Modified xylose reductase and uses thereof

Abstract

The present invention relates to a method for the production of D-fructose comprising the step of reducing D-glucuronate to D-fructose using a modified xylose reductase comprising the motif GX1GX2X3X4 or a functional fragment thereof, wherein the modified xylose reductase has a lower D-glucose specificity and a lower D-xylose specificity compared to the unmodified xylose reductase, wherein the modified xylose reductase comprises at least one mutation within the motif GX1GX2X3X4 present in the unmodified xylose reductase, X1 is leucine or phenylalanine, X2 is leucine or cysteine, X3 is tryptophan and X4 is lysine or arginine, wherein X3 in the modified xylose reductase is tyrosine or phenylalanine.

Description

Technical Field

[0001] This invention relates to the field of enzymes, and in particular to enzymes capable of reducing D-glucurone to D-fructose. Background Technology

[0002] D-glucose is abundant in various biopolymers such as starch and cellulose, which are part of renewable raw materials. D-glucose can be isomerized to D-fructose, which plays an important role in the sugar industry due to its higher sweetness, and can also be converted to 5-(hydroxymethyl)furfural (HMF), a precursor to 2,5-furandicarboxylic acid (FDCA). FDCA is used to synthesize furan polymers such as poly(ethylene)furanate (PEF), a sustainable alternative to fossil fuel-based PET (Milić et al., 2023).

[0003] D-fructose can also be reduced to D-mannitol and D-sorbitol. Both of these sugar alcohols are low-calorie and non-cariogenic sweeteners. In addition, D-mannitol is increasingly used in foods and pharmaceuticals for patients with diabetes and / or obesity (as a diuretic and in tablet formulations for the treatment of renal failure, or as a drug carrier for dry powder inhalers), and in the production of specialty chemicals (Grembecka, 2015).

[0004] There are several different methods for producing D-fructose from D-glucose. One stepwise method has been used for large-scale production, characterized by the hydrolysis of a polysaccharide to form D-glucose, followed by the isomerization of the D-glucose to D-fructose. This produces a mixture containing 42% D-fructose, 50% D-glucose, and 8% residual polysaccharide. Further accumulation of D-fructose is achieved through separation methods. Chromatography, such as that described in US 5,221,478, can be used. In particular, the production of high-purity D-fructose using chromatography is very complex and costly.

[0005] Another alternative method is to produce D-fructose from D-glucose by combining an enzymatic and a chemical step. US4,246,347 discloses a method for producing D-fructose in this manner, in which 2% D-glucose is initially converted to D-glucurone using pyranose-2-oxidase. In a second step, the D-glucurone is then converted to D-fructose via hydrogenation. Hydrogenation requires high pressure and high temperature, and the reaction must be carried out at low substrate concentrations.

[0006] In addition to using a combination of enzymatic and chemical steps, a two-step process incorporating enzymatic redox reactions on carbohydrates has been described. US 10,253,340 discloses the production of D-fructose from D-glucose using xylose reductase (XR) via D-sorbitol as an intermediate. XR first generates D-sorbitol, which is subsequently reduced by sorbitol dehydrogenase. Cofactor recycling can be achieved at each step.

[0007] XRs are NAD(P)-dependent reductases (Liang et al., 2007) that have a variety of different aldehyde-containing substrates, including D-xylose, D-glucose, or 2-keto-D-glucose (D-glucurone), but cannot reduce sugars containing ketone groups, such as D-fructose (Neuhauser et al., 1997). GXGXW motifs (such as...) Figure 1 The tryptophan (W) residue in the ketone (as defined in the ketone group) leads to aldehyde reduction (C1-carbonyl), however, mutations of W to Y or F result in a strong preference for ketone reduction (C2-carbonyl) (Kratzer et al., 2006). Therefore, when W is mutated to Y or F, the catalytic mechanism changes from C1-carbonyl reduction to C2-carbonyl reduction.

[0008] For example, XR is used in the production of deoxyketose (US 5,077,203) and xylitol (CN 110628835). Furthermore, redox methods that do not require xylose reductase activity are supplemented with external cofactors (CN 111876452).

[0009] Known methods for producing D-fructose suffer from various drawbacks, making them unattractive. Chemical catalytic processes are typically carried out under high pressure and high temperature, exhibiting low product yields and requiring laborious and costly separation processes.

[0010] Biocatalytic redox processes are widely used to replace more expensive chemical synthesis because they do not generate large amounts of waste and can be carried out in a shorter time under milder reaction conditions (atmospheric pressure and temperature).

[0011] US patent 10,113,192 discloses the production of D-fructose from D-glucose using XR via D-glucuronide as an intermediate. In a one-pot system, D-glucose is oxidized to D-glucuronide by pyranose-2-oxidase as an intermediate, and then D-glucuronide is used as a substrate for XR to generate D-fructose. One disadvantage of this enzymatic system is that the process is carried out at relatively low substrate concentrations (2.5% D-glucose substrate load is converted to 91% D-fructose within 48 hours). At higher substrate concentrations, up to 5%-10% of the byproduct (D-sorbitol) accumulates in the reaction vessel.

[0012] The bioconversion rate of D-glucose increases with increasing pressure, because using compressed air to increase pressure leads to a higher conversion rate (Karmali and Coelho, 2011).

[0013] One object of the present invention is to provide an efficient method for producing pure D-fructose from D-glucose using D-glucurone as an intermediate at high substrate concentrations by using modified XR. Summary of the Invention

[0014] Surprisingly, the modified xylose reductase or its functional fragments exhibited lower D-glucose and D-xylose specificity compared to the corresponding unmodified xylose reductase, making it suitable for efficient D-fructose production. The reduction in D-glucose and D-xylose specificity, achieved by simultaneously enhancing D-glucuronide activity, contributes to reducing or even eliminating the formation of the byproduct D-sorbitol. This reduction in D-glucose and D-xylose specificity can be achieved by mutating the conserved common sequence of the xylose reductase. The inserted mutation did not affect the catalytic mechanism compared to existing techniques (Kratzer et al., 2006).

[0015] As used in this article, the “unmodified xylose reductase” can reduce D-glucurone to D-fructose; however, compared with the corresponding modified xylose reductase, the unmodified xylose reductase exhibits higher D-glucose specificity and higher D-xylose specificity.

[0016] The present invention also relates to the economical operation of a xylose reductase (XR)-dependent redox process by applying xylose reductase at high substrate concentrations according to the disclosed common sequence.

[0017] Another aspect of the invention relates to a method for producing D-fructose, the method comprising the step of reducing D-glucurone to D-fructose using a modified xylose reductase comprising motifs GX1GX2X3X4 or a functional fragment thereof, wherein the modified xylose reductase has lower D-glucose specificity and lower D-xylose specificity compared to an unmodified xylose reductase, wherein the modified xylose reductase comprises at least one mutation in motif GX1GX2X3X4 compared to an unmodified xylose reductase, wherein in the unmodified xylose reductase, X1 is leucine or phenylalanine, X2 is leucine or cysteine, X3 is tryptophan, and X4 is lysine or arginine, wherein in the modified xylose reductase, X3 is tyrosine or phenylalanine.

[0018] Another aspect of the invention relates to a modified xylose reductase or a functional fragment thereof, wherein the modified xylose reductase has lower D-glucose specificity and lower D-xylose specificity compared to an unmodified xylose reductase.

[0019] Another aspect of the invention relates to a modified xylose reductase or a functional fragment thereof comprising the motif GX1GX2X3X4, wherein the modified xylose reductase has lower D-glucose specificity and lower D-xylose specificity compared to an unmodified xylose reductase, wherein the modified xylose reductase contains at least one mutation within the motif GX1GX2X3X4 compared to an unmodified xylose reductase, wherein in the unmodified xylose reductase, X1 is leucine or phenylalanine, X2 is leucine or cysteine, X3 is tryptophan, and X4 is lysine or arginine, wherein in the modified xylose reductase, X3 is tyrosine or phenylalanine.

[0020] Another aspect of the invention relates to a modified xylose reductase or a functional fragment thereof comprising the motif GX1GX2X3X4, wherein the modified xylose reductase has lower D-glucose specificity and lower D-xylose specificity compared to an unmodified xylose reductase, wherein the modified xylose reductase comprises an amino acid sequence selected from the following:

[0021] i) An amino acid sequence having at least 50% sequence identity with any of SEQ ID Nos. 1 to 5.

[0022] ii) An amino acid sequence encoded by a nucleic acid sequence having at least 50% identity with any of SEQ ID Nos. 6 to 10, and

[0023] iii) An amino acid sequence encoded by a nucleic acid that binds to a nucleic acid molecule complementary to any one of the nucleic acid sequences SEQ ID No. 6 to 10 under stringent conditions, wherein the stringent conditions include washing at 65°C and at a salt concentration of 0.1 to 2 times that of SSC.

[0024] Another aspect of the invention relates to a nucleic acid molecule encoding a modified xylose reductase according to the invention.

[0025] Another aspect of the present invention relates to a carrier comprising the nucleic acid molecule of the present invention.

[0026] Another aspect of the present invention relates to a host cell comprising the nucleic acid molecule or vector of the present invention.

[0027] Another aspect of the invention relates to the use of the protein or host cell or its lysate or homogenate according to the invention for the reduction of D-glucuronide to D-fructose.

[0028] The use of the xylose reductases of the present invention, possessing the unique specificity mentioned above, allows for the production of compositions with unique compositions because these xylose reductases are capable of producing D-fructose from D-glucuronide at a higher rate. Therefore, another aspect of the invention relates to a composition obtainable by the method of the present invention. Attached Figure Description

[0029] Figure 1 A comparison of different xylose reductases is shown to define the GxGxW motif.

