGTCTGTGATGTAACTAATCCCGATCAGGTGATGGCAATGGTGAAACATATTGTCAAAGAATGGGG

[0157] GACTTTGACTATAGGGGTTAACAACGCTGGCATGGGAATCTGGAGCGATGCAGTAACCATGCCCTAT

[0158] GAGATGTGGAGAAAGACCATGGCTTTAAACATGGATGGGATTTTTCTTTGTGCCAGGGAGGAGGCA

[0159] AAGTATATGATACGCGAGGGGTATGGGAAAATTATCAATACTGCTTCGATGAGCGCCCATATATCGA

[0160] ATACCCCACAGAATCAAGTAGCTTACAATGCATCCAAGGCGGGGGTTCTCCACATGACCAGATCTTTG

[0161] GCCGCAGAATGGGCTCCCTATGGCGTGAGGGTGAACTCCATCAGTCCCGGATATACAAGGACAGAG

[0162] CTGGTCGAAAAGCTGCTTGCAACTCCAGAGGGGAAAAAAATGGAATCAGACTGGTTGCAACTGATTC

[0163] CCCAAAAAAAGATGGCTACCGTCGAGGATTTGCAAGGCGCTTGCTTGTACCTTGCCTGCAGGGCTTC GGATTACACAACAGGTAGCGATATTCTCATAGATGGTGGGTATTGCTGTTGGTAA

[0164] SEQ ID Nr. 18:

[0165] MVDKILDRFSLKGKTALVTGGGQGIGRGYAFALGEAGAKVAIVDINGETAEATAKDLQAAGIASMSYVCD

[0166] VTNPDQVMAMVKHIVKEWGTLTIGVNNAGMGIWSDAVTMPYEMWRKTMALNM DGIFLCAREEAKY

[0167] M IREGYGKIINTASMSAHISNTPQNQVAYNASKAGVLHMTRSLAAEWAPYGVRVNSISPGYTRTELVEKL

[0168] LATPEGKKM ESDWLQLIPQKKMATVEDLQGACLYLACRASDYTTGSDILIDGGYCCW

[0169] SEQ ID Nr. 19: ATGGCAGAAAAAATTCTTGACAGATTCAAGCTGGACGGCAAGACCGCCCTGGTTACCGGAGGAGGG

[0170] CAGGGCATTGGCCAGGCCTACTGTTTCGCCCTGGGCGAAGCAGGCGCAAAGATAGCTGTGGTAGAC

[0171] ATCAACACCACCGTTGCGGAAGAGACCGCCCAGGCCCTGACAAAAAAAGGCATAGAGGCTATTGCG

[0172] ATAACCACAGATGTTACAAAAGAAGACGAAGTAATAAAGATGGTTAAAACCGTCATAGACAAATGGG GCTTCCTCACCATAGGCGTCAACAACGCCGGCATGGGTGTCTGGCGCGATGCGTTAACCCAGGACTTT GCCGAATGGCGGAAGATCCTCTCCCTCAACCTCGATTCGATCTTCCTCTGCTCTCGCACCGAAGCCGT

[0173] GGAAATGGCAAAGAAAGGCTATGGCAAGATCGTCAACACCGCTTCCATGTCCGCCCATATTTCCAAC ACCCCCCAGAACCAGGCAGCCTACAACAGCTCCAAAGCCGGTGTGCTCCATCTGACCCGCAGCCTCGC TGCCGAATGGGCGCCCAAAAATATCAGGGTAAACAGCATCTCCCCGGGTTATACAAAAACCGCCCTG

[0174] GTAGACAAGCTCCTCGAAACCCCGGAAGGCAAAACCATGCTGCCCAAGTGGCTGGAAAAAGTCCCCA

[0175] TGGGGCGCATGGCTACCGTGGAAGATCTGCAAGGCGCCGTTGTGTATCTGGCATCGCCCGCATCGGA TTATGCCACCGGCACAGATATTATCATTGACGGCGGATATTGCTGCTGGTAA

[0176] SEQ. ID Nr. 20:

[0177] MAEKILDRFKLDGKTALVTGGGQGIGQAYCFALGEAGAKIAVVDINTTVAEETAQALTKKGIEAIAITTDVT

[0178] KEDEVIKMVKTVIDKWGFLTIGVNNAGMGVWRDALTQDFAEWRKILSLNLDSIFLCSRTEAVEMAKKGY

[0179] GKIVNTASSMAHISNTPQNQAAYNSSKAGVLHLTRSLAAEWAPKNIRVNSISPGYTKTALVDKLLETPEGK TMLPKWLEKVPMGRMATVED LQG AVVYLAS PAS DYATGTD 111 DGG YCCW

[0180] SEQ. ID No. 21:

[0181] ATGGTTCTCTCTCAGCCCGAAAACAAGCATGTTATGAAAGCCTTCGATTTGACCGGCAAGGTCGCAGC

[0182] TGTGACAGGAGGAGCTCGCGGGATCGGTCTTGAGGTCTCCAGAGGGCTTGCAGAAGCTGGTGCGAA TGTTGCAGTCATCTACAGCTCGTCCAAGAATGCCGACGCCGTTGCAGCCGAGATCGCAGCTGCCAAC AACGTCAAGACAGCCGCATACAAGGCAGATGTAAGCACCAGGAGCAGATCGATCGATCCAGCCAG

[0183] CAGATAGCAAAGGACTTTGGAAAACTGGACATAATCGTGGCGAATTCTGGAATCGCAAGCACGCATC

[0184] CCGCTGAAGACTACACAGTGGAGGAGTGGAGAGATATCCTCAAAGTGAATCTAGATGGAGCGTTCTA

[0185] CACAGCGCAGGCGGCGGCAAGGATCTTCAAGACGCAAGGGCATGGAAACGTCATCTTCACGGCTTCT

[0186] GTCAGCGCAAGGCTGGTCAATGTCCCCCAGAAACAAGCTGCGTACAACGCCTCGAAAGCTGGTCTTG

[0187] TCCAGCTGGCAAAGTGCTTGTCAGTGGAATGGATAGACTATTGTCGTGTCAACTGCATTTCGCCTGGT

[0188] TTTATTGCGACAGAAATCTTGGATATCCCACCCCAAGGAGTGGAGAGAAAATGGCTCGACATGGTTC CCGCGCGTCGGATGGCTGCTACTTACGAGCTAAAGGGGGCGTATGTTTTCTGTGCTTCTGACGCGTCA AGCTACATGACCGGCGCTGATATAGTCATTGATGGTGGATATACGCTTCCGTAA

[0189] SEQ ID No. 22: MVLSQPENKHVMKAFDLTGKVAAVTGGARGIGLEVSRGLAEAGANVAVIYSSSKNADAVAAEIAAANNV KTAAYKADVSNQQDIESTIQQIAKDFGKLDIIVANSGIASTHPAEDYTVEEWRDILKVNLDGAFYTAQAA RIFKTQGHGNVIFTASVSARLVNVPQKQAAAYNASKAGLVQLAKCLSVEWIDYCRVNCISPGFIATEILDIHP KEWREKWLDMVPARRMAATYELKGAYVFCASDASSYMTGADIVIDGGYTLP

