Preparation of fructose from oligo- and / or polysaccharides

Abstract

The present invention relates to a method for converting at least one oligo- and / or polysaccharide into fructose, comprising the steps of: a) adding at least four enzymes to a composition comprising water, phosphate and at least one oligo- and / or polysaccharide; and b) subsequently enzymatically converting at least one oligo- and / or polysaccharide into fructose in the presence of at least four 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 sugars comprising 20 or fewer monosaccharide residues and / or combinations thereof; wherein in step a), at least four enzymes, preferably at least five enzymes, are selected from the group consisting of transferase, phosphorylase, mutase, isomerase, hydrolase, phosphatase, and combinations thereof; and in step a), at least one enzyme is phosphatase.

Description

Technology Field

[0001] The present invention relates to a method for converting at least one oligo- and / or polysaccharide into fructose, a composition for converting at least one oligo- and / or polysaccharide into fructose, and an aqueous composition comprising fructose. Additionally, the present invention relates to phosphatase and the use of phosphatase in the conversion of at least one oligo- and / or polysaccharide. Background Technology

[0002] Currently, the industrial manufacturing process of D-fructose is based particularly on a two-step process. In the first step, polysaccharides or oligosaccharides, such as starch, are cleaved into monosaccharides by hydrolysis. In the second step, isomerization is performed to obtain D-fructose in a yield of about 42% (corresponding to percentage). Subsequent work for the production of pure D-fructose is carried out by expensive chromatographic purification techniques.

[0003] An alternative industrial process is the hydrolysis of sucrose using enzymatic invertase. This yields an equimolar mixture of D-glucose and D-fructose. To enrich it with D-fructose, this must then be washed by an expensive chromatography procedure.

[0004] The industrial process has a significant disadvantage because only a D-fructose solution is formed, and D-fructose constitutes up to 50% of the sugars present in the solution. To obtain a higher D-fructose solution, expensive chromatographic purification must be performed, or expensive pure D-fructose, such as high fructose corn syrup (HFCS55) with a fructose content of 55%, must be added.

[0005] An alternative process for the production of pure D-fructose using D-glucose as a substrate is known and is described in detail in EP0028136B1 and AT513928B1. In this case, D-glucose is oxidized to D-glucosone by pyranose-2-oxidase and then reduced to D-fructose. Reduction can be carried out by chemical means, namely by homogeneous or heterogeneous catalysts, or by using biocatalysts.

[0006] This process has not yet been technically realized. It has the disadvantage that oxidation and reduction must operate separately, thus requiring additional process steps. Furthermore, if reduction occurs chemically, high-purity substrates are required, along with high pressure as well as high temperature, which can lead to the formation of undesirable byproducts. If reduction is performed enzymatically, very high costs are incurred for the reducing agent and the unstable coenzyme used, such as NADH.

[0007] The known process for the production of D-fructose has various disadvantages. High pressure and temperature are required in part for the efficient conversion of the substrate. Alternatively, depending on the substrate, only a yield of up to 50% is achieved, or the use of clean substrates is required to reduce by-products. Further concentration requires a very clean process stream to ensure the reuse of chromatography equipment.

[0008] There is a need for a method to convert oligo- and / or polysaccharides to D-fructose that overcomes the aforementioned disadvantages, such as the use of expensive coenzymes or adverse equilibria requiring additional chromatographic purification and concentration steps. The problem to be solved

[0009] The object of the present invention is to provide a method that allows for the production of D-fructose with high yield and with no or fewer by-products in relation to the substrate. In addition, the operation must be simplified and the use of organic coenzymes must not be necessary.

[0010] The above object is solved by a method for converting at least one oligo- and / or polysaccharide into fructose according to claim 1, a composition for converting at least one oligo- and / or polysaccharide into fructose according to claim 8, and an aqueous composition comprising fructose according to claim 10. Additionally, the object is solved by the use of phosphatase according to claim 13 and phosphatase in the conversion of at least one oligo- and / or polysaccharide according to claim 15. means of solving the problem

[0011] In a first aspect, the present invention

[0012] a) a step of adding at least four enzymes, preferably at least five enzymes, to a composition comprising water, phosphate, and at least one oligo- and / or polysaccharide, and

[0013] b) a step of subsequently enzymatically converting at least one oligo- and / or polysaccharide into fructose in the presence of at least four enzymes, preferably at least five enzymes.

[0014] The invention relates to a method for converting at least one oligo- and / or polysaccharide into fructose, comprising,

[0015] Herein, 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;

[0016] In step a), at least four enzymes, preferably at least five enzymes, are selected from the group consisting of transferase, phosphorylase, mutase, isomerase, hydrolase, phosphatase, and combinations thereof; and in step a), at least one enzyme is phosphatase.

[0017] Further preferred embodiments are described in the description as well as in the dependent claims.

[0018] In the context of the present invention, references to fructose should be understood as references to D-fructose.

[0019] The oligosaccharides according to the invention are sugars comprising at least two monosaccharide residues, wherein these monosaccharide residues may be the same or different.

