Process for the production of an aqueous solution containing l-sorbose

Abstract

A process for producing an aqueous solution containing L-sorbose by reducing D-glucose, which is dissolved in an aqueous solution, in vitro to D-sorbitol by treating the dissolved D-glocose with an NAD(P)H-dependent oxidoreductase, whereupon the D-sorbitol is oxidized in vitro with an NAD(P)+-dependent oxidoreductase to obtain L-sorbose, and then the two oxidoreductases are separated out.

Description

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

[0002] The present invention relates to the preparation of an aqueous solution of L-sorbose.

[0003] Background of the invention

[0004] The monosaccharide L-sorbose belongs to the group of ketohexoses and is a C5 epimer of D-fructose. L-sorbose can be used as a low-calorie sweetener (Shintani, 2019) and also serves as a starting material for the enzymatic synthesis of rare sugars such as L-tagatose (Itoh et al., 1996) or L-galactose (Leang et al., 2004) and sugar alcohols such as L-iditol (Vongsuvanlert & Tani, 1988). A recent study demonstrated the antitumor properties of L-sorbose, which induce apoptosis in cancer cells (Xu et al., 2023).

[0005] Furthermore, L-sorbose is an intermediate in the Reichstein synthesis of vitamin C (L-ascorbic acid). The route starts from D-glucose, which is first reduced to the sugar alcohol D-sorbitol (see below). D-sorbitol is then microbially oxidized to L-sorbose. Treatment with KMnO4 as an oxidant yields 2-keto-L-gulonic acid (using acetal protecting groups), which is converted to L-ascorbic acid upon addition of acid (Reichstein & Grüssner, 1934). L-sorbose can also be oxidized microbially or with molecular oxygen over Pt- or Pd-based catalysts to 2-keto-L-gulonic acid (Sugisawa et al., 1990; Bronnimann et al., 1994).

[0006] Several chemical processes are known for the production of L-sorbose. For example, D-glucose can be converted to L-sorbose using Pure Silicate Zeolite Beta with Lewis acid Ti 4+-centers are isomerized. However, the observed yields of L-sorbose are max. 12% (w / w) when using a 1% (w / w) D-glucose solution and a reaction temperature of 100 °C. Furthermore, D-fructose and D-mannose are found as byproducts (Gounder & Davis, 2013). This method has also been patented in US 9255120 B2 and US 11292806 B2.

[0007] L-sorbose can also be obtained by electrochemical oxidation of D-sorbitol on Pt electrodes with p-block metals (Bi, Sb, Pb, Sn, In) as promoters, but D-fructose is formed as a by-product (Kwon et al., 2014).

[0008] For the microbial oxidation of D-sorbitol, mainly acetic acid bacteria such as Gluconobacter or Acetobacter are used (Shintani, 2019; Zebiri et al., 2011; WO 2000 / 049133 A1; EP 0233050 B1; EP 0273648 B1; EP 0955358 B1; EP 1153120 A1; EP 0199548 A2; US 6664082 B1; US ​​4945048; JP H07102127 B2; KR 820001130 B1). US 4904588 A describes various Gluconobacter (sub)oxydans strains that can completely oxidize up to 300 g / l D-sorbitol within 24 h (Gluconobacter suboxydans strains) or 200 g / l D-sorbitol within 40 h (Gluconobacter oxydans strains) to L-sorbose. Liu et al. (2022) used an engineered G. oxydons strain to convert 300 g / l D-sorbitol to 298.61 g / l L-sorbose in 100 h.

[0009] The enzymes responsible for the oxidation of D-sorbitol to L-sorbose are the membrane-bound D-sorbitol dehydrogenase (mSLDH; EC 1.1.99.21), which has either pyrroloquinoline quinone (PQQ) or flavin adenine dinucleotide (FAD) as a cofactor (Toyama et al., 2005; Soemphol et al., 2007; Yang et al., 2008), the NADP-dependent D-sorbitol dehydrogenase from G. oxydans (GoSLDH) (Kim et al., 2019), and the NADP-dependent L-sorbose reductase (SR; EC 1.1.1.289) (Sugisawa et al., 1991; Shinjoh et al., 2002; Kubota et al., 2011). and mannitol 2-dehydrogenase (MDH; EC 1.1.1.67) from Pseudomonas fluorescens (Kavanagh et al., 2002) or Aspergillus fumigatus (Krahulec et al., 2011).

[0010] KR 102060253 Bl describes a recombinant E. coli strain expressing GoSLDH and an NAD(P)H oxidase (for cofactor regeneration) from Lactobacillus reuteri, which converted 10 mM (1.82 g / l) D-sorbitol to 92%.

[0011] The enzymatic oxidation of D-sorbitol can also be performed in cell-free processes (in vitro). WO 2014 / 171635 A1 describes a GoSLDH with which 10 mM (1.82 g / L) of D-sorbitol was converted to 7.5 mM L-sorbose within 3 h. Kim et al. (2016) also used a GoSLDH from G. oxydans G624 to oxidize 50 mM (9.11 g / L) of D-sorbitol to L-sorbose (47% conversion in 3 h). In both described processes, the cofactor NADP was used in stoichiometric amounts, meaning no cofactor regeneration system was used.

[0012] EP 0955358 A2 describes genetically modified Gluconobacter or Acetofaocter mutants lacking L-sorbose reductase activity. With the SR3 mutant, 80 g / l of D-sorbitol was oxidized to 75 g / l of L-sorbose within 24 hours, with the L-sorbose concentration in the medium decreasing to 70 g / l after 5 days. In the control strain (G. suboxydans IFO 3291) with intact L-sorbose reductase activity, only approximately 40 g / l of L-sorbose was detected after 24 hours, most of which was then further metabolized.

