Production of fructose from oligosaccharides and / or polysaccharides

Abstract

The present invention relates to a method for converting at least one oligosaccharide and / or polysaccharide into fructose, the method comprising the steps of: a) adding at least four enzymes to a composition comprising water, phosphate, and at least one oligosaccharide and / or polysaccharide; b) subsequently enzymatically converting said at least one oligosaccharide and / or polysaccharide to fructose in the presence of said at least four enzymes, In step a), at least one additional sugar is added, wherein said at least one additional sugar is selected from the group consisting of sugars containing 20 or fewer monosaccharide residues and / or combinations thereof; In step a), the at least four enzymes, preferably at least five enzymes, are selected from the group consisting of transferases, phosphorylases, mutases, isomerases, hydrolases, phosphatases, and combinations thereof; and At least one enzyme in step a) is a phosphatase.

Description

Technical Field

[0001] The present invention relates to a method for converting at least one oligosaccharide and / or polysaccharide into fructose, a composition for converting at least one oligosaccharide and / or polysaccharide into fructose, and an aqueous composition containing fructose. Further, the present invention relates to a phosphatase and the use of the phosphatase in the conversion of at least one oligosaccharide and / or polysaccharide.

Background Art

[0002] The current industrial production process of D-fructose is based particularly on a two-step process. In the first step, a polysaccharide or oligosaccharide, such as starch, is cleaved into monosaccharides by hydrolysis. In the second step, isomerization is carried out, whereby D-fructose is obtained in a yield of about 42% (corresponding to a percentage). The subsequent treatment for preparing pure D-fructose is carried out by costly chromatography purification techniques.

[0003] Another industrial process is the hydrolysis of sucrose using the enzyme invertase. This gives an equimolar mixture of D-glucose and D-fructose. To concentrate the D-fructose, it is necessary to purify it again by a costly chromatography operation.

[0004] The industrial processes have a major drawback in that only solutions of D-fructose are produced which occupy a maximum of 50% of the saccharides present in the solution. To obtain a higher D-fructose solution, it is necessary to carry out costly chromatography purification or add an expensive pure D-fructose such as high fructose corn syrup (HFCS55) with a fructose content of 55%.

[0005] Alternative processes for producing pure D-fructose using D-glucose as a substrate are known and are described in detail in EP0028136B1 and AT513928B1. In this case, D-glucose is oxidized by pyranose-2-oxidase to D-gluconic acid, which is then reduced to D-fructose. The reduction can be carried out by chemical means, i.e., homogeneous or heterogeneous catalysis, or by using a biocatalyst.

[0006] This process has not yet been technically realized. This is because there is a drawback that oxidation and reduction have to be carried out separately from each other, which requires additional process steps. Furthermore, when the reduction is carried out chemically, a high-purity substrate is required and high pressure and high temperature are required, which may lead to the formation of undesirable by-products. When the reduction is carried out enzymatically, very high costs are incurred for the reducing agent and the unstable coenzyme (e.g., NADH) used.

[0007] Known processes for the production of D-fructose have various drawbacks. To efficiently convert the substrate, high pressure and high temperature are partially required. Alternatively, depending on the substrate, a yield of only up to 50% can be achieved, or a clean substrate has to be used to reduce by-products. Furthermore, to concentrate, a very clean process stream is required to ensure the reuse of the chromatography equipment.

Summary of the Invention

Problems to be Solved by the Invention

[0008] There is a need for a method for converting oligosaccharides and / or polysaccharides to D-fructose that overcomes the above drawbacks such as the use of expensive cofactors or an unfavorable equilibrium that requires additional chromatographic purification and concentration steps.

Means for Solving the Problems

[0009] An object of the present invention is to provide a method for producing D-fructose with a high yield and no or few by-products with respect to a substrate. Further, the post-treatment should be simplified and the use of organic coenzymes should be made unnecessary.

Mode for Carrying Out the Invention

[0010] Said object is solved by a method for converting at least one oligosaccharide and / or polysaccharide according to claim 1 into fructose, by a composition for converting at least one oligosaccharide and / or polysaccharide according to claim 8 into fructose, and by an aqueous composition containing fructose according to claim 10. Further, said object is solved by a phosphatase according to claim 13 and by the use of said phosphatase in the conversion of at least one oligosaccharide and / or polysaccharide according to claim 15.

[0011] In a first aspect, the present invention relates to a method for converting at least one oligosaccharide and / or polysaccharide into fructose, said method comprising: a) adding at least four enzymes, preferably at least five enzymes, to a composition containing water, a phosphate, and at least one oligosaccharide and / or polysaccharide; b) subsequently, enzymatically converting said at least one oligosaccharide and / or polysaccharide into fructose in the presence of said at least four enzymes, preferably at least five enzymes; In step a), at least one additional sugar is added, said at least one additional sugar being selected from the group consisting of sugars containing 20 or fewer monosaccharide residues and / or combinations thereof, preferably 17 or fewer monosaccharide residues and / or combinations thereof; In step a), said at least four enzymes, preferably at least five enzymes, are selected from the group consisting of transferases, phosphorylases, mutases, isomerases, hydrolases, phosphatases, and combinations thereof, and at least one enzyme in step a) is a phosphatase.

[0012] Further preferred embodiments are described in the dependent claims as well as in the specification. In the present invention, references to fructose should be understood as references to D-fructose. The oligosaccharides according to the invention are sugars containing at least two monosaccharide residues, which may be the same or different.

[0013] Phosphatase should be understood as any enzyme belonging to the classification EC3.1.3. Transferase should be understood as any enzyme belonging to the classification EC2.4. Phosphorylase should be understood as any enzyme belonging to the classification EC2.4.1. Mutase should be understood as any enzyme belonging to the classification EC5.4.2. Isomerase should be understood as any enzyme belonging to the classification EC5.3.1. Hydrolase should be understood as any enzyme belonging to the classification EC3.2.

[0014] As substrates according to the invention, oligosaccharides and polysaccharides can be used. The improved yield can be obtained from sugars and sugar phosphates which can probably be prepared by cleavage of the fructose units already present in the sugars.

[0015] Surprisingly, a method for producing D-fructose has been found, which functions without high pressure or high temperature and better tolerates the presence of contaminants in the process. The choice of catalyst makes it possible to obtain a high yield of D-fructose in the process by generating intermediate sugar phosphates. As a result, the ratio based on the substrate (saccharide) can be made higher than that of the industrial production methods currently in use, and post-treatment becomes unnecessary. This eliminates costly cleaning operations. The sugar can be an oligosaccharide and / or a polysaccharide.

[0016] Preferably, the composition of step a) contains at least one oligosaccharide and / or polysaccharide of 35% or less on a dry weight basis. Preferably, the composition contains at least one oligosaccharide and / or polysaccharide of 0.5% to 35% on a dry weight basis.

[0017] More preferably, the oligosaccharide and / or polysaccharide is selected from the group consisting of glucose-based oligosaccharides and / or polysaccharides, preferably starch and its derivatives, hemicellulose and its derivatives, cellulose and its derivatives, and / or combinations thereof.

[0018] Each oligosaccharide and / or polysaccharide can be obtained via starch production or by hydrolysis of biomass, for example in pulp production or straw processing.

[0019] Preferably in step a), at least one additional sugar is selected from the group consisting of tetradecasaccharide, tridecasaccharide, dodecasaccharide, undecasaccharide, decasaccharide, nonasaccharide, octasaccharide, heptasaccharide, hexasaccharide, pentasaccharide, tetrasaccharide, trisaccharide, disaccharide, and / or combinations thereof, more preferably tetrasaccharide, trisaccharide, disaccharide, and combinations thereof, even more preferably selected from maltose, maltotriose, maltotetrose, and combinations thereof.

[0020] More preferably in step a), at least one additional sugar is maltodextrin. Maltodextrin is a saccharide composed of D-glucose units, mainly linked by α-1,4-glycosidic bonds and connected by chains of variable length. Usually, maltodextrin is composed of a mixture of chains with lengths of 3 to 17 glucose units. Maltodextrin is classified by DE (dextrose equivalent), and the DE is 3 to 20. The higher the DE value, the shorter the glucose chain.

