Modified xylose reductase and its use

Abstract

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

Description

[Technical Field]

[0001] The present invention relates to enzymes, particularly enzymes capable of reducing D-glucosone to D-fructose. [Background technology]

[0002] D-glucose is abundant in various biopolymers, including 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 because it is sweeter than D-glucose, and can also be converted to 5-(hydroxymethyl)furfural (HMF), a precursor to 2,5-franciocarboxylic acid (FDCA). FDCA is used for the synthesis of furan polymers such as poly(ethylene)furanate (PEF), a sustainable alternative to fossil fuel-based PET (Milic et al., 2023).

[0003] D-fructose is also reduced to D-mannitol and D-sorbitol. Both sugar alcohols are low-calorie and non-cariogenic sweeteners. Furthermore, D-mannitol is seeing increased use in foods for diabetic and / or obese patients, pharmaceuticals (as a diuretic and in the treatment of renal failure, for tablet formulations, or as a drug carrier in dry powder inhalers), and in the production of specialty chemicals (Grembecka, 2015).

[0004] Various methods exist for producing D-fructose from D-glucose. One method, used for bulk-scale production, is characterized by D-glucose formation by hydrolysis of polysaccharides and subsequent isomerization of D-glucose to D-fructose, thereby yielding a mixture of 42% D-fructose, 50% D-glucose, and 8% residual polysaccharides. Further accumulation of D-fructose is achieved by separation methods. For example, the chromatographic method described in U.S. Patent No. 5,221,478 can be used. In particular, the production of high-purity D-fructose using chromatographic methods is complex and very expensive.

[0005] An alternative method involves producing D-fructose from D-glucose by combining enzymatic and chemical steps. The production of D-fructose in this manner is disclosed in U.S. Patent No. 4,246,347, in which 2% D-glucose is first converted to D-glucosone using pyranose-2-oxidase. Then, in a second step, the D-glucosone is converted to D-fructose by hydrogenation. High pressure and high temperature are required to proceed with the hydrogenation, and the reaction is carried out at low substrate concentrations.

[0006] In addition to using a combination of enzymatic and chemical steps, a two-step process combining enzymatic redox reactions to carbohydrates is also described. U.S. Patent No. 10,253,340 discloses the xylose reductase (XR)-dependent production of D-fructose from D-glucose via D-sorbitol as an intermediate. XR first produces D-sorbitol, which is then reduced by sorbitol dehydrogenase. Cofactor recycling can be implemented for each step.

[0007] XR is an NAD(P)-dependent reductase (Liang et al., 2007) that utilizes a number of different aldehyde-containing substrates, particularly D-xylose, D-glucose, or 2-keto-D-glucose (D-glucosone), but does not use sugars containing keto groups, such as D-fructose (Neuhauser et al., 1997). The tryptophan (W) residue in the GXGXW motif (defined in Figure 1) leads to aldehyde reduction (C1-carbonyl group), but mutations of W to Y or F result in a strong preference for ketone reduction (C2-carbonyl group) (Kratzer et al., 2006). As a result, the catalytic mechanism switches from C1-carbonyl reduction to C2-carbonyl reduction when W is mutated to Y or F.

[0008] XR is implemented, for example, in the production processes of deoxyketose (U.S. Patent No. 5,077,203) and xylitol (Chinese Patent Application Publication No. 110628835). In addition, an external cofactor (Chinese Patent Application Publication No. 111876452) is added to redox methods that do not require xylose reductase activity.

[0009] Known methods for D-fructose production have various drawbacks that make them unattractive. Chemical catalytic processes are typically carried out under high pressure and high temperature, resulting in low product yields and requiring cumbersome and costly separation.

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

[0011] U.S. Patent No. 10,113,192 discloses the XR-dependent production of D-fructose from D-glucose via D-glucosone as an intermediate. In a one-pot setting, D-glucose is oxidized to D-glucosone as an intermediate by pyranose-2-oxidase, and XR then uses D-glucosone as a substrate to produce D-fructose. A drawback of this enzyme system is that the process is carried out at relatively low substrate concentrations (a substrate amount of 2.5% D-glucose is converted to 91% D-fructose in 48 hours). At higher substrate concentrations, a by-product (D-sorbitol) accumulates in the reaction vessel up to 5-10%.

[0012] The bioconversion rate of D-glucose increased with pressure. This is because increased pressure due to compressed air resulted in a higher conversion rate (Karmali & Coelho, 2011).

[0013] The object of the present invention is to provide an efficient method for producing pure D-fructose from D-glucose at a high substrate concentration using modified XR, with D-glucosone as an intermediate. [Overview of the project]

[0014] Surprisingly, it was found that D-fructose could be produced with high efficiency using modified xylose reductase enzymes or their functional fragments, exhibiting lower D-glucose specificity and lower D-xylose specificity compared to their respective unmodified counterparts. The reduced D-glucose and D-xylose specificity helps to reduce or even eliminate the formation of the byproduct D-sorbitol by simultaneously enhancing D-glucosone activity. The reduction in D-glucose and D-xylose specificity can be achieved by mutating the conserved consensus sequence of the xylose reductase. Compared to prior art (Kratzer et al., 2006), the introduced mutations did not affect the catalytic mechanism.

[0015] The "unmodified xylose reductase enzymes" used herein can reduce D-glucosone to D-fructose, but the unmodified xylose reductase enzymes exhibit higher D-glucose specificity and higher D-xylose specificity compared to their respective modified xylose reductase enzymes.