[0020] It should be understood that under phosphatase, all enzymes belonging to Classification EC 3.1.3. under transferase, all enzymes belonging to Classification EC 2.4. under phosphorylase, all enzymes belonging to Classification EC 2.4.1. under mutase, all enzymes belonging to Classification EC 5.4.2. under isomerase, all enzymes belonging to Classification EC 5.3.1. under hydrolase, all enzymes belonging to Classification EC 3.2.

[0021] Oligosaccharides and polysaccharides may be used as substrates according to the invention. The improved yield is due to sugar phosphates that can be produced by cutting the sugars and fructose units that may already be present in the sugars.

[0022] Surprisingly, a method for producing D-fructose has been discovered that operates without high pressure and temperature and tolerates the presence of contaminants better during the process. The selection of a catalyst enables the production of high-yield D-fructose in the process by forming the intermediate sugar-phosphate. As a result, a higher yield based on the substrate (sugars) can be achieved than in currently used industrial manufacturing methods, and subsequent operations are unnecessary. This eliminates costly cleaning procedures. The sugars can be oligosaccharides and / or polysaccharides.

[0023] Preferably, the composition of step a) comprises at least one oligo- and / or polysaccharide in an amount of 35% or less by dry weight. Preferably, the composition comprises at least one oligo- and / or polysaccharide in an amount of 0.5% to 35% by dry weight.

[0024] Additionally, preferably, the oligo- and / or polysaccharide is selected from glucose-based oligo- and / or polysaccharides, preferably from the group consisting of starch and its derivatives, hemicellulose and its derivatives, cellulose and its derivatives and / or combinations thereof.

[0025] Each oligo- and / or polysaccharide can be obtained through starch production or by the hydrolysis of biomass, for example, in pulp production or in the processing of straw.

[0026] Preferably, in step a), at least one additional sugar is selected from the group consisting of decasaccharides, thirsacharides, dodisaccharides, decasaccharides, quinasaccharides, octasaccharides, heptasaccharides, hexasaccharides, pentasaccharides, tetrasaccharides, trisaccharides, disaccharides and / or combinations thereof, more preferably tetrasaccharides, trisaccharides, disaccharides and / or combinations thereof, and more preferably selected from maltose, maltotriose, maltotetraose and combinations thereof.

[0027] Additionally, preferably, at least one additional sugar in step a) is maltodextrin. Maltodextrin is a sugar consisting of D-glucose units linked in chains of variable length, primarily linked by α-1,4-glycosidic bonds. Typically, maltodextrin consists of a mixture of various chains with lengths of 3 to 17 glucose units. Maltodextrin is classified by DE (dextrose equivalent) and has a DE of 3 to 20. The higher the DE value, the shorter the glucose chain.

[0028] Additionally, preferably, at least one additional sugar has at least one glycosidic bond that is at least partially identical to that of at least one oligo- and / or polysaccharide.

[0029] Preferably, the phosphatase comprises an amino acid sequence that is at least 90%, preferably at least 95%, more preferably at least 97%, and more preferably at least 98.5% identical to the sequence according to SEQ ID NO: 13.

[0030] 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 more preferably at least 99.5% identical to the sequence according to SEQ ID NO: 41.

[0031] According to a preferred embodiment, the phosphatase is selected from the group of phosphatases having a sequence according to 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.

[0032] Furthermore, in step a), preferably at least six enzymes are added to the composition.

[0033] 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.

[0034] Preferably, in step a), at least one transferase, preferably glycosyltransferase, more preferably glucanotransferase, more preferably alpha-glucanotransferase, is added;

[0035] Preferably, at least one phosphorylase, preferably a glucanphosphorylase, is added in step a) and / or;

[0036] Preferably, at least one mutase, preferably a phosphoglucomutase, is added in step a);

[0037] Preferably, at least one isomerase, preferably a phosphoglucoisomerase, is added in step a);

[0038] Preferably, at least one hydrolytic enzyme, preferably glucanohydrolytic enzyme, more preferably pullulanase, is added in step a).

[0039] According to a preferred embodiment of a method for converting at least one oligo- and / or polysaccharide into fructose, in step a), at least one additional sugar is selected from the group consisting of tetrasaccharides, trisaccharides, disaccharides, and combinations thereof, more preferably selected from maltose, maltotriose, maltotetraose, and combinations thereof; the composition comprises at least one oligo- and / or polysaccharide in an amount of 35% or less by dry weight in step a), preferably the composition comprises at least one oligo- and / or polysaccharide in an amount of 0.5% to 35% by dry weight; at least one oligo- and / or polysaccharide is selected from glucose-based oligo- and / or polysaccharides, preferably from the group consisting of starch and its derivatives, hemicellulose and its derivatives, cellulose and its derivatives, and / or combinations thereof; and in step a), at least six enzymes are added to the composition and / or At least one additional sugar has at least one glycosidic bond that is at least partially identical to that of at least one oligo- and / or polysaccharide.

[0040] Preferably, sugar phosphate is produced in the middle during step b).