[0190] SEQ ID No. 23:

[0191] ATGGCTGCGTCTCCCATCACCAACGGCCTCTTCAACCATGACAACTCCACCCCGCCCGAGCATCCAG CCTGTTCGCGCTGTTCTCGCTCAAGGGCAAGACCGCCATCGTCACGGGCGCCGGTGCTGGCATCGGC CTGCATGTGGCTCATGGTCTGGCTGAGGCTGGCCACCATCCAACCAACCTT GACGCCGGAGCGCGCTGCCGAGATCGAGCAGCAGTATGGCGTGAAGGCCAAGGCATACCAGGTCG ACGTCCGCGACGCCAAAGCTCGAGGAGACGGTCAACCAGGCCGTGCGACCTCAACGGCCGCC TGGACATCTTCATCGCCA CCACTACCAGGCCGTCGTGCAGACCGACCTGGACGGCGTCTTCTACTCGGCCAAGGCGGCGGCAGCC CACTGGCGGCGCCAGAAGGAGGAGGGCACCGACCTGTTCGGCAACAAGCTGCAGAACTTCACCTAC GGCAGCTTCGTGGCCACGGCGTCGATGAGCGGCCATATC ACAACGCCGCCAAGGCGGGCGTCATCCACATGGTCAAATCGTTTGCCGTCGAATGGGCGCGCTTCGC CCGTGCCAACTCGGTCTCTTCCTGGCTACATCGCCACGGATCTCCAACTTCGTTCCCGGAGACCA AGAAGCTCTGGCGGGACAAGACGCCGCTGGCCGCCAGGGCCAGGGCCGAG TACCTGTACCTGGCCAGCGATGCGGCCAGCTTCACGACGGGCGCGGACCTGGTGGTCGACGGAGGC TACACCCTGCCTTAA

[0192] SEQ. ID No. 24:

[0193] MAASPITNGLFNHDNSTPPEHPSLFALFSLKGKTAIVTGAGAGIGLHVAHGLAEAGANVALFYNTNTKTPE RAAEIEQQYGVKAKAYQVDVRDAKKLEETVNQAVRDLNGRLDIFIANAGIPWTKGPSVDGPLDHYQAVV QTDLDGVFYSAKAAAAHWRRQKEEGTDLFGNKLQNFTYGSFVATASMSGHIVNIPQLQAAYNAAKAGVI HMVKSFAVEWARFARANSVSPGYIATEISNFVPAETKKLWRDKTPLGREGLPQELKGAYLYLASDAASFTT GADLVVDGGYTLP

[0194] It has surprisingly been shown that an oxidoreductase having one of the amino acid sequences SEQ ID No. 2, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18, SEQ ID No. 20 is not only capable of reducing D-psicose to D-talitol, which reaction can preferably take place in the presence of NAD(P)H, but also of oxidizing D-talitol to D-psicose, which reaction can preferably take place in the presence of NAD(P) +can take place. The oxidoreductase for forming D-talitol from D-psicose or D-psicose from D-talitol preferably comprises an amino acid sequence which has an identity to SEQ ID No. 2, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18, SEQ ID No. 20, SEQ ID No. 22 or SEQ ID No. 24 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 oxidoreductase according to the invention for the formation of D-talitol from D-psicose comprises or consists of the amino acid sequence SEQ ID No. 2, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18 or SEQ ID No. 20.

[0195] Alternatively, the oxidoreductase for forming D-talitol from D-psicose or D-psicose from D-talitol preferably comprises an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 1, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 21 or SEQ ID No. 23 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 the formation of D-talitol from D-psicose comprises or consists of the nucleic acid sequence SEQ ID No. 1, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17 or SEQ ID No. 19.

[0196] 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.

[0197] 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 one 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 IVIatrix (Henikoff & Henikoff, 1989), alignments (B) of 50, expectation (E) of 10, M = 5, N = -4.

[0198] Alternatively, the oxidoreductase for forming D-talitol from D-psicose or D-psicose from D-talitol preferably comprises 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. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 21 or SEQ ID No. 23. As used herein, stringent conditions refer to conditions under which so-called specific hybrids, but not non-specific hybrids, are formed. For example, stringent conditions include hybridization in 6xSSC (sodium chloride / sodium citrate) at 45°C and then washing with 0.2 to 1xSSC, 0.1% SDS at 50 to 65°C; or such conditions may include hybridization in 1xSSC at 65 to 70°C and then washing with 0.3xSSC at 65 to 70°C.Hybridization can be 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).

[0199] A further aspect of the present invention relates to a process for the preparation of D-talitol or D-psicose, comprising the step of treating D-psicose or D-talitol with an oxidoreductase which comprises an amino acid sequence selected from the group consisting of: i) an amino acid sequence which has an identity to SEQ ID No. 2, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18 or 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. 1, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17 or SEQ ID No. 19 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid which anneals under stringent conditions to a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 1, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No.11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17 or SEQ ID NO: 19. A further aspect of the present invention relates to a process for the preparation of D-talitol comprising the step of treating D-psicose with an oxidoreductase which comprises an amino acid sequence selected from the group consisting of: i) an amino acid sequence which has an identity to SEQ ID NO: 22 or SEQ ID NO: 24 of at least 80%, ii) an amino acid sequence which is encoded by a nucleic acid which has an identity to SEQ ID NO: 21 or SEQ ID NO: 23 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: 21 or SEQ ID NO: 23.

[0200] Yet another aspect of the present invention relates to the use of an oxidoreductase for the reduction of D-psicose to D-talitol and / or for the oxidation of D-talitol to D-psicose comprising an amino acid sequence selected from the group consisting of: i) an amino acid sequence having identity to SEQ ID No. 2, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18 or SEQ ID No.

[0201] 20 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 1, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17 or SEQ ID No. 19 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid which anneals under stringent conditions to a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 1, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No.

[0202] 15, SEQ ID No. 17 or SEQ ID No. 19.

[0203] A further aspect of the present invention relates to the use of an oxidoreductase for reducing D-psicose to D-talitol comprising an amino acid sequence selected from the group consisting of: i) an amino acid sequence having an identity to SEQ ID No. 22 or SEQ ID No. 24 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 21 or SEQ ID No. 23 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. 21 or SEQ ID No. 23. The NAD(P) +-dependent oxidoreductase for the oxidation of D-talitol to D-tagatose preferably comprises an amino acid sequence selected from the group consisting of: i) an amino acid sequence having an identity to SEQ ID No. 4 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 3 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.

