Process for the production of an aqueous solution containing allose

Abstract

A process for producing an aqueous solution containing allose by forming D-psicose in vitro from D-fructose, which is dissolved in an aqueous solution, by treating the dissolved D-fructose with an epimerase, whereupon the D-psicose is reduced in vitro to allitol by treating the D-psicose with an NAD(P)H-dependent oxidoreductase, resulting in the formation of oxidized cofactor NAD(P)+, and then the allitol is enzymatically oxidized to obtain allose.

Description

[0001] Process for the preparation of an aqueous solution containing allose

[0002] The present invention relates to a process for producing an aqueous solution of the rare monosaccharide allose.

[0003] Background of the invention p-Allose

[0004] The aldohexose D-allose is the C3 epimer of D-glucose and, due to its low natural occurrence, is considered a rare sugar. It is found, for example, in certain seagrasses (Kannan et al., 2012) or as a component of glycosides in the South African plant Protect rubropilosa (sugarbush) (Perold et al., 1973).

[0005] D-allose is a sweet-tasting sugar (80% sweetness compared to sucrose), but unlike sucrose, it has few calories (Mooradian et al., 2017). In addition, D-allose has a wealth of other beneficial properties, such as antitumor (e.g., in bladder cancer (Tohi et al., 2022)), antioxidant (Ishihara et al., 2011), and anti-inflammatory (Gao et al., 2011) effects. Further examples and other physiological effects of D-allose are listed in review articles (Chen et al., 2018; Lim & Oh, 2011; Mijailovic et al., 2021).

[0006] Since the demand for D-allose cannot be covered by natural resources due to the wide range of possible applications, synthetic methods (chemical or biocatalytic) for the production of D-allose have been and are being developed starting from more common (and therefore cheaper) monosaccharides such as D-fructose or D-glucose.

[0007] US 9109266 B2 describes the conversion of D-fructose to D-allose using NaOH or strongly basic ion exchange resins (according to the mechanism of the Lobry-de-Bruyn-Alberda-van Ekenstein rearrangement), which, however, leads to a large number of by-products such as D-glucose, D-mannose, D-altose and D-psicose, which must be separated.

[0008] US 5433793 discloses the ammonium molybdate-catalyzed isomerization of D-glucose to D-allose, which is carried out at 130 °C in an acetic acid aqueous medium. This yields approximately 10 wt. % D-allose (based on dry mass), but product isolation requires activated carbon filtration, ion exchange, and simulated moving bed chromatography. Jumde et al. (2016) presented a chemical synthesis route starting from D-glucose that involves a palladium-catalyzed regioselective oxidation at the C3 position and a stereoselective reduction to D-allose using borohydride-based reagents.

[0009] WO 1997 / 042339 A1 describes a process for converting sucrose to D-allose and the corresponding sugar alcohol allitol. Fermentation with Agrobacterium tumefaciens oxidizes sucrose to 3-ketosucrose, which is then hydrogenated (on Raney nickel) to allosucrose. The cleavage of allosucrose can be carried out acid-catalyzed (cation exchange) or enzymatically by invertase or ß-fructosidase. The separation of the D-allose / D-fructose mixture can be achieved either by removing the D-fructose using yeast fermentation or by chromatographic methods (ion exchange). Allitol can be produced by hydrogenating D-allose (on Raney nickel).

[0010] Chemical processes such as those mentioned above are generally characterized by low conversions, complex reaction steps with poor atom economy and / or the formation of by-products and are therefore completely unsuitable for the production of D-allose on an industrial scale.

[0011] The inefficient chemical synthesis routes have now been replaced by more efficient biocatalytic processes.

[0012] Starting from D-glucose, three enzymatic steps lead to D-allose. D-glucose is first isomerized with a glucose / xylose isomerase (Gl / Xl) to D-fructose (Nam, 2022), which is then epimerized with a 3-ketose epimerase (D-tagatose 3-epimerase (DTE), D-psicose / allulose 3-epimerase (DPE / DAE), or L-ribulose 3-epimerase (LRE)) to D-psicose, also known as D-allulose (Zhang et al., 2016; Jiang et al., 2020). The final step (D-psicose D-allose) is accomplished by the action of various isomerases - L-rhamnose isomerase (L-RI, EC 5.3.1.14), D-ribose-5-phosphate isomerase (RPI, EC 5.3.1.6), D-galactose-6-phosphate isomerase (GaPI, EC 5.3.1.26) and D-glucose-6-phosphate isomerase (Gl PI, EC 5.3.1.9) (Chen et al., 2018; Lim & Oh, 2011).

[0013] L-rhamnose isomerases, by far the most important enzyme class for the isomerization of D-psicose and D-allose (Chen et al., 2018), are described, for example, in EP 1589102 Bl, EP 1788089 Bl, EP 1860195 Bl, US 7205141 B2, US 7501267 B2, US 7691619 B2 and US 8748589 B2.

[0014] The conversion of D-fructose to D-psicose by ketose 3-epimerase 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) (Zhang et al., 2016; Jiang et al., 2020). The subsequent isomerization is also an equilibrium reaction, with the equilibrium favoring D-psicose—depending on the conditions, the mass ratio ranges between 77:23 and 62.5:37.5 (D-psicose:D-allose) (Chen et al., 2018; Lim & Oh, 2011).

[0015] Lee et al. (2018) described a process for the conversion of D-fructose to D-allose using DPE from Flavonifractor plautii and RPI (R132E mutant) from Clostridium thermocellum. 600 g / L D-fructose was converted to 79 g / L D-allose within 2 h, corresponding to a conversion of 13%. In addition, 1 mM Co 2+ -ions are required as a cofactor for DPE.

[0016] Li et al. (2020) presented a similar method – DPE from Ruminococcus sp. and L-RI from Bacillus subtilis were expressed in Escherichia coli and immobilized on resins. After 5 h of reaction time, an equilibrium with a mass ratio of 66:24:10 (D-fructose:D-psicose:D-allose) was reached (using 500 g / L D-fructose as the substrate).

[0017] US 10689668 B2 describes fusion enzymes consisting of a DPE from Ensifer adhaerens or Clostridium scindens and an RPI from Persephonella marina EX-Hl (EDPE_RPI or CDPE_RPI), with which conversions of up to 13% can be achieved.

[0018] An alternative to the above-mentioned isomerases is a commercial glucose isomerase from Sweetzyme IT, which was used by Choi et al. to isomerize D-psicose to D-allose. The enzyme was immobilized in a fixed-bed reactor, allowing for easy separation of the product solution (Choi et al., 2021).

[0019] Since complete conversion cannot be achieved by combining epimerization and isomerization, D-allose must be separated from the resulting product mixtures.

[0020] Depending on the isomerase used, the situation is further complicated by the fact that D-psicose can also yield D-allose, the C2 epimer of D-allose (Chen et al., 2018; Lim & Oh, 2011). For example, using a recombinant L-RI from Pseudomonas stutzeri LL172, a product mixture consisting of 25% D-allose, 8% D-allose, and 67% D-psicose was obtained starting from D-psicose (Menavuvu et al., 2006).

[0021] Two methods are available for the isolation of D-allose from enzymatic conversions: 1) crystallization from a concentrated sugar solution by addition of ethanol (Menavuvu et al., 2006) or 2) SMB chromatography with subsequent crystallization from the syrup (Morimoto et al., 2006).

[0022] Furthermore, fermentative processes for the synthesis of D-allose are also known. Zheng et al. (2022) constructed a new metabolic pathway in E. coli based on Gl, DAE, and RPI to convert D-glucose to D-allose. 0.127 g / L D-allose could be obtained after 84 h, corresponding to a yield of 0.045 g D-allose per g D-glucose.

[0023] One way to circumvent the thermodynamically unfavorable epimerization and isomerization reactions is to use enzyme cascades with phosphorylated intermediates. The final step, dephosphorylation, is irreversible and thus drives the cascade (Li et al., 2021).

[0024] A cascade described in US 10745683 B2 and US 11236320 B2 features D-glucose-1-phosphate (G1P) as the central intermediate. G1P is first converted to D-glucose-6-phosphate (G6P) by phosphoglucomutase and then further to D-frucose-6-phosphate (F6P) by phosphoglucose isomerase. F6P is then epimerized to D-psicose-6-phosphate by D-psicose-6-phosphate 3-epimerase, which is subsequently converted to D-allose-6-phosphate by D-allose-6-phosphate isomerase. The final step is dephosphorylation to D-allose by D-allose-6-phosphate phosphatase.