[0041] Additionally, preferably, the enzymatic conversion in step b) is a one-pot reaction.

[0042] According to a preferred embodiment, in step b), sugar phosphate is produced in the middle, and the enzymatic conversion is a one-pot reaction.

[0043] The method of the invention allows the conversion of oligo- and polysaccharides into D-fructose by several catalytic steps. This results in an intermediate in the form of sugar phosphate, which is eventually cleaved into D-fructose and phosphate.

[0044] Preferably, the oligo- and / or polysaccharide added in step a) comprises a plurality of glucose units, more preferably D-glucose units. More preferably, the oligo- and / or polysaccharide added in step a) consists of a plurality of glucose units, more preferably D-glucose units.

[0045] A method of the invention according to one embodiment is an enzymatic process and a reaction sequence is applied that enables the cleavage of individual D-glucose molecules in the form of D-glucose-1-phosphate. Subsequently, these released D-glucose-1-phosphate molecules are converted to D-fructose-6-phosphate through additional sugar-phosphate intermediates. D-fructose-6-phosphate is subsequently cleaved, and D-fructose and phosphate are obtained.

[0046] During this conversion, the reaction step includes the following steps: the sugar molecule enzymatically cleaves D-glucose-1-phosphate, D-glucose-1-phosphate is enzymatically converted to D-glucose-6-phosphate, D-glucose-6-phosphate is enzymatically converted to D-fructose-6-phosphate, and D-fructose-6-phosphate is enzymatically cleaved into D-fructose and phosphate.

[0047] Phosphate is used in small amounts to form sugar phosphates in the middle, such as D-glucose-1-phosphate, D-glucose-6-phosphate, and D-fructose-6-phosphate. In the initial stage, phosphate is added to the D-glucose unit containing the mixture, and in the final stage, the phosphate is cleaved so that it can be reused for the initial stage.

[0048] This method of the present invention provides an enzymatic substitute for the production of D-fructose as known in the art and significantly simplifies the need to isolate and purify residual intermediates. Therefore, the present invention represents a significant improvement over currently used techniques in the production of D-fructose from sugars. In contrast to existing methods, the biocatalytic system used achieves enhanced enrichment of the product D-fructose.

[0049] The method is basically suitable for the use of oligosaccharides and polysaccharides.

[0050] Preferably, a D-fructose-rich hydrolysate can be produced from cellobiose, maltose, or starch. In this case, the initial step is carried out using cellobiose phosphorylase, maltose phosphorylase, or starch phosphorylase, followed by several catalytic steps. The method is particularly useful for the use of sucrose as a substrate, which can be obtained from sugar beets or cane. In this case, the initial step is carried out using sucrose phosphorylase. In this particular case, D-fructose or > 1 molar D-fructose is obtained with a yield of > 53% (by mass) per molar of sucrose.

[0051] The method of the present invention can be carried out in various solvent systems. Preferably, it is carried out in an aqueous solvent system.

[0052] It is also possible to supplement the aqueous system with a buffer system. Suitable buffers (systems) are known, and conventional buffers (systems), for example, acetate, potassium phosphate, Tris-HCl, glycylglycine and glycine buffers, or mixtures thereof.

[0053] Preferably, the buffer used in the method of the present invention has a pH of 3 to 12, preferably 4 to 12, and more preferably 4 to 11. Biocatalytic ions to be added, e.g., Mg 2+ For optimal activity, the use of stabilizers, such as glycerol, can allow for longer use of the biocatalyst.

[0054] 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, phosphate salt, polyphosphate, or a combination thereof is required.

[0055] Preferably, the method of the invention is carried out at a suitable temperature, which may vary, for example, depending on the enzyme used. Suitable 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.

[0056] Preferably, the temperature of step b) is 10 to 100°C, more preferably 20 to 90°C, and even more preferably 20 to 80°C.

[0057] Additionally, preferably, the pH of the composition is 3 to 12 and more preferably 4 to 10.

[0058] The advantage of the method of the invention is that all catalytic steps are performed in the same reaction batch without the need to separate intermediates. The method can be operated in a batch or continuous manner. In this case, all included enzymes may be added simultaneously, or only a portion of the enzymes may be added; for example, enzyme(s) may be added during step b), and another portion of the enzyme may be added later after a temporary or local delay.

[0059] Before the enzyme of Part 2 is added, the enzyme already present in the reaction mixture may be removed or inactivated, for example, physically (through filtration or immobilization). The latter may be carried out by increasing the temperature briefly, for example, to 80°C for 10 minutes. Alternatively, the reaction solution may also be passed through the reaction mixture several times for increased conversion and yield.

[0060] In the method of the invention, the cleavage of sugars to D-glucose-1-phosphate is enzymatic, that is, by an enzyme catalyst, and can be carried out according to known methods. The cleavage is preferably carried out by catalysis using a phosphorylase.

[0061] Preferably, at least 50% of the sugars present in the composition of step a) are converted into fructose after 24 hours of enzymatic conversion.