[0016] The present invention also relates to the economic operation of xylose reductase (XR)-dependent redox processes at high substrate concentrations by implementing xylose reductase in accordance with the disclosed consensus.

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

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

[0019] A further aspect of the present invention relates to a modified xylose reductase enzyme or a functional fragment thereof comprising the motif GX1GX2X3X4, wherein the modified xylose reductase enzyme has lower D-glucose specificity and lower D-xylose specificity as compared to an unmodified xylose reductase enzyme, the modified xylose reductase enzyme comprises at least one mutation within the motif GX1GX2X3X4 as compared to the unmodified xylose reductase enzyme comprising the motif GX1GX2X3X4, 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, and X3 in the modified xylose reductase enzyme is tyrosine or phenylalanine.

[0020] Another aspect of the present invention relates to a modified xylose reductase enzyme or a functional fragment thereof comprising the motif GX1GX2X3X4, wherein the modified xylose reductase enzyme has lower D-glucose specificity and lower D-xylose specificity as compared to an unmodified xylose reductase enzyme, and the modified xylose reductase enzyme is i) an amino acid sequence having at least 50% sequence identity with any one of SEQ ID NOs: 1 to 5, ii) an amino acid sequence encoded by a nucleic acid sequence having at least 50% identity to any one of SEQ ID NOs: 6 to 10, and iii) an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions to a nucleic acid molecule complementary to any one of the nucleic acid sequences of SEQ ID NOs: 6 to 10 (the stringent conditions comprise washing at 65°C and a salt concentration of 0.1 to 2×SSC) comprises an amino acid sequence selected from the group consisting of.

[0021] A further aspect of the present invention relates to a nucleic acid molecule encoding the modified xylose reductase enzyme according to the present invention.

[0022] Another aspect of the present invention relates to a vector comprising the nucleic acid molecule of the present invention.

[0023] A further aspect of the present invention relates to a host cell comprising the nucleic acid molecule or the vector according to the present invention.

[0024] Another aspect of the present invention relates to the use of the protein or the host cell according to the present invention, or a lysate or homogenate thereof, for reducing D-glucosone to D-fructose.

[0025] By using the xylose reductase enzyme of the present invention having the specific specificity as described above, a composition having a unique composition can be produced. This is because these xylose reductase enzymes can produce D-fructose from D-glucosone at a much higher ratio. Accordingly, a further aspect of the present invention relates to a composition obtainable by the method according to the present invention. [BRIEF DESCRIPTION OF THE DRAWINGS]

[0026] [Figure 1] A comparison of different xylose reductases for defining the GxGxW motif is shown. [Figure 2] The possible reduction products when D-glucosone is used as a substrate, namely D-glucose (by C-2-carbonyl reduction) or D-fructose (by C1-carbonyl reduction), are shown. [Figure 3] The production process of D-fructose is shown. [DETAILED DESCRIPTION OF THE INVENTION]

[0027] The present invention relates to a method for producing D-fructose, comprising the step of reducing D-glucosone to D-fructose using a modified xylose reductase enzyme or a functional fragment thereof, wherein the modified xylose reductase enzyme has lower D-glucose specificity and lower D-xylose specificity as compared to an unmodified xylose reductase enzyme.

[0028] The teachings of the present invention enable the modification of xylose reductase enzymes to exhibit lower D-glucose specificity and lower D-xylose specificity compared to their respective unmodified counterparts. The reduction in both activities results in a much higher degree of D-fructose formation compared to xylose reductase enzymes with unmodified D-glucose and / or D-xylose specificity.

[0029] As used herein, the term “functional fragment” of xylose reductase enzymes refers to a fragment of xylose reductase enzymes 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 especially 100% of their enzymatic activity with respect to their ability to reduce D-glucosone to D-fructose. A “functional fragment” may include one or more truncations of xylose reductase enzymes, preferably 1 to 20 amino acid residues, more preferably 2 to 15 amino acid residues, at the N-terminus. A “functional fragment” may also include one or more truncations of xylose reductase enzymes, preferably 1 to 50 amino acid residues, more preferably 2 to 25 amino acid residues, at the C-terminus.

[0030] As used herein, the terms “enzyme specificity” or “enzyme specificity” are intended to mean the degree to which an enzyme can catalyze a chemical reaction for two or more substrate molecules. An enzyme that can catalyze a reaction for exactly one molecular substrate but not for any other substrate is said to have very high specificity for that substrate. An enzyme that can catalyze a chemical reaction for many substrates is said to have low specificity. In some cases, enzyme specificity is described for one or more defined substrates.

[0031] Modified xylose reductase enzymes having "lower D-glucose specificity" and "lower D-xylose specificity" means that the modified xylose reductase enzymes catalyze 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%, and more preferably at least 95% less D-glucose and / or D-xylose compared to their respective unmodified xylose reductase enzymes.

[0032] According to a preferred embodiment of the present invention, the modified xylose reductase enzyme contains at least one mutation within the motif GX1GX2X3X4 present in the unmodified xylose reductase enzyme, where X1 is leucine or phenylalanine, X2 is leucine or cysteine, X3 is tryptophan, and X4 is lysine or arginine, and X3 in the modified xylose reductase enzyme is selected from the group consisting of tyrosine and phenylalanine, preferably an amino acid residue of phenylalanine.

[0033] According to a preferred embodiment of the present invention, the protein of the present invention comprises a polypeptide consisting of a GxGxW motif characterized by the alignment shown in Figure 1.