[0030] Figure 2 The possible reduction products when D-glucurone is used as a substrate are shown, namely D-glucose (reduced via C-2 carbonyl) or D-fructose (reduced via C1 carbonyl).

[0031] Figure 3 The process of D-fructose production is illustrated. Detailed Implementation

[0032] This invention relates to a method for producing D-fructose, the method comprising the step of reducing D-glucurone to D-fructose using a modified xylose reductase or a functional fragment thereof, wherein the modified xylose reductase has lower D-glucose specificity and lower D-xylose specificity compared to unmodified xylose reductase.

[0033] The teachings of this invention allow for the modification of xylose reductase to exhibit lower D-glucose specificity and lower D-xylose specificity compared to the corresponding unmodified xylose reductase. This reduction in both activities results in a much higher degree of D-fructose formation compared to xylose reductases with unmodified D-glucose and / or D-xylose specificity.

[0034] As used herein, the term "functional fragment" of xylose reductase refers to a fragment of xylose reductase that retains at least 50%, preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, and particularly 100% of its enzymatic activity in reducing D-glucuronide to D-fructose. The "functional fragment" may include one or more amino acid residues, preferably 1 to 20 amino acid residues, more preferably 2 to 15 amino acid residues, truncated at the N-terminus of xylose reductase. The "functional fragment" may also include one or more amino acid residues, preferably 1 to 50 amino acid residues, more preferably 2 to 25 amino acid residues, truncated at the C-terminus of xylose reductase.

[0035] As used herein, the term "enzyme specificity" or "enzyme specificity" is intended to indicate the degree to which an enzyme can catalyze a chemical reaction on more than one substrate molecule. An enzyme is said to have very high specificity for its substrate if it can catalyze a reaction on exactly one molecular substrate and not on any other substrate. Enzymes that can catalyze chemical reactions on many substrates are said to have low specificity. In some cases, enzyme specificity is described relative to one or more defined substrates.

[0036] The modified xylose reductase having "lower D-glucose specificity" and "lower D-xylose specificity" means that the modified xylose reductase catalyzes at least 10%, preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95% less D-glucose and / or D-xylose than the corresponding unmodified xylose reductase.

[0037] According to a preferred embodiment of the invention, the modified xylose reductase contains at least one mutation in the motif GX1GX2X3X4 present in the unmodified xylose reductase, wherein X1 is leucine or phenylalanine, X2 is leucine or cysteine, X3 is tryptophan and X4 is lysine or arginine, wherein X3 in the modified xylose reductase is an amino acid residue selected from tyrosine and phenylalanine, preferably phenylalanine.

[0038] According to a preferred embodiment of the present invention, the protein of the present invention comprises... Figure 1 The comparison shown is of polypeptides characterized by GxGxW motifs.

[0039] According to another preferred embodiment of the invention, the unmodified xylose reductase of the invention comprises GxGxW as a motif. The tryptophan residue of the unmodified enzyme motif may preferably be modified to tyrosine or phenylalanine to produce a modified xylose reductase that significantly prefers D-glucuronide over D-glucose and / or D-xylose as substrates.

[0040] Xylose reductase contains the motif GX1GX2X3X4. Results showed that modifications within this motif resulted in decreased specificity of the modified xylose reductase for D-glucose and / or D-xylose. However, the glycine residues at positions 1 and 3 of the motif remained unchanged.

[0041] The amino acid classification used in this article is as follows:

[0042] Polar amino acids are serine (Ser, S), threonine (Thr, T), cysteine ​​(Cys, C), glutamine (Gln, Q), and asparagine (Asn, N). Positively charged amino acids are lysine (Lys, K), arginine (Arg, R), and histidine (His, H). Negatively charged amino acids are aspartic acid (Asp, D) and glutamic acid (Glu, E). Nonpolar amino acids are glycine (Gly, G), alanine (Ala, A), valine (Val, V), leucine (Leu, L), isoleucine (Ile, I), methionine (Met, M), proline (Pro, P), phenylalanine (Phe, F), tyrosine (Tyr, Y), and tryptophan (Trp, W).

[0043] According to another preferred embodiment of the invention, the modified xylose reductase X2 and / or X4 are different from the unmodified xylose reductase X2 and / or X4, wherein X2 is selected from threonine, cysteine, valine, leucine, serine, isoleucine, methionine, glycine, proline, alanine, histidine, glutamine and asparagine, and / or X4 is lysine or arginine.

[0044] According to a preferred embodiment of the invention, in the modified xylose reductase, X2 and / or X3 of the motif GX1GX2X3X4 are replaced compared to the unmodified xylose reductase.

[0045] According to a particularly preferred embodiment of the invention, the modified xylose reductase contains the motif GX1GTFK (SEQ ID No. 29).

[0046] Modified xylose reductases containing the motif GX1GTFK are particularly preferred because such enzymes exhibit significantly reduced D-glucose and / or D-xylose specificity. These enzymes can be advantageously used to convert D-glucuronide to D-fructose, minimizing the formation of other reduction products besides D-fructose.

[0047] Xylose reductases capable of reducing D-glucurone to D-fructose may contain the motif GYRLFD (SEQ ID No. 30) at 15 to 20 amino acid residues, preferably 16 to 18 amino acid residues, in the C-terminal direction of the motif GX1GX2X3X4.

[0048] According to a preferred embodiment of the present invention, the modified xylose reductase comprises an amino acid sequence having at least 50% sequence identity with SEQ ID No. 1, wherein when compared with the sequence of SEQ ID No. 1, the amino acid sequence of the modified xylose reductase contains at least the mutation W24X3 compared with SEQ ID No. 1.

[0049] SEQ ID No. 1 (KlmXR) (Kluyveromyces marxianus)

[0050] MTYLAPTVTLNNGSKMPLVGLGCWKIPNEVCAEQVYEAIKLGYRLFDGAQDYANEKEVGQGINRAIKEGIVKREDLVVVSKLWNSFHHPDNVRTAVERTLNDLQLDYLDLFYIHFPLAFKFVPLDEKYPPGFYTGKDNFAKEIIEEEPVPILDTYRALEKLVDE GLIKSLGISNFSGALIQDLLRGARIKPVALQIEHHPYLVQDRLITYAQKVGLQVVAYSSFGPLSFVELNNEKALHTKTLFENDTIKAIAQKHNVTPSHVLLKWSTQRGIAVIPKSSKKERLLENLKIEETFTLSDEEIKEINGLDQGLRFNDPWDWLGNEFPPTFI

[0051] As used herein, the terms “sequence identity” and “identity” refer to the percentage of identical nucleotides or amino acids matched between at least two nucleotide or amino acid sequences aligned using a normalization algorithm. Such algorithms can insert gaps in the compared sequences in a normalized and reproducible manner to optimize the alignment between the two sequences, thereby enabling a more meaningful comparison.

[0052] The percentage of identity between sequences can be determined using one or more computer algorithms or programs known in the prior 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 preferably used. The BLAST software family contains several programs, including a tool called "BLAST 2 Sequences," which is used for direct pairwise comparisons of two nucleotide or amino acid sequences. "BLAST 2 Sequences" is also interactively accessible and usable via the NCBI World Wide Web page on the Internet. The blastn program (for nucleotide sequences) defaults to a word length (W) of 28, an expectation value (E) of 0.05, M = 1, N = -2, and a comparison of two strands. For amino acid sequences, the blastp program uses a default word length of 3, an expected value (E) of 0.05, a BLOSUM62 scoring matrix (Henikoff and Henikoff, 1989), an alignment (B) of 50, an expected value (E) of 0.05, M = 1, and N = -2.

[0053] According to another preferred embodiment of the invention, when compared with the sequence of SEQ ID No. 1, the amino acid sequence of the modified xylose reductase contains a further mutation C23X2 compared with SEQ ID No. 1, wherein X2 is selected from threonine, cysteine, valine, leucine, serine, isoleucine, methionine, glycine, proline, alanine, histidine, glutamine or asparagine, wherein threonine is particularly preferred.

[0054] This second modification allows for a further reduction in the D-xylose and D-glucose specificity of xylose reductase.

[0055] According to a preferred embodiment of the present invention, the modified xylose reductase comprises an amino acid sequence selected from the following:

[0056] i) An amino acid sequence having at least 50% sequence identity with any of SEQ ID Nos. 1 to 5.

[0057] ii) An amino acid sequence encoded by a nucleic acid sequence having at least 50% identity with any of SEQ ID Nos. 6 to 10, and

[0058] iii) An amino acid sequence encoded by a nucleic acid that binds to a nucleic acid molecule complementary to any one of the nucleic acid sequences SEQ ID No. 6 to 10 under stringent conditions, wherein the stringent conditions include washing at 65°C and at a salt concentration of 0.1 to 2 times the SSC (where 1 times the SSC is understood to be a mixture of 0.15 M sodium chloride / 0.015 M sodium citrate).