[0025] G1P can be produced directly by the action of phosphorylases on, for example, amylodextrin (obtained by the hydrolysis of starch) or sucrose, consuming phosphate. Since the terminal sugar monomers of the oligo- and polysaccharides cannot be phosphorylated by the corresponding phosphorylases, the polyphosphate glucokinase (D-glucose) must be used. G6P) or polyphosphate fructokinase (D-fructose F6P) can be used, whereby additional polyphosphates must be added as a phosphate source in order to increase the yields (US 10745683 B2; US 11236320 B2).

[0026] A major disadvantage of the route via phosphorylated sugar derivatives is the use of expensive, energy-rich phosphate compounds such as polyphosphate in stoichiometric amounts to introduce the phosphate groups. This problem can be partially circumvented by the use of phosphorylases, but terminal monosaccharides cannot be phosphorylated without the aid of energy-rich phosphate compounds. Furthermore, the remaining phosphate compounds and phosphate ions must be removed after the reaction.

[0027] L-allose

[0028] Unlike its enantiomer, D-allose, L-allose does not occur naturally. Only a few processes for the production of L-allose are known. A biocatalytic route was presented by Terami et al., starting from the equally rare monosaccharide L-psicose. This is isomerized to L-allose using an immobilized L-ribose isomerase from Cellulomonas parahominis MB426. At equilibrium, a mixture of 33:77 (L-allose:L-psicose) is present, which is separated chromatographically (Terami et al., 2015).

[0029] Allitol as an intermediate

[0030] By reduction at the Cl position of D-allose, the achiral sugar alcohol allitol can be obtained (see e.g. WO 1997 / 042339 Al), which, as an interface between the D- and L-hexoses, plays a central role in the so-called Izumoring strategy for the bioproduction of rare sugars (Izumori, 2006; Hassanin et al., 2017).

[0031] Allitol, in turn, can be produced from D-fructose and a combination of DTE, ribitol dehydrogenase (RDH), and formate dehydrogenase (FDH; as a regenerating enzyme for the cofactor nicotinamide adenine dinucleotide (NADH)). In this way, the equilibrium can be completely shifted toward the product side (D-psicose and subsequently allitol), and allitol can be obtained in high purity (Takeshita et al., 2000).

[0032] Wang et al. (2023) describe an E. coli whole-cell biocatalyst for the conversion of D-fructose to allitol. The E. coli cells used included a DPE from Clostridiales, a RDH from Providen a alcalifaciens, a FDH from Starkeya, and another DPE from Rhizobium straminoryzae. 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 (conversion 90.4%). Approximately 30 mM D-sorbitol (the reduction product of D-fructose) was formed as a byproduct.

[0033] Subsequent oxidation of the hydroxy group at the C-position of allitol yields D-allose and at the C6 position, L-allose. Currently, no biocatalytic processes are known for either oxidation.

[0034] Based on these findings, the present invention aims to provide an efficient enzymatic process for producing an aqueous solution containing allose.

[0035] Detailed description of the invention

[0036] This task is solved by 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 converted into phosphoric acid by treating it with an NAD(P)H-dependent oxidoreductase in vitro to form oxidized cofactor NAD(P) +to allitol, after which the allitol is enzymatically oxidized, preferably in vitro, to allose. A further preferred variant of the process according to the invention is characterized in that the epimerase and the NAD(P)H-dependent oxidoreductase are deactivated or removed from the aqueous solution by ultrafiltration before the allitol is enzymatically oxidized, preferably in vitro, to allose.

[0037] The reaction steps of the process according to the invention are schematically illustrated in the accompanying Figure 1. A stands for D-fructose, B for D-psicose, C for allitol, D for allose, E for 2-propanol, F for acetone, 1 for epimerization, 2 for reduction, 3 for alcohol dehydrogenase, 4 for oxidation, and 5 for NAD(P)H oxidase.

[0038] In a preferred variant of the process according to the invention, the oxidized cofactor NAD(P) produced by the reduction +reduced by an alcohol dehydrogenase and a secondary alcohol to form a ketone.

[0039] It has been shown that 2-propanol is particularly suitable as the secondary alcohol.

[0040] Furthermore, it is preferred if the enzymatic oxidation of allitol to allose is carried out with a corresponding NAD(P) + -dependent oxidoreductase.

[0041] It has been shown that the process according to the invention can be carried out as a one-pot reaction without isolation of intermediates.

[0042] The invention further relates to a process for producing an aqueous solution containing allose by oxidizing allitol, which process is characterized in that the oxidation is carried out enzymatically, preferably in vitro.

[0043] The enzymatic oxidation of allitol to allose can be very well achieved with a corresponding NAD(P) +- dependent oxidoreductase with formation of reduced cofactor NAD(P)H.

[0044] It has further been shown that the enzymes in the process according to the invention are present in a suspension, in the homogenate and / or in the lysate of the corresponding cells forming them, with lysates being particularly preferred.

[0045] 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 and Lysate Preparation for details).

[0046] 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.

[0047] In another variant, the enzymes can be in powder form, in lyophilized or spray-dried form.

[0048] The reduced cofactor NAD(P)H produced by oxidation can advantageously be converted to NAD(P) by means of an oxidase and oxygen. + be oxidized.

[0049] In a further preferred variant of the process according to the invention, the epimerase and the NAD(P)H-dependent oxidoreductase are deactivated by heat or removed from the aqueous solution by ultrafiltration.

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

[0051] The preferred concentration of allitol is 50 - 250 g / l.

[0052] The preferred temperature range for the process according to the invention is between 25 and 45 °C.

[0053] The preferred pH range for the process according to the invention is between 7 and 8.5.

[0054] In a further, particularly preferred embodiment of the process, only enzymes from the enzyme groups epimerases, dehydrogenases, reductases and oxidases are used for the conversion of the starting material, with one or more of these enzymes being selected from each of these groups.

[0055] 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.

[0056] The enzyme used to reduce D-psicose comes from the group of oxidoreductases, with ribitol dehydrogenase (EC 1.1.1.56) being particularly preferred.

[0057] The enzyme used for the oxidation of allitol to allose comes from the group of oxidoreductases, with a xylose reductase (EC 1.1.1.307, EC 1.1.1.430, EC 1.1.1.431) or aldo / keto reductase being particularly preferred. The oxidoreductase for the oxidation of allitol to allose preferably comprises an amino acid sequence selected from the group consisting of: i) an amino acid sequence having an identity SEQ ID No. 1, SEQ ID No. 3, 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. 23 of at least 75%, ii) an amino acid sequence encoded by a nucleic acid having identity to SEQ ID No. 2, SEQ ID No. 4, 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.24 of at least 75%, and iii) an amino acid sequence encoded by a nucleic acid which binds, under stringent conditions, to a complementary strand of a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 2, SEQ ID No. 4, 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. 24, wherein stringent conditions comprise one or more washing steps at 65°C and a salt concentration of 0.1x to 2x SSC.

[0058] SEQ ID No. 1:

[0059] MAPQIPNIKLSSGYDMPQVGFGLWKVDRSICADVVYNAIKIGYRLFDGACDYGNEVEAGQGIARAIKEGIVKR EELFIVSKLWNTFHDGDKVEPIVRKQLADWGIDYFDLYLVHFPVALEYVDPSVRYPPGWFYDGEKEIRPSKATI QETWTAMESLVEKGLARSIGVSNFQAQLLYDLLRYAKIRPATLQIEHHPFLVQQELLNLAKAEGIAVTAYSSFG PQSFLEFNM KHAVQLTPLFEDETIKKIAAKYNRPASQVLLRWATQRGLAIIPKSTRPEIMKSNLESIEFDLSEEDI ATISAFDRGLRFNQPTNYFPTEHLWIFG

[0060] SEQ ID No. 2:

[0061] ATGGCTCCCCAGATCCCCAACATTAAGCTCAGCAGCGGCTATGACATGCCCCAGGTGGGCTTTGGACTGT GGAAGGTCGACCGCTCGATCTGCGCCGACGTCGTCTACAACGCAATTAAGATTGGCTACCGCCTGTTTGA CGGCGCTTGCGACTATGGCAACGAAGTCGAGGCCGGCCAGGGCATTGCCCGCGCCATCAAGGAGGGCA TCGTGAAGCGTGAGGAGCTCTTCATCGTGTCCAAGCTCTGGAACACCTTCCACGACGGCGACAAGGTCG AGCCCATCGTCCGCAAGCAGCTCGCCGACTGGGGCATTGACTACTTCGACCTCTACCTCGTCCACTTCCCT GTCGCCCTCGAGTACGTCGACCCCTCGGTCCGCTACCCGCCCGGCTGGTTCTACGATGGCGAGAAGGAG ATCCGCCCCAGCAAGGCCACCATCCAGGAGACCTGGACCGCCATGGAGTCGCTCGTTGAGAAGGGTCTG GCCCGCAGCATTGGTGTCTCCAACTTCCAGGCCCAGCTCCTCTACGACCTGCTGCGCTACGCCAAGATCC GCCCGGCCACCCTTCAGATCGAGCATCACCCCTTCCTCGTCCAGCAGGAGCTGCTCAACCTGGCCAAGGC CGAGGGCATTGCCGTGACTGCCTACAGCTCATTCGGCCCTCAGAGCTTCCTCGAGTTCAACATGAAGCAC GCCGTGCAGCTCACCCCGCTCTTCGAGGACGAGACCATCAAGAAGATCGCCGCCAAGTACAACCGTCCTG CTTCGCAGGTTCTCCTGCGCTGGGCCACTCAGCGCGGTCTGGCCATTATCCCCAAGAGCACGCGCCCCGA

[0062] GATCATGAAGTCCAACCTCGAGAGCATCGAGTTCGACCTCAGCGAGGAAGATATTGCCACCATCTCGGCC

[0063] TTCGACCGCGGCCTGCGCTTCAACCAGCCCACAAACTACTTCCCCACCGAGCACCTCTGGATCTTTGGCTA A

[0064] SE ID Nr. 3:

[0065] MATPTIKLNSGYDMPLVGFGLWKVNKETCADQVYEAIKAGYRLFDGACDYGNEVEAGQGVARAIKEGIVKR

[0066] EDLFIVSKLWNTFHEADKVEPIARKQLADWGLDYFDLYLIHFPIALKYVDPAEIYPPGWTGTKKEVEFSNATIQE

[0067] TWQAM ETLVDKKLTRSIGISNFSAQLIMDLLRYARIRPATLQIEHHPYLTQQALVEYVQKEGIAVTAYSSFGPLS

[0068] FLELGHQVAKDTPLLFEHSTVKSIAEKHGKTPAQVLLRWATQRNIAVIPKSNNPGRLAQNLDVTAWDLEPADI

[0069] EALSALNKNLRFNNPPSYGLYIPIFA

[0070] SEQ ID No. 4:

[0071] ATGGCCACGCCTACTATCAAGCTGAACAGCGGCTATGACATGCCCCTGGTGGGCTTTGGTCTGTGGAAG

[0072] GTCAACAAGGAAACCTGCGCGGACCAGGTCTACGAGGCTATCAAGGCGGGCTACCGCTTGTTTGACGGT

[0073] GCGTGCGACTATGGCAACGAAGTTGAGGCCGGCCAGGGTGTCGCTCGCGCCATCAAGGAAGGCATTGT

[0074] GAAGCGTGAGGACCTCTTCATTGTGTCCAAGCTGTGGAACACGTTCCACGAGGCCGACAAGGTCGAGCC

[0075] GATCGCGCGGAAGCAGCTGGCCGACTGGGGCCTCGACTACTTTGACCTGTACCTCATCCACTTCCCGATC

[0076] GCGCTGAAGTACGTCGACCCGGCCGAGATCTACCCGCCGGGCTGGACGGGCACCAAGAAGGAGGTCGA

[0077] GTTCAGCAACGCGACGATCCAGGAGACGTGGCAGGCCATGGAGACCCTGGTCGACAAGAAGCTGACGC

[0078] GCAGCATCGGCATCAGCAACTTCAGCGCCCAGCTGATCATGGACCTGCTGCGGTACGCGCGCATCCGCCC

[0079] CGCGACCTTGCAGATCGAGCACCACCCGTACCTGACGCAGCAGGCGCTGGTCGAGTACGTGCAGAAGGA

[0080] GGGCATCGCCGTGACGGCGTACTCGTCCTTCGGCCCACTGAGCTTCCTGGAACTGGGCCACCAGGTCGCC

[0081] AAGGACACGCCGCTGCTCTTCGAGCACTCGACCGTCAAGTCGATCGCCGAGAAGCACGGCAAGACGCCC

[0082] GCCCAGGTGCTACTGCGCTGGGCCACCCAGCGCAACATCGCCGTCATCCCCAAGAGCAACAACCCGGGC

[0083] CGCCTGGCGCAGAACCTGGACGTGACGGCGTGGGATCTGGAGCCCGCCGACATTGAGGCCTTGAGCGC GCTGAACAAGAACCTTCGATTCAACAACCCACCTAGCTACGGACTGTACATCCCGATCTTCGCTTAA

[0084] SEQ ID Nr. 5:

[0085] MAPVIKLNSGYDMPQVGFGLWKVDNAVASDVVYNAIKAGYRLFDGACDYGNEVECGQGVARAISEGIVKRE

[0086] DLFIVSKLWNTFHDAERVEPIVKKQLADWGIEYFDLYLIHFPVALEWVDPAVRYPPGWHYDGKEEIRPSKATIQ

[0087] ETWTALESLVSKGLSKSIGISNFQAQLIYDLLRYAKIRPATLQVEHHPYLVQQELINLAKREGIAVTAYSSFGPASF

[0088] KEFNMKHADALAPLIEDETIKKIAAKHNRPASQVLLRWATQRGLAIIPKSTRPQIMAENFQSIDFDLSEEDIATIS

[0089] AFDRGIRFNQPSNYFPTELLWIFG SEQ ID Nr. 6:

[0090] ATGGCGCCGGTGATTAAACTGAACAGCGGCTATGATATGCCGCAGGTGGGCTTTGGCCTGTGGAAAGTG

[0091] GATAACGCGGTGGCGAGCGATGTGGTGTATAACGCGATTAAAGCGGGCTATCGCCTGTTTGATGGCGCG

[0092] TGCGATTATGGCAACGAAGTGGAATGCGGCCAGGGCGTGGCGCGCGCGATTAGCGAAGGCATTGTGAA

[0093] ACGCGAAGATCTGTTTATTGTGAGCAAACTGTGGAACACCTTTCATGATGCGGAACGCGTGGAACCGATT

[0094] GTGAAAAAACAGCTGGCGGATTGGGGCATTGAATATTTTGATCTGTATCTGATTCATTTTCCGGTGGCGC

[0095] TGGAATGGGTGGATCCGGCGGTGCGCTATCCGCCGGGCTGGCATTATGATGGCAAAGAAGAAATTCGCC

[0096] CGAGCAAAGCGACCATTCAGGAAACCTGGACCGCGCTGGAAAGCCTGGTGAGCAAAGGCCTGAGCAAA

[0097] AGCATTGGCATTAGCAACTTTCAGGCGCAGCTGATTTATGATCTGCTGCGCTATGCGAAAATTCGCCCGG

[0098] CGACCCTGCAGGTGGAACATCATCCGTATCTGGTGCAGCAGGAACTGATTAACCTGGCGAAACGCGAAG

[0099] GCATTGCGGTGACCGCGTATAGCAGCTTTGGCCCGGCGAGCTTTAAAGAATTTAACATGAAACATGCGG

[0100] ATGCGCTGGCGCCGCTGATTGAAGATGAAACCATTAAAAAAATTGCGGCGAAACATAACCGCCCGGCGA

[0101] GCCAGGTGCTGCTGCGCTGGGCGACCCAGCGCGGCCTGGCGATTATTCCGAAAAGCACCCGCCCGCAGA

[0102] TTATGGCGGAAAACTTTCAGAGCATTGATTTTGATCTGAGCGAAGAAGATATTGCGACCATTAGCGCGTT

[0103] TGATCGCGGCATTCGCTTTAACCAGCCGAGCAACTATTTTCCGACCGAACTGCTGTGGATTTTTGGCTAA

[0104] SEQ ID Nr. 7:

[0105] MSTTVNTPTIKLNSGYEMPLVGFGCWKVTNATAADQIYNAIKTGYRLFDGAEDYGNEKEVGEGINRAIKDGL

[0106] VKREELFITSKLWNNFHDPKNVETALNKTLSDLNLDYVDLFLIHFPIAAFKFVPIEEKYPPGFYCGDGDNFHYEDV

[0107] PLLDTWKALEKLVEAGKIKSIGISNFTGALIYDLIRGATIKPAVLQIEHHPYLQQPKLIEYVQKAGIAITGYSSFGPQ

[0108] SFLELESKRALNTPTLFEHETIKSIADKHGKSPAQVLLRWATQRNIAVIPKSNNPERLAQNLSWDFDLTKDDLD

[0109] NIAKLDIGLRFNDPWDWDNIPIFV

[0110] SE ID Nr. 8:

[0111] ATGAGCACGACCGTTAATACTCCGACGATCAAACTGAACTCCGGTTACGAGATGCCGCTGGTTGGCTTTG

[0112] GCTGTTGGAAAGTTACCAATGCGACGGCGGCGGACCAGATTTATAACGCTATTAAAACCGGTTACCGTCT

[0113] GTTCGATGGCGCAGAGGACTACGGTAACGAGAAAGAAGTGGGTGAAGGTATTAATCGCGCAATTAAAG

[0114] ACGGTCTGGTCAAACGTGAAGAGCTGTTTATTACCTCGAAACTGTGGAATAACTTCCATGATCCTAAGAA

[0115] CGTGGAGACTGCGCTGAATAAGACCCTGAGCGATCTGAACCTGGACTATGTGGATCTGTTTCTGATTCAC

[0116] TTCCCGATTGCTTTCAAATTTGTCCCGATCGAAGAAAAGTACCCACCGGGCTTCTACTGCGGTGACGGCG

[0117] ACAACTTCCATTATGAGGATGTTCCGCTGCTGGACACGTGGAAGGCGCTGGAGAAATTGGTGGAAGCCG

[0118] GTAAGATCAAGTCCATTGGCATTAGCAACTTCACCGGTGCCTTGATTTACGATTTGATCCGTGGTGCGACC

[0119] ATTAAACCGGCGGTTCTGCAGATCGAGCATCACCCGTATCTGCAACAGCCGAAACTGATCGAATACGTTC

[0120] AGAAAGCAGGTATCGCCATCACTGGCTATAGCAGCTTCGGTCCACAGAGCTTCCTGGAGCTGGAGAGCA AGCGTGCCCTGAATACCCCGACGTTGTTTGAACACGAAACCATCAAGTCTATCGCTGACAAACACGGTAA

[0121] GAGCCCTGCACAAGTCCTGCTGCGCTGGGCAACGCAACGTAATATTGCGGTTATTCCGAAGAGCAATAAC

[0122] CCGGAGCGTCTGGCGCAGAATCTGTCTGTCGTCGACTTTGATTTGACCAAGGATGACCTGGATAATATCG CGAAGCTGGACATCGGCCTGCGCTTCAACGATCCGTGGGATTGGGACAACATCCCGATCTTTGTGTA

[0123] SE ID Nr. 9:

[0124] MTYLAETVTLNNGEKMPLVGLGCWKMPNDVCADQIYEAIKIGYRLFDGAQDYANEKEVGQGVNRAIKEGLV

[0125] KREDLVVVSKLWNSFHHPDNVPRALERTLSDLQLDYVDIFYIHFPLAFKPVPFDEKYPPGFYTGKEDEAKGHIEE

[0126] EQVPLLDTWRALEKLVDQGKIKSLGISNFSGALIQDLLRGARIKPVALQIEHHPYLTQERLIKYVKNAGIQVVAYS

[0127] SFGPVSFLELENKKALNTPTLFEHDTIKSIASKHKVTPQQVLLRWATQNGIAIIPKSSKKERLLDNLRINDALTLTD DELKQISGLNQNIRFNDPWEWLDNEFPTFI

[0128] SEQ ID Nr. 10:

[0129] ATGACGTACTTAGCAGAAACAGTTACTTTAAACAATGGCGAAAAGATGCCGCTAGTCGGCTTAGGTTGCT

[0130] GGAAGATGCCCAACGACGTTTGTGCCGACCAAATTTACGAAGCCATTAAGATCGGATATCGTTTATTCGA

[0131] TGGTGCCCAAGATTACGCCAACGAGAAAGAAGTTGGACAGGGTGTCAACAGAGCCATCAAAGAAGGGC

[0132] TTGTTAAGAGAGAGGATTTAGTTGTTGTCTCCAAGCTATGGAACAGTTTCCACCATCCGGACAACGTACC

[0133] TCGTGCTTTGGAAAGAACTCTTTCCGATTTGCAATTGGACTATGTTGACATATTCTACATCCATTTCCCATT

[0134] GGCCTTCAAGCCTGTGCCATTCGATGAGAAGTATCCTCCAGGTTTCTACACCGGTAAGGAAGACGAAGCT

[0135] AAAGGTCACATTGAAGAAGAACAAGTACCACTATTGGACACTTGGAGGGCTTTAGAAAAACTAGTCGAC

[0136] CAAGGTAAGATCAAGTCCTTGGGTATTTCCAACTTTTCAGGTGCGTTGATCCAAGATTTGCTACGTGGTGC

[0137] TCGTATCAAGCCAGTTGCCCTACAAATCGAGCATCATCCATATTTGACACAGGAAAGATTGATTAAATAC

[0138] GTCAAGAATGCCGGCATTCAAGTGGTTGCCTACTCCTCGTTCGGTCCAGTGTCGTTCCTGGAATTGGAAA

[0139] ACAAGAAAGCTCTTAACACCCCTACTTTGTTTGAGCATGACACGATTAAGTCTATCGCTTCCAAGCACAAA

[0140] GTCACTCCACAACAAGTTCTGTTGAGATGGGCCACACAAAATGGCATTGCCATCATTCCAAAATCATCAA

[0141] AGAAGGAAAGATTGCTGGATAACTTGAGGATCAACGATGCATTGACTTTGACCGATGATGAATTGAAGC

[0142] AAATTTCCGGCTTGAATCAAAATATCAGATTCAATGACCCTTGGGAATGGCTAGACAACGAATTCCCAAC TTTCATCTAA

[0143] SEQ ID Nr. 11:

[0144] MPSPVIKLNSGYDMPQVGFGLWKVDNSVAADVVYNAIKAGYRLFDGACDYGNEVECGKGIARAISEGIVKRE

[0145] DLFIVSKLWNTFHDAERVEPIVKKQLADWGIDYFDLYLIHFPVALEYVDPSVRYPPGWFYDGKSEIRTSKATIQE

[0146] TWTAMESLVAKGLTKSIGISNFQAQLIYDLLRYAKIRPATLQIEHHPYLVQQELLNLAKQEGIAVTAYSSFGPASF

[0147] LEFNMQHAVKLTPLIEDETIKKIAAKYNREPAQVLLRWATQRGLAVIPKTTRPALMASNFQCTDFDLSEEDIATI

[0148] SSFDRGIRFNQPSNYFPTEYLWIFG SEQ. ID Nr. 12:

[0149] ATGCCGTCACCTGTCATCAAGCTCAACAGCGGCTATGACATGCCCCAGGTGGGCTTCGGCCTCTGGAAGG

[0150] TCGACAATTCCGTTGCCGCGGACGTCGTCTACAACGCCATCAAGGCCGGCTACCGCCTCTTCGATGGCGC

[0151] TTGCGACTACGGCAACGAGGTCGAGTGCGGCAAGGGTATCGCGCGCGCCATCTCGGAGGGCATTGTGA

[0152] AGCGTGAGGACCTCTTCATCGTGTCCAAGCTCTGGAACACCTTCCACGACGCCGAGCGCGTCGAGCCCAT

[0153] CGTCAAGAAGCAGCTCGCCGACTGGGGCATTGACTACTTCGACCTCTACCTCATCCACTTCCCTGTCGCCC

[0154] TCGAGTACGTCGACCCCTCGGTCCGCTACCCGCCCGGTTGGTTCTACGATGGCAAGAGCGAGATCCGCAC

[0155] CAGCAAGGCCACCATCCAGGAGACCTGGACTGCCATGGAGTCCCTTGTCGCCAAGGGCCTCACCAAGAG

[0156] CATCGGTATTTCCAACTTCCAGGCCCAGCTGATCTACGACCTCCTGCGGTACGCCAAGATCCGCCCGGCC

[0157] ACCCTCCAGATCGAGCACCACCCCTACCTCGTCCAGCAGGAGCTGCTCAACCTCGCCAAGCAGGAGGGCA

[0158] TCGCCGTCACCGCCTACAGCTCCTTCGGCCCCGCCAGCTTCCTCGAGTTCAACATGCAGCACGCCGTCAAG

[0159] CTCACCCCCCTTATCGAGGACGAGACCATCAAGAAGATCGCCGCCAAGTACAACCGCGAGCCCGCCCAA

[0160] GTCCTGCTCCGCTGGGCCACTCAGCGTGGCCTGGCCGTCATCCCCAAGACCACCCGCCCCGCCCTCATGG

[0161] CCTCCAACTTCCAGTGCACCGACTTTGATCTCTCCGAGGAGGACATCGCCACCATCTCGTCCTTCGACCGC

[0162] GGCATTCGCTTCAACCAGCCCTCCAACTACTTCCCCACCGAGTATCTCTGGATCTTTGGCTAA

[0163] SEQ ID Nr. 13:

[0164] MSTDTILIPGIDTPVSRVALGTWAIGGWMWGGPDDGNGVRTIHAALDDGINLIDTAPVYGFGHSEEVVGRA

[0165] LAEKPHKAFVATKLGLNWTDDKPEDRKVFRDSRPARIRQEVEDSLRRLRVETIDLEQIHWPDAKTPIDESAREL

[0166] QKLHQEGKIRALGVSNFSPEQMDIFREVAPLATIQPPLNLFERTCEKDILPYAKKNDAVVLAYGSLCRGLLSGK

[0167] MSKDTTFPKDDLRSGDPKFQKPKFESYLAAVEDFKKLAAKRNKSVLAFAVRWVLDQGPVIALWGARKPEQVS

[0168] GVRDVFGWSLTDEEKKAVDEILAHHVPEAIDPTFMGPPNRD

[0169] SEQ ID Nr. 14:

[0170] ATGTCCACCGACACCATTCTCATTCCAGGCATTGATACGCCCGTTTCTCGCGTTGCCCTTGGCACCTGGGC

[0171] TATCGGTGGCTGGATGTGGGGCGGCCCTGACGATGGAAACGGCGTCCGCACCATTCATGCCGCCCTTGA

[0172] TGATGGCATCAATCTGATTGATACCGCTCCGGTTTACGGCTTTGGACATTCCGAAGAAGTTGTGGGTCGC

[0173] GCACTGGCTGAGAAGCCGCACAAGGCCTTTGTCGCCACAAAGCTGGGCCTGAACTGGACGGATGACAAG

[0174] CCGGAAGACCGGAAGGTCTTTCGTGACTCCCGCCCCGCCCGTATCCGACAGGAAGTGGAAGACTCCCTG

[0175] CGCCGCCTGCGCGTTGAGACGATCGACCTTGAGCAGATTCACTGGCCGGATGCCAAGACCCCGATCGAC

[0176] GAAAGCGCGCGCGAGCTTCAGAAGCTGCATCAGGAAGGCAAGATCCGCGCTCTGGGCGTCAGCAACTTC

[0177] TCGCCCGAACAGATGGATATTTTCCGCGAAGTCGCGCCCCTCGCCACGATCCAGCCGCCGCTGAACCTGT

[0178] TCGAGCGTACCTGTGAAAAGGACATCCTGCCGTACGCGAAGAAGAACGATGCGGTTGTTCTGGCCTATG

[0179] GTTCGCTATGTCGCGGTCTGCTGTCCGGCAAAATGAGCAAGGACACGACGTTCCCGAAGGATGATCTGC GTTCGGGTGATCCGAAATTCCAGAAGCCGAAGTTCGAGAGCTATCTGGCCGCTGTCGAGGACTTCAAGA

[0180] AGCTGGCTGCCAAGCGGAACAAGTCCGTTCTCGCCTTTGCTGTCCGCTGGGTACTGGATCAGGGACCGG

[0181] TCATTGCTCTCTGGGGCGCTCGCAAGCCGGAGCAGGTCTCAGGCGTACGAGACGTTTTCGGCTGGTCTCT

[0182] GACGGATGAAGAAAAGAAGGCCGTAGACGAAATTCTGGCGCATCATGTGCCGGAGGCCATCGACCCGA

[0183] CCTTTATGGGGCCGCCGAACCGCGACTAA

[0184] SEQ ID Nr. 15:

[0185] MSSDTILIPGIDTPVSRVALGTWAIGGWMWGGPDDENGVRTIHAALDDGINFIDTAPVYGFGHSEEVVGRA

[0186] LAEKPNKAFVATKLGLNWTDDKPEDRKVFRDSRPARIRKEVEDSLRRLRVETIDLEQIHWPDDKTPIDESAREL

[0187] QKLHQEGKIRALGVSNFSPEQMDIFREVAPLATIQPPLNLFERTCEKDILPYAKKNNAVVLAYGALCRGLLSGK

[0188] MNKDTSFPKDDLRSGDPKFQKPKFESYLAAVEDFKELAAKRDKSVLAFAVRWVLDQGPVIALWGARKPEQVS

[0189] GVKDVFGWSLTDEEKKAVDEILARHVPDAIDPTFMGPPNRD

[0190] SEQ ID Nr. 16:

[0191] ATGTCCTCAGACACCATTCTCATTCCCGGCATCGACACTCCGGTTTCCCGCGTGGCTCTCGGCACCTGGGC

[0192] CATTGGCGGCTGGATGTGGGGCGGGCCGGACGATGAAAACGGCGTTCGCACCATCCATGCAGCTCTCGA

[0193] TGACGGCATCAATTTCATCGACACGGCACCCGTCTATGGCTTCGGGCACTCAGAAGAAGTTGTTGGCCGG

[0194] GCACTGGCCGAAAAGCCCAACAAGGCTTTCGTTGCGACAAAGCTGGGCCTGAACTGGACGGATGACAAA

[0195] CCCGAAGACCGCAAGGTTTTCCGTGATTCCCGCCCTGCCCGCATCCGCAAGGAAGTGGAAGACTCCCTGC

[0196] GCCGCCTGCGGGTCGAAACGATTGATCTCGAACAGATCCATTGGCCCGATGACAAGACTCCCATCGATGA

[0197] AAGCGCACGCGAGCTGCAGAAACTTCATCAGGAAGGCAAGATCCGGGCGCTCGGCGTTAGCAATTTCTC

[0198] GCCGGAGCAGATGGATATCTTTCGCGAAGTCGCCCCACTCGCAACAATTCAGCCCCCGCTGAACCTGTTC

[0199] GAACGCACCTGCGAAAAAGATATTCTCCCATACGCCAAGAAGAATAACGCCGTGGTGCTCGCTTATGGC

[0200] GCGCTCTGCCGCGGACTGCTCTCCGGCAAGATGAACAAGGATACGAGCTTCCCGAAAGATGACCTGCGT

[0201] TCAGGTGATCCGAAGTTCCAGAAGCCAAAGTTCGAGAGCTATCTCGCCGCAGTCGAAGACTTCAAGGAG

[0202] CTGGCCGCCAAGCGCGACAAGTCCGTTCTCGCCTTCGCTGTACGCTGGGTTCTGGATCAGGGGCCAGTCA

[0203] TCGCACTCTGGGGCGCACGCAAGCCTGAACAGGTCTCCGGCGTGAAGGATGTTTTCGGCTGGTCTCTGA

[0204] CCGACGAAGAAAAGAAGGCTGTGGACGAAATTCTGGCACGTTCACGTCCCGGACGCGATCGACCCGACCT

[0205] TCATGGGGCCACCGAACCGCGACTAA

[0206] SE ID Nr. 17:

[0207] MSSLVTLNNGLKMPLVGLGCWKIDKKVCANQIYEAIKLGYRLFDGACDYGNEKEVGEGIRKAISEGLVSRKDIF

[0208] VVSKLWNNFHHPDHVKLALKKTLSDMGLDYLDLYYIHFPIAFKYVPFEEKYPPGFYTGADDEKKGHITEAHVPII

[0209] DTYRALEECVDEGLIKSIGVSNFQGSLIQDLLRGCRIKPVALQIEHHPYLTQEHLVEFCKLHDIQVAYSSFGPQS FIEMDLQLACTTPTLFENDVIKKVSQNHPGSTTSQVLLRWATQRGIAVIPKSSKKKERLLGNLEKKFTEQFTLK

[0210] DISALNANIRFNDPWTWLDGKFPTFA

[0211] SEQ ID No. 18:

[0212] ATGTCTTCACTGGTTACTCTTAATAACGGTCTGAAAATGCCCCTAGTCGGCTTAGGGTGCTGGAAAATTG

[0213] ACAAAAAAGTCTGTGCGAATCAAATTTATGAAGCTATCAAATTAGGCTACCGTTTATTCGATGGTGCTTGC

[0214] GACTACGGCAACGAAAAGGAAGTTGGTGAAGGTATCAGGAAAGCCATCTCCGAAGGTCTTGTTTCTAGA

[0215] AAGGATATATTTGTTGTTTCAAAGTTATGGAACAATTTTCACCATCCTGATCATGTAAAATTAGCTTTAAA

[0216] GAAGACCTTAAGCGATATGGGACTTGATTATTTAGACCTGTATTATATTCACTTCCCAATCGCCTTCAAAT

[0217] ATGTTCCATTTGAAGAGAAATACCCTCCAGGATTCTATACGGGCGCAGATGACGAGAAGAAAGGTCACA

[0218] TCACCGAAGCACATGTACCAATCATAGATACGTACCGGGCTCTGGAAGAATGTGTTGATGAAGGCTTGAT

[0219] TAAGTCTATTGGTGTTTCCAACTTTCAGGGAAGCTTGATTCAAGATTTATTACGTGGTTGTAGAATCAAGC

[0220] CCGTGGCTTTGCAAATTGAACACCATCCTTATTTGACTCAAGAACACCTAGTTGAGTTTTGTAAATTACAC

[0221] GATATCCAAGTAGTTGCTTACTCCTCCTTCGGTCCTCAATCATTCATTGAGATGGACTTACAGTTGGCAAA

[0222] AACCACGCCAACTCTGTTCGAGAATGATGTAATCAAGAAGGTCTCACAAAAACCATCCAGGCAGTACCACT

[0223] TCCCAAGTATTGCTTAGATGGGCAACTCAGAGAGGCATTGCCGTCATTCCAAAATCTTCCAAGAAGGAAA

[0224] GGTTACTTGGCAACCTAGAAATCGAAAAAAAGTTCACTTTAACGGAGCAAGAATTGAAGGATATTTCTGC

[0225] ACTAAATGCCAACATCAGATTTAATGATCCATGGACCTGGTTGGATGGTAAATTCCCCACTTTTGCCTAA

[0226] SEQ ID No. 23:

[0227] MTYLAPTVTLNNGSKMPLVGLGCWKIPNEVCAEQVYEAIKLGYRLFDGAQDYANEKEVGQGINRAIKEGIVK

[0228] REDLVVVSKLWNSFHHPDNVRTAVERTLNDLQLDYLDLFYIHFPLAFKFVPLDEKYPPGFYTGKDNFAKEIIEEE

[0229] PVPILDTYRALEKLVDEGLIKSLGISNFSGALIQDLLRGARIKPVALQIEHHPYLVQDRLITYAQKVGLQVVAYSSF

[0230] GPLSFVELNNEKALHTKTLFENDTIKAIAQKHNVTPSHVLLKWSTQRGIAVIPKSSKKERLLENLKIEETFTLSDEE

[0231] IKEINGLDQGLRFNDPWDWLGNEFPTFI

[0232] SE ID Nr. 24:

[0233] ATGACATACCTCGCACCAACAGTTACCTTGAACAATGGATCCAAGATGCCGCTAGTCGGCTTGGGATGCT

[0234] GGAAAATCCCAAACGAAGTGTGTGCCGAACAGGTGTACGAAGCCATCAAGTTGGGCTACCGCTTGTTCG

[0235] ACGGCGCGCAGGACTACGCCAACGAAAAAGAGGTGGGCCAAGGTATTAACAGAGCCATCAAGGAAGGA

[0236] ATCGTCAAGAGAGAAGACTTGGTCGTCGTTTCTAAGTTGTGGAACAGTTTCCACCACCCAGACAACGTGC

[0237] GTACCGCAGTCGAAAGAACTTTGAACGACTTGCAATTGGACTACTTGGACTTGTTCTACATCCATTTCCCA

[0238] TTGGCTTTCAAGTTCGTGCCACTAGACGAGAAGTACCCTCCAGGTTTCTACACAGGTAAGGACAATTTCG

[0239] CCAAGGAAATCATCGAAGAGGAGCCTGTCCCAATCTTGGACACCTACAGAGCCCTTGAGAAGTTGGTCG

[0240] ACGAAGGTTTGATCAAATCTTTGGGTATCTCAAACTTTTCGGGTGCATTGATCCAGGACTTGTTGCGTGGC

[0241] GCCCGTATCAAGCCAGTCGCCTTGCAGATCGAACACCACCCATACTTGGTCCAGGACCGCTTGATCACGT ACGCCCAAAAGGTGGGCTTGCAAGTCGTCGCCTACTCCAGTTTCGGCCCACTATCCTTTGTCGAGTTGAA CAACGAAAAGGCCTTGCACACAAAGACTTTGTTCGAAAACGACACCATCAAGGCCATCGCTCAAAAACAC AACGTCACCCCATCCCACGTCTTGTTGAAGTGGTCCACCCAACGTGGTATCGCCGTCATTCCAAAGTCCTC CAAGAAGGAACGTCTCCTCGAGAACTTGAAGATCGAAGAGACCTTTACCTTGTCCGACGAAGAGATCAA GGAGATCAACGGCTTGGACCAGGGATTGAGATTTAACGACCCATGGGACTGGTTGGGCAACGAATTCCC AACCTTTATCTAA

[0242] The oxidoreductase for the oxidation of allitol to allose preferably comprises or consists of an amino acid sequence which has an identity to SEQ ID No. 1, SEQ ID No. 3, 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. 23 of at least 75%, even more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, in particular 100%. The oxidoreductase according to the invention for the oxidation of allitol to allose particularly preferably comprises the amino acid sequence SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No.

[0243] 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17 or SEQ ID No. 23.

[0244] Alternatively, the oxidoreductase for the oxidation of allitol to allose preferably comprises or consists of an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 2, SEQ ID No. 4, 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. 24 of at least 75%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%, in particular 100%. Particularly preferably, the nucleic acid which encodes the oxidoreductase according to the invention for the oxidation of allitol to allose comprises the nucleic acid sequence SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No.

[0245] 12, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18 or SEQ ID No. 24.

[0246] 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.

[0247] 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 28, an expectation (E) of 0.05, M = 1, N = -2, 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 0.05 and the BLOSUM62 scoring matrix (Henikoff & Henikoff, 1989), alignments (B) of 50, expectation (E) of 0.05, M = 1, N = -2.

[0248] Alternatively, the oxidoreductase for oxidizing allitol to allose preferably comprises or consists of an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a complementary strand of a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 2, SEQ ID No. 4, 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. 24. As used herein, stringent conditions refer to conditions under which so-called specific hybrids, but not non-specific hybrids, are formed.

[0249] For example, 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). Stringent conditions refer to washing at 65 °C and a salt concentration of 0.1x to 2x SSC (where 1xSSC is a mixture of 0.15 M sodium chloride / 0.015 M sodium citrate).

[0250] 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).

[0251] The NAD(P)-dependent alcohol dehydrogenase for cofactor regeneration (reduction of NAD(P) +to NAD(P)H) 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. 19 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 20 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid which binds under stringent conditions to a complementary strand of a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 20. Particularly suitable for cofactor regeneration in general is an alcohol dehydrogenase whose amino acid sequence is at least 80% identical to SEQ ID No. 19 or which is encoded by a nucleic acid which has an identity to SEQ ID No. 20 of at least 80% or which binds under stringent conditions to a complementary strand of a nucleic acid molecule with the nucleic acid sequence SEQ ID No. 20.