[0034] According to another preferred embodiment of the present invention, the unmodified xylose reductase enzyme of the present invention comprises GxGxW as a motif. By modifying the tryptophan residue of the motif of the unmodified enzyme, preferably to either tyrosine or phenylalanine, a modified xylose reductase enzyme can be obtained that significantly prefers D-glucosone to D-glucose and / or D-xylose as a substrate.

[0035] Xylose reductase contains the motif GX1GX2X3X4. Modifications within this motif were found to result in a modified xylose reductase enzyme with reduced D-glucose and / or D-xylose specificity. However, the glycine residues at positions 1 and 3 of the motif remained unchanged.

[0036] The amino acid classification used in this specification is as follows: 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) and arginine (Arg, R), as well as 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).

[0037] According to a more preferred embodiment of the present invention, X2 and / or X4 of the modified xylose reductase enzyme differ from X2 and / or X4 of the unmodified xylose reductase enzyme in that X2 is selected from the group consisting of threonine, cysteine, valine, leucine, serine, isoleucine, methionine, glycine, proline, alanine, histidine, glutamine, and asparagine, and / or X4 is lysine or arginine.

[0038] According to a preferred embodiment of the present invention, X2 and / or X3 of motif GX1GX2X3X4 are replaced in the modified xylose reductase enzyme compared to the unmodified xylose reductase enzyme.

[0039] According to a particularly preferred embodiment of the present invention, the modified xylose reductase enzyme contains the motif GX1GTFK (SEQ ID NO: 29).

[0040] Modified xylose reductase enzymes containing the motif GX1GTFK are particularly preferred because such enzymes exhibit significantly reduced D-glucose and / or D-xylose specificity. Such enzymes can be advantageously used in the conversion of D-glucosone to D-fructose, minimizing the formation of other reduction products besides D-fructose.

[0041] Xylose reductase enzymes capable of reducing D-glucosone to D-fructose may contain 15-20, preferably 16-18 amino acid residues in the C-terminal direction from motif GYRLFD (SEQ ID NO: 30) and motif GX1GX2X3X4.

[0042] According to a preferred embodiment of the present invention, the modified xylose reductase enzyme contains an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 1, and when the amino acid sequence of the modified xylose reductase enzyme is aligned with the sequence of SEQ ID NO: 1, it contains at least the mutant W24X3 compared to SEQ ID NO: 1.

[0043] Sequence ID 1 (KlmXR) (Kluyveromyces marxianus)

number

[0044] As used herein, the terms “sequence identity” and “identity” refer to the percentage of identical nucleotides or amino acids between at least two nucleotide or amino acid sequences aligned using a standardized algorithm. Such algorithms can optimize the alignment between two sequences by inserting gaps into the sequences being compared in a standardized and reproducible manner, thereby achieving a more meaningful comparison of the two sequences.

[0045] The percentage of identity between sequences is known in the prior art or can be determined using one or more computer algorithms or programs described herein. According to the present invention, preferably, the Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990) provided by the National Center for Biotechnology Information (NCBI) is used. The BLAST software series includes several programs, including a tool called "BLAST2 Sequences" used for direct pairwise comparison of two nucleotide or amino acid sequences. "BLAST2 Sequences" is also accessible and interactively usable over the internet via the NCBI's Worldwide Webpage. The blastn program (for nucleotide sequences) uses, by default, a word length (W) of 28, an expected value (E) of 0.05, M=1, N=-2, and a comparison of both strands. For amino acid sequences, the blastp program defaults to using a word length of 3 and an expected value of 0.05 (E), as well as the BLOSUM62 score matrix (Henikoff & Henikoff, 1989), alignment of 50 (B), expected value of 0.05 (E), M=1, and N=-2.

[0046] According to another preferred embodiment of the present invention, the amino acid sequence of the modified xylose reductase enzyme, when aligned to the sequence of SEQ ID NO: 1, contains a further mutation C23X2 compared to SEQ ID NO: 1, where X2 is selected from the group consisting of threonine, cysteine, valine, leucine, serine, isoleucine, methionine, glycine, proline, alanine, histidine, glutamine, or asparagine, with threonine being particularly preferred.

[0047] This second modification further reduces the D-xylose and D-glucose specificity of xylose reductase.

[0048] According to a preferred embodiment of the present invention, the modified xylose reductase enzyme is i) An amino acid sequence having at least 50% sequence identity with one of sequence numbers 1 to 5, ii) an amino acid sequence encoded by a nucleic acid sequence having at least 50% identity with any one of sequence numbers 6 to 10, and iii) The amino acid sequence encoded by the nucleic acid, which is bound to a nucleic acid molecule complementary to any one of the nucleic acid sequences of sequence numbers 6-10 under stringent conditions (stringent conditions include washing at 65°C with a salt concentration of 0.1-2 × SSC (where 1 × SSC is understood to be a mixture of 0.15 M sodium chloride / 0.015 M sodium citrate)). It contains an amino acid sequence selected from the group consisting of the following.

[0049] Sequence ID 2 (CgXR) (Chaetomium globosum)

number

[0050] Sequence ID 3 (CpXR) (Candida parapsilosis)

number

[0051] Sequence ID 4 (CtropXR) (Candida tropicalis)

number

[0052] Sequence ID 5 (CtenuisXR) (Candida_tenuis)

number

[0053] Sequence ID 6 (KlmXR) (Kluyveromyces marxianus)

number

[0054] Sequence ID 7 (CgXR) (Chaetomium globosum)

number

[0055] Sequence ID 8 (CpXR) (Candida parapsilosis)

number

[0056] Sequence ID 9 (CtropXR) (Candida tropicalis)

number

[0057] Sequence ID 10 (CtenuisXR) (Candida_tenuis)

number

[0058] Alternatively, the xylose reductase enzyme of the present invention preferably comprises an amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule complementary to the nucleic acid sequences of SEQ ID NOs. 6-10. As used herein, stringent conditions refer to conditions under which so-called specific hybrids are formed, but non-specific hybrids are not.