[0059] SEQ ID No. 2 (CgXR) (Chaetomium globosum)

[0060] MAPVIKLNSGYDMPQVGFGLWKVDNAVASDVVYNAIKAGYRLFDGACDYGNEVECGQGVARAISEGIVKREDLFIVSKLWNTFHDAERVEPIVKKQLADWGIEYFDLYLIHFPVALEWVDPAVRYPPGWHYDGKEEIRPSKATIQETWTALESLVSKGLSK SIGISNFQAQLIYDLLRYAKIRPATLQVEHHPYLVQQELINLAKREGIAVTAYSSFGPASFKEFNMKHADALAPLIEDETIKKIAAKHNRPASQVLLRWATQRGLAIIPKSTRPQIMAENFQSIDFDLSEEDIATISAFDRGIRFNQPSNYFPTELLWIFG

[0061] SEQ ID No. 3 (CpXR) (Candida parapsilosis)

[0062] MSIKLNSGHEMPIVGFGCWKVTNETAADQIYNAIKVGYRLFDGAQDYGNEKEVGEGINRAIDEGLVSRDELFVVSKLWNNYHDPKNVETALNKTLSDLNLEYLDLFLIHFPIAFKFVPIEEKYPPGFYCGDGDKFHYENVPLLDTWRALESLVQKGKI RSIGISNFNGGLIYDLVRGAKIKPAVLQIEHHPYLQQPRLIEFVQSQGIAITGYSSFGPQSFLELESKKALDTPTLFDHETIKSIASKHKKSSAQVLLRWATQRGIAVIPKSNNPDRLAQNLNVSDFELSKEDLEAINKLDKGLRFNDPWDWDHIPIFV

[0063] SEQ ID No. 4(CtropXR)(tropical yeast yeast(Candida tropicalis))

[0064] MSTTVNTPIKLNSGYEMPLVGFGCWKVTNATAADQIYNAIKTGYRLFDGAEDYGNEKEVGEGINRAIKDGLVKREELFITSKLWNNFHDPKNVETALNKTLSDLNLDYVDLFLIHFPIAFKFVPIEEKYPPGFYCGDGDNFHYEDVPLLDTWKALEKLVEAGKIKSIGISNFTGALIYDLIRGATIKPAVLQIEHHPYLQQPKLIEYVQKAGIAITGYSSFGPQSFLELESKRALNTPTLFEHETIKSIADKHGKSPAQVLLRWATQRNIAVIPKSNNPERLAQNLSVVDFDLTKDDLDNIAKLDIGLRFNDPWDWDNIPIFV

[0065] SEQ ID No. 5 (CtenuisXR) (Candida_tenuis)

[0066] MSASIPDIKLSSGHLMPSIGFGCWKLANATAGEQVYQAIKAGYRLFDGAEDYGNEKEVGDGVKRAIDEGLVKREEIFLTSKLWNNYHDPKNVETALNKTLADLKVDYVDLFLIHFPIAFKFVPIEEKYPPGFYCGDGNNFVYEDVPILETWKALEKLVAAGKIKSIGVSNFPGALLLDLRGATIKPAVLQVEHHPYLQQPKLIEFAQKAGVTITAYSSFGPQSFVEMNQGRALNTPTLFAHDTIKAIAAKYNKTPAEVLLRWAAQRGIAVIPKSNLPERLVQNRSFNTFDLTKEDFEEIAKLDIGLRFNDPWDWDNIPIFV

[0067] SEQ ID No. 6 (KlmXR)

[0068] ATGACATACCTCGCACCAACAGTTACCTTGAACAATGGATCCAAGATGCCGCTAGTCGGCTTGGGATGCTGGAAAATCCCAAACGAAGTGTGTGCCGAACAGGTGTACGAAGCCATCAAGTTGGGCTACCGCTTGTTCGACGGCGCGCAGGACTACGCCAACGAAAAAGAGGTGGGCCAAGGTATTAACAGAGCCATCAAGGAAGGAATCGTCAAGAGAGAAGACTTGGTCGTCGTTTCTAAGTTGTGGAACAGTTTCCACCACCCAGACAACGTGCGTACCGCAGTCGAAAGAACTTTGAACGACTTGCAATTGGACTACTTGGACTTGTTCTACATCCATTTCCCATTGGCTTTCAAGTTCGTGCCACTAGACGAGAAGTACCCTCCAGGTTTCTACACAGGTAAGGACAATTTCGCCAAGGAAATCATCGAAGAGGAGCCTGTCCCAATCTTGGACACCTACAGAGCCCTTGAGAAGTTGGTCGACGAAGGTTTGATCAAATCTTTGGGTATCTCAAACTTTTCGGGTGCATTGATCCAGGACTTGTTGCGTGGCGCCCGTATCAAGCCAGTCGCCTTGCAGATCGAACACCACCCATACTTGGTCCAGGACCGCTTGATCACGTACGCCCAAAAGGTGGGCTTGCAAGTCGTCGCCTACTCCAGTTTCGGCCCACTATCCTTTGTCGAGTTGAACAACGAAAAGGCCTTGCACACAAAGACTTTGTTCGAAAACGACACCATCAAGGCCATCGCTCAAAAACACAACGTCACCCCATCCCACGTCTTGTTGAAGTGGTCCACCCAACGTGGTATCGCCGTCATTCCAAAGTCCTCCAAGAAGGAACGTCTCCTCGAGAACTTGAAGATCGAAGAGACCTTTACCTTGTCCGACGAAGAGATCAAGGAGATCAACGGCTTGGACCAGGGATTGAGATTTAACGACCCATGGGACTGGTTGGGCAACGAATTCCCAACCTTTATCTAA

[0069] SEQ ID No. 7 (CgXR) (Chaetoceros globosa)

[0070] atggcgccggtgattaaactgaacagcggctatgatatgccgcaggtgggctttggcctgtggaaagtggataacgcggtggcgagcgatgtggtgtataacgcgattaaagcgggctatcgcctgtttgatggcgcgtgcgattatggcaacgaagtggaatgcggccagggcgtggcgcgcgcgattagcgaaggcattgtgaaacgcgaagatctgtttattgtgagcaaactgtggaacacctttcatgatgcggaacgcgtggaaccgattgtgaaaaaacagctggcggattggggcattgaatattttgatctgtatctgattcattttccggtggcgctggaatgggtggatccggcggtgcgctatccgccgggctggcattatgatggcaaagaagaaattcgcccgagcaaagcgaccattcaggaaacctggaccgcgctggaaagcctggtgagcaaaggcctgagcaaaagcattggcattagcaactttcaggcgcagctgatttatgatctgctgcgctatgcgaaaattcgcccggcgaccctgcaggtggaacatcatccgtatctggtgcagcaggaactgattaacctggcgaaacgcgaaggcattgcggtgaccgcgtatagcagctttggcccggcgagctttaaagaatttaacatgaaacatgcggatgcgctggcgccgctgattgaagatgaaaccattaaaaaaattgcggcgaaacataaccgcccggcgagccaggtgctgctgcgctgggcgacccagcgcggcctggcgattattccgaaaagcacccgcccgcagattatggcggaaaactttcagagcattgattttgatctgagcgaagaagatattgcgaccattagcgcgtttgatcgcggcattcgctttaaccagccgagcaactattttccgaccgaactgctgtggatttttggctaa

[0071] SEQ ID No. 8 (CpXR) (Candida parapsilosis)

[0072] atgtccattaagctgaatagcggtcacgagatgccaattgtgggttttggttgttggaaggtgaccaatgaaaccgcggccgatcaaatttataacgcgatcaaggtcggctaccgcttgttcgacggcgcccaagactacggcaacgagaaagaggttggcgagggcatcaaccgcgcgatcgatgagggtctggtgtctcgtgacgagctgttcgttgtcagcaagctgtggaataactatcacgacccgaagaatgtcgaaacggccctgaataaaacgctgagcgatttgaacctggaatacctggatctgttcctgatccactttccgatcgcgttcaaatttgtcccgatcgaagagaaataccctccgggcttttactgcggcgatggtgataaatttcactatgaaaacgttccgctgctggacacctggcgcgccttggaaagcctggtgcagaagggtaaaatccgttccattggtatcagcaatttcaacggtggcctgatttacgatctggttcgtggcgcaaagatcaagccggctgtgctgcagattgagcatcacccgtatctgcaacagcctcgcctgattgagttcgtccagagccagggtattgcaatcaccggttattctagcttcggtccacagagcttcctggagttggaaagcaagaaagcgctggataccccgaccctgttcgaccacgaaacgattaaatccatcgctagcaaacataagaagagcagcgcacaggtgctgctgcgttgggcgactcaacgtggtattgcggttattccgaaaagcaacaatccggaccgtctggcacaaaacctgaatgttagcgactttgagttgtcgaaagaggacctggaagcgatcaacaagctggataaaggtctgcgttttaatgacccgtgggactgggatcatattccaatctttgtttaa

[0073] SEQ ID. No. 9 (CtropXR) (Candida tropicalis)

[0074] atgagcacgaccgttaatactccgacgatcaaactgaactccggttacgagatgccgctggttggctttggctgttggaaagttaccaatgcgacggcggcggaccagatttataacgctattaaaaccggttaccgtctgttcgatggcgcagaggactacggtaacgagaaagaagtgggtgaaggtattaatcgcgcaattaaagacggtctggtcaaacgtgaagagctgtttattacctcgaaactgtggaataacttccatgatcctaagaacgtggagactgcgctgaataagaccctgagcgatctgaacctggactatgtggatctgtttctgattcacttcccgattgctttcaaatttgtcccgatcgaagaaaagtacccaccgggcttctactgcggtgacggcgacaacttccattatgaggatgttccgctgctggacacgtggaaggcgctggagaaattggtggaagccggtaagatcaagtccattggcattagcaacttcaccggtgccttgatttacgatttgatccgtggtgcgaccattaaaccggcggttctgcagatcgagcatcacccgtatctgcaacagccgaaactgatcgaatacgttcagaaagcaggtatcgccatcactggctatagcagcttcggtccacagagcttcctggagctggagagcaagcgtgccctgaataccccgacgttgtttgaacacgaaaccatcaagtctatcgctgacaaacacggtaagagccctgcacaagtcctgctgcgctgggcaacgcaacgtaatattgcggttattccgaagagcaataacccggagcgtctggcgcagaatctgtctgtcgtcgactttgatttgaccaaggatgacctggataatatcgcgaagctggacatcggcctgcgcttcaacgatccgtgggattgggacaacatcccgatctttgtgtaa