[0062] Also, preferably, at least 70% of the sugars present in the composition of step a) are converted into fructose after 48 hours of enzymatic conversion.

[0063] The method of the invention further allows for improved handling of highly concentrated oligo- and / or polysaccharides, such as expanded starch. For example, it is possible to carry out the method of the invention with a starting concentration of starch of 35% dry matter.

[0064] Preferably, step b) is performed at room temperature (25°C) for at least 24 hours.

[0065] In another aspect, the present invention relates to a method for converting at least one oligo- and / or polysaccharide into fructose, the method comprising: a) adding at least one enzyme to a composition comprising water, phosphate and at least one oligo- and / or polysaccharide; and b) subsequently enzymatically converting at least one oligo- and / or polysaccharide into fructose in the presence of at least one enzyme, 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 sugars comprising 20 or fewer monosaccharide residues and / or combinations thereof, preferably 17 or fewer monosaccharide residues and / or combinations thereof.

[0066] In relation to a method for converting at least one oligo- and / or polysaccharide into fructose, all references, particularly regarding the enzyme and oligo- and / or polysaccharide mentioned so far, also apply to the present method where applicable.

[0067] Preferably, at least one enzyme in step a) is phosphatase.

[0068] Furthermore, in step a), preferably at least 4 enzymes, more preferably at least 5 enzymes, and even more preferably at least 6 enzymes are added to the composition.

[0069] In a second aspect, the invention relates to a composition for converting at least one oligo- and / or polysaccharide into fructose, wherein the composition is

[0070] - water;

[0071] - Phosphate;

[0072] - At least four enzymes, preferably at least five enzymes, in which at least one enzyme is phosphatase;

[0073] - comprising at least one oligo- and / or polysaccharide;

[0074] 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

[0075] The composition further comprises at least one additional sugar, 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.

[0076] All references regarding a method for converting at least one oligo- and / or polysaccharide into fructose, particularly regarding the enzyme and oligo- and / or polysaccharide mentioned so far, also apply to a composition for converting at least one oligo- and / or polysaccharide into fructose 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 more preferably at least 98.5% identical to the sequence according to SEQ ID NO: 13.

[0078] 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 more preferably at least 99.5% identical to the sequence according to SEQ ID NO: 41.

[0079] According to a preferred embodiment, the phosphatase is selected from the group of phosphatases having an amino acid sequence according to 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.

[0080] 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.

[0081] According to a preferred embodiment, the composition comprises a transferase, preferably glycosyltransferase, more preferably glucanotransferase, and more preferably alpha-glucanotransferase; and / or

[0082] The composition further comprises a phosphorylase, preferably a glucanphosphorylase is added;

[0083] The composition comprises a mutase, preferably a phosphoglucomutase;

[0084] The composition comprises an isomerase, preferably a phosphoglucoisomerase;

[0085] The composition includes a hydrolytic enzyme, preferably a glucanohydrolytic enzyme, more preferably a pullulanase.

[0086] In another aspect, the invention relates to a composition for converting at least one oligo- and / or polysaccharide into fructose, comprising water; phosphate; at least five enzymes in which at least one enzyme is phosphatase; and at least one oligo- and / or polysaccharide; wherein the phosphatase comprises an amino acid sequence that is at least 90%, preferably at least 95%, more preferably at least 97%, and more preferably at least 98.5% identical to the sequence according to SEQ ID NO: 13.

[0087] With respect to a method for converting at least one oligo- and / or polysaccharide into fructose, in particular with respect to the enzyme and oligo- and / or polysaccharide and the composition for converting at least one oligo- and / or polysaccharide into fructose mentioned so far, all references also apply to this aspect where applicable.

[0088] Preferably, at least five enzymes are selected from the group consisting of phosphatase, transferase, phosphorylase, mutase, isomerase and / or hydrolase.

[0089] In a third aspect, the invention

[0090] - At least 50% fructose by dry weight;

[0091] - 0.001 to 25% glucose based on dry weight, preferably 0.005 to 20% glucose;

[0092] - 0.01 to 22% of phosphate based on dry weight, preferably 0.05 to 20% of phosphate; and

[0093] - The invention relates to an aqueous composition comprising at least four enzymes, preferably at least five enzymes, in an amount of 0.001 to 2% by dry weight, preferably at least four enzymes, preferably at least five enzymes, in an amount of 0.005 to 1% by dry weight, and

[0094] Herein, at least four enzymes, preferably at least five enzymes, are selected from the group consisting of transferase, phosphorylase, mutase, isomerase, hydrolase, phosphatase, and combinations thereof; and

[0095] At least one enzyme is phosphatase.

[0096] In relation to a method for converting at least one oligo- and / or polysaccharide into fructose, all references, particularly regarding the enzyme and oligo- and / or polysaccharide mentioned so far, also apply to aqueous compositions where applicable.

[0097] 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 more preferably at least 98.5% identical to the sequence according to SEQ ID NO: 13.

[0098] 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 more preferably at least 99.5% identical to the sequence according to SEQ ID NO: 41.