[0013] When using an mSLDH (from G. suboxydans IFO 3255), an electron acceptor such as phenazine methosulfate (7.7 mM) must be added for the oxidation of D-sorbitol (38 mM = 6.95 g / l) (formation rate of L-sorbose: 1.3 mg / h), as described in EP 0728840 B1 and US 5747301. The sugar alcohol D-sorbitol also occurs naturally in small amounts, for example, in the fruits of the rowan tree (Sorbus aucuparia L.) (Lohmar, 1957), but is mostly produced chemically by hydrogenation on nickel surfaces. Industrially widely used Raney nickel catalysts are characterized by their high activity and low price, but suffer from nickel leaching during the process (Jonas & Silveira, 2004; Zhao et al., 2020; Garcia et al., 2021), which requires nickel residues to be removed from the product in subsequent purification steps.Contact with nickel can trigger a number of diseases such as allergies, cardiovascular and kidney diseases, pulmonary fibrosis, and lung and nasal cancer (Genchi et al., 2020).

[0014] Biotechnological processes for the production of D-sorbitol are also known.

[0015] D-sorbitol can be produced by fermentation with Zymomonas mobilis. The responsible enzyme is glucose-fructose oxidoreductase (GFOR, EC 1.1.99.28), which converts mixtures of D-glucose and D-fructose into D-gluconolactone (or D-gluconate by enzymatic hydrolysis) and D-sorbitol (Barrow et al., 1984; Zachariou & Scopes, 1986). Processes using Zymomonas mobilis are described, for example, in US Pat. No. 4,755,467, US Pat. No. 5,190,869, EP 0427150 A1, and EP 0212517 A2.

[0016] EP 0132557 B1 describes a process for converting a D-glucose / D-fructose mixture into D-gluconate and sorbitol using immobilized glucose dehydrogenase from Bacillus megaterium and sorbitol dehydrogenase from sheep liver. Ikemi et al. described a similar system using only D-glucose as the substrate. The reduction was carried out using an aldose reductase from Candida tropicalis IAM 12202, with the regeneration of the cofactor NADPH being accomplished with a glucose dehydrogenase (Ikemi et al., 1990a; Ikemi et al., 1990b).

[0017] The aldol reaction of L-glyceraldehyde and dihydroxyacetone phosphate (DHAP), catalyzed by a fructose-l,6-diphosphate aldolase or a tagatose-l,6-diphosphate aldolase, followed by removal of the terminal phosphate group, yields a mixture of L-psicose and L-sorbose. The reaction cascade can be carried out both in vitro and fermentatively in an engineered Corynebacterium glutamicum strain (Yang et al., 2015). Yang et al. (2016) further developed the reaction cascade so that glycerol can also be used as a starting material for the fermentative production of L-sorbose. The core of the cascade is also the aldol reaction of L-glyceraldehyde and DHAP.

[0018] L-sorbose can also be produced directly from mixtures of D-glucose and D-fructose. In the first step, D-fructose is reduced to D-sorbitol using permeabilized Z. mobilis cells, which is then oxidized to L-sorbose by G. suboxydans in a second step. This oxidation step is inhibited by the D-gluconic acid formed by Z. mobilis from D-glucose, which is why the process must be carried out as a two-step fed-batch procedure (Kim et al., 1994).

[0019] Most biotechnological processes for the production of D-sorbitol start from D-fructose. Only the process by Ikemi et al. (Ikemi et al., 1990a; Ikemi et al., 1990b) starts from the cheaper raw material D-glucose, which is also used for the large-scale production of D-sorbitol by hydrogenation. The disadvantage of this process, however, is that half of the D-glucose must be oxidized to D-gluconic acid to provide the necessary reducing equivalents for the production of D-sorbitol.

[0020] According to the current state of the art, no one-pot process, i.e. a process without isolation of intermediate products, starting from D-glucose can be described for L-sorbose.

[0021] For D-fructose (the C5 epimer of L-sorbose), enzymatic processes are known in the literature (e.g., US 11339415 B2) that proceed via the intermediate D-sorbitol. D-glucose was first reduced to D-sorbitol using a xylose reductase from Candida tropicalis in combination with an alcohol dehydrogenase (ADH) from Lactobacillus kefir for cofactor regeneration (oxidizes 2-propanol to acetone). Both enzymes were added in the form of cell lysates. After deactivation of the enzymes at 60 °C under vacuum (to remove 2-propanol and acetone), the oxidation step to D-fructose was carried out with a sorbitol dehydrogenase from Bacillus subtilis, with cofactor regeneration being achieved either with a lactate dehydrogenase from rabbit muscle (and pyruvate as cosubstrate) or with an NADH oxidase from Leuconostoc mesenteroides.

[0022] This is where the object of the present invention comes in. The invention aims to provide an efficient process for producing an aqueous solution of L-sorbose with high product concentration and high conversion starting from D-glucose, which can be carried out in a one-pot process and in which no deactivation is required between the reduction and oxidation steps.

[0023] Detailed description of the invention

[0024] The present invention consists in reducing D-glucose, which is dissolved in an aqueous solution, to D-sorbitol by treatment with an NAD(P)H-dependent oxidoreductase in vitro, after which the D-sorbitol is reacted with an NAD(P) + -dependent oxidoreductase in vitro to L-sorbose, after which the two oxidoreductases are separated.

[0025] A preferred variant of the process according to the invention consists in that the oxidized cofactor NAD(P) produced by the reduction + by means of an alcohol dehydrogenase and a secondary alcohol to form a ketone. Methods for regenerating cofactors are known, for example, from EP 2812439 B1.

[0026] 2-Propanol is particularly used as a secondary alcohol.

[0027] The reduced cofactor NAD(P)H produced by oxidation is best converted to NAD(P) using an NAD(P)H oxidase and oxygen. + oxidized.