[0075] SEQ ID No. 10 (Ctenuis XR) (Candida filamentosa)

[0076] ATGAGCGCAAGTATCCCAGACATCAAATTGAGCTCCGGCCACTTAATGCCTTCCATCGGTTTCGGCTGTTGGAAGCTCGCCAACGCTACCGCCGGTGAACAAGTCTACCAAGCCATCAAGGCCGGTTACAGATTGTTCGACGGTGCCGAGGACTACGGTAACGAAAAGGAAGTCGGTGACGGTGTCAAGAGAGCCATCGACGAAGGTCTTGTCAAGAGAGAAGAGATCTTCCTCACCTCCAAGTTGTGGAACAACTACCACGACCCAAAGAACGTCGAGACCGCCTTGAACAAAACCCTCGCCGACCTTAAGGTTGACTACGTTGACTTGTTCTTGATCCATTTCCCAATTGCCTTCAAGTTCGTCCCAATCGAGGAGAAGTACCCACCAGGATTCTACTGTGGTGACGGTAACAACTTCGTCTACGAAGACGTTCCAATCTTGGAGACCTGGAAGGCCCTCGAGAAGTTGGTTGCTGCCGGTAAGATTAAGTCCATCGGTGTCTCTAACTTCCCAGGTGCTTTGCTCTTGGACTTGCTCAGAGGTGCTACCATCAAGCCAGCTGTCTTGCAAGTTGAGCACCACCCATACTTGCAACAACCAAAGTTGATCGAGTTCGCTCAAAAGGCCGGTGTTACCATCACCGCCTACTCTTCTTTCGGTCCTCAATCTTTCGTTGAGATGAACCAAGGTAGAGCTTTGAACACCCCAACCTTGTTCGCACACGACACCATCAAGGCTATTGCTGCCAAGTACAACAAGACCCCAGCTGAGGTTTTATTGAGATGGGCCGCCCAAAGAGGTATCGCTGTCATTCCAAAGTCTAACCTCCCAGAGAGATTAGTCCAAAACAGAAGTTTCAACACCTTCGACTTGACCAAGGAGGACTTCGAGGAAATCGCCAAGTTGGACATCGGCTTGAGATTCAACGACCCATGGGACTGGGACAACATTCCAATCTTCGTTTAA

[0077] Alternatively, the xylose reductase of the present invention preferably comprises an amino acid sequence encoded by a nucleic acid that, under stringent conditions, binds to a nucleic acid molecule complementary to any of the nucleic acid sequences SEQ ID No. 6 to 10. As used herein, stringent conditions refer to conditions under which a so-called specific hybrid is formed without the formation of a non-specific hybrid.

[0078] Hybridization can be performed using conventional, known procedures, such as those described in J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory (1989). Strict conditions refer to washing at 65°C and with a salt concentration of 0.1 to 2 times the amount of SSC (where 1 times the amount of SSC is understood as a mixture of 0.15 M sodium chloride / 0.015 M sodium citrate).

[0079] The xylose reductase of the present invention can be modified once, twice, three times, four times, or five times by a water-soluble polymer. For example, the water-soluble polymer can be polyethylene glycol. According to the present invention, the binding of polyethylene glycol preferably occurs at the N-terminus of the protein. The protein of the present invention can also bind to solids such as polyethylene, polystyrene, polysaccharides, cellulose, or cellulose derivatives.

[0080] The xylose reductase of the present invention (preferably obtained through recombinant expression in Escherichia coli) can be thermoprecipitated to remove E. coli background enzymes in order to achieve food enzyme compliance after ultrafiltration and DNA removal. For example, heat incubation can be carried out at, for example, 45°C, but not limited to, 30 minutes.

[0081] Furthermore, the modified xylose reductase of the present invention can be part of a fusion protein. Such a fusion protein may contain at least another polypeptide immediately adjacent to the N-terminus and / or C-terminus of the enzyme of the present invention. For example, the fusion protein can be more easily isolated from other proteins or expressed in larger quantities in cells.

[0082] According to a preferred embodiment of the invention, the modified xylose reductase comprises an amino acid sequence encoded by a nucleic acid sequence having at least 60% identity, preferably at least 70% identity, more preferably at least 80% identity, more preferably at least 90% identity, and particularly at least 95% identity with SEQ ID No. 1.

[0083] According to another preferred embodiment of the invention, the modified xylose reductase comprises or consists of an amino acid sequence selected from or composed of SEQ ID No. 11 to 28.

[0084] SEQ ID No. 11 (CtropXR W26Y) (Candida tropicalis)

[0085] MSTTVNTPTIKLNSGYEMPLVGFGC Y KVTNATAADQIYNAIKTGYRLFDGAEDYGNEKEVGEGINRAIKDGLVKREELFITSKLWNNFHDPKNVETALNKTLSDLNLDYVDLFLIHFPIAFKFVPIEEKYPPGFYCGDGDNFHYEDVPLLDTWKALEKLVEAGKIKSIGISNFTGALIYDLIRGATIKPAVLQIEHHPYLQQPKLIEYVQKAGIAITGYSSFGPQSFLELESKRALNTPTLFEHETIKSIADKHGKSPAQVLLRWATQRNIAVIPKSNNPERLAQNLSVVDFDLTKDDLDNIAKLDIGLRFNDPWDWDNIPIFV

[0086] SEQ ID No. 12 (KlmXR W24Y) (Kluyveromyces marxianus)

[0087] MTYLAPTVTLNNGSKMPLVGLGC Y KIPNEVCAEQVYEAIKLGYRLFDGAQDYANEKEVGQGINRAIKEGIVKREDLVVVSKLWNSFHHPDNVRTAVERTLNDLQLDYLDLFYIHFPLAFKFVPLDEKYPPGFYTGKDNFAKEIIEEEPVPILDTYRALEKLVDEGLIKSLGISNFSGALIQDLLRGARIKPVALQIEHHPYLVQDRLITYAQKVGLQVVAYSSFGPLSFVELNNEKALHTKTLFENDTIKAIAQKHNVTPSHVLLKWSTQRGIAVIPKSSKKERLLENLKIEETFTLSDEEIKEINGLDQGLRFNDPWDWLGNEFPTFI

[0088] SEQ ID No. 13 (KlmXR C23T W24F) (Kluyveromyces marxianus)

[0089] MTYLAPTVTLNNGSKMPLVGLG TFKIPNEVCAEQVYEAIKLGYRLFDGAQDYANEKEVGQGINRAIKEGIVKREDLVVVSKLWNSFHHPDNVRTAVERTLNDLQLDYLDLFYIHFPLAFKFVPLDEKYPPGFYTGKDNFAKEIIEEEPVPPILDTYRALEKLVDEGLIKFSSLKFS GALIQDLLRGARIKPVALQIEHHPYLVQDRLITYAQKVGLQVVAYSSFGPLSFVELNNEKALHTKTLFENDTIKAIAQKHNVTPSHVLLKWSTQRGIAVIPKSSKKERLLENLKIEEETTFTLSDEEIKEINGLDQGLRFNDPWDWLGNEFPTFIFI

[0090] SEQ ID No. 14 (CgXR W21Y)

[0091] MAPVIKLNSGYDMPQVGFGL Y KVDNAVASDVVYNAIKAGYRLFDGACDYGNEVECGQGVARAISEGIVKREDLFIVSKLWNTFHDAERVEPIVKKQLADWGIEYFDLYLIHFPVALEWVDPAVRYPPGWHYDGKEEIRPSKATIQETWTALESLVSKGLSKSIGISGIQQAQ LIYDLLRYAKIRPATLQVEHHPYLVQQELINLAKREGIAVTAYSSFGPASFKEFNMKHADALAPLIEDETIKKIAAKHNRPASQVLLRWATQRGLAIIPKSTRPQIMAENFQSIDFDLSEEDIATISAFDRGIRFNQPSNYFPTELLWIFG

[0092] SEQ ID No. 15 (CpXR C18T W19F)

[0093] MSIKLNSGHEMPIVGFG TFKVTNETAADQIYNAIKVGYRLFDGAQDYGNEKEVGEGINRAIDEGLVSRDELFVVSKLWNNYHDPKNVETALNKTLSDLNLEYLDLFLIHFPIAAFKFVPIEEKYPPGFYCGDGDKFHYENVPLLDTWRALESLVQKGGISIGNKIRSIGNGNG GLIYDLVRGAKIKPAVLQIEHHPYLQQPRLIEFVQSQGIAITGYSSFGPQSFLELESKKALDTPTLFDHETIKSIASKKKSSAQLLRWATQRGIAVIPKSNNPDRLAQNLNVSDFELSKEDLEAINKLDKGLRFNDPWDWDHIPIFV

[0094] SEQ ID No. (KlmXR C23T W24F K25R)

[0095] MTYLAPTVTLNNGSKMPLVGLG TFR IPNEVCAEQVYEAIKLGYRLFDGAQDYANEKEVGQGINRAIKEGIVKREDLVVVSKLWNSFHHPDNVRTAVERTLNDLQLDYLDLFYIHFPLAFKFVPLDEKYPPGFYTGKDNFAKEIIEEEPVPPILDTYRALEKLVDEGLIKNFISSLGNFSG ALIQDLLRGARIKPVALQIEHHPYLVQDRLITYAQKVGLQVVAYSSFGPLSFVELNNEKALHTKTLFENDTIKAIAQKHNVTPSHVLLKWSTQRGIAVIPKSSKKERLLENLKIEEETTFTLSDEEIKEIKEINGLDQGLRFNDPWDWLGNEFPTFI

[0096] SEQ ID No. 17 (KlmXR C23V W24F)