[0099] According to a preferred embodiment, the phosphatase is selected from the group of phosphatases having an amino acid sequence according to 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.

[0100] 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.

[0101] According to a preferred embodiment, the composition comprises a transferase, preferably glycosyltransferase, more preferably glucanotransferase, and more preferably alpha-glucanotransferase; and / or

[0102] The composition further comprises a phosphorylase, preferably a glucanphosphorylase is added;

[0103] The composition comprises a mutase, preferably a phosphoglucomutase;

[0104] The composition comprises an isomerase, preferably a phosphoglucoisomerase;

[0105] The composition includes a hydrolytic enzyme, preferably a glucanohydrolytic enzyme, more preferably a pullulanase.

[0106] In the fourth aspect, the invention

[0107] - At least 50% fructose by dry weight;

[0108] - 0.001 to 25% glucose based on dry weight, preferably 0.005 to 20% glucose;

[0109] - 0.01 to 22% of phosphate based on dry weight, preferably 0.05 to 20% of phosphate; and

[0110] - The invention relates to an aqueous composition comprising at least four enzymes, preferably at least five enzymes, in an amount of 0.001 to 2% by dry weight, preferably at least four enzymes, preferably at least five enzymes, in an amount of 0.005 to 1% by dry weight, and

[0111] Herein, at least four enzymes, preferably at least five enzymes, are selected from the group consisting of transferase, phosphorylase, mutase, isomerase, hydrolase, phosphatase, and combinations thereof; and

[0112] At least one enzyme is phosphatase, and

[0113] The composition can be obtained by the method of the invention described above.

[0114] In relation to a method for converting at least one oligo- and / or polysaccharide into fructose, all references, particularly regarding the enzyme and oligo- and / or polysaccharide mentioned so far, also apply to an aqueous composition obtainable by the method of the invention where applicable.

[0115] 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 more preferably at least 98.5% identical to the sequence according to SEQ ID NO: 13.

[0116] 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 more preferably at least 99.5% identical to the sequence according to SEQ ID NO: 41.

[0117] According to a preferred embodiment, the phosphatase is selected from the group of phosphatases having an amino acid sequence according to 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.

[0118] 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.

[0119] According to a preferred embodiment, the composition comprises a transferase, preferably glycosyltransferase, more preferably glucanotransferase, and more preferably alpha-glucanotransferase; and / or

[0120] The composition further comprises a phosphorylase, preferably a glucanphosphorylase is added;

[0121] The composition comprises a mutase, preferably a phosphoglucomutase;

[0122] The composition comprises an isomerase, preferably a phosphoglucoisomerase;

[0123] The composition includes a hydrolytic enzyme, preferably a glucanohydrolytic enzyme, more preferably a pullulanase.

[0124] In another aspect, the invention relates to an aqueous composition comprising at least 50% fructose by dry weight; less than 25% glucose by dry weight, preferably less than 20% glucose; less than 22% phosphate by dry weight, preferably less than 20% phosphate; and less than 2% of at least four enzymes by dry weight, preferably less than 1% of at least four enzymes, wherein one enzyme is phosphatase.

[0125] In relation to a method for converting at least one oligo- and / or polysaccharide into fructose, all references, particularly regarding the enzyme and oligo- and / or polysaccharide mentioned so far, also apply to aqueous compositions where applicable.

[0126] According to a preferred embodiment, the phosphatase comprises at least 90%, preferably at least 95%, more preferably at least 97%, and more preferably at least 98.5% identical amino acids to the sequence according to SEQ ID NO: 13.

[0127] According to a preferred embodiment, the phosphatase is selected from the group of phosphatases having an amino acid sequence according to 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.

[0128] According to a preferred embodiment, the composition comprises a transferase, preferably glycosyltransferase, more preferably glucanotransferase, more preferably alpha-glucanotransferase, and / or; the composition further comprises a phosphorylase, preferably glucanphosphorylase, and / or; the composition comprises a mutase, preferably phosphoglucomutase, and / or; the composition comprises an isomerase, preferably phosphoglucoisomerase, and / or; and the composition comprises a hydrolase, preferably glucanohydrolase, more preferably pullulanase.

[0129] In another aspect, the invention relates to an aqueous composition comprising at least 50% fructose by dry weight; less than 25% glucose by dry weight, preferably less than 20% glucose; less than 22% phosphate by dry weight, preferably less than 20% phosphate; and less than 2% of at least four enzymes by dry weight, preferably less than 1% of at least four enzymes, wherein one enzyme is phosphatase, and the composition can be obtained by the method of the invention described above.

[0130] All references regarding the method of converting at least one oligo- and / or polysaccharide into fructose, in particular regarding the enzyme and oligo- and / or polysaccharide and the composition for converting at least one oligo- and / or polysaccharide into fructose mentioned so far, also apply to the aqueous composition obtainable by the method of the invention where applicable.

[0131] 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 more preferably at least 98.5% identical to the sequence according to SEQ ID NO: 13.

[0132] According to a preferred embodiment, the phosphatase is selected from the group of phosphatases having an amino acid sequence according to 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.