[0021] More preferably, at least one additional sugar has at least partially the same glycosidic bond as at least one oligosaccharide and / or polysaccharide.

[0022] Preferably, the phosphatase comprises an amino acid sequence that is at least 90%, preferably at least 95%, more preferably at least 97%, and even more preferably at least 98.5% identical to the sequence of SEQ ID NO: 13.

[0023] In a preferred embodiment, the phosphatase comprises an amino acid sequence that is at least 98%, preferably at least 98.5%, more preferably at least 99%, and even more preferably at least 99.5% identical to the sequence of SEQ ID NO: 41.

[0024] In a preferred embodiment, the phosphatase is selected from the group of phosphatases having the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, or SEQ ID NO: 49.

[0025] Furthermore, in step a), preferably at least 6 enzymes are added to the composition.

[0026] Preferably at least 4 enzymes, more preferably 5 enzymes, and even more preferably 6 enzymes are selected from the group consisting of phosphatase, transferase, phosphorylase, mutase, isomerase, and / or hydrolase.

[0027] Preferably in step a), at least 1 transferase, preferably glycosyltransferase, more preferably glucanotransferase, and even more preferably alpha - glucanotransferase is added; and / or Preferably in step a), at least 1 phosphorylase, preferably glucan phosphorylase is added; and / or Preferably in step a), at least 1 mutase, preferably phosphoglucomutase is added; and / or Preferably in step a), at least 1 isomerase, preferably phosphoglucose isomerase is added; and / or Preferably in step a), at least 1 hydrolase, preferably glucanohydrolase, more preferably pullulanase is added.

[0028] According to a preferred embodiment of the method for converting at least one oligosaccharide and / or polysaccharide into fructose, at least one additional sugar in step a) is selected from the group consisting of tetrasaccharides, trisaccharides, disaccharides, and combinations thereof, more preferably selected from maltose, maltotriose, maltotetraose, and combinations thereof; and / or the composition of step a) contains at least one oligosaccharide and / or polysaccharide of 35% or less based on dry weight, preferably the composition contains at least one oligosaccharide and / or polysaccharide of 0.5% to 35% based on dry weight; and / or at least one oligosaccharide and / or polysaccharide is selected from the group consisting of glucose-based oligosaccharides and / or polysaccharides, preferably starch and its derivatives, hemicellulose and its derivatives, cellulose and its derivatives, and / or combinations thereof; and / or in step a), at least six enzymes are added to the composition; and / or at least one additional sugar has at least a partially same glycosidic bond as at least one oligosaccharide and / or polysaccharide.

[0029] Preferably in step b), sugar phosphate is produced intermmediately. More preferably in step b), the enzymatic conversion is a one-pot reaction. According to a preferred embodiment of step b), sugar phosphate is produced intermmediately and the enzymatic conversion is a one-pot reaction.

[0030] The method of the present invention enables the conversion of oligosaccharides and polysaccharides into D-fructose through several catalytic steps. Thereby, an intermediate in the form of sugar phosphate is produced and finally cleaved into D-fructose and phosphate.

[0031] Preferably, the oligosaccharide and / or polysaccharide added in step a) contains a plurality of glucose units, more preferably D-glucose units. Even more preferably, the oligosaccharide and / or polysaccharide added in step a) consists of a plurality of glucose units, more preferably D-glucose units.

[0032] In one embodiment, the method of the present invention is an enzymatic process to which a reaction sequence is applied and which enables individual D-glucose molecules to be cleaved into the form of D-glucose-1-phosphate. Subsequently, these released D-glucose-1-phosphate molecules are converted into D-fructose-6-phosphate via additional sugar phosphate intermediates. Subsequently, D-fructose-6-phosphate is split to obtain D-fructose and phosphate.

[0033] The reaction steps during this conversion include the following steps: The sugar molecule is enzymatically cleaved into D-glucose-1-phosphate, D-glucose-1-phosphate is enzymatically converted into D-glucose-6-phosphate, D-glucose-6-phosphate is enzymatically converted into D-fructose-6-phosphate, and D-fructose-6-phosphate is enzymatically cleaved into D-fructose and phosphate.

[0034] Phosphate is used only in a small catalytic amount to form sugar phosphates such as D-glucose-1-phosphate, D-glucose-6-phosphate, D-fructose-6-phosphate, etc. intermmediately. In the first step, phosphate is added to the D-glucose unit-containing mixture, and in the last step, the phosphate is cleaved off and can be reused in the first step.

[0035] This method of the present invention provides an enzymatic alternative for the production of D-fructose as known in the art, which greatly simplifies the need to separate and purify residual intermediates. Thus, the present invention represents a significant improvement in the production of D-fructose from sugars compared to currently used technologies. In contrast to existing methods, the biocatalytic system used achieves an enhanced concentration of the product D-fructose.

[0036] This method is basically suitable for the use of oligosaccharides and polysaccharides.

[0037] Preferably, a hydrolyzate rich in D-fructose can be produced from cellobiose, maltose, or starch. In this case, the first step is carried out using cellobiose phosphorylase, maltose phosphorylase, or starch phosphorylase, followed by several catalytic steps. This method is particularly useful in the use as a substrate of sucrose obtainable from sugar beet or sugar cane. In this case, the first step is carried out using sucrose phosphorylase. In this particular case, D-fructose with a yield of more than 53% (based on mass), or more than 1 mol of D-fructose per 1 mol of sucrose, is obtained.

[0038] The method of the present invention can be carried out in various solvent systems. Preferably, this is carried out in an aqueous solvent system.

[0039] It is also possible to supplement the aqueous system with a buffer system. Suitable buffers (systems) are known and include conventional buffers (systems) such as acetate, potassium phosphate, Tris-HCl, glycylglycine, and glycine buffers, or mixtures thereof.

[0040] Preferably, the buffer used in the method of the present invention has a pH of 3 to 12, preferably pH 4 to 12, more preferably pH 4 to 11. For the optimal activity of the biocatalyst, ions such as Mg 2+ are added. The use of stabilizers, such as glycerol, may allow the biocatalyst to be used for a longer time.

[0041] In the method of the present invention, phosphate is required. To ensure a sufficient amount of phosphate in the process, external addition in the form of phosphoric acid, salts of phosphoric acid, polyphosphates, or combinations thereof is necessary.

[0042] Preferably, the method of the present invention is carried out at an appropriate temperature, for example, different temperatures depending on the enzyme used. Appropriate temperatures include 10 to 100 °C, preferably 10 to 90 °C, more preferably 20 to 90 °C, and even more preferably 20 to 80 °C. Preferably, the temperature in step b) is from 10 to 100 °C, more preferably from 20 to 90 °C, and even more preferably from 20 to 80 °C. Even more preferably, the pH of the composition is from 3 to 12, and more preferably from 4 to 10.

[0043] An advantage of the method of the present invention is that all catalytic steps are carried out in the same reaction batch without the need to isolate the intermediates. The method can be operated batchwise or continuously. In this case, all the enzymes involved can be added simultaneously or only a part of the enzymes can be added. For example, the enzyme can be added during step b), and another part of the enzyme can be added later with a time or local delay.

[0044] Before adding the second part of the enzyme, the enzyme already present in the reaction mixture can be removed, for example physically (by filtration or immobilization), or inactivated. The latter can be done, for example, by a short-term temperature increase up to 80 °C for 10 minutes. Alternatively, the reaction solution can be passed through the reaction mixture to increase the conversion rate and increase the yield several times.

[0045] The cleavage of the sugar to D-glucose-1-phosphate in the method of the present invention is enzymatic, i.e., by enzyme catalysis, and can be carried out according to known methods. The cleavage is preferably carried out by the catalysis of phosphorylase.