[0097] MTYLAPTVTLNNGSKMPLVGLG VFKIPNEVCAEQVYEAIKLGYRLFDGAQDYANEKEVGQGINRAIKEGIVKREDLVVVSKLWNSFHHPDNVRTAVERTLNDLQLDYLDLFYIHFPLAFKFVPLDEKYPPGFYTGKDNFAKEIIEEEPVPPILDTYRALEKLVDEGLIKSLGISNFSGALIQDLLRGARIKPVALQIEHHPYLVQDRLITYAQKVGLQVVAYSSFGPLSFVELNNEKALHTKTLFENDTIKAIAQKHNVTPSHVLLKWSTQRGIAVIPKSSKKERLLENLKIEETFTLSDEEIKEINGLDQGLRFNDPWDWLGNEFPTFI

[0098] SEQ ID No. 18 (KlmXR C23L W24F)

[0099] MTYLAPTVTLNNGSKMPLVGLG LF KIPNEVCAEQVYEAIKLGYRLFDGAQDYANEKEVGQGINRAIKEGIVKREDLVVVSKLWNSFHHPDNVRTAVERTLNDLQLDYLDLFYIHFPLAFKFVPLDEKYPPGFYTGKDNFAKEIIEEEPVPPILDTYRALEKLVDEGLIKSLGISNFSGALIQDLLRGARIKPVALQIEHHPYLVQDRLITYAQKVGLQVVAYSSFGPLSFVELNNEKALHTKTLFENDTIKAIAQKHNVTPSHVLLKWSTQRGIAVIPKSSKKERLLENLKIEETFTLSDEEIKEINGLDQGLRFNDPWDWLGNEFPTFI

[0100] SEQ ID No. 19 (KlmXR C23S W24F)

[0101] MTYLAPTVTLNNGSKMPLVGLG SFKIPNEVCAEQVYEAIKLGYRLFDGAQDYANEKEVGQGINRAIKEGIVKREDLVVVSKLWNSFHHPDNVRTAVERTLNDLQLDYLDLFYIHFPLAFKFVPLDEKYPPGFYTGKDNFAKEIIEEEPVPPILDTYRALEKLVDEGLIKFSSLKFS GALIQDLLRGARIKPVALQIEHHPYLVQDRLITYAQKVGLQVVAYSSFGPLSFVELNNEKALHTKTLFENDTIKAIAQKHNVTPSHVLLKWSTQRGIAVIPKSSKKERLLENLKIEEETTFTLSDEEIKEINGLDQGLRFNDPWDWLGNEFPTFIFI

[0102] SEQ ID No. 20 (KlmXR C23I W24F)

[0103] MTYLAPTVTLNNGSKMPLVGLG IF KIPNEVCAEQVYEAIKLGYRLFDGAQDYANEKEVGQGINRAIKEGIVKREDLVVVSKLWNSFHHPDNVRTAVERTLNDLQLDYLDLFYIHFPLAFKFVPLDEKYPPGFYTGKDNFAKEIIEEEPVPPILDTYRALEKLVDEGLIKFSSLKFS GALIQDLLRGARIKPVALQIEHHPYLVQDRLITYAQKVGLQVVAYSSFGPLSFVELNNEKALHTKTLFENDTIKAIAQKHNVTPSHVLLKWSTQRGIAVIPKSSKKERLLENLKIEEETTFTLSDEEIKEINGLDQGLRFNDPWDWLGNEFPTFIFI

[0104] SEQ ID No. (KlmXR C23M W24F)

[0105] MTYLAPTVTLNNGSKMPLVGLG MFKIPNEVCAEQVYEAIKLGYRLFDGAQDYANEKEVGQGINRAIKEGIVKREDLVVVSKLWNSFHHPDNVRTAVERTLNDLQLDYLDLFYIHFPLAFKFVPLDEKYPPGFYTGKDNFAKEIIEEEPVPPILDTYRALEKLVDEGLIKSLGISNFSGALIQDLLRGARIKPVALQIEHHPYLVQDRLITYAQKVGLQVVAYSSFGPLSFVELNNEKALHTKTLFENDTIKAIAQKHNVTPSHVLLKWSTQRGIAVIPKSSKKERLLENLKIEETFTLSDEEIKEINGLDQGLRFNDPWDWLGNEFPTFI

[0106] SEQ ID No. 22 (KlmXR W24F)

[0107] MTYLAPTVTLNNGSKMPLVGLGC F KIPNEVCAEQVYEAIKLGYRLFDGAQDYANEKEVGQGINRAIKEGIVKREDLVVVSKLWNSFHHPDNVRTAVERTLNDLQLDYLDLFYIHFPLAFKFVPLDEKYPPGFYTGKDNFAKEIIEEEPVPPILDTYRALEKLVDEGLIKSLGISNFSGALIQDLLRGARIKPVALQIEHHPYLVQDRLITYAQKVGLQVVAYSSFGPLSFVELNNEKALHTKTLFENDTIKAIAQKHNVTPSHVLLKWSTQRGIAVIPKSSKKERLLENLKIEETFTLSDEEIKEINGLDQGLRFNDPWDWLGNEFPTFI

[0108] SEQ ID No. 23 (KlmXR C23G W24F)

[0109] MTYLAPTVTLNNGSKMPLVGLG GFKIPNEVCAEQVYEAIKLGYRLFDGAQDYANEKEVGQGINRAIKEGIVKREDLVVVSKLWNSFHHPDNVRTAVERTLNDLQLDYLDLFYIHFPLAFKFVPLDEKYPPGFYTGKDNFAKEIIEEEPVPPILDTYRALEKLVDEGLIKFSSLKFS GALIQDLLRGARIKPVALQIEHHPYLVQDRLITYAQKVGLQVVAYSSFGPLSFVELNNEKALHTKTLFENDTIKAIAQKHNVTPSHVLLKWSTQRGIAVIPKSSKKERLLENLKIEEETTFTLSDEEIKEINGLDQGLRFNDPWDWLGNEFPTFIFI

[0110] SEQ ID No. 24 (KlmXR C23P W24F)

[0111] MTYLAPTVTLNNGSKMPLVGLG PF KIPNEVCAEQVYEAIKLGYRLFDGAQDYANEKEVGQGINRAIKEGIVKREDLVVVSKLWNSFHHPDNVRTAVERTLNDLQLDYLDLFYIHFPLAFKFVPLDEKYPPGFYTGKDNFAKEIIEEEPVPPILDTYRALEKLVDEGLIKFSSLKFS GALIQDLLRGARIKPVALQIEHHPYLVQDRLITYAQKVGLQVVAYSSFGPLSFVELNNEKALHTKTLFENDTIKAIAQKHNVTPSHVLLKWSTQRGIAVIPKSSKKERLLENLKIEEETTFTLSDEEIKEINGLDQGLRFNDPWDWLGNEFPTFIFI

[0112] SEQ ID No. 25 (KlmXR C23A W24F)

[0113] MTYLAPTVTLNNGSKMPLVGLG AFKIPNEVCAEQVYEAIKLGYRLFDGAQDYANEKEVGQGINRAIKEGIVKREDLVVVSKLWNSFHHPDNVRTAVERTLNDLQLDYLDLFYIHFPLAFKFVPLDEKYPPGFYTGKDNFAKEIIEEEPVPPILDTYRALEKLVDEGLIKFSSLKFS GALIQDLLRGARIKPVALQIEHHPYLVQDRLITYAQKVGLQVVAYSSFGPLSFVELNNEKALHTKTLFENDTIKAIAQKHNVTPSHVLLKWSTQRGIAVIPKSSKKERLLENLKIEEETTFTLSDEEIKEINGLDQGLRFNDPWDWLGNEFPTFIFI

[0114] SEQ ID No. (KlmXR C23H W24F)

[0115] MTYLAPTVTLNNGSKMPLVGLG HF KIPNEVCAEQVYEAIKLGYRLFDGAQDYANEKEVGQGINRAIKEGIVKREDLVVVSKLWNSFHHPDNVRTAVERTLNDLQLDYLDLFYIHFPLAFKFVPLDEKYPPGFYTGKDNFAKEIIEEEPVPPILDTYRALEKLVDEGLIKFSSLKFS GALIQDLLRGARIKPVALQIEHHPYLVQDRLITYAQKVGLQVVAYSSFGPLSFVELNNEKALHTKTLFENDTIKAIAQKHNVTPSHVLLKWSTQRGIAVIPKSSKKERLLENLKIEEETTFTLSDEEIKEINGLDQGLRFNDPWDWLGNEFPTFIFI

[0116] SEQ ID No. (KlmXR C23Q W24F)

[0117] MTYLAPTVTLNNGSKMPLVGLG QFKIPNEVCAEQVYEAIKLGYRLFDGAQDYANEKEVGQGINRAIKEGIVKREDLVVVSKLWNSFHHPDNVRTAVERTLNDLQLDYLDLFYIHFPLAFKFVPLDEKYPPGFYTGKDNFAKEIIEEEPVPILDTYRALEKLVDEGLIKSLGISNFS GALIQDLLRGARIKPVALQIEHHPYLVQDRLITYAQKVGLQVVAYSSFGPLSFVELNNEKALHTKTLFENDTIKAIAQKHNVTPSHVLLKWSTQRGIAVIPKSSKKERLLENLKIEETFTLSDEEIKEINGLDQGLRFNDPWDWLGNEFPPTFI

[0118] SEQ ID No. 28 (KlmXR C23N W24F) (Kluyveromyces martensii)

[0119] MTYLAPTVTLNNGSKMPLVGLG NF KIPNEVCAEQVYEAIKLGYRLFDGAQDYANEKEVGQGINRAIKEGIVKREDLVVVSKLWNSFHHPDNVRTAVERTLNDLQLDYLDLFYIHFPLAFKFVPLDEKYPPGFYTGKDNFAKEIIEEEPVPILDTYRALEKLVDEGLIKSLGISNFS GALIQDLLRGARIKPVALQIEHHPYLVQDRLITYAQKVGLQVVAYSSFGPLSFVELNNEKALHTKTLFENDTIKAIAQKHNVTPSHVLLKWSTQRGIAVIPKSSKKERLLENLKIEETFTLSDEEIKEINGLDQGLRFNDPWDWLGNEFPPTFI

[0120] According to a preferred embodiment of the present invention, D-glucurone can be obtained by enzymatic oxidation of D-glucose (preferably by using pyranose-2-oxidase).