[0133] According to a preferred embodiment, the composition comprises a transferase, preferably glycosyltransferase, more preferably glucanotransferase, more preferably alpha-glucanotransferase, and / or; the composition further comprises a phosphorylase, preferably glucanphosphorylase, and / or; the composition comprises a mutase, preferably phosphoglucomutase, and / or; the composition comprises an isomerase, preferably phosphoglucoisomerase, and / or; and the composition comprises a hydrolase, preferably glucanohydrolase, more preferably pullulanase.

[0134] In another aspect, the invention relates to fructose that can be obtained by the method of the invention described above.

[0135] All references regarding the method of converting at least one oligo- and / or polysaccharide into fructose, in particular regarding the enzyme and oligo- and / or polysaccharide and the composition for converting at least one oligo- and / or polysaccharide into fructose mentioned so far, also apply to fructose obtainable by the method of the invention where applicable.

[0136] In a fifth aspect, the invention relates to a phosphatase comprising an amino acid sequence identical to the sequence according to SEQ ID NO: 41 by at least 98%, preferably at least 98.5%, more preferably at least 99%, and more preferably at least 99.5%.

[0137] All references to a method for converting at least one oligo- and / or polysaccharide into fructose, in particular to the enzyme and oligo- and / or polysaccharide and the composition for converting at least one oligo- and / or polysaccharide into fructose mentioned so far, also apply to phosphatase where applicable.

[0138] Preferably, phosphatase has an amino acid sequence according to 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.

[0139] Such phosphatases provide a higher yield of D-fructose. The phosphatases of the invention provide improved selectivity with respect to fructose-6-phosphate. Due to higher activity, it is possible to reduce the amount of enzyme applied. Therefore, these phosphatases are particularly suitable for industrial processes.

[0140] In another aspect, the invention relates to a phosphatase comprising an amino acid sequence identical to the sequence according to SEQ ID NO: 13 by at least 90%, preferably at least 95%, more preferably at least 97%, and more preferably at least 98.5%.

[0141] Preferably, phosphatase has an amino acid sequence according to 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.

[0142] In relation to the method of converting at least one oligo- and / or polysaccharide into fructose, all references, particularly in relation to the enzyme and oligo- and / or polysaccharide mentioned so far, also apply to phosphatase where applicable.

[0143] In a sixth aspect, the invention relates to the use of the phosphatase of the invention in the conversion of at least one oligo- and / or polysaccharide into fructose.

[0144] In relation to the method of converting at least one oligo- and / or polysaccharide into fructose, all references, particularly in relation to the enzyme and oligo- and / or polysaccharide mentioned so far, also apply to the use of the phosphatase of the invention where applicable. Brief explanation of the drawing

[0145] FIG. 1 is a schematic diagram of the process of the invention that starts with starch and consequently produces fructose; FIG. 2 illustrates an intermediate step of the process illustrated in FIG. 1; glucan phosphorylase (3) uses inorganic phosphate (G) to cleave the α-1,4 bond between the terminal glucose residue of the polymer (BA) and the rest, releasing glucose-1-phosphate (C); FIG. 3 illustrates an intermediate step of the process illustrated in FIG. 1; glucose-1-phosphate (C) is converted to glucose-6-phosphate (D) by the action of phosphoglucomutase (4); FIG. 4 illustrates an intermediate step of the process illustrated in FIG. 1; glucose-6-phosphate is converted to fructose-6-phosphate (E) using phosphoglucoisomerase (5); FIG. 5 illustrates an intermediate step of the process illustrated in FIG. 1; fructose-6-phosphate is cleaved into fructose (F) and phosphate (G) by the action of phosphatase (6); FIG. 6 illustrates an intermediate step of the process shown in FIG. 1; all α-1,6 bonds contained in starch (A - a mixture of amylose (B) and amylopectin (H)) are cleaved by the action of pullulanase; FIG. 7 illustrates an intermediate step of the process illustrated in FIG. 1; α-glucanotransferase catalyzes the transfer of a segment of 1,4-α-D-glucan (B) to a new position in an acceptor carbohydrate (B) to generate a new α-1,4 bond; Figure 8 is a diagram showing the progress of an optimized process using an improved phosphatase, namely P46T, and maltose (0.1 mM) for faster conversion of natural starch within 48 hours; Figure 9 is a graph showing the results of an experimental environment demonstrating the beneficial effects of short oligosaccharides on the breakdown of natural starch using pullulanase, α-glucanotransferase, and glucan phosphorylase, and the detection of the generated G1P; Figure 10 is a graph showing a comparison between wild-type phosphatase and nine variants (P46T, P46TE47Y, P46TE47YG50L, Y23HP46TE47Y, P46TE47YG50I, Y23HP46TE47YG50L, Y23HP46TE47YG50I, Y23SP46TE47YG50L, Y23SP46TE47YG50I) regarding improved catalytic performance in the context of three different sugar-phosphates (G6P and F6P) generated in the process; Figure 11 is a graph showing a comparison between wild-type phosphatase and six variants (HTYL, Y47T, Y47F, V45M, V45Q, and V45R) regarding improved catalytic performance in the context of three different sugar-phosphates (G6P and F6P) generated in the process; Figure 12 is a graph showing a comparison between six phosphatase variants (V45R, HTFL, HTTLV45R, HTFLV45R, HTFLV45M, and HTFLV45Q) regarding improved catalytic performance in the context of three different sugar-phosphates (G6P and F6P) generated in the process; Figure 13 is a graph showing a comparison between wild-type phosphatase and two variants (TY and HTYL) regarding improved catalytic performance in the context of the conversion of sucrose to fructose. Specific details for implementing the invention