[0204] SEQ ID No. 3:

[0205] ATGGCTGCCAACGTCCCCAAGACCATGAAGGCTCTCAGATATGAGAAGCCTGAGACGTTCTCCATCGT CGACATTCCCGTTCCCACTCTGCGTGAGAACGATGTCTTGATCAAGGTCAAGGCTTGCGGTGTCTGTG GTACCGACCTGCACATTCACGAGGGAGAATTCCTTGCCAAGTTCCCTCTCGTTCCTGGCCACGAGACT GTCGGTGTTGTTGCCGCAGTTGGACCCAAGGTCAAGGGTTTCGAGATCGGTGACCGTGTTGTTGCCG ACAACTCCGAGCTTTGCGGCCAATGCTTCTACTGCCGACGAGGAGAGGAGTTGCTCTGCGAGCACTT CGAAGCTCACGGTGTCACGATGAACGGCGGTTTCGCTGAGTACTGCGCCTACCCTGCCGGCCGTGTC TTCAAGATCAAGAACCTCTCTGACGTGGACGCCACTCTGCTTGAGCCCGCGTCCTGCGCCGCTCACGG TCTGGACAAGATTGCCCCCAAGATGGGCTCGTCCGTCCTGGTGTTCGGCGCCGGTCCCACCGGTCTG GTCCTTGCTCAGATGCTCCGTCTGAACGGAGGATGCCGCGTCGTCGTCGCTGCGCCCGAGGGTCTGA AGATGGACCTGGCCCAGAAGCTCGGCGCTGGTGATGAATACGTTGCTCTTTCTCGCACGAACCCTCA GGCTCAGTTTGACAAGCTGAAGGCCGACAACCCGTACGGCTTCGACATTGTCGTCGAGGCTACCGGC AATGCCAAGATCCTGGAGGATGCCATCAACTATGTCCGCCGTGGAGGCAAACTGGTCGTGTACGGTG TGTACGCGAACAAGGACCGCGTCTCGTGGCCCCCGAGCAAGATCTTCGGTGACGAAATCACCATTCT GGGTAGCTTCTCCGAGACCTACAAGTTCCCCGCCGCCATCGACTACCTGGACTCCGGCAAGGTGAAGGTCCAGGGCATCGTGAACAAGACCTTCCGGCTGGAGCAGTGGGAGGAGTGTCTGGCGTCGTTGAAG AACAAGAGCGCCATCAAGGCGGCGATCGTCTTTGACTAA

[0206] SEQ ID No. 4:

[0207] MAANVPKTMKALRYEKPETFSIVDIPVPTLRENDVLIKVKACGVCGTDLHIHEGEFLAKFPLVPGHETVGV VAAVGPKVKGFEIGDRVVADNSELCGQCFYCRRGEELLCEHFEAHGVTM NGGFAEYCAYPAGRVFKIKNL SDVDATLLEPASCAAHGLDKIAPKMGSSVLVFGAGPTGLVLAQMLRLNGGCRVVVAAPEGLKMDLAQKL GAGDEYVALSRTNPQAQFDKLKADNPYGFDIVVEATGNAKILEDAINYVRRGGKLVVYGVYANKDRVSW PPSKIFGDEITILGSFSETYKFPAAIDYLDSGKVKVQGIVNKTFRLEQWEECLASLKNKSAIKAAIVFD It has surprisingly been shown that an oxidoreductase with one of the above-mentioned amino acid sequences is able to oxidize D-talitol to D-tagatose, wherein the reaction preferably takes place in the presence of NAD(P) + can take place.

[0208] The oxidoreductase for forming D-tagatose from D-talitol preferably comprises an amino acid sequence that has an identity to SEQ ID No. 4 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 oxidoreductase according to the invention for forming D-tagatose from D-talitol comprises or consists of the amino acid sequence SEQ ID No. 4.

[0209] Alternatively, the oxidoreductase for forming D-tagatose from D-talitol preferably comprises an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 3 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 forming D-tagatose from D-talitol comprises or consists of the nucleic acid sequence SEQ ID No. 3.

[0210] Alternatively, the oxidoreductase for forming D-tagatose from D-talitol preferably comprises an amino acid sequence encoded by a nucleic acid that binds under stringent conditions (see definition above) to a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 3.

[0211] A further aspect of the present invention relates to a process for the preparation of D-tagatose comprising the step of treating D-talitol with an oxidoreductase comprising an amino acid sequence selected from the group consisting of: i) an amino acid sequence having an identity to SEQ ID No. 4 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 3 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.

[0212] Yet another aspect of the present invention relates to the use of oxidoreductase for the oxidation of D-talitol to D-tagatose comprising an amino acid sequence selected from the group consisting of: i) an amino acid sequence having an identity to SEQ ID No. 4 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 3 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.

[0213] 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).

[0214] The NAD(P) formed during the reduction of D-psicose to D-talitol +is reduced to NAD(P)H using formate and a formate dehydrogenase with the formation of CO2 (cofactor regeneration).

[0215] Particularly preferably, a formate dehydrogenase is used which comprises or consists of the amino acid sequence SEQ ID No. 26, or a functional fragment of this formate dehydrogenase. The preferably used formate dehydrogenase is preferably encoded by the nucleic acid sequence SEQ ID No. 25. 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. 26, 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.

[0216] SEQ ID No. 25:

[0217] ATGGCGAAAATACTTTGCGTTCTCTATGACGATCCGGTCGACGGCTACCCGAAGACCTATGCGCGCG ACGACCTGCCGAAGATCGACCACTATCCGGGCGGGCAGACGCTGCCCACGCCCAAGGCGATCGACTT CACGCCGGGCGCGCTGCTCGGCTCGGTCTCCGGCGAGCTCGGCCTGCGCAAATACCTGGAAGCCAAC GGCCATACCTTCGTCGTCACCTCCGATAAGGACGGCCCGGATTCGGTGTTCGAGAGGGAACTCGTCG ACGCCGACGTGGTGATCTCGCAGCCCTTCTGGCCGGCCTATCTGACGCCCGAGCGCATCGCCAAGGC GAAGAACCTGAAGCTCGCGCTCACCGCCGGCATCGGCTCCGATCATGTCGATCTTCAGTCAGCTATCG ACCGTGGCATCACTGTGGCCGAAGTCACATATTGCAACTCGATCAGCGTCGCCGAGCACGTGGTGAT GATGATCCTCGGCCTGGTACGAAACTACATTCCCTCGCATGACTGGGCGCGCAAGGGCGGCTGGAAC ATAGCCGACTGCGTAGAGCACTCCTACGACCTCGAGGGCATGACCGTCGGCTCGGTGGCCGCCGGCC GCATCGGCCTCGCCGTGCTGCGCCGCCTCGCGCCGTTCGACGTGAAGCTGCACTATACCGACCGCCA CCGTCTGCCAGAAGCGGTCGAGAAGGAGCTGGGCCTCGTCTGGCACGATACCCGCGAGGACATGTA CCCGCATTGCGACGTGGTCACGCTCAACGTGCCGCTGCACCCCGAAACCGAGCACATGATCAATGAC GAGACGCTGAAGCTGTTCAAGCGCGGCGCCTATATCGTCAACACCGCCCGCGGCAAGCTCGCCGACC GCGACGCCATCGTCCGCGCGATCGAGAGCGGGCAGCTCGCGGGCTATGCCGGCGACGTGTGGTTCCCGCAGCCGGCTCCGAAGGACCACCCCTGGCGCACCATGAAGTGGGAAGGCATGACGCCGCACATCT CCGGCACCTCGCTCTCTGCCCAGGCGCGCTACGCGGCGGGCACGCGCGAGATCCTCGAATGCTTCTT CGAGGGCCGGCCGATCCGCGACGAGTACCTGATCGTGCAGGGCGGCGCCGCCGGCACCGGCGC GCATTCCTACTCGAAGGGCAATGCGACCGGCGTTCGGAAGGGCCGCGAAGTTCAAAAGGCTGG CTGA