[0028] The process according to the invention can be carried out as a one-pot reaction without isolation of intermediates.

[0029] The reaction scheme of the process according to the invention is shown in the attached Figure 1. A stands for D-glucose, B for D-sorbitol, C for L-sorbose, D for 2-propanol, E for acetone, 1 for reduction, 2 for alcohol dehydrogenase, 3 for oxidation, and 4 for NAD(P)H oxidase.

[0030] The preferred concentration of D-glucose is 50 - 250 g / l.

[0031] The preferred temperature range for the reduction step is between 25 and 55 °C, and for the oxidation step between 20 and 40 °C.

[0032] The preferred pH range for both steps is between 7.0 and 9.0.

[0033] In a further preferred variant of the method according to the invention, the enzymes are present in a suspension, in the homogenate and / or in the lysate of the corresponding cells forming them, with lysates being particularly preferred.

[0034] 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 lack 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).

[0035] In another variant, the enzymes can also be immobilized on a solid support material. Separation of the enzymes can be achieved, for example, by centrifugation or ultrafiltration.

[0036] The enzyme used to reduce D-glucose comes from the group of oxidoreductases, with xylose reductase being particularly preferred.

[0037] The enzyme used for the oxidation of D-sorbitol comes from the group of oxidoreductases, with mannitol dehydrogenase being particularly preferred.

[0038] 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).

[0039] 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.

[0040] 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. 7 or SEQ ID No. 9 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 8 or SEQ ID No. 10 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 or to a nucleic acid molecule with the nucleic acid sequence SEQ ID No. 8 or SEQ ID No. 10, wherein stringent conditions preferably comprise one or more washing steps at 65°C and a salt concentration of 0.1x to 2x SSC.

[0041] SEQ ID No. 7:

[0042] MKVWVGCTHAGTAAVKTILNEHPDASVSVYERNDNVSFLSCGIALYVGGVVKDPAGLFYSSPEELASMGAKIN

[0043] MEHNVKNIDNENKVVVIENLKTGETFEESYDKLVMTTGSWPIIPPIDGINSENILLCKNYNQANEIIKESKNAKKIV IVGGGYIGIELVEAFAESGKQVTLVDGLDRILNKYLDAEFTSVLEHDLQERGVTLNQTVEKFVAVKVK TPVGEYEADLVILCVGFKPNTDLLKDKVEM LPNGAIVVDEYMRTSDEAIFAAGDCAVHYNPTGGSAYIPLATN AVRMGALVGKNIVSPTVKYRGTQATSGLYLFGFNIGSTGLTENSAPHFGVVVVVEDNYRPEFMPTTEKVT MKLVYEVGTNRIVGGQIMSKYDVTQSANTLSLCVQNKMTIEDLAYVDFFFQPHFDRPWNYLNILAQAAVEQER KLAK SE ID Nr. 8:

[0044] ATGAAAGTAGTAGTAGTAGGCTGTACACATGCAGGAACAGCGGCAGTTAAGACGATTTTAAATGAACATC

[0045] CAGATGCATCAGTATCAGTATATGAGCGTAATGACAATGTCTCATTTCTATCTTGTGGGATTGCGTTGTATG

[0046] TTGGTGGAGTTGTGAAAGATCCTGCAGGTTTGTTTTATTCAAGTCCAGAAGAACTTGCATCAATGGGCGCG

[0047] AAAATTAACATGGAACACATGTGAAAATATAGATAATGAATAAGGTCGTAGTAATTGAATTTTAAA

[0048] AACAGGCGAAACATTTGAAGAAAGCTATGATAAGTTGGTAATGACAACTGGATCATGGCCAATTATTCCTC

[0049] CAATTGATGGAATCAATAGTGAAAATATTCTTTTGTGTAAAAACTATAACCAAGCAAATGAATTATTAAAG

[0050] AATCAAAAAAATGCTAAAAGATTGTCATTGTTGGTGGTGGCTATATTGGAATTGAATTAGTTGAGGCATTT

[0051] GCAGAATCTGGCAAGCAAGTGACGCTAGTTGATGGATTAGATCTGTATTTAAACAAATATTTAGTGCTGA

[0052] ATTCACTTCTGTTTTAGAGCATGATTTACAAGAAAGAGGCGTTACGCTAGCTTTAAACCAAACCGTCGAGAA

[0053] ATTTGTTGCCAATGAATCAGGTGCTGTGACAGCTGTGAAAACACCAGTTGGAGAATATGAGGCTGATTTAG

[0054] TTATTTTATGTGTTGGATTTAAACCAAATACTGATTTGTTGAAGGATAAAGTAGAGATGTTGCCAAATGGTG

[0055] CCATCGTAGTGGATGAATATATGAGAACAAGCGATGAAGCGATTTTTGCTGCTGGCGATAGTTGCGCGGTT

[0056] CATTATAATCCAACTGGAGGCTCTGCGTAATTCCGTTAGCTACAAATGCAGTTTAGATGGGAGCTTTAGTT

[0057] GGGAAAAAATTGTTTCTCCCAACAGTTAAATATCGTGGCACGCAAGCAACTTCTGGTTTATATTTTATTGGT

[0058] TTTAATATAGGTTCAACCGGATTGACTGAAAATAGCGCTCCTCATTTTGGCGTAGAGGTTCGTTCAGTAGTT

[0059] GTAGAAGATAATTATCGTCCAGAGTTTATGCCGCAACAGAGAAAGTAACGATGAAATTAGTTTATGAAGT

[0060] AGGAACGAATCGGATTGTTGGAGGTCAAATCATGTCAAAATATGATGTGACACAATCTGCCAATACGTTAT

[0061] CTTTATGTGTTCCAAATAAAATGACGATTGAGGATTTGGCTTATGTAGATTTCTTCTTCCAACCTCACTTTGA TCGTCCTTGGAACTATTTAATATTTTAGCGCAAGCTGTTGAGCAAGAGCGTAAACTAGCAAAATAA