[0059] Hybridization can be carried out by conventionally known procedures, such as those described in J. Sambrook et al, Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory (1989). Stringent conditions are those in which washing is performed at 65°C with a salt concentration of 0.1–2x SSC (where 1x SSC is understood to be a mixture of 0.15 M sodium chloride / 0.015 M sodium citrate).

[0060] The xylose reductase enzyme of the present invention can be modified once, twice, three times, four times, or five times by a water-soluble polymer. The water-soluble polymer is, for example, polyethylene glycol. The binding of polyethylene glycol preferably occurs at the N-terminus of the protein according to the present invention. The protein of the present invention can also be bound to solids such as polyethylene, polystyrene, polysaccharides, cellulose, or cellulose derivatives.

[0061] To achieve enzyme compliance for DNA removal food after ultrafiltration, the xylose reductase enzyme of the present invention, preferably obtained by recombinant expression in Escherichia coli (E. coli), can be subjected to thermal precipitation of the E. coli-derived enzyme. For example, the thermal incubation may be carried out at, for example, 45°C for 30 minutes, but is not limited to this.

[0062] Furthermore, the modified xylose reductase enzyme of the present invention may be part of a fusion protein. Such a fusion protein may contain at least one further polypeptide at its N-terminus and / or C-terminus, adjacent to the enzyme of the present invention. The fusion protein may, for example, be more easily isolated from other proteins or be expressed in larger quantities within cells.

[0063] According to a preferred embodiment of the present invention, the modified xylose reductase enzyme comprises an amino acid sequence encoded by a nucleic acid sequence having at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 90%, and particularly at least 95% identity with respect to SEQ ID NO: 1.

[0064] According to a more preferred embodiment of the present invention, the modified xylose reductase enzyme comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 11 to 28.

[0065] Sequence ID 11 (CtropXR W26Y) (Candida tropicalis)

number

[0066] Sequence ID 12 (KlmXR W24Y) (Kluyveromyces marxianus)

number

[0067] Sequence ID 13 (KlmXR C23T W24F) (Kluyveromyces marxianus)

number

[0068] Sequence ID 14 (CgXR W21Y) (Chaetomium globosum)

number

[0069] Sequence ID 15 (CpXR C18T W19F) (Candida parapsilosis)

number

[0070] Sequence ID 16 (KlmXR C23T W24F K25R) (Kluyveromyces marxianus)

number

[0071] Sequence ID 17 (KlmXR C23V W24F) (Kluyveromyces marxianus)

number

[0072] Sequence ID 18 (KlmXR C23L W24F) (Kluyveromyces marxianus)

number

[0073] Sequence ID 19 (KlmXR C23S W24F) (Kluyveromyces marxianus)

number

[0074] Sequence ID 20 (KlmXR C23I W24F) (Kluyveromyces marxianus)

number

[0075] Sequence ID 21 (KlmXR C23M W24F) (Kluyveromyces marxianus)

number

[0076] Sequence ID 22 (KlmXR W24F) (Kluyveromyces marxianus)

number

[0077] Sequence ID 23 (KlmXR C23G W24F) (Kluyveromyces marxianus)

number

[0078] Sequence ID 24 (KlmXR C23P W24F) (Kluyveromyces marxianus)

number

[0079] Sequence ID 25 (KlmXR C23A W24F) (Kluyveromyces marxianus)

number

[0080] Sequence ID 26 (KlmXR C23H W24F) (Kluyveromyces marxianus)

number

[0081] Sequence ID 27 (KlmXR C23Q W24F) (Kluyveromyces marxianus)

number

[0082] Sequence ID 28 (KlmXR C23N W24F) (Kluyveromyces marxianus)

number

[0083] According to a preferred embodiment of the present invention, D-glucosone can be obtained by enzymatically oxidizing D-glucose, preferably using pyranose-2-oxidase.

[0084] In the method of the present invention, D-glucosone is reduced to D-fructose using the modified xylose reductase enzyme of the present invention. D-glucosone can be obtained from any available source. However, it has been found to be advantageous to use D-glucosone that can be obtained by enzymatic oxidation of D-glucose, preferably by using pyranose-2-oxidase. This makes it possible to produce D-fructose using any source of D-glucose.

[0085] The D-glucose present in the reaction mixture has no effect on the reduction of D-glucosone to D-fructose. This is because the modified xylose reductase enzyme of the present invention has reduced D-glucose specificity. This property makes it possible to reduce almost all, or even all, of the D-glucosone present in the reaction mixture to D-fructose.

[0086] Due to the specificity of the modified xylose reductase of the present invention, D-glucose from any source can be used for D-glucosone production. Suitable sources of D-glucose in the method according to the present invention are, for example, enzymatic or non-enzymatic hydrolysates of starch, particularly maize starch, enzymatic or non-enzymatic hydrolysates of saccharose, or enzymatic or non-enzymatic hydrolysates of cellulose. Cellulose that can be used in the method according to the present invention may be obtained, for example, from biomass, preferably lignocellulosic biomass, such as wood, straw, such as wheat straw, maize straw, bagasse, sisal, or energy grass. For example, amylase can be used for the enzymatic hydrolysis of maize starch. For example, invertase is suitable for the enzymatic cleavage of saccharose. For example, cellulase can be used for the enzymatic cleavage of cellulose. Acid-catalyzed cleavage is suitable, for example, for the non-enzymatic cleavage of the aforementioned sugars.