[0218] SEQ. ID Nr. 26:

[0219] MAKILCVLYDDPVDGYPKTYARDDLPKIDHYPGGQTLPTPKAIDFTPGALLGSVSGELGLRKYLEANGHTFV VTSDKDGPDSVFERELVDADVVISQPFWPAYLTPERIAKAKNLKLALTAGIGSDHVDLQSAIDRGITVAEVT YCNSISVAEHVVMM ILGLVRNYIPSHDWARKGGWNIADCVEHSYDLEGMTVGSVAAGRIGLAVLRRLAP FDVKLHYTDRHRLPEAVEKELGLVWHDTREDMYPHCDVVTLNVPLHPETEHM INDETLKLFKRGAYIVNT ARGKLADRDAIVRAIESGQLAGYAGDVWFPQPAPKDHPWRTMKWEGMTPHISGTSLSAQARYAAGTR EILECFFEGRPIRDEYLIVQGGALAGTGAHSYSKGNATGGSEEAAKFKKAG

[0220] 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. 26 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 25 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. 25.

[0221] The formate dehydrogenase preferably comprises an amino acid sequence having an identity to SEQ ID No. 26 of at least 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%, in particular 100%.

[0222] Alternatively, the formate dehydrogenase preferably comprises an amino acid sequence defined by a

[0223] Nucleic acid is encoded which has an identity to SEQ ID No. 25 of at least 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%, in particular 100%.

[0224] The NAD(P)H oxidase used for cofactor regeneration 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.

[0225] 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. 28, SEQ ID No. 30 or SEQ ID No. 32 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 11, SEQ ID No. 29 or SEQ ID No. 31 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. 27, SEQ ID No. 29 or SEQ ID No. 31.

[0226] SEQ ID No. 27:

[0227] ATGAAAGTAGTAGTAGTAGGCTGTACACATGCAGGAACAGCGGCAGTTAAGACGATTTTAAATGAAC ATCCAGATGCATCAGTATCAGTATATGAGCGTAATGACAATGTCTCATTTCTATCTTGTGGGATTGCGT TGTATGTTGGTGGAGTTGTGAAAGATCCTGCAGGTTTGTTTTATTCAAGTCCAGAAGAACTTGCATCA ATGGGCGCGAAAATTAACATGGAACACAATGTGAAAAATATAGATAATGAGAATAAGGTCGTAGTA ATTGAGAATTTAAAAACAGGCGAAACATTTGAAGAAAGCTATGATAAGTTGGTAATGACAACTGGAT CATGGCCAATTATTCCTCCAATTGATGGAATCAATAGTGAAAATATTCTTTTGTGTAAAAACTATAACC AAGCAAATGAAATTATTAAAGAATCAAAAAATGCTAAAAAGATTGTCATTGTTGGTGGTGGCTATATT GGAATTGAATTAGTTGAGGCATTTGCAGAATCTGGCAAGCAAGTGACGCTAGTTGATGGATTAGATC GTATTTTAAACAAATATTTAGATGCTGAATTCACTTCTGTTTTAGAGCATGATTTACAAGAAAGAGGC GTTACGCTAGCTTAAACCAAACCGTCGAGAAATTTGTTGCCAATGAATCAGGTGCTGTGACAGCTGT GAAAACACCAGTTGGAGAATATGAGGCTGATTTAGTTATTTTATGTGTTGGATTTAAACCAAATACTG ATTTGTTGAAGGATAAAGTAGAGATGTTGCCAAATGGTGCCATCGTAGTGGATGAATATATGAGAAC AAGCGATGAAGCGATTTTTGCTGCTGGCGATAGTTGCGCGGTTCATTATAATCCAACTGGAGGCTCTG CGTATATTCCGTTAGCTACAAATGCAGTTAGAATGGGAGCTTTAGTTGGGAAAAATATTGTTTCTCCAACAGTTAAATATCGTGGCACGCAAGCAACTTCTGGTTTATATTTATTTGGTTTTAATATAGGTTCAACC GGATTGACTGAAAATAGCGCTCCTCATTTTGGCGTAGAGGTTCGTTCAGTAGTTGTAGAAGATAATTA TCGTCCAGAGTTTATGCCGACAACAGAGAAAGTAACGATGAAATTAGTTTATGAAGTAGGAACGAAT CGGATTGTTGGAGGTCAAATCATGTCAAAATATGATGTGACACAATCTGCCAATACGTTATCTTTATG

[0228] TGTTCAAAATAAAATGACGATTGAGGATTTGGCTTATGTAGATTTCTTCTTCCAACCTCACTTTGATCG

[0229] TCCTTGGAACTATTTAAATATTTTAGCGCAAGCAGCTGTTGAGCAAGAGCGTAAACTAGCAAAATAA

[0230] SEQ. ID No. 28:

[0231] M KVVVVGCTHAGTAAVKTILNEHPDASVSVYERNDNVSFLSCGIALYVGGVVKDPAGLFYSSPEELASMG

[0232] AKINMEHNVKNIDNENKVVVIENLKTGETFEESYDKLVMTTGSWPIIPPIDGINSENILLCKNYNQANEIIKE

[0233] SKNAKKMVGGGYIGIELVEAFAESGKQVTLVDGLDRILNKYLDAEFTSVLEHDLQERGVTLALNQTVEKFV

[0234] ANESGAVTAVKTPVGEYEADLVILCVGFKPNTDLLKDKVEMLPNGAIVVDEYM RTSDEAIFAAGDSCAVH

[0235] YNPTGGSAYIPLATNAVRMGALVGKNIVSPTVKYRGTQATSGLYLFGFNIGSTGLTENSAPHFGVEVRSVV

[0236] VEDNYRPEFM PTTEKVTMKLVYEVGTNRIVGGQIMSKYDVTQSANTLSLCVQNKMTIEDLAYVDFFFQP

[0237] HFDRPWNYLNILAQAAVEQERKLAK

[0238] SEQ. ID Nr. 29:

[0239] ATGAGCAAAATTGTTATCGTGGGTGCAAATCATGCAGGCACCGCAGCAATTAATACCATTCTGGATAA

[0240] TTATGGCAGCGAAAATGAAGTGGTTGTGTTTGATCAGAATAGCAACATTAGCTTTCTGGGTTGTGGTA

[0241] TGGCACTGTGGATTGGTAAACAAATTAGCGGTCCGCAGGGTCTGTTTTATGCAGATAAAGAAAGCCT

[0242] GGAAGCAAAAGGTGCCAAAATCTATATGGAAAGTCCGGTTACCGCCATTGATTATGATGCAAAACGT

[0243] GTTACCGCACTGGTTAATGGTCAAGAACATGTTGAAAGCTACGAGAAACTGATTCTGGCAACCGGTA

[0244] GCACCCCGATTCTGCCTCCGATTAAAGGTGCAGCCATTAAAGAAGGTAGTCGCGATTTTGAAGCAACC

[0245] CTGAAAAATCTGCAGTTCGTGAAACTGTATCAGAATGCCGAAGATGTGATTAACAAACTGCAGGATA

[0246] AAAGCCAGAATCTGAATCGTATTGCAGTTGTTGGTGCAGGTTATATTGGTGTTGAACTGGCAGAAGC

[0247] ATTTAAACGTCTGGGTAAAGAAGTGATTCTGATTGACGTTGTTGATACCTGTCTGGCAGGTTATTATG

[0248] ATCAGGATCTGAGCGAAATGATGCGTCAGAATCTGGAAGATCATGGTATCGAACTGGCATTTGGTGA

[0249] AACCGTTAAAGCAATTGAAGGTGATGGTAAAGTGGAACGTATTGTTACCGATAAAGCAAGCCATGAT

[0250] GTGGATATGGTTATTCTGGCAGTTGGTTTTCGTCCGAATACAGCACTGGGTAATGCAAAACTGAAAAC

[0251] CTTTCGTAATGGTGCCTTTCTGGTGGATAAAAAACAAGAAACCAGCATCCCGGATGTTTATGCAATTG

[0252] GTGATTGTGCAACCGTGTATGATAATGCCATTAACGACACCAACTATATTGCACTGGCAAGCAATGCA

[0253] CTGCGTAGCGGTATTGTTGCAGGTCATAATGCAGCCGGTCATAAACTGGAAAGTCTGGGTGTTCAGG

[0254] GTAGCAATGGTATTTCAATTTTTGGCCTGAATATGGTTAGCACCGGTCTGACCCAAGAAAAAGCCAAA

[0255] CGTTTTGGTTATAATCCGGAAGTTACCGCCTTTACCGATTTTCAGAAAGCCAGCTTTATCGAGCATGAT

[0256] AACTATCCGGTTACGCTGAAAATTGTGTATGACAAAGATAGCCGTCTGGTTCTGGGTGCACAGATGG

[0257] CCAGCAAAGAAGATATGAGCATGGGTATTCACATGTTTAGCCTGGCCATTCAAGAGAAAGTTACCATT

[0258] GAACGTCTGGCCCTGCTGGATTATTTCTTTCTGCCGCATTTTAATCAGCCGTACAACTATATGACCAAA

[0259] GCAGCACTGAAAGCCAAATAA SEQ ID NO. 30:

[0260] MSKIVIVANGHAGTAAINTILDYGSENEVVVFDQNSNISFLGCGMALWIGKQISGPQGLFYADKESLEAK

[0261] GAKIYMESPVTAIDYDAKRVTALVNGQEHVESYEKLILATGSTPILPPIKGAAIKEGSRDFEATLKNLQFVKLY

[0262] QNAEDVINKLQDKSQNLNRIAVVGAGYIGVELAEAFKRLGKEVILIDVVDTCLAGYYDQDLSEMMRQNLE

[0263] DHGIELAFGETVKAIEGDGKVERIVTDKASHDVDMVILAVGFRPNTALGNAKLKTFRNGAFLVDKKQETSI

[0264] PDVYAIGDCATVYDNAINDTNYIALASNALRSGIVAGHNAAGHKLESLGVQGSNGISIFGLNMVSTGLTQE

[0265] KAKRFGYNPEVTAFTDFQKASFIEHDNYPVTLKIVYDKDSRLVLGAQMASKEDMSMGIHMFSLAIQEKVTI

[0266] ERLALLDYFFLPHFNQPYNYMTKAALKAK

[0267] SEQ ID No. 31:

[0268] ATGAAAGTAGTAGTAGTAGGCTGTACACATGCAGGAACAGCGGCAGTTAAGACGATTTTAAATGAAC

[0269] ATCCAGATGCATCAGTATCAGTATATGAGCGTAATGACAATGTCTCATTTCTATCTTGTGGGATTGCGT

[0270] TGTATGTTTGGTGGAGTTGTGAAAGATCCTGCAGGTTTGTTTTTATTCAAGTCCAGAAGAACTTGCATCA

[0271] ATGGGCGCGAAAATTAACATGGAACACAATGTGAAAAATATAGATAATGAGAATAAGGTCGTAGTA

[0272] ATTGAGAATTTAAAAACAGGCGAAACATTTGAAGAAAGCTATGATAAGTTGGTAATGACAACTGGAT

[0273] CATGGCCAATTATTCCTCCAATTGATGGAATCAATAGTGAAAATATTCTTTTGTGTAAAAACTATAACC

[0274] AAGCAAATGAAATTATTAAAGAATCAAAAAATGCTAAAAAGATTGTCATTGTTGGTGGTGGCTATATT

[0275] GCGATTGAATTAGTTGAGGCATTTGCAGAATCTGGCAAGCAAGTGACGCTAGTTGCGCGTAGCGATC

[0276] GTATTTTACGTAAATATTTAGATGCTGAATTCACTTCTGTTTTAGAGCATGATTTACAAGAAAGAGGCG

[0277] TTACGCTAGCTTTAAACCAAACCGTCGAGAAATTTGTTGCCAATGAATCAGGTGCTGTGACAGCTGTG

[0278] AAAACACCAGTTGGAGAATATGAGGCTGATTTAGTTATTTTATGTGTTGGATTTAAACCAAATACTGA

[0279] TTTGTTGAAGGATAAAGTAGAGATGTTGCCAAATGGTGCCATCGTAGTGGATGAATATATGAGAACA

[0280] AGCGATGAAGCGATTTTTGCTGCTGGCGATAGTTGCGCGGTTCATTATATCAACTGGAGGCTCTGC

[0281] GTATATTCCGTTAGCTACAAATGCAGTTAGAATGGGAGCTTTAGTTGGGAAAAATATTGTTTCTCCAA

[0282] CAGTTAAATATCGTGGCACGCAAGCAACTTCTGGTTTATATTTTTGGTTTTAATATAGGTTCAACCG

[0283] GATTGACTGAAAATAGCGCTCCTCATTTTGGCGTAGAGGTTCGTTCAGTAGTTGTAGAAGATAATTTAT

[0284] CGTCCAGAGTTTATGCCGACAACAGAGAAAGTAACGATGAAATTAGTTTGAAGTAGGAACGAATC

[0285] GGATTGTTGGAGGTCAAATCATGTCAAAATATGATGTGACACAATCTGCCAATACGTTATCTTTATGT

[0286] GTTCAAAATAAAATGACGATTGAGGATTTGGCTTATGTAGATTTCTTCTTCCAACCTCACTTTGATCGT

[0287] CCTTGGAACTATTTAATATTTTAGCGCAAGCAGCTGTTGAGCAAGAGCGTAAACTAGCAAAATAA

[0288] SE ID Nr. 32:

[0289] M KVVVVGCTHAGTAAVKTILNEHPDASVSVYERNDNVSFLSCGIALYVGGVVKDPAGLFYSSPEELASMG

[0290] AKINMEHNVKNIDNENKVVVIENLKTGETFEESYDKLVMTTGSWPIIPPIDGINSENILLCKNYNQANEIIKE SKNAKKIVIVGGGYIAIELVEAFAESGKQVTLVARSDRILRKYLDAEFTSVLEHDLQERGVTLALNQTVEKFV ANESGAVTAVKTPVGEYEADLVILCVGFKPNTDLLKDKVEMLPNGAIVVDEYM RTSDEAIFAAGDSCAVH YNPTGGSAYIPLATNAVRMGALVGKNIVSPTVKYRGTQATSGLYLFGFNIGSTGLTENSAPHFGVEVRSVV VEDNYRPEFM PTTEKVTMKLVYEVGTNRIVGGQIMSKYDVTQSANTLSLCVQNKMTIEDLAYVDFFFQP HFDRPWNYLNILAQAAVEQERKLAK

[0291] The preferably used F O-forming NAD(P)H oxidase preferably comprises or consists of an amino acid sequence that has an identity to SEQ ID No. 28, SEQ ID No. 30 or SEQ ID No. 32 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. 28, SEQ ID No. 30 or SEQ ID No. 32.

[0292] 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. 11, SEQ ID No. 29 or SEQ ID No. 31 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. 11, SEQ ID No. 29 or SEQ ID No. 31.

[0293] 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. 28, SEQ ID No. 30 or SEQ ID No. 32 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 11, SEQ ID No. 29 or SEQ ID No. 31 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. 27, SEQ ID No. 29 or SEQ ID No. 31.

[0294] Materials

[0295] D-Tagatose and D-Talitol were purchased from TCI, D-Psicose was purchased from TCI and Hunan Garden Naturals Inc. (China), D-Fructose, NADPH tetrasodium salt, acetone, 2-propanol and methanol were purchased from PanReac AppliChem (ITW Reagents), zinc chloride, IPTG (isopropyl-ß-D-thiogalactopyranoside) were purchased from Sigma-Aldrich, magnesium chloride hexahydrate, potassium dihydrogen phosphate, di-potassium hydrogen phosphate, NAD + , NADH disodium salt, NADP + Disodium salt and sodium dodecyl sulfate (SDS) were purchased from Carl Roth, sodium formate was purchased from Fluka, and triethanolamine (TEA) was purchased from Chem-Lab NV.

[0296] Production of enzymes & preparation of lysates

[0297] General information on the expression of recombinant enzymes in E. coli

[0298] 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.

[0299] 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.

[0300] 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.

[0301] 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).

[0302] Preparation of cell lysates using sonifier disruption

[0303] 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.

[0304] 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).

[0305] 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.

[0306] Table 1. Enzyme classes and donor organisms for the enzymes used in the examples.

[0307] Analytical methods

[0308] High Performance Liquid Chromatography

[0309] An Agilent HPLC 1260 Infinity II Series system was used to quantify D-psicose, D-tagatose, and D-fructose, as well as the intermediate D-talitol, 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.

[0310] Determination of enzyme activities (optical-enzymatic assay)

[0311] Enzyme activities in the lysates were determined using a Shimadzu UV-1900 spectrophotometer. The formation and consumption of NAD(P)H were 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 NAD(P)H 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).

[0312] 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. Example 1

[0313] Production of D-talitol from D-fructose - cofactor regeneration with alcohol dehydrogenase

[0314] 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.

[0315] Initially, 50 ml of a D-fructose solution (500 g / l), 238.7 ml of deionized water, and 89 ml of a 200 mM TEA-HCl buffer (pH 7.5) were placed in the reactor and heated to 35 °C while stirring.

[0316] To start the reaction, 25 ml of D-psicose-3-epimerase lysate was added. Then, 35 ml of oxidoreductase I lysate, 8 kJ of alcohol dehydrogenase I lysate, 10 ml of 10 mM NAD + -solution and 40 ml of 2-propanol.

[0317] 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).

[0318] After 24 h and 42 h of running time, 15 ml of 2-propanol were added.

[0319] After 67 h, 98.0% of the D-fructose was converted to D-talitol (found concentration: 45.1 g / l).

[0320] The reactor contents were then heated to 70 °C with stirring for 30 min. The hot mixture was filtered through a pleated filter (Schleicher & Schuell 595%, 185 mm). The filtrate was stirred with activated carbon for 30 min at 50 °C, after which the activated carbon was filtered off (pleated filter Schleicher & Schuell 595%, 185 mm). The colorless solution was treated with a mixed-bed ion exchange resin (Amberlite MB20) for 30 min at 50 °C, which was then removed by filtration (pleated filter Schleicher & Schuell 595%, 185 mm). The solution was concentrated on a rotary evaporator (60 °C, 150-60 mbar) to a concentration of approximately 600 g / l D-talitol. The resulting syrup was incubated overnight at 4 °C in a refrigerator. Since no crystal formation was observed, a seed crystal was added and the syrup was again stored overnight in the refrigerator at 4 °C.The syrup was mixed with the same volume of IPA and stored in a freezer at -20 °C to initiate crystallization. The colorless crystals were filtered through a glass frit (P4, 10-16 pm) after applying a vacuum and dried for 24 h at 50 °C in a vacuum oven. HPLC analysis of the crystals (50% yield) revealed that the product was D-talitol (94% purity).

[0321] Example 2

[0322] Production of D-talitol from D-fructose - cofactor regeneration with formate dehydrogenase

[0323] 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.

[0324] Initially, 17.5 g of D-fructose, 125 ml of deionized water and 4.2 ml of a 500 mM TEA-HCl buffer (pH 7.5) were placed in the reactor and brought to 35 °C while stirring.

[0325] To start the reaction, 17.5 ml of D-psicose-3-epimerase lysate was added. Then, 4.7 kJ of oxidoreductase I lysate, 5.5 kJ of formate dehydrogenase lysate, 3.5 ml of 10 mM NAD + solution and 45.5 ml of a 5 M sodium formate solution. Air was bubbled into the reaction mixture to remove the CO2 produced by the formate dehydrogenase.

[0326] 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).

[0327] After 26 h, 99% of the D-fructose (50 g / l) was converted to D-talitol (found concentration: 34.4 g / l).

[0328] The reactor contents were then heated to 70 °C with stirring for 30 min. The hot mixture was filtered through a pleated filter (Schleicher & Schuell 595%, 185 mm). The filtrate was stirred with activated carbon for 30 min at 50 °C, after which the activated carbon was filtered off (pleated filter Schleicher & Schuell 595%, 185 mm). The colorless solution was treated with a mixed-bed ion exchange resin (Amberlite MB20) for 30 min at 50 °C, which was then removed by filtration (pleated filter Schleicher & Schuell 595%, 185 mm). The solution was concentrated on a rotary evaporator (60 °C, 150-60 mbar) to a concentration of approximately 600 g / l D-talitol. The resulting syrup was incubated overnight at 4 °C in a refrigerator. Since no crystal formation was observed, a seed crystal was added and the syrup was again stored overnight in the refrigerator at 4 °C.The syrup was mixed with the same volume of IPA and stored in a freezer at -20 °C to initiate crystallization. The colorless crystals were filtered through a glass frit (P4, 10-16 pm) after applying a vacuum and dried for 24 h at 50 °C in a vacuum oven. HPLC analysis of the crystals (50% yield) revealed that the product was D-talitol (94% purity).