[0046] Preferably, at least 50% of the sugar present in the composition of step a) is converted to fructose after 24 hours of enzymatic conversion. Also preferably, at least 70% of the sugar present in the composition of step a) is converted to fructose after 48 hours of enzymatic conversion.

[0047] The method of the present invention further enables the handling of high concentrations of oligosaccharides and / or polysaccharides, such as swollen starch. For example, it is possible to carry out the method of the present invention at an initial concentration of 35% dry matter starch. Preferably, step b) is carried out at room temperature (25 °C) for at least 24 hours.

[0048] In another aspect, the present invention relates to a method for converting at least one oligosaccharide and / or polysaccharide into fructose, the method comprising: a) adding at least one enzyme to a composition comprising water, phosphate, and at least one oligosaccharide and / or polysaccharide; and b) subsequently enzymatically converting at least one oligosaccharide and / or polysaccharide into fructose in the presence of at least one enzyme, wherein at least one additional sugar is added in step a), and the at least one additional sugar is selected from the group consisting of sugars comprising 20 or fewer monosaccharide residues and / or combinations thereof, preferably 17 or fewer monosaccharide residues and / or combinations thereof.

[0049] All of the foregoing descriptions of the method for converting at least one oligosaccharide and / or polysaccharide into fructose, particularly with respect to the enzyme and the oligosaccharide and / or polysaccharide, apply mutatis mutandis to this method where applicable.

[0050] Preferably, the at least one enzyme in step a) is phosphatase. Furthermore, in step a), preferably at least 4 enzymes, more preferably at least 5 enzymes, even more preferably at least 6 enzymes are added to the composition.

[0051] In a second aspect, the present invention relates to a composition for converting at least one oligosaccharide and / or polysaccharide into fructose, the composition comprising - water, - phosphate, - at least 4 enzymes, preferably at least 5 enzymes (wherein at least one enzyme is phosphatase), - comprising at least one oligosaccharide and / or polysaccharide, Here, at least four enzymes, preferably at least five enzymes, are selected from the group consisting of transferases, phosphorylases, mutases, isomerases, hydrolases, phosphatases, and combinations thereof, and The composition further comprises at least one additional sugar, and the at least one additional sugar is selected from the group consisting of sugars containing 20 or fewer monosaccharide residues and / or combinations thereof, preferably 17 or fewer monosaccharide residues and / or combinations thereof.

[0052] All descriptions regarding a method for converting at least one oligosaccharide and / or polysaccharide to fructose, particularly all descriptions regarding the aforementioned enzymes and oligosaccharides and / or polysaccharides, apply also to the composition for converting at least one oligosaccharide and / or polysaccharide, where applicable.

[0053] According to a preferred embodiment, the phosphatase comprises an amino acid sequence that is at least 90%, preferably at least 95%, more preferably at least 97%, and even more preferably at least 98.5% identical to the sequence of SEQ ID NO: 13.

[0054] According to another preferred embodiment, the phosphatase comprises an amino acid sequence that is at least 98%, preferably at least 98.5%, more preferably at least 99%, and even more preferably at least 99.5% identical to the sequence of SEQ ID NO: 41.

[0055] According to a preferred embodiment, the phosphatase is selected from the group of phosphatases having the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, or SEQ ID NO: 49.

[0056] Preferably, at least four enzymes, more preferably five enzymes, and even more preferably six enzymes are selected from the group consisting of phosphatase, transferase, phosphorylase, mutase, isomerase, and / or hydrolase.

[0057] According to a preferred embodiment, the composition comprises a transferase, preferably a glycosyl transferase, more preferably a glucano transferase, even more preferably an alpha - glucano transferase; and / or The composition further comprises a phosphorylase, preferably a glucan phosphorylase is added; and / or The composition comprises a mutase, preferably a phosphoglucomutase; and / or The composition comprises an isomerase, preferably a phosphoglucose isomerase; and / or The composition comprises a hydrolase, preferably a glucanohydrolase, more preferably a pullulanase.

[0058] In another aspect, the present invention relates to a composition for converting at least one oligosaccharide and / or polysaccharide into fructose, the composition comprising water, phosphate, at least five enzymes (wherein at least one enzyme is a phosphatase), wherein the phosphatase comprises an amino acid sequence that is at least 90%, preferably at least 95%, more preferably at least 97%, and even more preferably at least 98.5% identical to the sequence of SEQ ID NO: 13.

[0059] All of the foregoing descriptions regarding the method for converting at least one oligosaccharide and / or polysaccharide into fructose, particularly the enzymes, and the oligosaccharide and / or polysaccharide, and the composition for converting at least one oligosaccharide and / or polysaccharide into fructose apply, mutatis mutandis, to this aspect where applicable.

[0060] Preferably, at least five enzymes are selected from the group consisting of phosphatase, transferase, phosphorylase, mutase, isomerase, and / or hydrolase.

[0061] In a third aspect, the present invention relates to - at least 50% fructose on a dry weight basis, - 0.001 to 25% glucose on a dry weight basis, preferably 0.005 to 20% glucose, - 0.01 to 22% phosphate on a dry weight basis, preferably 0.05 to 20% phosphate, and - an aqueous composition containing, on a dry weight basis, 0.001 to 2% of at least 4 enzymes, preferably at least 5 enzymes, preferably 0.005 to 1% of at least 4 enzymes, preferably at least 5 enzymes, wherein the at least 4 enzymes, preferably at least 5 enzymes, are selected from the group consisting of transferases, phosphorylases, mutases, isomerases, hydrolases, phosphatases, and combinations thereof, and wherein at least one enzyme is a phosphatase.

[0062] All of the above-described methods for converting at least one oligosaccharide and / or polysaccharide to fructose, particularly all descriptions regarding enzymes and oligosaccharides and / or polysaccharides, apply mutatis mutandis to the aqueous composition where applicable.

[0063] According to a preferred embodiment, the phosphatase comprises an amino acid sequence that is at least 90%, preferably at least 95%, more preferably at least 97%, and even more preferably at least 98.5% identical to the sequence of SEQ ID NO: 13.

[0064] According to another preferred embodiment, the phosphatase comprises an amino acid sequence that is at least 98%, preferably at least 98.5%, more preferably at least 99%, and even more preferably at least 99.5% identical to the sequence of SEQ ID NO: 41.

[0065] According to a preferred embodiment, the phosphatase is selected from the group of phosphatases having the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, or SEQ ID NO: 49.

[0066] Preferably, at least four enzymes, more preferably five enzymes, and even more preferably six enzymes are selected from the group consisting of phosphatase, transferase, phosphorylase, mutase, isomerase, and / or hydrolase.

[0067] According to a preferred embodiment, the composition comprises a transferase, preferably a glycosyltransferase, more preferably a glucanotransferase, and even more preferably an alpha-glucanotransferase; and / or The composition further comprises a phosphorylase, preferably a glucan phosphorylase is added; and / or The composition comprises a mutase, preferably a phosphoglucomutase; and / or The composition comprises an isomerase, preferably a phosphoglucose isomerase; and / or The composition comprises a hydrolase, preferably a glucanohydrolase, more preferably a pullulanase.

[0068] In a fourth aspect, the present invention - at least 50% fructose on a dry weight basis - 0.001 to 25% glucose on a dry weight basis, preferably 0.005 to 20% glucose, - 0.01 to 22% phosphate on a dry weight basis, preferably 0.05 to 20% phosphate, and - Regarding an aqueous composition containing at least 4 enzymes, preferably at least 5 enzymes, preferably at least 4 enzymes of 0.001 to 2% on a dry weight basis, preferably at least 5 enzymes, wherein the at least 4 enzymes, preferably at least 5 enzymes, are selected from the group consisting of transferases, phosphorylases, mutases, isomerases, hydrolases, phosphatases, and combinations thereof, and wherein at least 1 enzyme is a phosphatase, which is obtained by the method of the present invention as described above.