[0121] In the method of the present invention, D-glucuronide is reduced to D-fructose using the modified xylose reductase of the present invention. D-glucuronide can be provided from any available source. However, results have shown that it is advantageous to use D-glucuronide obtained by enzymatic oxidation of D-glucose (preferably using pyranose-2-oxidase). This allows for the production of D-fructose using any source of D-glucose.

[0122] The presence of D-glucose in the reaction mixture has no effect on the reduction of D-glucurone to D-fructose because the modified xylose reductase of the present invention has reduced D-glucose specificity. Due to this property, almost or even all of the D-glucurone present in the reaction mixture can be reduced to D-fructose.

[0123] Due to the specificity of the modified xylose reductase of the present invention, D-glucose from any source can be used to produce D-glucurone. Suitable sources of D-glucose in the method according to the invention include, for example, enzymatic or non-enzymatic hydrolysates of starch (especially corn starch), enzymatic or non-enzymatic hydrolysates of sucrose, or enzymatic or non-enzymatic hydrolysates of cellulose. Cellulose that can be used in the method according to the invention can be obtained from, for example, biomass, preferably lignocellulosic biomass, such as, for example, wood, straw (such as wheat straw, corn straw), bagasse, sisal, and energy grass. For example, amylase can be used for the enzymatic hydrolysis of corn starch. For example, invertase is suitable for the enzymatic cleavage of sucrose. For example, cellulase can be used for the enzymatic cleavage of cellulose. For example, acid-catalyzed cleavage is suitable for the non-enzymatic cleavage of the polysaccharide.

[0124] For example, pyranose-2-oxidases used to oxidize D-glucose to D-glucurone require oxygen. As is known in the art, the oxygen can be introduced into the reaction mixture and can be obtained, for example, by contact with ambient air or by increasing the oxygen supply (e.g., by compressed air or by injecting pure oxygen).

[0125] During the execution of the method of the present invention, when additionally using pyranose-2-oxidase, it is preferable to use an overpressure of air / oxygen. As used herein, "overpressure" means exceeding ambient pressure, preferably exceeding 1.1 bar, more preferably exceeding 1.2 bar, more preferably exceeding 1.5 bar, more preferably exceeding 2 bar, and more preferably exceeding 5 bar. The upper limit of the overpressure is preferably not exceeding 20 bar, preferably not exceeding 10 bar, and more preferably not exceeding 5 bar.

[0126] According to a preferred embodiment of the present invention, catalase is used to remove newly generated H2O2.

[0127] During the reaction of an oxidase (e.g., pyranose-2-oxidase), H2O2 is formed, which is preferably removed from the reaction mixture. The removal of H2O2 can be carried out according to conventional methods, and is preferably enzymatically promoted, for example by means of catalase. For example, catalase is added to the reaction mixture. Suitable catalases are known and can be obtained from, for example, *Aspergillus*, *Corynebacterium glutamicum*, or bovine liver.

[0128] The oxidation reaction for the formation of D-glucurone and the reduction reaction for the formation of D-fructose are preferably carried out in the same reaction batch without the need to separate intermediates, particularly where the two enzymatic redox reactions involved in product formation and the enzyme system for cofactor regeneration are carried out in one reaction batch without the need to separate intermediates, referred to herein as "one-pot synthesis". In this process, all the enzymes involved can be added simultaneously, or a portion of the enzymes, such as one or more enzymes from step (a), can be added first, followed by a delayed addition of another portion of the enzymes, such as one or more enzymes from step (b). Before adding the second portion of the enzymes, the enzymes already present in the reaction batch can be inactivated, for example, by conventional methods (such as heating to, for example, 65°C for 10 minutes).

[0129] According to another preferred embodiment of the invention, NAD(P)H / NAD(P) is added to the reaction mixture during the reduction of D-glucurone to D-fructose and / or during the oxidation of D-glucose to D-glucurone. + .

[0130] According to another embodiment of the invention, NAD(P)H / NAD(P) + Recycling is achieved through a suitable cofactor regeneration system, which requires the presence of a suitable cosubstrate that is consumed during the regeneration of the redox cofactor. Suitable cosubstrates include, for example, alcohols such as isopropanol (2-propanol, IPA), lactic acid and its salts, oxygen, hydrogen, and / or formic acid and its salts.

[0131] To regenerate redox cofactors, oxidoreductases are used. When using the redox cofactor NAD(P)H / NAD(P)... + In this context, the oxidoreductases considered include, for example, dehydrogenases, such as alcohol dehydrogenase, lactate dehydrogenase, formate dehydrogenase, with alcohol dehydrogenase being preferred. Methods for regenerating cofactors as outlined above have been disclosed, for example, in US 2015 / 353978, which also discloses the reduction of D-glucuronide to D-fructose using xylose reductase.

[0132] At the start of the conversion of D-glucose to D-fructose, the conversion process can be carried out under overpressure or without overpressure (as outlined above) and may involve enzymes such as alcohol dehydrogenases. If IPA or other alcohols are used as co-substrate, the IPA level or the level of other alcohols can be 5% to 15% (v / v), preferably 8% to 12% (v / v), more preferably about 10% (v / v). As the process proceeds, for example, the IPA level decreases and acetone accumulates. Notably, the xylose reductase of the present invention is unexpectedly tolerant of organic solvents throughout the process, which typically results in a fructose conversion rate >99% at the end of the operation. Another advantage is that an organic solvent concentration (a mixture of isopropanol and acetone) of at least 5% in the product solution inhibits microbial growth.

[0133] Another aspect of the invention relates to a modified xylose reductase or a functional fragment thereof, wherein the modified xylose reductase has lower D-glucose specificity and lower D-xylose specificity compared to an unmodified xylose reductase.

[0134] Another aspect of the invention relates to a nucleic acid molecule encoding a modified xylose reductase according to the invention. The nucleic acid molecule may be a DNA or RNA molecule.

[0135] Another aspect of the invention relates to a carrier comprising a nucleic acid molecule according to the invention.

[0136] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid linked to it. Useful vectors are those capable of autonomously replicating and / or expressing the nucleic acid linked to them. Vectors capable of directing the expression of a gene operatively linked to them are referred to herein as "expression vectors." Expression vectors used in recombinant DNA technologies are typically in the form of "plasmids," which generally refer to a circular double-stranded DNA loop that does not bind to a chromosome when in vector form. As used herein, "plasmid" and "vector" are used interchangeably because plasmids are the most commonly used form of vector. However, other forms of expression vectors that perform equivalent functions and are subsequently known in the art are also included.

[0137] Suitable cloning vectors for overexpressing and producing the proteins of the present invention include, for example, pKK223-3, pTrc99a, pUC, pTZ, pSK, pBluescript, pGEM, pQE, pET, PHUB, pPLc, pKC30, pRM1 / pRM9, pTrxFus, pAS1, pGEx, pMAL, or pTrx.

[0138] The nucleic acid molecule of the present invention encoding the protein of the present invention can be operatively linked to a promoter. As used herein, the term "operatively linked" means that the selected nucleotide sequence (such as that encoding the protein described herein) is closely adjacent to the promoter to allow the promoter to regulate the expression of the selected nucleic acid molecule. Furthermore, in both transcriptional and translational directions, the promoter is upstream of the selected nucleotide sequence. "Operationally linked" also means that the nucleotide sequence and the regulatory sequence are linked in a manner that enables the expression of the nucleic acid molecule / gene when a suitable molecule (such as a transcriptional activator protein) binds to the regulatory sequence.

[0139] Suitable expression promoters include, for example, the trp-lac (tac) promoter, the trp-lac (trc) promoter, the lac promoter, the T5 promoter, the T7 promoter, and the λpL promoter.

[0140] Another aspect of the invention relates to a host cell containing a nucleic acid molecule or vector according to the invention.

[0141] Host cells containing the nucleic acid molecule or vector according to the invention can be used to produce the modified xylose reductase of the invention. Suitable host cells include bacteria such as Escherichia coli and yeast cells such as Komagataella phaffii.

[0142] Another aspect of the invention relates to the use of the modified xylose reductase or host cell, or cell suspension, lysis buffer or homogenate, or in immobilized, lyophilized or spray-dried form according to the invention.

[0143] The modified xylose reductase of the present invention can be used in the methods of the present invention as whole cells, cell homogenates, cell lysates, in a completely purified (at least 95%, preferably at least 98%) or partially purified state, or as a functional fragment thereof. The methods of the present invention are carried out using the protein according to the present invention or with cells containing the protein according to the present invention. In this process, the cells used can be provided in a native, permeabilized, or lysed state.

[0144] The present invention will be further illustrated by the following embodiments, but is not limited thereto.

[0145] Example

[0146] Material

[0147] D-fructose, tetrasodium NADPH and NADP +The D-glucose and IPTG (isopropyl-β-D-thiogalactopyranoside) were obtained from PanReac AppliChem (ITW Reagents). The D-glucurone was prepared in-house, and the triethanolamine (TEA) was purchased from Chem-Lab NV.

[0148] Enzyme production and lysis buffer production

[0149] General information about the expression of recombinases in Escherichia coli.