[0329] Example 3

[0330] Oxidation of D-talitol to D-tagatose (cofactor regeneration with NAD(P)H oxidase)

[0331] The following components were mixed in a 2 ml glass vial: 147.2 μl of deionized water, 125 μl of a 400 mM TEA-HCl buffer (pH 8.5), 150 μl of a D-talitol solution (168 g / l), 35 μl of oxidoreductase II lysate, 10 U of NAD(P)H oxidase lysate and 5 μl of a 5 mM NADP + -solution. The mixture was incubated in an Eppendorf Thermomixer with continuous shaking (24 °C, 800 rpm) for 20 h.

[0332] 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).

[0333] In this way, 81.7% of the D-talitol was converted to D-tagatose (found concentration: 47.7 g / l).

[0334] Example 4

[0335] Oxidation of D-talitol to D-tagatose (cofactor regeneration with alcohol dehydrogenase)

[0336] The reaction was carried out in a Labfors 5 benchtop bioreactor (Infors AG). A glass reactor (volume 3.4 l) with an attached stirrer and pH electrode was used as the vessel. pH control was achieved by adding 1M NaOH or 1M H2SO4. Initially, 43.8 ml of a D-talitol solution (571 g / l), 379.5 ml of deionized water, and 43.4 ml of a 500 mM TEA-HCl buffer (pH 8) were placed in the reactor and brought to 30 °C with stirring.

[0337] To start the reaction, 25 ml of oxidoreductase II lysate, 12 ml of alcohol dehydrogenase II lysate, and 25 ml of acetone were added. Additionally, overpressure was applied to minimize evaporation losses.

[0338] 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).

[0339] After 14 h, 25 ml of oxidoreductase II-lysate, 12 kJ of alcohol dehydrogenase II-lysate and 5 ml of a 10 mM NADP + -solution introduced.

[0340] After 34 h, 43% of the D-talitol was converted to D-tagatose (found concentration: 18.1 g / l).

[0341] Example 5

[0342] Production of D-tagatose from D-fructose (one-pot process)

[0343] The reaction was carried out in a Labfors 5 benchtop bioreactor (Infors AG). A glass reactor (volume 3.4 l) with a stirrer, pH electrode, and O2 sensor was used as the vessel. pH control was achieved by adding 1M NaOH or 1M H2SO4.

[0344] Initially, 50 ml of a D-fructose solution (500 g / l), 238.8 ml of deionized water, and 89 ml of a 200 mM TEA-HCl buffer (pH 7.5) were placed in the reactor and heated to 35 °C while stirring.

[0345] To start the reaction, 25 ml of D-psicose-3-epimerase lysate was added. Then, 35 ml of oxidoreductase I lysate, 8 kU of alcohol dehydrogenase I lysate, 10 ml of 10 mM NAD + -solution and 40 ml of 2-propanol.

[0346] 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).

[0347] After 24 h of operation, 15 ml of 2-propanol was added.

[0348] After 47 h, 94.0% of the D-fructose was converted to D-talitol (found concentration: 41.3 g / l).

[0349] After cooling the reactor contents to 24 °C, 15 ml of oxidoreductase II lysate, 5 kJ of NAD(P)H oxidase lysate and 10 ml of a 5 mM NADP + solution was added. Additionally, the pH of the reactor solution was increased to 8.

[0350] After a total runtime of 97 h, the reactor contents were heated to 70 °C, the pH value was adjusted to 4, and the mixture was stirred for 30 min at 70 °C. The precipitate was filtered through a glass frit (P3).

[0351] 29.9 g / l D-tagatose was detected in the filtrate.

[0352] The filtrate was concentrated on a rotary evaporator to a D-tagatose concentration of 257 g / l.

[0353] Example 6

[0354] Oxidation of D-talitol to D-psicose

[0355] The following components were mixed in a 2 ml glass vial: 30 μl deionized water, 250 μl of a 200 mM TEA-HCl buffer (pH 8), 125 μl of a D-talitol solution (200 g / l), 50 μl of oxidoreductase I lysate, 40 μl of NADH oxidase lysate and 5 μl of a 10 mM NAD + -solution. The mixture was incubated in an Eppendorf Thermomixer with continuous shaking (30 °C, 800 rpm) for 20 h.

[0356] 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).

[0357] In this way, 75.3% of the D-talitol was converted to D-psicose (found concentration: 39.3 g / l).

[0358] Example 7 Production of D-psicose from D-fructose (one-pot process)

[0359] The following components were mixed in a 2 ml glass vial: 73.8 μl of deionized water, 250 μl of a 200 mM TEA-HCl buffer (pH 7.5), 50 μl of a D-fructose solution (500 g / l), and 25 μl of D-psicose-3-epimerase lysate. Then, 35 μl of oxidoreductase I lysate, 8 U of alcohol dehydrogenase I lysate, and 5 μl of a 10 mM NAD +solution and 50 μl of 2-propanol were added to the mixture. The mixture was incubated with continuous shaking (35 °C, 800 rpm) for a total of 20 h.

[0360] The mixture was then heated to 70 °C for 60 min. After cooling, 25 μl of oxidoreductase I lysate, 10 U of NADH oxidase lysate, 5 μl of 5 mM NAD + solution and 100 ml of deionized water were added. The mixture was incubated in an Eppendorf Thermomixer with continuous shaking (24 °C, 800 rpm) for a further 20 h.

[0361] 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).

[0362] In this way, 76.9% of the D-fructose (50 g / l) was converted to D-psicose (found concentration: 39.9 g / l).

[0363] Example 8

[0364] Reduction of D-Psicose to D-Talitol

[0365] 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.

[0366] Initially, 98.8 ml of a D-psicose solution (508 g / l), 270.9 ml of deionized water, and 41.2 ml of a 500 mM TEA-HCl buffer (pH 7.5) were placed in the reactor and heated to 35 °C while stirring.

[0367] To start the reaction, 35 ml of oxidoreductase VII lysate, 12 ml of alcohol dehydrogenase II lysate, 5 ml of a 10 mM NADP + -solution and 40 ml of 2-propanol.

[0368] 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).

[0369] After 24 h and 48 h of running time, 20 ml of 2-propanol were added.

[0370] After 25 h, 94% of D-psicose (100 g / l) could be reduced to D-talitol - after 52 h the reduction was complete (see Figure 2).

[0371] To separate the enzymes, the reactor contents were heated to 70 °C, the pH was adjusted to 4, and stirred for 30 min at 70 °C. The precipitate was filtered through a glass frit (P3).