[0069] All the foregoing descriptions of the method for converting at least one oligosaccharide and / or polysaccharide to fructose, particularly regarding enzymes and oligosaccharides and / or polysaccharides, apply, where applicable, to the aqueous composition obtained by the method of the present invention.

[0070] According to a preferred embodiment, the phosphatase comprises an amino acid sequence that is at least 90%, preferably at least 95%, more preferably at least 97%, and even more preferably at least 98.5% identical to the sequence of SEQ ID NO: 13.

[0071] According to another preferred embodiment, the phosphatase comprises an amino acid sequence that is at least 98%, preferably at least 98.5%, more preferably at least 99%, and even more preferably at least 99.5% identical to the sequence of SEQ ID NO: 41.

[0072] According to a preferred embodiment, the phosphatase is selected from the group of phosphatases having the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, or SEQ ID NO: 49.

[0073] Preferably, at least four enzymes, more preferably five enzymes, and even more preferably six enzymes are selected from the group consisting of phosphatases, transferases, phosphorylases, mutases, isomerases, and / or hydrolases.

[0074] According to a preferred embodiment, the composition comprises a transferase, preferably a glycosyltransferase, more preferably a glucanotransferase, and even more preferably an alpha - glucanotransferase; and / or The composition further comprises a phosphorylase, preferably glucan phosphorylase is added; and / or The composition comprises a mutase, preferably phosphoglucomutase; and / or The composition comprises an isomerase, preferably phosphoglucose isomerase; and / or The composition comprises a hydrolase, preferably a glucanohydrolase, more preferably pullulanase.

[0075] In another aspect, the present invention relates to an aqueous composition comprising at least 50% fructose on a dry weight basis, less than 25% glucose on a dry weight basis, preferably less than 20% glucose, less than 22% phosphate on a dry weight basis, preferably less than 20% phosphate, and less than 2% of at least four enzymes on a dry weight basis, preferably less than 1% of at least four enzymes, wherein one enzyme is a phosphatase.

[0076] All of the foregoing methods for converting at least one oligosaccharide and / or polysaccharide to fructose, particularly all descriptions regarding enzymes and oligosaccharides and / or polysaccharides, apply mutatis mutandis to the aqueous composition where applicable.

[0077] According to a preferred embodiment, the phosphatase comprises an amino acid sequence that is at least 90%, preferably at least 95%, more preferably at least 97%, and even more preferably at least 98.5% identical to the sequence of SEQ ID NO: 13.

[0078] According to a preferred embodiment, the phosphatase is selected from the group of phosphatases having the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, or SEQ ID NO: 49.

[0079] According to a preferred embodiment, the composition comprises a transferase, preferably a glycosyltransferase, more preferably a glucanotransferase, even more preferably an alpha - glucanotransferase, and / or the composition further comprises a phosphorylase, preferably a glucan phosphorylase is added, and / or the composition comprises a mutase, preferably a phosphoglucomutase, and / or the composition comprises an isomerase, preferably a phosphoglucose isomerase, and / or the composition comprises a hydrolase, preferably a glucanohydrolase, more preferably a pullulanase.

[0080] In another aspect, the present invention relates to an aqueous composition comprising at least 50% fructose on a dry weight basis, less than 25% glucose on a dry weight basis, preferably less than 20% glucose, less than 22% phosphate on a dry weight basis, preferably less than 20% phosphate, and less than 2% of at least four enzymes on a dry weight basis, preferably less than 1% of at least four enzymes, wherein one enzyme is the phosphatase obtained by the method of the present invention as described above.

[0081] All of the foregoing methods for converting at least one oligosaccharide and / or polysaccharide to fructose, particularly all descriptions regarding enzymes and oligosaccharides and / or polysaccharides, apply mutatis mutandis to the aqueous composition obtained by the method of the present invention, where applicable.

[0082] According to a preferred embodiment, the phosphatase comprises an amino acid sequence that is at least 90%, preferably at least 95%, more preferably at least 97%, and even more preferably at least 98.5% identical to the sequence of SEQ ID NO: 13.

[0083] According to a preferred embodiment, the phosphatase is selected from the group of phosphatases having the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, or SEQ ID NO: 49.

[0084] According to a preferred embodiment, the composition comprises a transferase, preferably a glycosyltransferase, more preferably a glucanotransferase, even more preferably an alpha - glucanotransferase; and / or the composition further comprises a phosphorylase, preferably with the addition of a glucan phosphorylase; and / or the composition comprises a mutase, preferably a phosphoglucomutase; and / or the composition comprises an isomerase, preferably a phosphoglucose isomerase; and / or the composition comprises a hydrolase, preferably a glucanohydrolase, more preferably a pullulanase.

[0085] In another aspect, the present invention relates to fructose obtained by the method of the present invention as described above.

[0086] All descriptions above regarding the method of converting at least one oligosaccharide and / or polysaccharide into fructose, particularly regarding the enzyme and the oligosaccharide and / or polysaccharide, apply mutatis mutandis to the fructose obtained by the method of the present invention, where applicable.

[0087] In a fifth aspect, the present invention relates to a phosphatase comprising an amino acid sequence that is at least 98%, preferably at least 98.5%, more preferably at least 99%, and even more preferably at least 99.5% identical to the sequence of SEQ ID NO: 41.

[0088] All descriptions of the foregoing method for converting at least one oligosaccharide and / or polysaccharide to fructose, particularly those regarding the enzyme and the oligosaccharide and / or polysaccharide, apply, where applicable, also to the phosphatase.

[0089] Preferably, the phosphatase has the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, or SEQ ID NO: 49.

[0090] These phosphatases provide a higher yield of D-fructose. The phosphatase of the present invention provides improved selectivity with respect to fructose-6-phosphate. Due to these higher activities, it is possible to reduce the amount of enzyme applied. Therefore, these phosphatases are particularly suitable for industrial processes.

[0091] In another aspect, the present invention relates to a phosphatase comprising an amino acid sequence that is at least 90%, preferably at least 95%, more preferably at least 97%, and even more preferably at least 98.5% identical to the sequence of SEQ ID NO: 13.

[0092] Preferably, the phosphatase has the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, or SEQ ID NO: 49.

[0093] The foregoing method for converting at least one oligosaccharide and / or polysaccharide into fructose, in particular all explanations regarding enzymes and oligosaccharides and / or polysaccharides, apply, where applicable, also to phosphatases.

[0094] In a sixth aspect, the invention relates to the use of the phosphatase of the invention in converting at least one oligosaccharide and / or polysaccharide into fructose.

[0095] The foregoing method for converting at least one oligosaccharide and / or polysaccharide into fructose, in particular all explanations regarding enzymes and oligosaccharides and / or polysaccharides, apply, where applicable, also to the use of the phosphatase of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0096]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

[0097] The abbreviations and enzymes used in the figures and / or tables are explained below. 1 Pullulanase 2 α-Glucanotransferase 3 Glucan phosphorylase 4 Phosphoglucomutase 5 Phosphoglucose isomerase 6 Phosphatase A Starch B Amylose B-A Amylose with one glucose unit removed C Glucose-1-phosphate (G1P) D Glucose-6-phosphate (G6P) E Fructose-6-phosphate (F6P) F Fructose G Phosphate H Amylopectin WT Wild type; SEQ ID NO: 13

[0098] T Phosphatase variant with an amino acid exchange from proline to threonine at position 46 (P46T); SEQ ID NO: 15; TY Phosphatase variant with an additional amino acid exchange from proline to threonine at position 46 and from glutamate to tyrosine at position 47 (P46TE47Y); SEQ ID NO: 17; TYL Phosphatase variant with amino acid exchanges from proline to threonine at position 46, from glutamate to tyrosine at position 47, and from glycine to leucine at position 50 (P46TE47YG50L); SEQ ID NO: 19;