[0087] For example, pyranose-2-oxidase, used to oxidize D-glucose to form D-glucosone, requires oxygen. This oxygen can be introduced into the reaction mixture as is known in the art, for example, through contact with ambient air, or made available by an increased oxygen supply, such as the injection of compressed air or pure oxygen.

[0088] Preferably, when pyranose-2-oxidase is used in addition, it is preferable to use air / oxygen overpressure when carrying out the method of the present invention. As used herein, “overpressure” means a pressure higher than the ambient pressure, preferably greater than 1.1 bar, more preferably greater than 1.2 bar, more preferably greater than 1.5 bar, more preferably greater than 2 bar, and more preferably greater than 5 bar. The upper limit of the overpressure is preferably not greater than 20 bar, preferably not greater than 10 bar, and more preferably not greater than 5 bar.

[0089] According to a preferred embodiment of the present invention, the newly formed H2O2 is removed using catalase.

[0090] During the reaction with an oxidase (e.g., pyranose-2-oxidase), H2O2 (preferably removed from the reaction mixture) is formed. The removal of H2O2 may be carried out according to conventional methods, preferably enzymatically with the help of catalase, for example. For example, catalase is added to the reaction mixture. Suitable catalases are known and can be obtained, for example, from Aspergillus species, Corynebacterium glutamicum, or from bovine liver.

[0091] The oxidation reaction for forming D-glucosone and the reduction reaction for forming D-fructose are preferably carried out in the same reaction batch without isolating intermediates, and in particular, the two enzymatic redox reactions and the enzyme system for cofactor regeneration involved in product formation are carried out in a single reaction batch without isolating intermediates, which is referred to herein as "one-pot synthesis". In the process, all enzymes involved can be added simultaneously, or a portion of the enzymes, e.g., the enzyme(s) for step (a) are added first, and then another portion of the enzymes, e.g., the enzyme(s) for step (b) are added after a certain period of time. Before the addition of the second portion of the enzymes, any enzymes already present in the reaction batch may be inactivated, for example, by conventional methods, e.g., by raising the temperature, e.g., up to 65°C over 10 minutes.

[0092] According to another preferred embodiment of the present invention, during the reduction of D-glucosone to D-fructose and / or the oxidation of D-glucose to D-glucosone, NAD(P)H / NAD(P) + However, it is added to the reaction mixture.

[0093] According to a further embodiment of the present invention, NAD(P)H / NAD(P) + These are recycled by a suitable cofactor regeneration system that requires the presence of a suitable comatrix that is depleted during the regeneration of the redox cofactor. Suitable comatrixes include, for example, alcohols, e.g., isopropyl alcohol (2-propanol, IPA), lactic acid and its salts, oxygen, hydrogen, and / or formic acid and its salts.

[0094] Oxidoreductases are used to regenerate redox cofactors. Redox cofactor NAD(P)H / NAD(P) +Examples of oxidoreductases that may be considered when used include, for example, dehydrogenases such as alcohol dehydrogenase, lactate dehydrogenase, formate dehydrogenase, preferably alcohol dehydrogenase. A method for regenerating the cofactors outlined above is disclosed, for example, in U.S. Patent Application Publication No. 2015 / 353978, which also discloses the use of xylose reductase to reduce D-glucosone to D-fructose.

[0095] At the initiation of the conversion of D-glucose to D-fructose (which can be carried out with or without pressure and may involve the aforementioned enzymes such as alcohol dehydrogenase, as outlined above), if IPA or other alcohols are used as co-substrates, the IPA level or other alcohol level may be 5–15% (v / v), preferably 8–12% (v / v), more preferably about 10% (v / v). As the process progresses, for example, the IPA level decreases and acetone accumulates simultaneously. It is noteworthy that the xylose reductase of the present invention is remarkably resistant to organic solvents throughout the entire process, which typically results in over 99% fructose conversion at the end of the operation. A further advantage is that the concentration of at least 5% organic solvents (a mixture of isopropanol and acetone) in the product solution inhibits microbial growth.

[0096] Another aspect of the present invention relates to a modified xylose reductase enzyme or a functional fragment thereof, wherein the modified xylose reductase enzyme has lower D-glucose specificity and lower D-xylose specificity compared to the unmodified xylose reductase enzyme.

[0097] Further aspects of the present invention relate to nucleic acid molecules encoding a modified xylose reductase enzyme according to the present invention. The nucleic acid molecule may be a DNA or RNA molecule.

[0098] A further aspect of the present invention relates to a vector comprising a nucleic acid molecule according to the present invention.

[0099] As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is ligated. Useful vectors are those capable of autonomous replication and / or expression of the nucleic acid ligated to them. A vector capable of operably directing the expression of a ligated gene is referred to herein as an “expression vector.” Expression vectors used in recombinant DNA technology often take the form of a “plasmid,” which, when in vector form, usually refers to a circular double-stranded DNA loop that does not bind to a chromosome. As used herein, plasmids are the most commonly used form of vectors, so “plasmid” and “vector” are used interchangeably. However, other forms of expression vectors that perform equivalent functions and are later known in the art are also included.

[0100] Suitable cloning vectors for overexpression and for the production of the protein 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.