[0372] The time course of D-talitol concentration is shown in Figure 2. Comparison with the time courses in the publication (Figure 6) by Sasahara et al. (1998) shows that the reduction of D-psicose (10% = 100 g / L) to D-talitol can be achieved in approximately 1 / 10 of the time using an in vitro system.

[0373] Example 9

[0374] Production of D-talitol from D-glucose (one-pot process)

[0375] In this example, D-fructose is generated in situ from D-glucose using glucose isomerase and further converted to D-talitol.

[0376] The following components were mixed in a 2 ml glass vial: 95.2 μl of deionized water, 100 μl of a 500 mM TEA-HCl buffer (pH 8), 100 μl of a D-glucose solution (500 g / l), 50 μl of a 5 mM MgSCU solution and 15 mg of glucose isomerase (Streptomyces murinus; Sigma-Aldrich G4166).

[0377] Then, first 25 μl of D-psicose-3-epimerase lysate was added, followed by 35 μl of oxidoreductase IX lysate, 12 U of alcohol dehydrogenase I lysate, 5 μl of 10 mM NAD + solution and 50 μl of 2-propanol were added. The mixture was incubated with continuous shaking (40 °C, 800 rpm) for a total of 20 h.

[0378] 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). After 20 h, 100 g / l of D-glucose had been converted to 60 g / l D-talitol.

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Claims

Patent claims 1. A process for the preparation of an aqueous solution containing D-talitol by forming D-psicose from D-fructose dissolved in an aqueous solution by treatment with an epimerase in vitro, after which the D-psicose is reduced to D-talitol by treatment with an NAD(P)H-dependent oxidoreductase in vitro and the epimerase and the oxidoreductase are separated or deactivated.

2. A process for the preparation of D-talitol, characterized in that the aqueous solution prepared according to claim 1 is concentrated and the D-talitol is crystallized.

3. Process according to one of claims 1 and 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 2-propanol.

5. Process according to one of claims 1 and 2, characterized in that the oxidized cofactor NAD(P) produced by the reduction + by means of a formate dehydrogenase and formate to form carbon dioxide.

6. The method according to any one of claims 1 to 5, characterized in that the NAD(P)H-dependent oxidoreductase for the reduction of D-psicose to D-talitol 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, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ. ID No. 14, SEQ ID No. 16, SEQ ID No. 18, SEQ ID No. 20, SEQ ID No. 22 or SEQ ID No. 24 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 1, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 21 or SEQ ID No. 23 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid which anneals under stringent conditions to a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 1, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No.9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 21 or SEQ ID No.

23.

7. A process for the preparation of D-talitol or D-psicose comprising the step of treating D-psicose or D-talitol with an oxidoreductase comprising an amino acid sequence selected from the group consisting of: i) an amino acid sequence having identity to SEQ ID No. 2, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18 or 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. 1, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17 or SEQ ID No. 19 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid which anneals under stringent conditions to a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 1, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17 or SEQ ID No.

19.

8. A process for the preparation of D-talitol comprising the step of treating D-psicose with an oxidoreductase comprising an amino acid sequence selected from the group consisting of: i) an amino acid sequence having an identity to SEQ ID No. 22 or SEQ ID No. 24 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 21 or SEQ ID No. 23 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. 21 or SEQ ID No.

23.

9. Use of an oxidoreductase for the reduction of D-psicose to D-talitol or for the oxidation of D-talitol to D-psicose comprising an amino acid sequence selected from the group consisting of: i) an amino acid sequence having identity to SEQ ID No. 2, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18 or 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. 1, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17 or SEQ ID No. 19 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 #1, SEQ ID #5, SEQ ID #7, SEQ ID #9, SEQ ID #11, SEQ ID #13, SEQ ID #15, SEQ. ID No. 17 or SEQ ID No. 19 binds.

10. Use of an oxidoreductase for the reduction of D-psicose to D-talitol comprising an amino acid sequence selected from the group consisting of: i) an amino acid sequence having an identity to SEQ ID No. 22 or SEQ ID No. 24 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 21 or SEQ ID No. 23 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. 21 or SEQ ID No.

23.

11. A process for the preparation of an aqueous solution of D-tagatose or D-psicose, characterized in that the aqueous solution containing D-talitol prepared by the process according to claims 1 to 6 is reacted in vitro with an NAD(P) + -dependent oxidoreductase to form reduced cofactor NAD(P)H to oxidize D-talitol to D-tagatose or D-talitol to D-psicose, after which the oxidoreductase is separated.

12. The method according to claim 11, characterized in that the reduced cofactor NAD(P)H produced by the oxidation is converted by means of an oxidase and oxygen to NAD(P) + is oxidized.

13. The method according to claim 11, characterized in that the reduced cofactor NAD(P)H produced by the oxidation is converted to NAD(P) by means of an alcohol dehydrogenase and a ketone. + is oxidized.

14. The process according to claim 13, characterized in that the ketone is acetone.

15. The method according to claim 12, characterized in that the oxidase is used to oxidize cofactor NAD(P)H to NAD(P) + comprises an amino acid sequence selected from the group consisting of: i) an amino acid sequence having at least 80% identity to SEQ ID No. 28, SEQ ID No. 30 or SEQ ID No. 32, ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity to SEQ ID No. 11, SEQ ID No. 29 or SEQ ID No. 31, 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. 27, SEQ ID No. 29 or SEQ ID No.

31.

16. Process according to one of claims 1 to 14, characterized in that it is carried out as a one-pot reaction without isolation of intermediate products.

17. Method according to one of claims 1 to 16, characterized in that the enzymes are present as a homogenate or lysate, lyophilisate or spray-dried enzyme product of the corresponding cells forming them, the enzymes are modified with a water-soluble polymer, preferably polyethylene glycol, at the N-terminus, or the enzymes are immobilized in or on a solid matrix or are part of a fusion protein.

18. Method according to one of claims 11 to 17, characterized in that the NAD(P) +- dependent oxidoreductase for the oxidation of D-talitol to D-tagatose 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 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 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.

19. Method according to one of claims 11 to 17, characterized in that the NAD(P) + - dependent oxidoreductase for the oxidation of D-talitol to D-psicose comprises an amino acid sequence selected from the group consisting of: i) an amino acid sequence which has identity to SEQ ID No. 2, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ. ID No. 12, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18 or 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. 1, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17 or SEQ ID No. 19 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid which anneals under stringent conditions to a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 1, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17 or SEQ ID No.

19.

20. A process for the preparation of D-tagatose comprising the step of treating D-talitol with an oxidoreductase comprising an amino acid sequence selected from the group consisting of: i) an amino acid sequence having an identity to SEQ ID No. 4 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 3 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.

21. Use of an oxidoreductase for the oxidation of D-talitol to D-tagatose comprising an amino acid sequence selected from the group consisting of: i) an amino acid sequence having an identity to SEQ ID No. 4 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 3 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.

22. The method according to any one of claims 5 to 8 and 11 to 20, characterized in that the formate dehydrogenase comprises an amino acid sequence selected from the group consisting of: i) an amino acid sequence having at least 80% identity to SEQ ID No. 26, ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity to SEQ ID No. 25, 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. 25.

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