[0099] HTY Phosphatase variant with amino acid exchanges from proline to threonine at position 46, from glutamate to tyrosine at position 47, and from tyrosine to histidine at position 23 (Y23HP46TE47Y); SEQ ID NO: 23; Phosphatase variant (P46TE47YG50I) having amino acid exchanges from proline to threonine at position 46, from glutamate to tyrosine at position 47, and from glycine to isoleucine at position 50; SEQ ID NO: 21; Phosphatase variant (Y23HP46TE47YG50L) having amino acid exchanges from proline to threonine at position 46, from glutamate to tyrosine at position 47, from glycine to leucine at position 50, and from tyrosine to histidine at position 23; SEQ ID NO: 25;

[0100] Phosphatase variant (Y23HP46TE47YG50I) having amino acid exchanges from proline to threonine at position 46, from glutamate to tyrosine at position 47, from glycine to isoleucine at position 50, and from tyrosine to histidine at position 23; SEQ ID NO: 27; Phosphatase variant (Y23SP46TE47YG50L) having amino acid exchanges from proline to threonine at position 46, from glutamate to tyrosine at position 47, from glycine to leucine at position 50, and from tyrosine to serine at position 23; SEQ ID NO: 29; Phosphatase variant (Y23SP46TE47YG50I) having amino acid exchanges from proline to threonine at position 46, from glutamate to tyrosine at position 47, from glycine to isoleucine at position 50, and from tyrosine to serine at position 23; SEQ ID NO: 31;

[0101] Phosphatase variant (Y23HP46TE47TG50L) having amino acid exchanges from proline to threonine at position 46, from glutamate to threonine at position 47, from glycine to leucine at position 50, and from tyrosine to histidine at position 23; SEQ ID NO: 33; Phosphatase variant (Y23HP46TE47FG50L) having amino acid exchanges from proline to threonine at position 46, from glutamate to phenylalanine at position 47, from glycine to leucine at position 50, and from tyrosine to histidine at position 23; SEQ ID NO: 35; Phosphatase variant (Y23HV45MP46TE47YG50L) having amino acid exchanges from proline to threonine at position 46, from glutamate to tyrosine at position 47, from glycine to leucine at position 50, from tyrosine to histidine at position 23, and from valine to methionine at position 45; SEQ ID NO: 37;

[0102] Phosphatase variant (Y23HV45QP46TE47YG50L) having amino acid exchanges from proline to threonine at position 46, from glutamate to tyrosine at position 47, from glycine to leucine at position 50, from tyrosine to histidine at position 23, and from valine to glutamine at position 45; SEQ ID NO: 39; Phosphatase variant (Y23HV45RP46TE47YG50L) having amino acid exchanges from proline to threonine at position 46, from glutamate to tyrosine at position 47, from glycine to leucine at position 50, from tyrosine to histidine at position 23, and from valine to arginine at position 45; SEQ ID NO: 41; Phosphatase variant (Y23HV45RP46TE47TG50L) having amino acid exchanges from proline to threonine at position 46, from glutamate to threonine at position 47, from glycine to leucine at position 50, from tyrosine to histidine at position 23, and from valine to arginine at position 45; SEQ ID NO: 43;

[0103] Phosphatase variant (Y23HV45RP46TE47FG50L) having amino acid exchanges from proline to threonine at position 46, from glutamate to phenylalanine at position 47, from glycine to leucine at position 50, from tyrosine to histidine at position 23, and from valine to arginine at position 45; SEQ ID NO: 45; Phosphatase variant (Y23HV45MP46TE47FG50L) having amino acid exchanges from proline to threonine at position 46, from glutamate to phenylalanine at position 47, from glycine to leucine at position 50, from tyrosine to histidine at position 23, and from valine to methionine at position 45; SEQ ID NO: 47; Phosphatase variant (Y23HV45QP46TE47FG50L) having amino acid exchanges from proline to threonine at position 46, from glutamate to phenylalanine at position 47, from glycine to leucine at position 50, from tyrosine to histidine at position 23, and from valine to glutamine at position 45; SEQ ID NO: 49.

[0104] Figure 1 shows a schematic diagram of the process of the present invention, which begins with starch (a mixture of A-amylose and amylopectin) being converted to amylose (B) using pullulanase (1) and α-glucanotransferase (2). In the next step, inorganic phosphate (G) is used and catalyzed by phosphorylase (glucan phosphorylase (3)) to cleave the α-1,4 bond between the terminal glucose residue of the polymer and the remaining part, releasing glucose-1-phosphate (C). C is converted to glucose-6-phosphate (D) by the action of phosphoglucomutase (4). Glucose-6-phosphate is converted to fructose-6-phosphate (E) using phosphoglucose isomerase (5). In the final step, fructose-6-phosphate is cleaved to fructose (F) and phosphate (G) by the action of phosphatase (6).

[0105] Figure 2 shows an intermediate process step of the general process shown in Figure 1. Using glucan phosphorylase (3) and inorganic phosphate (G), the α-1,4 bond between the terminal glucose residue and the rest of the polymer (B-A) is cleaved, and glucose-1-phosphate (C) is released.

[0106] Figures 3 to 7 show further intermediate steps of the process shown in Figure 1.

[0107] Figures 8 to 13 present graphs of experimental data that will be further discussed in the following experimental section.

[0108] The present invention has been described with reference to specific preferred embodiments, but it will be understood by those skilled in the art that various changes can be made without departing from the scope of the invention and equivalents can be substituted for its elements. Furthermore, many modifications can be made to adapt a particular situation or material to the teachings of the invention without departing from its essential scope. Accordingly, the invention is not limited to the specific embodiments described, but is intended to cover all embodiments included within the scope of the appended claims.

[0109] Experimental section

[0110] 1. Materials All chemicals were of analytical grade or higher quality, purchased from Sigma-Aldrich, Carbosynth, or VWR, and used without further purification. Glucidex 12 and Glucidex 19 are maltodextrin compounds obtained from Roquette Freres (France).

[0111] The molecular weight of the glucose polymer can be calculated using the formula (180×n - 18×(n - 1)), where n is the DP (degree of polymerization) of the glucose polymer. DE (dextrose equivalent) can be calculated as 100×(180 / molecular weight (glucose polymer)). DP can be calculated using the formula DP = 111 / DE (Balto, Amy S., et al. "On the use of differential solubility in aqueous ethanol solutions to narrow the DP range of food-grade starch hydrolysis products." Food chemistry 197 (2016): 872-880). Glucidex 12 with a DE of 11 - 14 is assumed to have an average DP of 8 - 10. Glucidex 19 with a DE of 18 - 20 is assumed to have an average DP of 5 - 6. However, since these preparations are obtained by enzymatic treatment, they also contain smaller and larger maltodextrins.

[0112] The following strains were used in this test: Escherichia coli XL1 Blue, Escherichia coli BL21(DE3). All constructs were normally expressed using auto-induction medium at 37°C.

[0113] 2. Methods Analysis The analysis of the complete process shown in Figure 1 was performed using an HPLC Dionex Ultimate 3000 system equipped with an autosampler (WPS 3000TRS), a column compartment (TCC3000RS), and a light scattering detector. Separation by gradient elution (15 - 85%) was carried out using a YMC Triart Diol Hilic column (100×2 mm, 1.9 μm, 12 nm) at 7°C, with 0.1% formic acid (pH 4.5) and 0.1% formic acid in ACN as the mobile phase at a flow rate of 0.45 ml / min. The sample was diluted with water / acetonitrile at a ratio of 3:7. The data was analyzed using Dionex Chromeleon software.

[0114] The detection of the entire ketose (fructose and fructose-6-phosphate) was performed using an assay based on triphenyltetrazolium chloride (TTC). The detection method is based on the difference in the reduction rates of aldose and ketose. In a 2 ml test tube, add 0.05 ml of the sample, 0.01 ml of 1% aqueous TTC, and 0.04 ml of 6N NaOH. Exactly 5 minutes later, add 1.5 ml of acetic acid:ethanol (1:9) and vortex mix the contents of the test tube. Use water as a blank and measure the absorbance at 480 nm using a spectrophotometer (Multiskan GO, Thermo Fisher). Glucose and glucose-6-phosphate reduce TTC to a red dye (triphenylformazan). This is approximately 100 times slower than an equivalent amount of fructose.