[0062] SEQ ID No. 9:

[0063] MSKMVGANHAGTAAINTILDNYGSENEVVVFDQNSNISFLGCGMALWIGKQISGPQLFYADKESLEAKGAKI

[0064] YM ESPVTAIDYDAKRVTALVNGQEHVESYEKLILATGSTPILPPIKGAAIKEGSRDFEATLKNLQFVKLYQNAEDVI

[0065] NKLQDKSQNLNRIAVVGAGYIGVELAEAFKRLGKEVILIDVVDTCLAGYYDQDLSEMM RQNLEDHGIELAFGET

[0066] VKAIEGDGKVERIVTDKASHDVDMVILAVGFRPNTALGNAKLKTFRNGAFLVDKKQETSIPDVYAIGDCATVYD

[0067] NAINDTNYIALASNALRSGIVAGHNAAGHKLESLGVQGSNGISIFGLNMVSTGLTQEKAKRFGYNPEVTAFTDF

[0068] QKASFIEHDNYPVTLKIVYDKDSRLVLGAQMASKEDMSMGIHM FSLAIQEKVTIERLALLDYFFLPHFNQPYNY MTKAALKAK

[0069] SE ID Nr. 10:

[0070] ATGAGCAAAATTGTTATCGTGGGTGCAAATCATGCAGGCACCGCAGCAATTAATACCATTCTGGATAATTA

[0071] TGGCAGCGAAAATGAAGTGGTTGTGTTTGATCAGAATAGCAACATTAGCTTTCTGGGTTGTGGTATGGCAC

[0072] TGTGGATTGGTAAACAAATTAGCGGTCCGCAGGGTCTGTTTTATGCAGATAAAGAAAGCCTGGAAGCAAA

[0073] AGGTGCCAAAATCTATATGGAAAGTCCGGTTACCGCCATTGATTATGATGCAAAACGTGTTACCGCACTGG TTAATGGTCAAGAACATGTTGAAAGCTACGAGAAACTGATTCTGGCAACCGGTAGCACCCCGATTCTGCCT CCGATTAAAGGTGCAGCCATTAAAGAAGGTAGTCGCGATTTTGAAGCAACCCTGAAAAATCTGCAGTTCGT GAAACTGTATCAGAATGCCGAAGATGTGATTAACAAACTGCAGGATAAAAGCCAGAATCTGAATCGTATT GCAGTTGTTGGTGCAGGTTATATTGGTGTTGAACTGGCAGAAGCATTTAAACGTCTGGGTAAAGAAGTGA TTCTGATTGACGTTGTTGATACCTGTCTGGCAGGTTATTATGATCAGGATCTGAGCGAAATGATGCGTCAG AATCTGGAAGATCATGGTATCGAACTGGCATTTGGTGAAACCGTTAAAGCAATTGAAGGTGATGGTAAAG TGGAACGTATTGTTACCGATAAAGCAAGCCATGATGTGGATATGGTTATTCTGGCAGTTGGTTTTCGTCCG AATACAGCACTGGGTAATGCAAAACTGAAAACCTTTCGTAATGGTGCCTTTCTGGTGGATAAAAAACAAGA AACCAGCATCCCGGATGTTTATGCAATTGGTGATTGTGCAACCGTGTATGATAATGCCATTAACGACACCA ACTATATTGCACTGGCAAGCAATGCACTGCGTAGCGGTATTGTTGCAGGTCATAATGCAGCCGGTCATAAA CTGGAAAGTCTGGGTGTTCAGGGTAGCAATGGTATTTCAATTTTTGGCCTGAATATGGTTAGCACCGGTCT GACCCAAGAAAAAGCCAAACGTTTTGGTTATAATCCGGAAGTTACCGCCTTTACCGATTTTCAGAAAGCCAGCTTTATCGAGCATGATAACTATCCGGTTACGCTGAAAATTGTGTATGACAAAGATAGCCGTCTGGTTCTG GGTGCACAGATGGCCAGCAAAGAAGATATGAGCATGGGTATTCACATGTTTAGCCTGGCCATTCAAGA AAGTTACCATTGAACGTCTGGCCCTGCTGGATTATTTCTTTCTGCCGCATTTTAATCAGCCGTACAACTATAT GACCAAAGCAGCACTGAAAGCCAAATAA

[0074] The preferably used HjO-forming NAD(P)H oxidase preferably comprises or consists of an amino acid sequence that has an identity to SEQ ID No. 7 or SEQ ID No. 9 of at least 80%, even more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, in particular 100%. Particularly preferably, the HjO-forming NAD(P)H oxidase comprises or consists of the amino acid sequence SEQ ID No. 7 or SEQ ID No. 9.

[0075] 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. 8 or SEQ ID No. 10 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. 8 or SEQ ID No. 10.

[0076] 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. 7 or SEQ ID No. 9 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 8 or SEQ ID No. 10 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 or to a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 8 or SEQ ID No. 10, wherein stringent conditions preferably comprise one or more washing steps at 65°C and a salt concentration of 0.1x to 2x SSC.

[0077] The oxidoreductase used according to the invention to form D-sorbitol from D-glucose is preferably a xylose reductase. It has been shown that xylose reductases are capable of converting D-glucose to D-sorbitol in the presence of the cofactor NAD(P)H. Accordingly, a further aspect of the present invention relates to the use of xylose reductases for reducing D-glucose to D-sorbitol in the presence of the cofactor NAD(P)H.