[0146] The abbreviations and enzymes used in the drawings and / or tables are explained below:

[0147] 1. Fluranase

[0148] 2 α-glucanotransferase

[0149] 3. Glucan Phosphorylase

[0150] 4. Phosphoglucomutase

[0151] 5 Phosphoglucoisomerase

[0152] 6 Phosphatase

[0153] A starch

[0154] B. Amylose

[0155] BA amylose minus 1 glucose unit

[0156] C Glucose-1-phosphate (G1P)

[0157] D Glucose-6-phosphate (G6P)

[0158] E Fructose-6-phosphate (F6P)

[0159] F fructose

[0160] G phosphate

[0161] H amylopectin

[0162] WT wild type; Sequence number: 13;

[0163] Phosphatase variant with an amino acid exchange from proline to threonine at T position 46 (P46T); Sequence No.: 15;

[0164] Phosphatase variant with amino acid exchange from proline to threonine at TY position 46 and from glutamate to tyrosine at additional position 47 (P46TE47Y); SEQ ID: 17;

[0165] Phosphatase variant with amino acid exchange from proline to threonine at TYL position 46, from glutamate to tyrosine at position 47, and from glycine to leucine at position 50 (P46TE47YG50L); SEQ ID NO: 19;

[0166] Phosphatase variant with amino acid exchange 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: 23;

[0167] Phosphatase variant with amino acid exchange from proline to threonine at position 46, from glutamate to tyrosine at position 47, and from glycine to isoleucine at position 50 (P46TE47YG50I); SEQ ID NO: 21;

[0168] Phosphatase variant with amino acid exchange 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 (Y23HP46TE47YG50L); SEQ ID No.: 25;

[0169] Phosphatase variant with 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 (Y23HP46TE47YG50I); SEQ ID No.: 27;

[0170] Phosphatase variant with amino acid exchange 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 (Y23SP46TE47YG50L); SEQ ID No.: 29;

[0171] Phosphatase variant with 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 (Y23SP46TE47YG50I); SEQ ID No.: 31;

[0172] Phosphatase variant with amino acid exchange 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 (Y23HP46TE47TG50L); SEQ ID: 33;

[0173] Phosphatase variant with amino acid exchange 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 (Y23HP46TE47FG50L); SEQ ID NO: 35;

[0174] V45M Phosphatase variant with amino acid exchange 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 (Y23HV45MP46TE47YG50L); Sequence No.: 37;

[0175] V45Q Phosphatase variant with amino acid exchange 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 (Y23HV45QP46TE47YG50L); SEQ ID No.: 39;

[0176] Phosphatase variant with amino acid exchange 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 (Y23HV45RP46TE47YG50L); Sequence No.: 41;

[0177] Phosphatase variant with 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 (Y23HV45RP46TE47TG50L); SEQ ID No.: 43;

[0178] HTFLV45R Phosphatase variant with amino acid exchange 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 (Y23HV45RP46TE47FG50L); SEQ ID No.: 45;

[0179] HTFLV45M Phosphatase variant with amino acid exchange 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 (Y23HV45MP46TE47FG50L); SEQ ID No.: 47;

[0180] HTFLV45Q Phosphatase variant 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 (Y23HV45QP46TE47FG50L); SEQ ID: 49.

[0181] FIG. 1 illustrates a schematic diagram of the process of the invention starting with starch (A - a mixture of amylose and amylopectin) which is converted into amylose (B) by the use of pullulanase (1) and α-glucanotransferase (2); the next step involves catalyzing the α-1,4 bond between the terminal glucose residue of the polymer and the remainder using an inorganic phosphate (G) and a phosphorylase (glucan phosphorylase (3)) to cleave the α-1,4 bond, thereby releasing glucose-1-phosphate (C), which is converted into glucose-6-phosphate (D) by the action of phosphoglucomutase (4), glucose-6-phosphate is converted into fructose-6-phosphate (E) using a phosphoglucoisomerase (5), and in the final step, fructose-6-phosphate is converted into fructose (F) and by the action of phosphatase (6). It is converted into phosphate (G).

[0182] Figure 2 illustrates an intermediate process step of the general process shown in Figure 1. Glucan phosphorylase (3) and inorganic phosphate (G) are used to cleave the α-1,4 bond between the terminal glucose residue of the polymer (BA) and the rest, releasing glucose-1-phosphate (C).