[0115] Glucose is detected using an assay based on glucose oxidase. Mix 50 μl of the sample or diluted sample with 50 μl of master mix (0.75 mM ABTS, 2 U / ml glucose oxidase, 0.1 U / ml peroxidase, 20 mM KPi, pH 6.0), incubate at 30 °C for 30 minutes, and then measure at 418 nm using a spectrophotometer (Multiskan GO, Thermo Fisher).

[0116] Protein expression Optimized protein expression is exemplarily described for one enzyme and the same procedure was performed for all proteins.

[0117] E. coli BL21(DE3) containing the plasmid of interest (a pET28 derivative having one gene encoding one of the enzymes described in the process) was grown in 250 ml of auto-induction medium (Studier, F. William. "Protein production by auto-induction in high-density shaking cultures." Protein expression and purification 41.1 (2005): 207-234). The preculture was incubated overnight at 37 °C with a rotary shaker (180 rpm) in 4 ml of LB medium containing 100 μg / ml kanamycin. The expression culture was inoculated with a 1:100 dilution of the overnight culture. Incubation was carried out at 37 °C for 24 h. Cells were harvested by centrifugation and resuspended in 50 mM Tris-HCl (pH 8.0). The crude extract was prepared using a Basic-Z cell disruptor (IUL Constant Systems) or an ultrasonic device, followed by addition of MgCl2 to a final concentration of 2.5 mM in combination with DNaseI (1 μg / ml), and then incubation at room temperature (25 °C) for 20 min to degrade DNA. Next, all crude extracts were heat-treated at 70 °C for 30 min. The insoluble fraction of the lysate was removed by centrifugation (20,000 rpm, 40 min at 4 °C). The supernatant was filtered through a 0.45 μm syringe filter and used as the purified enzyme preparation. An aliquot of each purified preparation was subjected to 12% SDS-Page as described in Laemmli Ulrich K. "Cleavage of structural proteins during the assembly of the head of bacteriophage T4." nature 227.5259 (1970): 680.

[0118] The enzyme corresponds to the following sequences: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49.

[0119] Enzyme activity test The activity of pullulanase from Bacillus flavocaldarius (SEQ ID NO: 1) was tested using pullulan as the substrate and 3,5-dinitrosalicylic acid (DNS) for detection.

[0120] Pullulanase was incubated at 65 °C with 1% (w / v) pullulan in 50 mM potassium phosphate buffer (pH 7.0) and 5 mM MgCl2. At various time points, 24 μl aliquots were transferred to 96 μl of DNS reagent (10 g of 3,5-dinitrosalicylic acid, 300 g of potassium sodium tartrate, 16 g of sodium hydroxide per liter). It was heated to 95 °C for 5 minutes, then 100 μl was transferred to a flat-bottom microtiter plate and measured at 540 nm with a spectrophotometer (Multiskan GO, Thermo Fisher).

[0121] The α-glucanotransferase from Meiothermus ruber (SEQ ID NO: 3) was tested for activity using maltotriose as the substrate and for its ability to construct larger oligosaccharides. Detection of activity was performed using TLC (isopropanol:ethyl acetate:water (3:1:1)) and a thymol spray reagent (0.5 g thymol, 95 ml ethanol, 5 ml sulfuric acid 97%). The α-glucanotransferase from Meiothermus ruber was incubated at 65 °C with 50 mM maltotriose in 50 mM potassium phosphate buffer (pH 7.0) and 5 mM MgCl2.

[0122] The α-glucan phosphorylase (SEQ ID NO: 5) of Thermotoga naphthophila was tested for activity using an enzyme combination, and the product α-D-glucose-1-phosphate was detected. α-D-glucose-1-phosphate was converted to α-D-glucose-6-phosphate and subsequently oxidized to α-D-gluconic acid-6-phosphate by α-D-glucose-6-phosphate dehydrogenase. The NADH produced was used to convert WST-1 using 1-methoxy-5-methylphenazinium methyl sulfate (PMS) as an electron mediator. The α-glucan phosphorylase was incubated at 65 °C with 1% (w / v) soluble starch, 5 mM MgCl2, and 0.1 mM pyridoxal-5-phosphate in 50 mM potassium phosphate buffer (pH 7.0). At various time points, 10 μl aliquots were transferred to 190 μl of a detection solution containing 0.1 U of phosphoglucomutase, 0.1 U of α-D-glucose-6-phosphate dehydrogenase, 0.1 mM WST-1, and 0.005 mM PMS. The mixture was incubated for 30 minutes and measured at 440 nm with a spectrophotometer (Multiskan GO, ThermoFisher).

[0123] The sucrose phosphorylase (SEQ ID NO: 51) of Bifidobacterium adolescentis was tested for activity using sucrose as a substrate and 3,5-dinitrosalicylic acid for detection. In the assay using 3,5-dinitrosalicylic acid for detection, only the fructose of the product generates a signal. The sucrose phosphorylase was incubated at 50 °C with 100 mM sucrose in 100 mM potassium phosphate buffer (pH 7.0) and 5 mM MgCl2. At various time points, 24 μl aliquots were transferred to 96 μl of DNS reagent (10 g of 3,5-dinitrosalicylic acid, 300 g of potassium sodium tartrate, 16 g of sodium hydroxide per liter). It was heated to 95 °C for 5 minutes, then 100 μl was transferred to a flat-bottom microtiter plate and measured at 540 nm with a spectrophotometer (Multiskan GO, Thermo Fisher).

[0124] The phosphoglucomutase (SEQ ID NO: 7) of Saccharolobus sulfataricus P2 or the phosphoglucomutase (SEQ ID NO: 9) of Clostridium thermocellum was tested for activity using α-D-glucose-1-phosphate as a substrate and 3,5-dinitrosalicylic acid for detection. In the assay using 3,5-dinitrosalicylic acid for detection, only the α-D-glucose-6-phosphate of the product generates a signal. The phosphoglucomutase was incubated at 65 °C with 50 mM α-D-glucose-1-phosphate in 50 mM potassium phosphate buffer (pH 7.0) and 5 mM MgCl2. At various time points, 24 μl aliquots were transferred to 96 μl of DNS reagent (10 g of 3,5-dinitrosalicylic acid, 300 g of potassium sodium tartrate, 16 g of sodium hydroxide per liter). It was heated to 95 °C for 5 minutes, then 100 μl was transferred to a flat-bottom microtiter plate and measured at 540 nm with a spectrophotometer (Multiskan GO, Thermo Fisher).

[0125] The phosphoglucose isomerase of Thermotoga maritima (SEQ ID NO: 11) was tested for activity using fructose-6-phosphate as the substrate and α-D-glucose-6-phosphate dehydrogenase for detection. The generated NADH was used to convert WST-1 using 1-methoxy-5-methylphenazinium methyl sulfate (PMS) as the electron mediator. The phosphoglucose isomerase was incubated at 65°C with 50 mM fructose-6-phosphate in 50 mM potassium phosphate buffer (pH 7.0) and 5 mM MgCl2. At various time points, 10 μl aliquots were transferred to 190 μl of a detection solution containing 0.1 U of α-D-glucose-6-phosphate dehydrogenase, 0.1 mM WST-1, and 0.005 mM PMS. The mixture was incubated for 30 minutes and measured at 440 nm with a spectrophotometer (Multiskan GO, ThermoFisher).

[0126] The phosphatases of Thermotoga naphthophila (SEQ ID NO: 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49) were tested for activity using fructose-6-phosphate as the substrate in a phosphate assay. The phosphate assay consists of three solutions: solution A (12% (w / v) l-ascorbic acid in 1 N HCl solution), solution B (2% (w / v) Na2MoO4·2H2O in ddH2O), and solution F (2% (w / v) citric acid and 2% (w / v) acetic acid in ddH2O). Solution D is a 2:1 mixture of solution A and solution B and is freshly prepared before measurement. 75 μl of solution D is pipetted into the required wells of a flat-bottom microtiter plate. In the next step, 25 μl of the sample or a defined concentration of the standard is added and incubated at room temperature for 5 minutes. In the final step, 75 μl of solution F is added and an additional incubation step at room temperature for 15 minutes is added to stop the reaction. The samples are measured at 655 nm with a spectrophotometer (Multiskan GO, Thermo Fisher).