[0078] The oxidoreductase for the formation of D-sorbitol from D-glucose preferably 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. 1 or 3 of at least 80%, ii) an amino acid sequence which is encoded by a nucleic acid which has an identity to SEQ ID No. 2 or 4 of at least 80%, and iii) an amino acid sequence which is encoded by a nucleic acid which binds under stringent conditions to a complementary strand of a nucleic acid molecule or to a nucleic acid molecule with the nucleic acid sequence SEQ ID No. 2 or 4, wherein stringent conditions preferably comprise one or more washing steps at 65°C and a salt concentration of 0.1x to 2x SSC.

[0079] SEQ ID No. 1:

[0080] MATPTIKLNSGYDMPLVGFGLWKVNRETCADQVYEAIKAGYRLFDGACDYGNEVEAGQGVARAIKEGIVKR EDLFIVSKLWNTFHEADKVEPIARKQLADWGLDYFDLYLIHFPIALKYVDPAEIYPPGWTGTKKEVEFSNATIQE TWQAM ETLVDKKLTRSIGISNFSAQLIMDLLRYARIRPATLQIEHHPYLTQQALVEYVQKEGIAVTAYSSFGPLS FLELGHQVAKDTPLLFEHSTVKSIAEKHGKTPAQVLLRWATQRNIAVIPKSNNPGRLAQNLDVTAWDLEPADI EALSALNKNLRFNNPPSYGLYIPIFA

[0081] SEQ ID Nr. 2:

[0082] ATGGCCACGCCTACTATCAAGCTGAACAGCGGCTATGACATGCCCCTGGTGGGCTTTGGTCTGTGGAAG GTCAACAAGGAAACCTGCGCGGACCAGGTCTACGAGGCTATCAAGGCGGGCTACCGCTTGTTTGACGGT GCGTGCGACTATGGCAACGAAGTTGAGGCCGGCCAGGGTGTCGCTCGCGCCATCAAGGAAGGCATTGT

[0083] GAAGCGTGAGGACCTCTTCATTGTGTCCAAGCTGTGGAACACGTTCCACGAGGCCGACAAGGTCGAGCC

[0084] GATCGCGCGGAAGCAGCTGGCCGACTGGGGCCTCGACTACTTTGACCTGTACCTCATCCACTTCCCGATC

[0085] GCGCTGAAGTACGTCGACCCGGCCGAGATCTACCCGCCGGGCTGGACGGGCACCAAGAAGGAGGTCGA

[0086] GTTCAGCAACGCGACGATCCAGGAGACGTGGCAGGCCATGGAGACCCTGGTCGACAAGAAGCTGACGC

[0087] GCAGCATCGGCATCAGCAACTTCAGCGCCCAGCTGATCATGGACCTGCTGCGGTACGCGCGCATCCGCCC

[0088] CGCGACCTTGCAGATCGAGCACCACCCGTACCTGACGCAGCAGGCGCTGGTCGAGTACGTGCAGAAGGA

[0089] GGGCATCGCCGTGACGGCGTACTCGTCCTTCGGCCCACTGAGCTTCCTGGAACTGGGCCACCAGGTCGCC

[0090] AAGGACACGCCGCTGCTCTTCGAGCACTCGACCGTCAAGTCGATCGCCGAGAAGCACGGCAAGACGCCC

[0091] GCCCAGGTGCTACTGCGCTGGGCCACCCAGCGCAACATCGCCGTCATCCCCAAGAGCAACAACCCGGGC

[0092] CGCCTGGCGCAGAACCTGGACGTGACGGCGTGGGATCTGGAGCCCGCCGACATTGAGGCCTTGAGCGC

[0093] GCTGAACAAGAACCTTCGATTCAACAACCCACCTAGCTACGGACTGTACATCCCGATCTTCGCTTAA

[0094] SEQ ID Nr. 3:

[0095] MAPQIPNIKLSSGYDMPQVGFGLWKVDRSICADVVYNAIKIGYRLFDGACDYGNEVEAGQGIARAIKEGIVKR

[0096] EELFIVSKLWNTFHDGDKVEPIVRKQLADWGIDYFDLYLVHFPVALEYVDPSVRYPPGWFYDGEKEIRPSKATI

[0097] QETWTAMESLVEKGLARSIGVSNFQAQLLYDLLRYAKIRPATLQIEHHPFLVQQELLNLAKAEGIAVTAYSSFG

[0098] PQSFLEFNM KHAVQLTPLFEDETIKKIAAKYNRPASQVLLRWATQRGLAIIPKSTRPEIMKSNLESIEFDLSEEDI

[0099] ATISAFDRGLRFNQPTNYFPTEHLWIFG

[0100] SEQ ID Nr. 4:

[0101] ATGGCTCCCCAGATCCCCAACATTAAGCTCAGCAGCGGCTATGACATGCCCCAGGTGGGCTTTGGACTGT

[0102] GGAAGGTCGACCGCTCGATCTGCGCCGACGTCGTCTACAACGCAATTAAGATTGGCTACCGCCTGTTTGA

[0103] CGGCGCTTGCGACTATGGCAACGAAGTCGAGGCCGGCCAGGGCATTGCCCGCGCCATCAAGGAGGGCA

[0104] TCGTGAAGCGTGAGGAGCTCTTCATCGTGTCCAAGCTCTGGAACACCTTCCACGACGGCGACAAGGTCG

[0105] AGCCCATCGTCCGCAAGCAGCTCGCCGACTGGGGCATTGACTACTTCGACCTCTACCTCGTCCACTTCCCT

[0106] GTCGCCCTCGAGTACGTCGACCCCTCGGTCCGCTACCCGCCCGGCTGGTTCTACGATGGCGAGAAGGAG

[0107] ATCCGCCCCAGCAAGGCCACCATCCAGGAGACCTGGACCGCCATGGAGTCGCTCGTTGAGAAGGGTCTG

[0108] GCCCGCAGCATTGGTGTCTCCAACTTCCAGGCCCAGCTCCTCTACGACCTGCTGCGCTACGCCAAGATCC

[0109] GCCCGGCCACCCTTCAGATCGAGCATCACCCCTTCCTCGTCCAGCAGGAGCTGCTCAACCTGGCCAAGGC

[0110] CGAGGGCATTGCCGTGACTGCCTACAGCTCATTCGGCCCTCAGAGCTTCCTCGAGTTCAACATGAAGCAC

[0111] GCCGTGCAGCTCACCCCGCTCTTCGAGGACGAGACCATCAAGAAGATCGCCGCCAAGTACAACCGTCCTG

[0112] CTTCGCAGGTTCTCCTGCGCTGGGCCACTCAGCGCGGTCTGGCCATTATCCCCAAGAGCACGCGCCCCGA

[0113] GATCATGAAGTCCAACCTCGAGAGCATCGAGTTCGACCTCAGCGAGGAAGATATTGCCACCATCTCGGCC TTCGACCGCGGCCTGCGCTTCAACCAGCCCACAAACTACTTCCCCACCGAGCACCTCTGGATCTTTGGCTA A

[0114] The oxidoreductase for forming D-sorbitol from D-glucose preferably comprises or consists of an amino acid sequence having an identity to SEQ ID No. 1 or 3 of at least 80%, even more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 98%, even more preferably 99%, in particular 100%. Particularly preferably, the oxidoreductase according to the invention for forming D-sorbitol from D-glucose comprises or consists of the amino acid sequence SEQ ID No. 1 or 3.

[0115] Alternatively, the oxidoreductase for forming D-sorbitol from D-glucose preferably comprises or consists of an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 2 or 4 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 nucleic acid encoding the oxidoreductase according to the invention for forming D-sorbitol from D-glucose comprises or consists of the nucleic acid sequence SEQ ID No. 2 or 4.

[0116] 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.

[0117] 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 B LOS UM 62 scoring matrix (Henikoff & Henikoff, 1989), alignments (B) of 50, expectation (E) of 0.05, M = 1, N = -2.

[0118] Alternatively, the oxidoreductase for forming L-sorbose from D-glucose 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 or to a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 2 or 4. As used herein, the stringent conditions refer to conditions under which so-called specific hybrids, but not non-specific hybrids, are formed.

[0119] 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).

[0120] The oxidoreductase used according to the invention for the oxidation of D-sorbitol to L-sorbose is preferably a mannitol dehydrogenase. Mannitol dehydrogenases have been shown to be capable of oxidizing D-sorbitol in the presence of the cofactor NAD(P). + to L-sorbose. Accordingly, a further aspect of the present invention relates to the use of mannitol dehydrogenases for the oxidation of D-sorbitol to L-sorbose in the presence of the cofactor NAD(P) +.

[0121] The oxidoreductase for forming L-sorbose from D-sorbitol preferably comprises an amino acid sequence selected from the group consisting of: i) an amino acid sequence having an identity to SEQ ID No. 5 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 6 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 or to a nucleic acid molecule having the nucleic acid sequence SEQ ID No. 6, wherein stringent conditions preferably comprise one or more washing steps at 65°C and a salt concentration of 0.1x to 2x SSC.

[0122] SEQ ID No. 5: MATKFSLATLDAVKATAGVPNYGRHDLRAGIVHFGVGNFHRAHQAVYLDDLFNLGRDRDWAIIGAGVLPSD KVM RDKLEAQDFLTTVVEQDNNRTGAHVTGAM IAYLEPGDTPAIVAQLASPLIRIVSLTITEGGYFIDPASGVF DPAHPAIVEDARDPAAPKTVFGLILAGLAERRAKGIPPFTIMSCDNIPGNGEVTHAAVSGLARLSDPGFADWI DANVAFPNGMVDRITPATGAREIGIVASQYGIDDAWPVFCEEFKQWVLEDRFPQGRPALEEVGVQFVPDVA PYEHM KIRILNGGHAAIAYPAALLDIHFVHEAM EEPLIRAFLSKLEHDEIIPVIPPVPDTDLKDYYKLIETRFSNPKI GDTVARLAQDGSNRQPKFILPSTADRLRRGEDVVGLSLVSALWCRYFAGKSDSGKEIVFNDANADRHAAAV AAKDDPMAFLALSDIFGDVAQSDLFRRRFAHALKVLWEKGTRATLQLYLDGNLGE

[0123] SEQ ID No. 6:

[0124] ATGGCGACCAAATTCTCCCTTGCCACGCTCGACGCGGTCAAAGCCACGGCCGGCGTCCCGAACTACGGCC GGCATGACCTTCGGGCCGGCATCGTGCACTTCGGCGTCGGCAATTTCCACCGCGCGCATCAGGCGGTCTA TCTCGATGACCTTTTCAACCTGGGACGCGATCGCGATTGGGCGATCATCGGCGCGGGTGTCCTGCCATCG GACAAGGTCATGCGCGACAAGCTCGAAGCGCAGGATTTCCTGACGACGGTGGTCGAGCAGGACAACAA CCGCACGGGCGCGCATGTCACCGGCGCCATGATCGCCTACCTCGAGCCCGGCGACACGCCCGCGATCGT GGCGCAGCTCGCGAGCCCGCTCATCCGCATCGTCTCCCTGACGATCACCGAAGGCGGCTATTTCATCGAT CCGGCATCCGGCGTCTTCGACCCGGCGCATCCGGCGATCGTCGAGGATGCGCGCGATCCGGCGGCTCCA AAAACCGTCTTCGGCCTCATCCTGGCAGGGCTCGCCGAACGCCGCGCCAAGGGTATTCCGCCGTTCACGA TCATGTCCTGCGACAATATTCCTGGAAACGGGGAGGTCACCCACGCTGCCGTTTCCGGTCTGGCGCGCCT TTCCGATCCAGGCTTTGCGGACTGGATCGATGCCAATGTCGCCTTTCCGAACGGCATGGTCGATCGCATC ACGCCGGCGACCGGTGCCCGCGAGATCGGCATCGTCGCCTCGCAATACGGAATCGACGATGCCTGGCCG GTCTTTTGCGAGGAATTCAAGCAATGGGTGCTGGAAGACCGCTTCCCGCAAGGCCGCCCGGCGCTGGAA GAGGTGGGCGTTCAGTTCGTGCCCGACGTGGCGCCGTACGAGCACATGAAGATCCGTATCCTCAACGGC GGCCATGCGGCGATCGCCTATCCGGCGGCACTGCTCGACATCCATTTCGTTCATGAGGCGATGGAGGAGCCGTTGATCCGCGCCTTCCTGTCAAAGCTGGAGCACGACGAGATCATCCCGGTGATACCGCCCGTTCCGG ACACGGATCTGAAGGATTATTACAAGCTCATCGAGACGCGTTTCTCCAATCCGAAGATCGGCGACACGGT CGCGCGGCTGGCGCAGGACGGCTCCAACCGACAGCCGAAATTCATCCTGCCGTCGACGGCCGACCGCCT GCGCCGCGGCGAAGACGTCGTCGGGTTGTCGCTCGTTTCGGCGCTGTGGTGCCGCTATTTCGCCGGCAA GTCGGACAGCGGCAAGGAGATCGTTTTCAACGACGCCAACGCCGACCGGCTGCATGCCGCCGCGGTTGC AGCGAAGGACGATCCAATGGCGTTCCTTGCGCTTTCCGACATCTTCGGCGATGTCGCGCAATCCGATCTC TTCCGCCGCCGTTTCGCTCACGCGTTGAAAGTGCTTTGGGAAAAGGGTACGCGTGCCACGCTCCAGCTTT ATCTGGACGGAAACCTCGGGGAATAA

[0125] The oxidoreductase for forming L-sorbose from D-sorbitol preferably comprises or consists of an amino acid sequence having an identity to SEQ ID No. 5 of at least 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%, and in particular 100%. Particularly preferably, the oxidoreductase according to the invention for forming L-sorbose from D-sorbitol comprises or consists of the amino acid sequence SEQ ID No. 5.

[0126] Alternatively, the oxidoreductase for forming L-sorbose from D-sorbitol preferably comprises or consists of an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 6 of at least 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%, in particular 100%. Particularly preferably, the nucleic acid encoding the oxidoreductase according to the invention for forming L-sorbose from D-sorbitol comprises or consists of the nucleic acid sequence SEQ ID No. 6.

[0127] Materials

[0128] L-Sorbose was purchased from TCI or Sigma-Aldrich, NAD + , NADH disodium salt, NADP +-Disodium salt, NADPH tetrasodium salt, and methanol were purchased from PanReac AppliChem (ITW Reagents), D-glucose, zinc chloride, IPTG (isopropyl-ß-D-thiogalactopyranoside) and 2-propanol were purchased from Sigma-Aldrich, D-sorbitol, 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.

[0129] Production of enzymes & preparation of lysates

[0130] General information on the expression of recombinant enzymes in E. coli

[0131] 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.

[0132] The results of the cloning step were verified by restriction enzyme digestion and DNA sequencing. The resulting construct carries the target gene under the IPTG-inducible T5 promoter. 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.

[0133] 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).

[0134] Preparation of cell lysates using sonifier disruption

[0135] 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.

[0136] 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).

[0137] 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.

[0138] Table 1. Enzyme classes and donor organisms for the enzymes used in the examples.

[0139] * Note: This protein is used as xylose reductase in US 10106824 B2.

[0140] Analytical methods

[0141] High Performance Liquid Chromatography

[0142] An Agilent HPLC 1260 Infinity II Series system was used to quantify D-glucose, L-sorbose, and D-sorbitol using HPLC (high-performance liquid chromatography). Detection was performed using a refractive index detector (RI detection). A Phenomenex Rezex RCM-Monosaccharide Ca+2 (8%) column with a corresponding precolumn was used for the measurement and eluted isocratically with 3.5% (v / v) 2-propanol in ultrapure water.

[0143] Determination of enzyme activities (optical-enzymatic assay)

[0144] 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).

[0145] 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 method described above.

[0146] Example 1

[0147] Reduction of D-glucose to D-sorbitol

[0148] The reduction reactions were carried out in 2 ml glass vials containing D-glucose (final concentration 50 g / l) dissolved in 50 mM TEA-HCl buffer (pH 8). The solutions also contained 5 U of alcohol dehydrogenase lysate and 50 μl of 2-propanol for cofactor regeneration. To start the reaction, 0.5 U of xylose reductase (XR) lysate (see Table 2 below) was added. The mixtures were incubated in an Eppendorf Thermomixer with continuous shaking (30 °C, 800 rpm) for 24 h.

[0149] For analysis, 50 μl of a sample was mixed with 200 μl MeOH and incubated in an Eppendorf Thermomixer at 60 °C and 1200 rpm for 15 min. The sample was briefly centrifuged, mixed with 750 μl deionized water, vortexed, and then centrifuged for 5 min at max. 1 g. 150 μl of the supernatant was transferred to an HPLC vial with an insert and analyzed by HPLC (RI detection). The results are presented in Table 2 below.