[0101] The nucleic acid molecule of the present invention encoding the protein of the present invention can be operably ligated to a promoter. As used herein, the term “operably ligated” means that a selected nucleotide sequence (e.g., encoding the protein described herein) is close to a promoter, and the promoter enables the expression of the selected nucleic acid molecule. Furthermore, the promoter is located upstream of the selected nucleotide sequence in the direction of transcription and translation. “Operatably ligated” means that when an appropriate molecule (e.g., a transcription activator protein) binds to the regulatory sequence, the nucleotide sequence and the regulatory sequence are ligated in a manner that allows the nucleic acid molecule / gene to be expressed.

[0102] 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.

[0103] Another aspect of the present invention relates to a host cell comprising a nucleic acid molecule or vector according to the present invention.

[0104] Host cells containing nucleic acid molecules or vectors according to the present invention can be used to produce the modified xylose reductase enzyme of the present invention. Suitable host cells include bacteria such as Escherichia coli and yeast cells such as Komagataella phaffii.

[0105] Further aspects of the present invention relate to the use of the modified xylose reductase enzyme or host cells according to the present invention, or cell suspensions, lysates or homogenates or immobilized products or freeze-dried products or spray-dried products.

[0106] The modified xylose reductase enzyme of the present invention can be used in the methods of the present invention in any form, either as whole cells, cell homogenates, cell lysates, in a completely (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 using cells containing the protein according to the present invention. In this way, the cells used can be prepared in a natural, permeable or lysed state.

[0107] The present invention is further illustrated by, but is not limited to, the following examples.

[0108] example material D-fructose, tetrasodium NADPH salt, and NADP +The following were obtained from PanReac AppliChem (ITW Reagents). D-glucose and IPTG (isopropyl-β-D-thiogalactopyranoside) were obtained from Sigma-Aldrich. D-glucosone was prepared in-house, and triethanolamine (TEA) was purchased from Chem-Lab NV.

[0109] Enzyme production and lysate production General information regarding recombinant enzyme expression in Escherichia coli (E. coli). To produce recombinant enzymes in Escherichia coli strains, the gene to be expressed was first amplified by PCR using genomic DNA or an equivalent synthetically fitted to the codon usage frequency of E. coli as a template, along with a specific oligonucleotide further containing the recognition sequence of the restriction endonuclease. The resulting mixture was then isolated. After nucleic acid digestion with restriction enzymes SphI and HindIII, the gene fragment encoding the target enzyme was ligated to the SphI and HindIII-cleaved backbone of the expression vector pQE70-Kan. The ligation product was transformed into chemically competent E. coli cells Top10F, and the resulting colonies were used for plasmid isolation and restriction analysis.

[0110] The results of the cloning step were verified by restriction enzyme digestion and DNA sequencing. The resulting construct contains the target gene under an IPTG-inducible T5 promoter.

[0111] To overexpress the enzyme in Escherichia coli (E. coli), the obtained 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 assays.

[0112] The following day, the expression culture was subjected to an optical density (OD) of 0.02. 550 Inoculated with 0.3 OD 550It was shaken at 37°C until it reached 0.5 OD. Then the temperature was lowered to 25°C and 0.5 OD. 550 Once the target was reached, the culture was induced with 0.5 mM IPTG. After 22 hours, the culture was harvested (separated from the medium in the form of a cell pellet by centrifugation), and recombinant enzyme expression was analyzed by SDS gel electrophoresis and activity assay (using in-use assay or optical enzyme assay).

[0113] Production of cell lysates by GEA disruption To prepare a cell suspension (over 50 ml in volume), the cell pellet prepared according to the above procedure was weighed into a suitable vessel and dissolved in buffer (100 mM triethanolamine (TEA-HCl) pH 7). The mass fraction of biomass is typically 20%, with the remainder being buffer.

[0114] The GEA laboratory homogenizer PandaPLUS 2000 was used for cell disruption (at a pressure of approximately 1000 bar).

[0115] The homogenate was centrifuged at 4°C and 16,000 rpm for 10 minutes (Hermle Z 446 K centrifuge) to separate insoluble cell fragments and obtain a lysate.

[0116] Kinetic measurements The activity of the enzyme preparation was measured as follows: A specified volume (10 μl) of enzyme dilution was mixed in a 1 mL cuvette containing 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 is an indicator of enzyme activity according to Lambert-Beer's law (ΔA = εΔc·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 substrate / min under specified conditions. The activity given below is expressed as U / g (wet weight) of biomass. Unless otherwise specified, the substrate concentrations used were 200 mM D-glucose, 20 mM D-glucosone, and 200 mM D-fructose.

[0117] PCR-mediated mutagenesis XR mutations were generated using standard overlap extension PCR (as described, for example, by Hilgarth and Lanigan (2019)).

[0118] High-performance liquid chromatography (HPLC) D-fructose, D-glucose, D-glucosone, and D-sorbitol were quantified using HPLC (High-Performance Liquid Chromatography). Detection was performed using a refractive index detector. A Phenomenex Rezex RCM-Monosaccharide Ca2+ column (temperature: 80°C) containing the corresponding pre-column was used for the measurement, and elution was performed with 3.5% isopropanol (flow rate: 0.5 ml / min).

[0119] Example 1 Kinetic characteristics of XR (optical-enzyme assay) [Table 1]

[0120] This example clearly demonstrates that the mutant XR preferentially uses D-glucosone as a substrate.