[0127] All the enzymes tested met the requirement of catalyzing one of the steps in the process and were stable under the process conditions (65 °C, 50 mM potassium phosphate buffer (pH 7.0), 5 mM MgCl2, 0.005 mM PLP).

[0128] 3. Conversion from starch to fructose (Example 1 of the present invention; IE1) The conversion experiment was carried out on a 15 ml scale using 50 ml Falcon tubes containing 7 g of native starch. The native starch was pretreated with 6 ml of water and buffer and heated to 95 °C for 30 minutes to swell it almost completely. Then, the swollen starch was cooled to 65 °C and the enzyme was added. To 7 g of native starch, 10.5 ml of buffer, salts, and enzyme were added. Further, maltose was added to a final concentration of 10 mM. The final mixture contained 50 mM KPi (pH 7.0), 5 mM MgCl2, 0.1 mM PLP, 10 mM maltose, 0.4 mg pullulanase, 1.5 mg α-glucanotransferase, 30 mg α-glucan phosphorylase, 1.4 mg phosphoglucomutase, 1.2 mg phosphoglucose isomerase, and 14 mg phosphatase. Enzymes from Bacillus flavocaldarius, Mayosermus ruber, Thermotoga naphthophila (phosphorylase and phosphatase), Clostridium thermocellum, and Thermotoga maritima were used. Starting from 0 hours of incubation, samples were taken regularly and filtered using a 10 kDa spin filter (VWR 82031-350). If necessary, the samples were diluted with water / acetonitrile in a ratio of 3:7 for further analysis.

[0129] The results of the conversion from starch to fructose according to Example 1 (IE1) of the present invention (35% dry matter starch corresponds to a monosaccharide concentration of 2153.5 mM) are shown in Table 1 and Figure 8.

Table 1

[0130] It can be seen from Table 1 that fructose can be obtained at a yield of about 70% after 48 hours.

[0131] 4. Conversion of starch to fructose in the presence of maltose (IE2 - IE5), maltotriose (IE6 - IE9), maltotetraose (IE10 - IE12), maltodextrin Glucidex 12 (IE13 - IE15), or maltodextrin Glucidex 19 (IE16 - IE18) 50 mM potassium phosphate buffer (pH 7.0), 5 mM MgCl2, 0.005 mM PLP, 1% (w / v) swollen native starch, maltose (0.01 mM, 0.1 mM, 1.0 mM, or 10 mM; IE2 - IE5), or maltotriose (0.01 mM, 0.1 mM, 1.0 mM, or 10 mM; IE6 - IE9), or maltotetraose (0.01 mM, 0.1 mM, or 1.0 mM; IE10 - IE12), or maltodextrin Glucidex 12 (0.0342% (w / v), 0.00342% (w / v), or 0.000342% (w / v); IE13 - IE15), or maltodextrin Glucidex 19 (0.0342% (w / v), 0.00342% (w / v), or 0.000342% (w / v); IE16 - IE18), glucan phosphorylase (0.0025 mg / ml), alpha - glucanotransferase (0.075 mg / ml), pullulanase (0.02 mg / ml), a total of 5 ml was incubated at 65 °C for 1 hour, and aliquots were taken out and checked for glucose - 1 - phosphate. The glucose - 1 - phosphate assay is described in the activity test of the above enzymes.

[0132] Comparative Examples 1 - 5 (CE1 - CE5) were each carried out by adding only 1 mM of maltose, maltotriose, maltotetraose, Glucidex 12, or Glucidex 19 without adding starch. Comparative Example 6 was carried out without adding additional saccharides such as maltose, maltotriose, maltotetraose, Glucidex 12, or Glucidex 19.

[0133] Table 2 shows the results of tests on the effect of additional sugars, such as maltose, maltotriose, maltotetraose, Glucidex 12, or Glucidex 19, short oligosaccharides, on the conversion rate to glucose-1-phosphate. Each figure is shown in Figure 9.

Table 2

[0134] The conversion of large polysaccharides, such as starch or equivalent substrates like cellulose in the process, involves one major rate-limiting step, for example, the production of glucose-1-phosphate molecules catalyzed by glucan phosphorylase. Phosphorylase requires an accessible end of the polysaccharide to catalyze the cleavage of the α-1,4 bond between the terminal glucose residue of the polymer and the rest using inorganic phosphate to release glucose-1-phosphate. The combined action of pullulanase, α-glucanotransferase, and short oligosaccharides, such as maltose (IE2 - IE5), maltotriose (IE6 - IE9), maltotetraose (IE10 - IE12), maltodextrin Glucidex 12 (IE13 - IE15), or maltodextrin Glucidex 19 (IE16 - IE18) (see Table 2), shows that the conversion rate improves when short oligosaccharides are added. This results in higher production of glucose-1-phosphate and thus enables a more rapid conversion from starch to fructose within the process of the present invention.

[0135] The conversion rates of all Comparative Examples 1 - 6 are lower compared to Examples 2 - 18 of the present invention.

[0136] 5. Phosphate Assay A phosphate assay was performed to track the different activities of the different phosphatases present in the process and the variants of the two sugar phosphate intermediates.

[0137] The reaction mixture contained 50 mM Tris-HCl buffer (pH 7.0), 2.5 mM MgCl2, 10 mM of G1P, G6P, or F6P, and one phosphatase (0.003 mg / ml) in a total volume of 1 ml. The mixture was incubated at 65 °C, aliquots were removed at regular time intervals, and tested for released phosphate. The phosphate assay is described in more detail above.

[0138] The results of the phosphate assay are shown in Table 3 and Figure 10. The amounts of G6P and F6P were detected and the F6P / G6P ratio was determined. The F6P / G6P ratio indicates the selectivity of the phosphatase tested.

Table 3

[0139] Introduction of the P46T (IE19(T)) mutation into the phosphatase of Thermotoga naphthophila increases the overall activity of the enzyme and decreases the activity of the enzyme against glucose-6-phosphate (G6P), as can be seen from Table 3 and Figure 10. Thereby, from the perspective of reducing the amount of enzyme and improving the overall fructose production, the economy of the process is improved. P46TE47Y (IE20(TY)) has slightly improved activity against F6P compared to the wild type (CE7(WT)), shows almost the same activity against G6P, and the unnecessary by-products such as glucose are reduced. P46TE47YG50L (IE21(TYL)) has improved activity against F6P compared to the wild type (CE7(WT)), the activity against G6P is decreased, and the unnecessary by-products such as glucose are also reduced. Y23HP46TE47Y (IE23(HTY)) also has improved activity against F6P compared to CE7(WT), and the activity against G6P is slightly increased. However, in relation to both activities, this variant also shows improved performance of the process of the present invention, enabling an improved process that reduces unwanted by-products such as glucose. P46TE47YG50I (IE22(TYI)) has improved selectivity against F6P. Y23HP46TE47YG50L (IE24(HTYL)) has improved activity against F6P compared to CE7(WT), and the activity against G6P is decreased in the situation where the overall activity is increased. Y23HP46TE47YG50I (IE25(HTYI)) has improved activity against F6P, and the activity against G6P is decreased in the situation where the overall activity is increased. Y23SP46TE47YG50L (IE26(STYL)) has improved activity against F6P compared to CE7(WT), and the activity against G6P is decreased in the situation where the overall activity is increased. Y23SP46TE47YG50I (IE27(STYI)) has improved selectivity against F6P.

[0140] The phosphatase of the present invention (see Table 3 and Figure 10) shows improved activity, improved selectivity, or both.