[0252] SEQ ID No. 19:

[0253] M KAAVVEQFKKPLQVKEVEKPKISYGEVLVRIKACGVCHTDLHAAHGDWPVKPKLPLIPGHEGVGVIEEV GPGVTHLKVGDRVGIPWLYSACGHCDYCLSGQETLCERQQNAGYSVDGGYAEYCRAAADYVVKIPDNLS FEEAAPIFCAGVTTYKALKVTGAKPGEWVAIYGIGGLGHVAVQYAKAMGLNVVAVDLGDEKLELAKQLG ADLVVNPKHDDAAQWIKEKVGGVHATVVTAVSKAAFESAYKSIRRGCACVLVGLPPEEIPIPIFDTVLNGV KIIGSIVGTRKDLQEALQFAAEGKVKTIVEVQPLENINDVFDRM LKGQINGRVVLKVD

[0254] SEQ ID No. 20:

[0255] ATGAAAGCTGCAGTTGTGGAACAATTTAAAAAAGCCGTTACAAGTGAAAGAAGTGGAAAAACCTAAG ATTCCATACGGGGAAGTATTAGTGCGCATCAAAGCGTGTGGGTATGCCATACAGACTTGCATGCCG CACATGGCGACTGGCCTGTAAAGCCTAAACTGCCTCTCATTCCTGGCCATGAAGGCGTCGGGTGTAATT GAAAAGTAGGTCCTGGGGTAAACATTTAAAAGTTGGAGATCGCGTAGGTATCCCTTGGCTTATTC GGCGTGCGGTCATTGTGACTATTGCTTAAGCGGACAAGAAACATTATGCGAACGTCAACAAAACGCT GGCTATTCCGTCGATGGTGGTTATGCTGAATATTGCCGTGCTGCAGCCGATTATGTCGTAAAAAATTCC TGATAACTTATCGTTTGAAGAAGCCGCTCCAATCTTTGCGCTGGTGTAACAACATATAAAGCGCTCA AAGTAACAGCGCAAAACCAGGTGAATGGGGTAGCCATTTACGGTATCGGCGGGCTTGGACATGTC CAGTCCAATACGCAAAGGCGATGGGGTTAAACGTCGTTGCTGTCGATTTAGGTGATGAAAAACTTGA GCTTGCTAAACAACTTGGTGCAGATCTTGTCGTCAATCCGAAACATGATGATGCAGCACAATGGATAA AAGAAAAAGTGGGCGGTGTGCCATGCGACTGTCGTCACAGCTGTTTCAAAAGCCGCGTTCGAATCAGC CTACAAATCCATTCGTCGCGGTGGTGCTTGCGTACTCGTCGGATTACCGCCGGAAGAATACCTATTC CAATTTTCGATACAGTATTAAATGGAGTAAAAAATTATTGGTTCTATCGTTGGTACGCGCAAAGACTTA CAAGAGGCACTTCAATTTGCAGCAGAAGGAAAAGTAAAAACAATTGTCGAAGTGCAACCGCTTGAAAACATTAACGACGTATTCGATCGTATGTTAAAAGGGCAAATTAACGGCCGCGTCGTGTTAAAAGTAGA TTAA

[0256] The alcohol dehydrogenase for cofactor regeneration mentioned here preferably comprises an amino acid sequence that has an identity to SEQ ID No. 19 of at least 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%, and in particular 100%. The alcohol dehydrogenase for cofactor regeneration according to the invention particularly preferably comprises or consists of the amino acid sequence SEQ ID No. 19.

[0257] 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. 20 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. 20.

[0258] A further aspect of the present invention relates to the use of an alcohol dehydrogenase for cofactor regeneration (reduction of NAD(P) +to NAD(P)H), 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. 19 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 20 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a complementary strand of a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 20.

[0259] 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.

[0260] A particularly preferred H2O-forming NAD(P)H oxidase for cofactor regeneration (oxidation of NAD(P)H to NAD(P) + ) 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. 21 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 22 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid which binds under stringent conditions to a complementary strand of a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 22.

[0261] SEQ ID No. 21:

[0262] MKVVVGCTHAGTAAVKTILNEHPDASVSVYERNDNVSFLSCGIALYVGGVVKDPAGLFYSSPEELASMGAKIN MEHNVKNIDNENKVVVIENLKTGETFEESYDKLVMTTGSWPIIPPIDGINSENILLCKNYNQANEIIKESKNAKKIV IVGGGYIAIELVEAFAESGKQVTLVARSDRILRKYLDAEFTSVLEHDLQERGVTLALNQTVEKFVANESGAVTAVK TPVGEYEADLVILCVGFKPNTDLLKDKVEM LPNGAIVVDEYMRTSDEAIFAAGDSCAVHYNPTGGSAYIPLATN AVRMGALVGKNIVSPTVKYRGTQATSGLYLFGFNIGSTGLTENSAPHFGVEVRSVVEDNYRPEFMPTTEKVT MKLVYEVGTNRIVGGQIMSKYDVTQSANTLSLCVQNKMTIEDLAYVDFFFQPHFDRPWNYLNILAQAAVEQER KLAK

[0263] SEQ ID No. 22:

[0264] ATGAAAGTAGTAGTAGTAGGCTGTACACATGCAGGAACAGCGGCAGTTAAGACGATTTTAAATGAACATC CAGATGCATCAGTATCAGTATATGAGCGTAATGACAATGTCTCATTTCTATCTTGTGGGATTGCGTTGTATG TTGGTGGAGTTGTGAAAGATCCTGCAGGTTTGTTTTATTCAAGTCCAGAAGAACTTGCATCAATGGGCGCG AAAATTAACATGGAACACAATGTGAAAAATATAGATAATGAGAATAAGGTCGTAGTAATTGAGAATTTAAA AACAGGCGAAACATTTGAAGAAAGCTATGATAAGTTGGTAATGACAACTGGATCATGGCCAATTATTCCTC CAATTGATGGAATCAATAGTGAAAATATTCTTTTGTGTAAAAACTATAACCAAGCAAATGAAATTATTAAAG AATCAAAAAATGCTAAAAAGATTGTCATTGTTGGTGGTGGCTATATTGCGATTGAATTAGTTGAGGCATTT GCAGAATCTGGCAAGCAAGTGACGCTAGTTGCGCGTAGCGATCGTATTTTACGTAAATATTTAGATGCTGA ATTCACTTCTGTTTTAGAGCATGATTTACAAGAAAGAGGCGTTACGCTAGCTTTAAACCAAACCGTCGAGAA ATTTGTTGCCAATGAATCAGGTGCTGTGACAGCTGTGAAAACACCAGTTGGAGAATATGAGGCTGATTTAG TTATTTTATGTGTTGGATTTAAACCAAATACTGATTTGTTGAAGGATAAAGTAGAGATGTTGCCAAATGGTG CCATCGTAGTGGATGAATATATGAGAACAAGCGATGAAGCGATTTTTGCTGCTGGCGATAGTTGCGCGGTT CATTATAATCCAACTGGAGGGCTCTGCGTATATTCCGTTAGCTACAAATGCAGTTAGAATGGGAGCTTTAGTTGGGAAAAATATTGTTTCTCCAACAGTTAAATATCGTGGCACGCAAGCAACTTCTGGTTTATATTTTATTGGT TTTAATATAGGTTCAACGGATTGACTGAAAATAGCGCTCCTCATTTTGGCGTAGAGGTTCGTTCAGTAGTT GTAGAAGATAATTATCGTCCAGAGTTTATGCCGACAACAGAGAAAGTAACGATGAAATTAGTTTATGAAGT AGGAACGAATCGGATTGTTGGAGGTCAAATCATGTCAAAATATGATGTGACACAATCTGCCAATACGTTAT CTTTATGTGTTCAAAATAAAATGACGATTGAGGATTTGGCTTATGTAGATTTCTTCTTCCAACCTCACTTTGA TCGTCCTTGGAACTATTTAAATATTTTAGCGCAAGCAGCTGTTGAGCAAGAGCGTAAACTAGCAAAATAA

[0265] 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. 21 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 HjO-forming NAD(P)H oxidase comprises or consists of the amino acid sequence SEQ ID No. 21.

[0266] 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. 22 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. 22.

[0267] A further aspect of the present invention relates to the use of a HjO-forming NAD(P)H oxidase for cofactor regeneration (oxidation of 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. 21 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 22 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid which binds under stringent conditions to a complementary strand of a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 22.

[0268] The enzymatic strategy presented here in combination with cofactor regeneration enables a biocatalytic, environmentally friendly and highly efficient production process for allose.

[0269] Materials

[0270] D-Psicose was supplied by TCI and Hunan Garden Naturals Inc. (China), D-Allose and Allitol were supplied by TCI, D-Fructose, Lysozyme, NAD +, NADH disodium salt, NADP + -Disodium salt, NADPH tetrasodium salt, 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 and sodium dodecyl sulfate (SDS) were purchased from Carl Roth, and triethanolamine (TEA) was purchased from Chem-Lab NV.

[0271] Production of enzymes & preparation of lysates General information on the expression of recombinant enzymes in E. coli

[0272] 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.

[0273] 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.

[0274] 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.

[0275] 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).

[0276] Preparation of cell lysates using sonifier disruption

[0277] 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.