[0150] For the production of recombinases in *E. coli* strains, genomic DNA or its synthetic equivalent adapted to *E. coli* codons is first used as a template. The gene to be expressed is amplified in PCR along with specific oligonucleotides carrying recognition sequences for restriction endonucleases, and then isolated from the reaction mixture. After digestion of the nucleic acid with restriction enzymes SphI and HindIII, the gene fragment encoding the target enzyme is ligated to the backbone of the expression vector pQE70-Kan, which is digested with SphI and HindIII. The ligation product is transformed into chemically competent *E. coli* cells Top10F, and the resulting colonies are used for plasmid isolation and restriction analysis.

[0151] The cloning results were validated by restriction enzyme digestion and DNA sequencing. The resulting construct carried the target gene under the IPTG-inducible T5 promoter.

[0152] To overexpress the enzyme in *E. coli*, the resulting expression plasmid was transformed into competent expression cells RB791. After incubation at 37°C for 24 hours, the resulting colonies were inoculated into LB medium for expression assay.

[0153] The following day, with an optical density OD of 0.02... 550 Inoculate the expression culture and incubate it with shaking at 37°C until OD. 550 Reaching 0.3. Then the temperature was lowered to 25°C, and when OD... 550 When the concentration reached 0.5, the culture was induced with 0.5 mM IPTG. After 22 hours, the culture was harvested (separated from the culture medium as a cell pellet by centrifugation) and the expression of the recombinant enzyme was analyzed by SDS gel electrophoresis and activity assays (applied assays or optical enzymology assays).

[0154] Cell lysate production using the GEA disruption method

[0155] To prepare a cell suspension (volume > 50 ml), weigh the cell pellet prepared according to the above procedure into a suitable container and dissolve it in buffer (100 mM triethanolamine (TEA-HCl), pH 7). The biomass mass fraction is typically 20%, with the remainder being buffer.

[0156] Cell disruption was performed using a GEA Lab Homogenizer PandaPLUS 2000 (pressure approximately 1000 bar).

[0157] The homogenate was centrifuged at 4°C and 16,000 rpm for 10 minutes (using a Hermle Z 446 K centrifuge) to separate insoluble cell debris and obtain lysate.

[0158] Dynamic measurement

[0159] The activity of enzyme preparations was measured as follows:

[0160] A defined volume (10 µL) of enzyme dilution was mixed in a cuvette with a total volume of 1 mL consisting of 0.2 mM cofactors (NADH and NADPH, respectively) and 100 mM TEA-HCl (pH 7.0). The change in absorbance (ΔA) measured at 340 nm according to the Lambert-Beer law is a measure of enzyme activity (ΔA = ε∙Δc∙d; where ε is 6.22 l / (mmol·cm) for NAD(P)H; Δc is the change in concentration, and d is the optical path length of the cuvette (1 cm)). One enzyme unit U corresponds to the amount of enzyme that catalyzes the conversion of 1 μmol of substrate per minute under defined conditions. The activities given below are expressed in U / g biomass (wet weight). Unless otherwise specified, the substrate concentrations used are: D-glucose, 200 mM; D-glucuronide, 20 mM; D-fructose, 200 mM.

[0161] PCR-mediated mutagenesis

[0162] The XR mutation was generated using standard overlap extension PCR (e.g., as described by Hilgarth and Lanigan (2019)).

[0163] High-performance liquid chromatography (HPLC)

[0164] D-fructose, D-glucose, D-glucuronide, and D-sorbitol were quantified using HPLC (high performance liquid chromatography). Detection was performed using a refractive index detector. Measurements were taken using a Phenomenex Rezex RCM-Monosaccharide Ca2+ column (temperature: 80°C) with the appropriate pre-column, and eluted with 3.5% isopropanol (flow rate: 0.5 ml / min).

[0165] Example 1

[0166] Kinetic properties of XR (optical enzymological assay)

[0167] Table 1. Results of kinetic measurements.

[0168]

[0169] This example clearly demonstrates that mutant XR preferentially uses D-glucuronide as a substrate.

[0170] Example 2

[0171] In a solution containing 15 µM NADP + In TEA-HCl aqueous buffer (50 mM, pH 8.0), different sugars (D-fructose, 50 g / L, or D-glucose, 50 g / L, or D-glucurone, 60 g / L) were incubated with alcohol dehydrogenase (ADH) (Methanobacterium veterum; GenBank accession number MCZ3365290) (5.5 U) in the absence or presence of different xylose reductase (XR) (1.0 U, based on D-glucurone activity or using the same biomass when D-glucose or D-fructose is used as substrate). The reaction was carried out at 30°C with continuous shaking (1200 rpm) for 16 h in a final volume of 500 µL containing 10% isopropanol. During the reaction, the ADH-mediated cofactor recycling system consumed isopropanol to regenerate cofactors and produce acetone.

[0172] In this embodiment, the redox cofactor NADP + Recycling via ADH / isopropanol to form acetone and NADPH+H + The recirculating cofactor NADPH+H + It can be used by XR for reduction reactions.

[0173] When D-glucurone is used as a substrate, different products may be generated, including D-glucose reduced by C-2-carbonyl or D-fructose reduced by C1-carbonyl. Figure 2 ).

[0174] Table 2. Products detected after XR-mediated reduction of different substrates.

[0175]

[0176] <LOD – Below the limit of detection

[0177] <LOQ – Below the limit of quantification

[0178] Results

[0179] Neither Klm WT nor the GxGxW motif mutants Klm W24Y and Klm C23T W24F produced D-sorbitol from D-fructose. In addition, D-glucose reduction was significantly decreased when the GxGxW motif mutants Klm W24Y and Klm CW(23,24)TF were tested compared to Klm WT. When D-glucuronate was used as a substrate, the XR mutants of the GxGxW motif (Klm W24Y and Klm C23T W24F) unexpectedly produced D-fructose, indicating that these mutants catalyze aldehyde reduction (C1-carbonyl) rather than ketone reduction (C2-carbonyl) in D-glucuronate. This finding is inconsistent with the data published by Kratzer et al. (2006), which showed that the tryptophan residue in the GXGXW motif (as defined in Kratzer et al. (2006)) Figure 1 led to aldehyde reduction (C1-carbonyl), whereas mutation to Y or F led to a strong preference for ketone reduction (C2-carbonyl).

[0180] Thus, it is shown that the GxGxW motif mutants display a significantly altered substrate specificity towards D-glucuronate without changing the catalytic mechanism.

[0181] Example 3

[0182] Kinetic properties of the screened XR mutants (optical enzymatic assay)

[0183] Table 3. Results of kinetic measurements of the screened XR

[0184]

[0185] This example clearly shows that the mutated XR (marked in bold) preferentially uses D-glucuronate as a substrate and has an activity similar to that of Klm W24Y and Klm C23T W24F. It should also be noted that Klm C23T W24F K25R has a similar substrate preference to Klm C23T W24F.

[0186] Example 4

[0187] D-fructose produced from invert sugar using a mutant XR.

[0188] A mixture of D-glucose / D-fructose (51% D-glucose, 49% D-fructose) (as described in Table 4, total final sugar concentration 200–300 g / L) was dissolved in TEA-HCl aqueous buffer (50 mM, pH 8.0) with pyranose-2-oxidase (Trametes versicolor; Uniprot accession number P79076) (1.6 U), catalase (Micrococcus luteus; Uniprot accession number P29422) (1600 U), various xylose reductases (XR) (2.7 U), and alcohol dehydrogenase (ADH) (Methanobacterium archaeogenides; GenBank accession number MCZ3365290) (30 U). The reaction was carried out in a total volume of 500 µL containing 50 µL isopropanol (final 10%) at 30°C with continuous shaking (1200 rpm) for 24 hours. During the reaction, the ADH-mediated cofactor recycling system consumes isopropanol to regenerate the cofactor and produces volatile acetone. Figure 3 ).

[0189] In this embodiment, the redox cofactor NADP + Recycling via ADH / isopropanol to form NADPH+H + And volatile acetone. XR uses recycled cofactor NADPH+H + To mediate the reduction of D-glucuronide to form D-fructose and NADP + .

[0190] Table 4. Results of the D-fructose production experiment.

[0191]

[0192] This example clearly demonstrates that during D-fructose production, the mutant XR preferentially uses D-glucurone as a substrate and does not form byproducts from D-glucose.

[0193] Example 5

[0194] XR preparation via thermal incubation

[0195] 20% GEA lysis buffer (as described in the Methods section) was heat-incubated in an Eppendorf Thermomixer C (45°C, 30 min, 300 UpM) and centrifuged (10 min; 20,000 g; 4°C). Using 100 mM D-glucuronide (instead of 20 mM) as the substrate, the clarified lysis buffer (referred to as “heat-incubated”) and the control lysis buffer before heat incubation (referred to as “control”) were characterized by kinetic measurements (Table 5).

[0196] Table 5. Kinetic characteristics of heat-incubated XR.

[0197]

[0198] This example clearly demonstrates that enzyme preparation (including heat incubation) does not significantly affect D-glucuronide activity.

[0199] literature

[0200] Milić, M., de María, PD, and Kara, S. (2023). A patent survey on the biotechnological production of 2,5-furandicarboxylic acid (FDCA): Currenttrends and challenges. EFB Bioeconomy Journal, 100050. https: / / doi.org / 10.1016 / j.bioeco.2023.100050

[0201] Grembecka, M. (2015). Sugar alcohols—their role in the modern world of sweeteners: a review. European Food Research and Technology, 241, 1-14. https: / / doi.org / 10.1007 / s00217-015-2437-7

[0202] Karmali A, Coelho J. Bioconversion of D-glucose into D-glucosone by glucose 2-oxidase from Coriolus versicolor at moderate pressures. Appl Biochem Biotechnol. 2011 Apr;163(7):906-17. doi: 10.1007 / s12010-010-9094-x.