[0121] Example 2 Different sugars (50 g / L D-fructose, or 50 g / L D-glucose, or 60 g / L D-glucosone) were mixed with 15 μM NADP + in an aqueous TEA-HCl buffer (50 mM, pH 8.0), in the absence or presence of different xylose reductase (XR) (defined as 1.0 U based on D-glucosone activity, and the same biomass was used when D-glucose or D-fructose was used as the substrate), incubated together with alcohol dehydrogenase (ADH) (Methanobacterium veterum, GenBank Accession No. MCZ3365290) (5.5 U). The reaction was carried out in a total volume of 500 μl containing a final isopropanol concentration of 10% at 30°C under continuous shaking (1200 rpm) for 16 hours. During the reaction, the ADH-mediated cofactor recycling system consumes isopropanol to regenerate the cofactor and produce acetone.

[0122] In this example, the redox cofactor NADP + is recycled via ADH / isopropanol to form acetone and NADPH+H + . The recycled cofactor NADPH+H + can be used by XR to perform the reduction.

[0123] When D-glucosone is used as the substrate, different products are possible: D-glucose after C-2-carbonyl reduction or D-fructose by C1-carbonyl reduction (Figure 2).

[0124] [Table 2]

[0125] Results Neither Klm WT nor the GxGxW motif mutants Klm W24Y and Klm C23T W24F produced D-sorbitol from D-fructose. Furthermore, compared to Klm WT, the reduction of D-glucose was dramatically reduced when the GxGxW motif mutants Klm W24Y and Klm CW(23,24)TF) were tested. Surprisingly, when D-glucosone was used as a substrate, the XR mutants of the GxGxW motif (Klm W24Y and Klm C23T W24F) produced D-fructose, suggesting that the mutants catalyze aldehyde reduction (C1-carbonyl group) but not ketone reduction (C2-carbonyl group) at D-glucosone. This finding is inconsistent with the published data by Kratzer et al. (2006), which showed that the tryptophan residue in the GXGXW motif (defined in Figure 1 of Kratzer et al. (2006)) leads to aldehyde reduction (C1-carbonyl group), while mutations to Y or F result in a strong preference for ketone reduction (C2-carbonyl group).

[0126] Therefore, it can be seen that the GxGxW motif mutant exhibits a significant change in substrate specificity for D-glucosone without altering the catalytic mechanism.

[0127] Example 3 Kinetic characteristics of screened XR mutants (optical-enzyme assay) [Table 3]

[0128] This example clearly demonstrates that mutant XR (marked in bold) preferentially uses D-glucosone as a substrate and exhibits similar activity to Klm W24Y and Klm C23T W24F. It should also be noted that Klm C23T W24F K25R exhibits similar substrate preference to Klm C23T W24F.

[0129] Example 4 D-fructose production from invert sugar using mutant XR A D-glucose / D-fructose mixture (51% D-glucose, 49% D-fructose) (total final sugar concentration, 200-300 g / L as listed in Table 4) was dissolved in aqueous TEA-HCl buffer (50 mM, pH 8.0) together with pyranose-2-oxidase (Trametes versicolor, uniprot accession number P79076) (1.6 U), catalase (Micrococcus luteus, uniprot accession number P29422) (1600 U), different xylose reductases (XR) (2.7 U), and alcohol dehydrogenase (ADH) (Methanobacterium veterum, GenBank accession number MCZ3365290) (30 U). The reaction was carried out for 24 hours at 30°C under continuous shaking (1200 rpm) in a total volume of 500 μl containing 50 μl of isopropanol (final 10%). During the reaction, the ADH-mediated cofactor recycling system consumed isopropanol to regenerate the cofactor and produce volatile acetone (Figure 3).

[0130] In this example, the redox cofactor NADP + It is recycled via ADH / isopropanol, and NADPH+H + and forms volatile acetone. XR is recycled cofactor NADPH+H + Using D-glucosone reduction, D-fructose and NADP are mediated. + It forms.

[0131] [Table 4]

[0132] This example clearly demonstrates that mutant XR preferentially uses D-glucosone as a substrate during D-fructose production without the formation of byproducts from D-glucose.

[0133] Example 5 XR preparation by heat incubation The 20% GEA solution (described in the Methods section) was subjected to heat incubation (45°C, 30 min, 300 UpM) in an Eppendorf thermomixer C and centrifuged (10 min, 20,000 g, 4°C). The clarified solution (referred to as "heat-incubated") and the control solution before heat incubation (referred to as "control") were characterized by kinetic measurements using 100 mM D-glucosone (instead of 20 mM) as the substrate (Table 5).

[0134] [Table 5]

[0135] This example clearly demonstrates that enzyme preparation, including heat incubation, did not significantly affect D-glucosone activity.

[0136] literature JPEG2026536517000034.jpg248163

Claims

1. Motif GX １ GX ２ X ３ X ４ A method for producing D-fructose, comprising the step of reducing D-glucosone to D-fructose using a modified xylose reductase enzyme or a functional fragment thereof, wherein the modified xylose reductase enzyme has lower D-glucose specificity and lower D-xylose specificity compared to an unmodified xylose reductase enzyme, and the modified xylose reductase enzyme has a motif GX present in the unmodified xylose reductase enzyme １ GX ２ X ３ X ４ (X １ is leucine or phenylalanine, X ２ is leucine or cysteine, X ３ is tryptophan, and X ４ is lysine or arginine), wherein the modified xylose reductase enzyme comprises at least one mutation within said sequence, and X in the modified xylose reductase enzyme ３ is tyrosine or phenylalanine, the method for producing D-fructose.

2. X in the modified xylose reductase enzyme ３ The method according to claim 1, wherein the other is phenylalanine.

3. X of the modified xylose reductase enzyme ２ and / or X ４ However, the X of the unmodified xylose reductase enzyme ２ and / or X ４ Unlike X ２ but selected from the group consisting of threonine, cysteine, valine, leucine, serine, isoleucine, methionine, glycine, proline, alanine, histidine, glutamine, and asparagine, and / or X ４ The method according to claim 1 or 2, wherein the other is lysine or arginine.