[0141] The results of further phosphate assays testing additional phosphatases, compared to wild-type phosphatase, are shown in Table 4 and Figure 11. Since the phosphatases used showed improved activity, the time intervals for measurement were shortened.

Table 4

[0142] Y23HP46TE47YG50L (HTYL) has improved activity against F6P compared to the wild type, and its activity against G6P is decreased in the situation where the overall activity is increased. Therefore, the enzyme enables an improved process and reduces unwanted by-products such as glucose. The same applies to the variants Y23HP46TE47TG50L (Y47T), Y23HP46TE47TG50L (Y47F), Y23HV45MP46TE47TG50L (V45M), Y23HV45QP46TE47TG50L (V45Q), Y23HV45RP46TE47TG50L (V45R).

[0143] The results of another phosphate assay testing additional phosphatases are shown in Table 5 and Figure 12. Since the phosphatases used showed improved activity, the time intervals for measurement were also shortened.

Table 5

[0144] The variants Y23HV45RP46TE47TG50L (V45R), Y23HP46TE47FG50L (HTFL), Y23HV45RP46TE47TG50L (HTTLV45R), Y23HV45MP46TE47FG50L (V45M), and Y23HV45QP46TE47FG50L (V45Q) all have improved activity against F6P, and their activity against G6P is decreased in the situation where the overall activity is increased. Therefore, the enzyme enables an improved process that reduces unwanted by-products such as glucose.

[0145] 6. Conversion of Sucrose to Fructose Using Improved Phosphatase Enzymes 50 mM potassium phosphate buffer (pH 7.0), 5 mM MgCl2, 1000 mM sucrose, sucrose phosphorylase (0.25 mg / ml), phosphoglucomutase (0.04 mg / ml), phosphoglucose isomerase (0.075 mg / ml), and phosphatase (0.18 mg / ml) were incubated at 50 °C for 48 h in a total volume of 10 ml, and aliquots were taken out to check the concentrations of glucose and fructose. Enzymes from Bifidobacterium adolescentis (sucrose phosphorylase), Thermotoga naphthophila (phosphatase), Clostridium thermocellum (phosphoglucomutase), and Thermotoga maritima (phosphoglucose isomerase) were used. Fructose was determined as described in the method.

[0146] Table 6 and Figure 13 show the results of the conversion of sucrose to fructose using three different phosphatase enzymes. [Table 6]

[0147] As is clear from Table 6, the conversion rates of the phosphatase TY (IE40) and HTYL (IE41) of the present invention are higher compared to the wild-type phosphatase WT (CE9).

Claims

1. A method for converting at least one oligosaccharide and / or polysaccharide into fructose, comprising: a) adding at least six enzymes to a composition comprising water, phosphate, and at least one oligosaccharide and / or polysaccharide; b) subsequently, enzymatically converting the at least one oligosaccharide and / or polysaccharide into fructose in the presence of the at least six enzymes, wherein in step a), at least one additional sugar is added, and the at least one additional sugar is selected from the group consisting of tetradecasaccharide, tridecasaccharide, dodecasaccharide, undecasaccharide, decasaccharide, nonasaccharide, octasaccharide, heptasaccharide, hexasaccharide, pentasaccharide, tetrasaccharide, trisaccharide, disaccharide, and / or combinations thereof; the at least one additional sugar has at least a partially identical glycosidic bond with the at least one oligosaccharide and / or polysaccharide; in step a), the at least six enzymes are selected from the group consisting of phosphatase, transferase, phosphorylase, mutase, isomerase, and / or hydrolase; at least one enzyme in step a) is phosphatase; the at least one oligosaccharide and / or polysaccharide is selected from glucose-based oligosaccharides and / or polysaccharides; and the phosphatase comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO:

41.

2. The method according to claim 1, wherein in step a), the at least one additional sugar is selected from the group consisting of tetrasaccharide, trisaccharide, disaccharide, and combinations thereof; and / or the composition in step a) comprises 35% or less of the at least one oligosaccharide and / or polysaccharide on a dry weight basis; and / or the at least one oligosaccharide and / or polysaccharide is selected from the group consisting of starch and its derivatives, hemicellulose and its derivatives, cellulose and its derivatives, and / or combinations thereof.

3. The method according to claim 2, wherein the tetrasaccharide, trisaccharide, disaccharide, and combinations thereof are maltose, maltotriose, maltotetraose, and combinations thereof.

4. The method according to any one of claims 1 to 3, wherein in step b), sugar phosphate is formed intermmediately; and / or in step b), the step of enzymatically converting is a one-pot reaction step.

5. The method according to any one of claims 1 to 4, wherein In step a), at least one transferase is added, and / or, In step a), at least one phosphorylase is added, and / or, In step a), at least one mutase is added, and / or, In step a), at least one isomerase is added, and / or, In step a), at least one hydrolase is added.

6. The transferase is a glycosyltransferase, and / or, The phosphorylase is a glucan phosphorylase, and / or, The mutase is a phosphoglucomutase, and / or, The isomerase is a phosphoglucose isomerase, and / or, The hydrolase is a glucanohydrolase, The method according to claim 5.

7. Step b) is carried out at room temperature (25 °C) for at least 24 hours, the method according to any one of claims 1 to 6.

8. The method according to any one of claims 1 to 7, At least 50% of the sugar present in the composition of step a) is converted to fructose after 24 hours of enzymatic conversion, and / or, At least 70% of the sugar present in the composition of step a) is converted to fructose after 48 hours of enzymatic conversion.

9. The method according to any one of claims 1 to 6 and 8, The temperature of step b) is 10 to 100 °C, and / or, The pH of the composition is 3 to 12.

10. The temperature of step b) is 20 to 90 °C, and / or The pH of the composition is 4 to 10, the method according to claim 9.

11. A composition for converting at least one oligosaccharide and / or polysaccharide to fructose, comprising: - water, - phosphate, - at least six enzymes (where at least one enzyme is a phosphatase), - at least one oligosaccharide and / or polysaccharide, The at least six enzymes are selected from the group consisting of phosphatase, transferase, phosphorylase, mutase, isomerase, and / or hydrolase, The composition further comprises at least one additional sugar, and the at least one additional sugar is selected from the group consisting of tetradecasaccharide, tridecasaccharide, dodecasaccharide, undecasaccharide, decasaccharide, nonasaccharide, octasaccharide, heptasaccharide, hexasaccharide, pentasaccharide, tetrasaccharide, trisaccharide, disaccharide, and / or combinations thereof. The at least one additional sugar has at least a partially same glycosidic bond as the at least one oligosaccharide and / or polysaccharide, the at least one oligosaccharide and / or polysaccharide is selected from glucose-based oligosaccharides and / or polysaccharides, and the phosphatase comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 41, a composition.

12. The composition according to claim 11, wherein the phosphatase comprises an amino acid sequence that is at least 98.5% identical to the sequence of SEQ ID NO: 41, or the phosphatase has an amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, or SEQ ID NO: 49, a composition.

13. The composition according to claim 12, wherein the phosphatase comprises an amino acid sequence that is at least 99% identical to the sequence of SEQ ID NO:

41.

14. - at least 50% fructose on a dry weight basis, - 0.001 to 25% glucose on a dry weight basis, - 0.01 to 22% phosphate on a dry weight basis, and - an aqueous composition comprising 0.001 to 2% of at least six enzymes on a dry weight basis, wherein the at least six enzymes are selected from the group consisting of phosphatase, transferase, phosphorylase, mutase, isomerase, and / or hydrolase, at least one enzyme is phosphatase; and the phosphatase comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 41, an aqueous composition.

15. The aqueous composition according to claim 14, wherein the phosphatase comprises an amino acid sequence that is at least 98.5% identical to the sequence of SEQ ID NO: 41, or the phosphatase has an amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, or SEQ ID NO: 49, an aqueous composition.

16. The aqueous composition according to claim 15, wherein the phosphatase comprises an amino acid sequence that is at least 99% identical to the sequence of SEQ ID NO: 41.

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