[0203] Kratzer, R., Leitgeb, S., Wilson, D. K., and Nidetzky, B. (2006). Probing the substrate binding site of Candida tenuis xylose reductase (AKR2B5) with site-directed mutagenesis. Biochemical Journal, 393(1), 51-58. https: / / doi.org / 10.1042 / BJ20050831

[0204] Liang, L., Zhang, J., and Lin, Z. (2007). Altering coenzyme specificity of Pichia stipitis xylose reductase by the semi-rational approach CASTing. Microbial Cell Factories, 6, 36. https: / / doi.org / 10.1186 / 1475-2859-6-36

[0205] Neuhauser, W., Haltrich, D., Kulbe, K. D., and Nidetzky, B. (1997). NAD(P)H-dependent aldose reductase from the xylose-assimilating yeast Candidatenuis. Isolation, characterization and biochemical properties of the enzyme. Biochemical Journal, 326(3), 683 - 92. https: / / doi.org / 10.1042 / bj3260683

[0206] Hilgarth, R.S., and Lanigan, T. M. (2019). Optimization of overlapextension PCR for efficient transgene construction. MethodsX, 7, 100759. https: / / doi.org / 10.1016 / j.mex.2019.12.001

[0207] Henikoff, S., and Henikoff, J. G. (1992). Amino acid substitutionmatrices from protein blocks. Proceedings of the National Academy of Sciencesof the United States of America, 89(22), 10915–10919. https: / / doi.org / 10.1073 / pnas.89.22.10915

[0208] Sambrook, J., Fritsch, E. R., and Maniatis, T. (1989). Molecular Cloning:A Laboratory Manual (2nd ed.). Cold Spring Harbor, NY: Cold Spring HarborLaboratory Press.

[0209] Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990). Basic local alignment search tool. Journal of Molecular Biology, 215(3), 403–410. https: / / doi.org / 10.1016 / S0022-2836(05)80360-2

Claims

1. A method for producing D-fructose, comprising the step of reducing D-glucurone to D-fructose using a modified xylose reductase comprising motifs GX1GX2X3X4 or a functional fragment thereof, wherein the modified xylose reductase has lower D-glucose specificity and lower D-xylose specificity compared to an unmodified xylose reductase, wherein the modified xylose reductase contains at least one mutation in the motifs GX1GX2X3X4 present in the unmodified xylose reductase, wherein X1 is leucine or phenylalanine, X2 is leucine or cysteine, X3 is tryptophan, and X4 is lysine or arginine, wherein X3 in the modified xylose reductase is tyrosine or phenylalanine.

2. The method according to claim 2, wherein X3 in the modified xylose reductase is phenylalanine.

3. The method according to claim 1 or 2, wherein the modified xylose reductase X2 and / or X4 is different from the unmodified xylose reductase X2 and / or X4, wherein X2 is selected from threonine, cysteine, valine, leucine, serine, isoleucine, methionine, glycine, proline, alanine, histidine, glutamine, and asparagine, and / or X4 is lysine or arginine.

4. The method according to any one of claims 1 to 3, wherein the modified xylose reductase comprises the motif GX1GTFK.

5. The method according to any one of claims 1 to 4, wherein the modified xylose reductase further comprises the motif GYRLFD.

6. The method according to any one of claims 1 to 5, wherein the modified xylose reductase comprises an amino acid sequence having at least 50% sequence identity with SEQ ID No. 1, wherein when compared with the sequence of SEQ ID No. 1, the amino acid sequence of the modified xylose reductase contains at least the mutation W24X3 compared with SEQ ID No.

1.

7. The method according to claim 6, wherein when compared with the sequence of SEQ ID No. 1, the amino acid sequence of the modified xylose reductase contains a further mutation C23X2 compared with SEQ ID No. 1, wherein X2 is selected from threonine, cysteine, valine, leucine, serine, isoleucine, methionine, glycine, proline, alanine, histidine, glutamine, or asparagine.

8. The method according to any one of claims 1 to 7, wherein the modified xylose reductase comprises an amino acid sequence selected from the following: i) An amino acid sequence having at least 50% sequence identity with any of SEQ ID Nos. 1 to 5. ii) An amino acid sequence encoded by a nucleic acid sequence having at least 50% identity with any of SEQ ID Nos. 6 to 10, and iii) An amino acid sequence encoded by a nucleic acid that binds to a nucleic acid molecule complementary to any one of the nucleic acid sequences SEQ ID No. 6 to 10 under stringent conditions, wherein the stringent conditions include washing at 65°C and at a salt concentration of 0.1 to 2 times that of SSC.

9. The method according to any one of claims 1 to 8, wherein the modified xylose reductase comprises an amino acid sequence encoded by a nucleic acid sequence having at least 60% identity, preferably at least 70% identity, more preferably at least 80% identity, more preferably at least 90% identity, particularly at least 95% identity with SEQ ID No.

1.

10. The method according to any one of claims 1 to 9, wherein the modified xylose reductase comprises, or is composed of, an amino acid sequence selected from, SEQ ID No. 11 to 29.

11. The method according to any one of claims 1 to 10, wherein the D-glucurone can be obtained by enzymatically, preferably by using pyranose-2-oxidase to oxidize D-glucose.

12. The method of claim 11, wherein the newly generated H2O2 is removed by catalase.

13. The method according to any one of claims 1 to 12, wherein NAD(P)H / NAD(P) is added to the reaction mixture during the reduction of D-glucurone to D-fructose and / or during the oxidation of D-glucose to D-glucurone. + .

14. The method of claim 13, wherein NAD(P)H / NAD(P) + Recycling is performed through a suitable cofactor regeneration system, wherein the cofactor regeneration system includes a cosubstrate that is consumed.

15. The method of claim 14, wherein the co-substrate is selected from alcohols, lactic acid and its salts, pyruvic acid and its salts, oxygen, hydrogen and / or formic acid and its salts.

16. A modified xylose reductase or a functional fragment thereof comprising the motif GX1GX2X3X4, wherein the modified xylose reductase has lower D-glucose specificity and lower D-xylose specificity compared to an unmodified xylose reductase, wherein the modified xylose reductase contains at least one mutation in the motif GX1GX2X3X4 present in the unmodified xylose reductase, wherein X1 is leucine or phenylalanine, X2 is leucine or cysteine, X3 is tryptophan, and X4 is lysine or arginine, wherein X3 in the modified xylose reductase is tyrosine or phenylalanine.

17. The modified xylose reductase according to claim 16, wherein X3 in the modified xylose reductase is phenylalanine.

18. The modified xylose reductase according to claim 16 or 17, wherein X2 and / or X4 of the modified xylose reductase are different from X2 and / or X4 of the unmodified xylose reductase, wherein X2 is selected from threonine, cysteine, valine, leucine, serine, isoleucine, methionine, glycine, proline, alanine, histidine, glutamine, and asparagine, and / or X4 is lysine or arginine.

19. The modified xylose reductase according to any one of claims 16 to 18, wherein the modified xylose reductase comprises the motif GX1GTFK.

20. The modified xylose reductase according to any one of claims 16 to 19, wherein the modified xylose reductase further comprises the motif GYRLFD.

21. The modified xylose reductase according to any one of claims 16 to 20, wherein the modified xylose reductase comprises an amino acid sequence having at least 50% sequence identity with SEQ ID No. 1, wherein when compared with the sequence of SEQ ID No. 1, the amino acid sequence of the modified xylose reductase contains at least the mutation W24X3 compared with SEQ ID No.

1.

22. The modified xylose reductase according to claim 21, wherein when compared with the sequence of SEQ ID No. 1, the amino acid sequence of the modified xylose reductase contains a further mutation C23X2 compared with SEQ ID No. 1, wherein X2 is selected from threonine, cysteine, valine, leucine, serine, isoleucine, methionine, glycine, proline, alanine, histidine, glutamine, or asparagine.

23. The modified xylose reductase according to any one of claims 16 to 22, wherein the modified xylose reductase comprises an amino acid sequence selected from: i) An amino acid sequence having at least 50% sequence identity with any of SEQ ID Nos. 1 to 5. ii) An amino acid sequence encoded by a nucleic acid sequence having at least 50% identity with any of SEQ ID Nos. 6 to 10, and iii) An amino acid sequence encoded by a nucleic acid that binds to a nucleic acid molecule complementary to any one of the nucleic acid sequences SEQ ID No. 6 to 10 under stringent conditions, wherein the stringent conditions include washing at 65°C and at a salt concentration of 0.1 to 2 times that of SSC.

24. The modified xylose reductase according to any one of claims 16 to 23, wherein the modified xylose reductase comprises an amino acid sequence encoded by a nucleic acid sequence having at least 60% identity, preferably at least 70% identity, more preferably at least 80% identity, more preferably at least 90% identity, particularly at least 95% identity with any of SEQ ID No. 6 to 10.

25. The modified xylose reductase according to any one of claims 16 to 24, wherein the modified xylose reductase comprises, or is composed of, an amino acid sequence selected from, SEQ ID No. 11 to 28.

26. A nucleic acid molecule encoding a modified xylose reductase according to any one of claims 16 to 25.

27. A vector comprising the nucleic acid molecule according to claim 26.

28. A host cell comprising the nucleic acid molecule of claim 26 or the vector of claim 27.

29. Use of the modified xylose reductase according to any one of claims 16 to 25 or the host cell or its lysate or homogenate according to claim 28, whether or not heat-incubated, for the reduction of D-glucuronide to D-fructose.

30. A composition obtainable by the method according to any one of claims 1 to 15.

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