[0278] 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). The resulting homogenate was centrifuged for 10 min at 4 °C and 16,000 rpm (Eppendorf Centrifuge 5417R) to separate the insoluble cell fragments and obtain the lysate.

[0279] Table 1. Enzyme classes and donor organisms for the enzymes used in the examples.

[0280] Analytical methods

[0281] High Performance Liquid Chromatography

[0282] An Agilent HPLC 1260 Infinity II Series system was used to quantify D-psicose, D-fructose, and the intermediate 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.

[0283] High Performance Anion Exchange Chromatography

[0284] HPAEC was used to determine substrate conversions and product concentrations during the oxidation of allitol to allose. The analytes were detected using pulsed amperometric detection (PAD). A Dionex™ CarboPac™ PA210-fast-4pm column with a corresponding precolumn was used, eluted with a NaOH gradient.

[0285] Determination of enzyme activities (optical-enzymatic assay)

[0286] 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 NADH / NADPH at 340 nm (E = 6220 L mol 1 cm 1), the enzyme activity of the lysate can be determined in U / ml (based on the volume of the lysate) or U / g (based on the biomass used for production). 1 U represents 1 pmol of substrate turnover per minute (1 U = 1 pmol / min = l.67-10 _8 cat).

[0287] 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

[0288] Oxidation of allitol to allose (with cofactor regeneration)

[0289] The oxidation reactions were carried out in 2 ml glass vials containing allitol (final concentration 50 g / l) and NADP +(final concentration 0.05 mM) were dissolved in 100 mM TEA-HCl buffer (pH 8). Additionally, the solutions each contained 10 U of NAD(P)H oxidase lysate for cofactor regeneration. To start the reaction, 50 μl of oxidoreductase lysate (see Table 2 below) were added. The vials were incubated in an Eppendorf Thermomixer with continuous shaking (30 °C, 800 rpm) for 20 h.

[0290] For analysis, 40 μl of a sample 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 760 μl of ultrapure water, vortexed, and then centrifuged for 5 min at max. 1 g. The supernatant was diluted 1:400 with ultrapure water and analyzed using HPAEC (PAD). The results are presented in Table 2 below.

[0291] Table 2

[0292] Example 2

[0293] Oxidation of allitol to allose (without cofactor regeneration)

[0294] The following components were mixed in two 2 ml glass vials (preparations 1 and 2): 225 μl of deionized water, 50 μl of a 500 mM TEA-HCl buffer (pH 8), 25 μl of a 100 g / l allitol solution (final concentration 5 g / l) and 275 μl of a 10 mM NADP + solution (final concentration 5.5 mM). To start the reaction, 25 μl of oxidoreductase lysate (see Table 3 below) was added to each vial. The vials were incubated with continuous shaking (30 °C, 800 rpm) for a total of 20 h.

[0295] For analysis, 100 μl of a sample 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. The supernatant was diluted 1:100 with ultrapure water and analyzed using HPAEC (PAD). The results are presented in Table 3 below.

[0296] Table 3

[0297] The results in Table 3 show that for the conversion of allitol to allose, cofactor regeneration (see Example 1) is advantageous in terms of yield, but not absolutely necessary.

[0298] Example 3

[0299] Production of allose from D-fructose

[0300] The following components were mixed in three 2 ml glass vials (batches 1 to 3): 210.4 μl of deionized water, 100 μl of a 500 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. Then, 35 μl of ribitol dehydrogenase lysate, 12 U of alcohol dehydrogenase lysate, and 5 μl of a 10 mM NAD + solution and 50 μl of 2-propanol were added to the samples. The samples were incubated with continuous shaking (35 °C, 800 rpm) for a total of 20 h.

[0301] Then, batches 1 to 3 were heated to 70 °C for 60 min. After cooling, 25 μl of oxidoreductase lysate (see Table 4 below), 10 U of NAD(P)H oxidase lysate and 5 μl of 5 mM NADP +-solution was added. The mixtures were incubated in an Eppendorf Thermomixer with continuous shaking (24 °C, 800 rpm) for 24 h. In three additional 2 ml glass vials (mixtures 4 to 6), the following components were mixed: 142.7 μl of deionized water, 100 μl of a 500 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. Then, 35 μl of ribitol dehydrogenase lysate, 12 U of alcohol dehydrogenase lysate, 5 μl of a 10 mM NAD + -solution, 50 μl 2-propanol, 25 μl oxidoreductase lysate (see Table 4 below), 10 U NAD(P)H oxidase lysate and 5 μl of a 5 mM NADP + solution was added to the mixture. The mixture was incubated with continuous shaking (30 °C, 800 rpm) for a total of 44 h.

[0302] For analysis, 50 μl of a sample 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 350 μl of deionized water, vortexed, and then centrifuged for 5 min at max. 200 μl of the supernatant was transferred to an HPLC vial with insert and analyzed by HPLC (RI detection). The supernatant was diluted 1:1000 with ultrapure water and analyzed by HPAEC (PAD).

[0303] Table 4

[0304] Table 4 shows that the conversion of D-fructose in a one-pot process can be carried out both with and without thermal deactivation of the enzymes of the first step (epimerase and ribitol dehydrogenase), with the conversion without deactivation providing better yields.

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Claims

Patent claims 1. A process for the preparation of an aqueous solution containing allose, by forming D-psicose from D-fructose, which is dissolved in an aqueous solution, by treatment with an epimerase in vitro, after which the D-psicose is converted into oxidized cofactor NAD(P) by treatment with an NAD(P)H-dependent oxidoreductase in vitro to form + is reduced to allitol, after which the allitol is enzymatically oxidized to allose.

2. Process according to claim 1, characterized in that the epimerase and the NAD(P)H-dependent oxidoreductase are deactivated or removed from the aqueous solution by ultrafiltration before the allitol is enzymatically oxidized to allose.

3. Process according to one of claims 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 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. A process for preparing an aqueous solution containing allose by oxidizing allitol, characterized in that the oxidation is carried out enzymatically.

7. A process according to any one of claims 1 to 6, characterized in that the enzymatic oxidation of allitol to allose with a corresponding NAD(P) + -dependent oxidoreductase with formation of reduced cofactor NAD(P)H.

8. Method according to one of claims 1 to 7, characterized in that the enzymes are present as a lysate of the corresponding cells producing them.

9. A method according to claim 7, characterized in that the reduced cofactor NAD(P)H produced by the oxidation is converted to NAD(P) by means of an NAD(P)H oxidase and oxygen. + is oxidized.

10. Process according to one of claims 1 to 9, characterized in that the enzymatic oxidation of allitol to allose with an NAD(P) +-dependent oxidoreductase comprising an amino acid sequence selected from the group consisting of: i) an amino acid sequence having an identity to SEQ ID No. 1, SEQ ID No. 3, 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. 23 of at least 75%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 2, SEQ ID No. 4, 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. 24 of at least 75%, and iii) an amino acid sequence encoded by a nucleic acid which binds under stringent conditions to a complementary strand of a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 2, SEQ ID No. 4, 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.

24.

11. Method according to one of claims 3 or 4, characterized in that the alcohol dehydrogenase is used to reduce the cofactor NAD(P) + comprises or consists of an amino acid sequence selected from the group consisting of: i) an amino acid sequence having at least 80% identity to SEQ ID No. 19, ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity to SEQ ID No. 20, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a complementary strand of a nucleic acid molecule having the nucleic acid sequence SEQ ID No.

20.

12. The method according to claim 9, characterized in that the NAD(P)H oxidase is used to oxidize NAD(P)H to NAD(P) +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. 21 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 22 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a complementary strand of a nucleic acid molecule having the nucleic acid sequence SEQ ID No.

22.

13. Use of an NAD(P) +-dependent oxidoreductase comprising an amino acid sequence selected from the group consisting of: i) an amino acid sequence having an identity to SEQ ID No. 1, SEQ ID No. 3, 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. 23 of at least 75%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 2, SEQ ID No. 4, 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. 24 of at least 75%, and iii) an amino acid sequence encoded by a nucleic acid which anneals under stringent conditions to a complementary strand of a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 2, SEQ ID No. 4, 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.24 binds to oxidize allitol to allose.

14. Use of an alcohol dehydrogenase to reduce NAD(P) + to NAD(P)H, 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. 19 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity to SEQ ID No. 20, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a complementary strand of a nucleic acid molecule having the nucleic acid sequence SEQ ID No.

20.

15. Use of a HjO-forming NAD(P)H oxidase to oxidize 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. 21 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 22 of at least 80%, and iii) an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a complementary strand of a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 22.

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