4. The modified xylose reductase enzyme is motif GX １ The method according to any one of claims 1 to 3, comprising GTFK.

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

6. The modified xylose reductase enzyme contains an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 1, and when the amino acid sequence of the modified xylose reductase enzyme is aligned with the sequence of SEQ ID NO: 1, it exhibits at least the W24X mutation compared to SEQ ID NO:

1. ３ The method according to any one of claims 1 to 5, including the method described in any one of claims 1 to 5.

7. When aligned to the sequence of SEQ ID NO: 1, the amino acid sequence of the modified xylose reductase enzyme shows a further mutation C23X compared to SEQ ID NO:

1. ２ Includes X ２ The method according to claim 6, wherein the selected element is from the group consisting of threonine, cysteine, valine, leucine, serine, isoleucine, methionine, glycine, proline, alanine, histidine, glutamine, or asparagine.

8. The modified xylose reductase enzyme described above, i) An amino acid sequence having at least 50% sequence identity with any one of sequence numbers 1 to 5, ii) an amino acid sequence encoded by a nucleic acid sequence having at least 50% identity with any one of sequence numbers 6 to 10, and iii) An amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule complementary to any one of the nucleic acid sequences of sequence numbers 6 to 10, wherein the stringent conditions include washing at 65°C and a salt concentration of 0.1 to 2 × SSC. The method according to any one of claims 1 to 7, comprising an amino acid sequence selected from the group consisting of the following.

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

1.

10. The method according to any one of claims 1 to 9, wherein the modified xylose reductase enzyme comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 11 to 28.

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

12. The (nascent) H produced ２ O ２ The method according to claim 11, wherein the substance is removed using catalase.

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

14. NAD(P)H / NAD(P) ＋ The method according to claim 13, wherein the cofactor is recycled by a suitable cofactor regeneration system, the cofactor regeneration system consuming the cofactor substrate.

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

16. Motif GX １ GX ２ X ３ X ４ A modified xylose reductase enzyme or a functional fragment thereof, wherein the modified xylose reductase enzyme has lower D-glucose specificity and lower D-xylose specificity compared to the unmodified xylose reductase enzyme, and the modified xylose reductase enzyme contains the motif GX present in the unmodified xylose reductase enzyme. １ GX ２ X ３ X ４ (X １ However, it is leucine or phenylalanine, X ２ However, it is leucine or cysteine, X ３ However, it is tryptophan, X ４ The modified xylose reductase enzyme contains at least one mutation in the lysine or arginine (which is X) ３ A modified xylose reductase enzyme or a functional fragment thereof, which is tyrosine or phenylalanine.

17. X in the modified xylose reductase enzyme ３ The modified xylose reductase enzyme according to claim 16, wherein the enzyme is phenylalanine.

18. X of the modified xylose reductase enzyme ２ and / or X ４ However, the X of the unmodified xylose reductase enzyme ２ and / or X ４ Unlike X ２ but selected from the group consisting of threonine, cysteine, valine, leucine, serine, isoleucine, methionine, glycine, proline, alanine, histidine, glutamine, and asparagine, and / or X ４ The modified xylose reductase enzyme according to claim 16 or 17, wherein the enzyme is lysine or arginine.

19. The modified xylose reductase enzyme is motif GX １ A modified xylose reductase enzyme according to any one of claims 16 to 18, comprising GTFK.

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

21. The modified xylose reductase enzyme contains an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 1, and when the amino acid sequence of the modified xylose reductase enzyme is aligned with the sequence of SEQ ID NO: 1, it exhibits at least the W24X mutation compared to SEQ ID NO:

1. ３ A modified xylose reductase enzyme according to any one of claims 16 to 20, comprising:

22. When aligned to the sequence of SEQ ID NO: 1, the amino acid sequence of the modified xylose reductase enzyme shows a further mutation C23X compared to SEQ ID NO:

1. ２ Includes X ２ The modified xylose reductase enzyme according to claim 21, wherein the enzyme is selected from the group consisting of threonine, cysteine, valine, leucine, serine, isoleucine, methionine, glycine, proline, alanine, histidine, glutamine, or asparagine.

23. The modified xylose reductase enzyme described above, i) An amino acid sequence having at least 50% sequence identity with any one of sequence numbers 1 to 5, ii) an amino acid sequence encoded by a nucleic acid sequence having at least 50% identity with any one of sequence numbers 6 to 10, and iii) An amino acid sequence encoded by a nucleic acid that binds under stringent conditions to a nucleic acid molecule complementary to any one of the nucleic acid sequences of sequence numbers 6 to 10, wherein the stringent conditions include washing at 65°C and a salt concentration of 0.1 to 2 × SSC. A modified xylose reductase enzyme according to any one of claims 16 to 22, comprising an amino acid sequence selected from the group consisting of the following.

24. A modified xylose reductase enzyme according to any one of claims 16 to 23, comprising an amino acid sequence encoded by a nucleic acid sequence having at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 90%, and particularly at least 95% identity with any one of sequence numbers 6 to 10.

25. The modified xylose reductase enzyme according to any one of claims 16 to 24, wherein the modified xylose reductase enzyme comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 11 to 28.

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

27. A vector comprising the nucleic acid molecule described in claim 26.

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

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

30. A composition that can be obtained by the method described in any one of claims 1 to 15.

Images