[0150] Table 2

[0151] Example 2

[0152] Oxidation of D-sorbitol to L-sorbose (without cofactor regeneration)

[0153] The following components were mixed in a 2 ml glass vial: 250 μl of deionized water, 50 μl of a 500 mM TEA-HCl buffer (pH 8), 50 μl of a 100 g / l D-sorbitol solution (final concentration 10 g / l) and 50 μl of a 100 mM NADP +solution (final concentration 10 mM). To start the reaction, 25 μl of mannitol dehydrogenase I lysate was added to the mixture. The mixture was incubated with continuous shaking (30 °C, 800 rpm) for a total of 20 h.

[0154] For analysis, 400 μl of the mixture was mixed with 200 μl MeOH and incubated in an Eppendorf Thermomixer at 60 °C and 1200 rpm for 15 min. The sample was briefly centrifuged, mixed with 400 μl deionized water, vortexed, and then centrifuged for 5 min at max. 1 g. 150 μl of the supernatant was transferred to an HPLC vial with an insert and analyzed by HPLC (RI detection).

[0155] In this way, 87% of the D-sorbitol could be oxidized to L-sorbose.

[0156] Example 3

[0157] Oxidation of D-sorbitol to L-sorbose (with cofactor regeneration)

[0158] The oxidation reactions were carried out in 2 ml glass vials containing D-sorbitol (final concentration 50 g / l) and NAD + (final concentration 0.1 mM) in 100 mM TEA-HCl buffer (pH 8). Additionally, the solutions each contained 10 U of NADH oxidase lysate for cofactor regeneration. To start the reaction, 50 μl of mannitol dehydrogenase lysate (see Table 3 below) were added. The mixtures were incubated in an Eppendorf Thermomixer with continuous shaking (30 °C, 800 rpm) for 20 h.

[0159] For analysis, 100 μl of a sample was mixed with 200 μl of acetonitrile and incubated in an Eppendorf Thermomixer at 85 °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. 100 μl of the supernatant was transferred to an HPLC vial with an insert and analyzed by HPLC (RI detection). The results are presented in Table 3 below.

[0160] Table 3

[0161] Example 4

[0162] Production of L-sorbose from D-glucose

[0163] The reaction was carried out in a Labfors 5 benchtop bioreactor (Infors AG). A glass reactor (volume 3.4 l) equipped with a stirrer, pH electrode, and C sensor was used as the vessel. pH control was achieved by adding 1M NaOH or 1M H2SO4.

[0164] Initially, 100 ml of a D-glucose solution (500 g / l), 198.2 ml of deionized water, and 118 ml of a 200 mM TEA-HCl buffer (pH 8) were placed in the reactor and heated to 50 °C while stirring.

[0165] To start the reaction and to form D-sorbitol, 25 ml of xylose reductase II lysate, 8 kJ of alcohol dehydrogenase lysate, 5 ml of a 5 mM NADP + -solution and 50 ml of 2-propanol.

[0166] After 26 h, the reactor was cooled to 24 °C and 25 ml of mannitol dehydrogenase I lysate, 10 kU NADH oxidase lysate and 5 ml of 10 mM NAD + -solution added.

[0167] After a total runtime of 72 h (conversion: 98.0%), the reactor contents were heated to 70 °C, the pH was adjusted to 4, and the mixture was stirred for 60 min at 70 °C. The precipitate was filtered through a glass frit (P3).

[0168] 92.4 g / l L-sorbose was detected in the filtrate. Continuous samples were taken from the reactor solution during operation and analyzed as follows: 100 μl of the reaction mixture was mixed with 200 μl of acetonitrile and incubated in an Eppendorf Thermomixer at 85 °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. 100 μl of the supernatant was transferred to an HPLC vial with insert and analyzed by HPLC (RI detection).

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Claims

Patent claims 1. A process for the preparation of an aqueous solution containing L-sorbose, in which D-glucose, which is dissolved in an aqueous solution, is reduced to D-sorbitol by treatment with an NAD(P)H-dependent oxidoreductase in vitro, after which the D-sorbitol is reacted with an NAD(P) + -dependent oxidoreductase in vitro to L-sorbose, after which the two oxidoreductases are separated.

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

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

4. A method according to any one of claims 1 to 3, 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.

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

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

7. The method according to any one of claims 1 to 6, characterized in that the NAD(P)H-dependent oxidoreductase for reducing D-glucose to D-sorbitol is a xylose reductase and preferably comprises an amino acid sequence selected from the group consisting of: i) an amino acid sequence having an identity to SEQ ID No. 1 or 3 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 2 or 4 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. 2 or 4.

8. Method according to one of claims 1 to 7, characterized in that the NAD(P) +- dependent oxidoreductase for the oxidation of D-sorbitol to L-sorbose is a mannitol dehydrogenase and preferably 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. 5 of at least 80%, ii) an amino acid sequence which is encoded by a nucleic acid which has an identity to SEQ ID No. 6 of at least 80%, and iii) an amino acid sequence which is 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.

6.

9. Method according to one of claims 4 to 8, 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 at least 80% identity to SEQ ID No. 7 or SEQ ID No. 9, ii) an amino acid sequence encoded by a nucleic acid having at least 80% identity to SEQ ID No. 8 or SEQ ID No. 10, 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. 8 or SEQ ID No.

10.

10. Use of an NAD(P)H-dependent oxidoreductase for the reduction of D-glucose to D-sorbitol, wherein the NAD(P)H-dependent oxidoreductase comprises an amino acid sequence selected from the group consisting of: i) an amino acid sequence having an identity to SEQ ID No. 1 or 3 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 2 or 4 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. 2 or 4.

1. Use of an NAD(P) + -dependent oxidoreductase to oxidize D-sorbitol to L-sorbose, whereby the NAD(P) +-dependent oxidoreductase comprises an amino acid sequence selected from the group consisting of: i) an amino acid sequence having an identity to SEQ ID No. 5 of at least 80%, ii) an amino acid sequence encoded by a nucleic acid having an identity to SEQ ID No. 6 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. 6.

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