[0183] FIGS. 3 to 7 illustrate additional intermediate steps of the process illustrated in FIG. 1.

[0184] Figures 8 to 13 provide diagrams of test data that will be discussed further in the following experiment section.

[0185] Although the invention has been described by reference to specific preferred embodiments, those skilled in the art will understand that various variations may be made and equivalents may be substituted for elements without departing from the scope of the invention. Furthermore, many modifications may be made to adapt specific situations or materials to the teachings of the invention without departing from the essential scope of the invention. Therefore, the invention is not limited to specific embodiments, but is intended to include all embodiments that fall within the scope of the appended claims.

[0186] Experiment Section

[0187] 1. Matter

[0188] All chemicals were analytical grade or higher, purchased from Sigma-Aldrich, Carbosynth, or VWR, and used without further purification.

[0189] Glusidex 12 and Glusidex 19 are maltodextrin compounds obtained from Roquette Freres (France).

[0190] The molecular mass of the glucose polymer can be calculated using the formula (180*n - 18*(n-1)), where n is the degree of polymerization (DP) of the glucose polymer. The dextrose equivalent (DE) can be calculated as 100*(180 / molecular mass (glucose polymer)). The 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.). Glusidex 12, with a DE of 11 to 14, is assumed to have an average DP of 8 to 10. Glusidex 19, with a DE of 18 to 20, is assumed to have an average DP of 5 to 6. However, since these preparations are induced by enzymatic treatment, smaller or larger maltodextrin is also included.

[0191] The following strains were used during this work: Escherichia coli XL1 Blue, Escherichia coli BL21(DE3), and all components were successfully expressed using auto-induction medium at 37°C.

[0192] 2. Method

[0193] Analysis

[0194] The analysis of the complete process illustrated in Fig. 1 was performed using an HPLC Dionex Ultimate 3000 system equipped with an autosampler (WPS 3000TRS), column compartment (TCC3000RS), and light scattering detector. At 7°C, a YMC Triart Diol Hilic column (100 * 2 mm, 1.9 μm, 12 nm) was used for separation by gradient elution (15-85%) into 0.1% formic acid in ACN as the mobile phase at pH 4.5 and 0.45 ml / min. Samples were diluted with water / acetonitrile in a 3:7 ratio. Data were analyzed using Dionex Chromeleon software.

[0195] The detection of total ketoses (fructose and fructose-6-phosphate) was performed using a assay based on triphenyltetrazolium chloride (TTC). The detection method is based on the differential reduction rates between aldoses and ketoses. 0.05 ml of the sample, plus 0.01 ml of 1% aqueous TTC, and plus 0.04 ml of 6 N NaOH were added to a 2 ml test tube. Exactly 5 minutes later, 1.5 ml of acetic acid:ethanol (1:9) was added, and the contents of the test tube were swirled. Water was used as a blank, and absorbance was measured at 480 nm using a spectrophotometer (Multiskan GO, Thermo Fisher). Glucose and glucose-6-phosphate reduce TTC to the red pigment triphenylformazan approximately 100 times more slowly than an equivalent amount of fructose.

[0196] Glucose is detected using a glucose oxidase-based assay. 50 μl of a sample or diluted sample is mixed with 50 μl of a master mixture (0.75 mM ABTS, 2 U / ml glucose oxidase, 0.1 U / ml peroxidase, 20 mM KPi pH 6.0) and incubated at 30°C for 30 minutes, then measured at 480 nm using a spectrophotometer (Multiskan GO, Thermo Fisher).

[0197] Protein expression

[0198] Optimized protein expression was described as an example for one enzyme, and performed for all proteins by the same process.

[0199] Escherichia coli BL21(DE3) containing the plasmid of interest (a pET28 derivative containing a single 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.). Pre-cultures were incubated overnight at 37°C in 4 ml of LB medium containing 100 μg / ml kanamycin on a rotary shaker (180 rpm). Expression cultures were inoculated with an overnight culture diluted 1:100. Incubated at 37°C for 24 hours. Cells were obtained by centrifugation and resuspended in 50 mM TRIS-HCl (pH 8.0). Unpurified extracts were prepared using a Basic-Z cell disruptor (IUL Constant Systems) or an ultrasonic device, followed by the addition of MgCl2 to a final concentration of 2.5 mM along with DNaseI (1 μg / ml) and subsequent DNA degradation by incubation at room temperature (25°C) for 20 minutes. All unpurified extracts were then heat-treated at 70°C for 30 minutes. The insoluble fraction of the lysate was removed by centrifugation (20,000 rpm at 4°C for 40 minutes). The supernatant was filtered through a 0.45 μm syringe filter and used as the purified enzyme preparation. 12% SDS-PAGE as described by Laemmli Ulrich K. was applied to subsamples of each purified product ("Cleavage of structural proteins during the assembly of the head of bacteriophage T4." nature 227.5259(1970): 680.).

[0200] The enzyme corresponds to the following sequences: sequence number: 1, sequence number: 3, sequence number: 5, sequence number: 7, sequence number: 9, sequence number: 11, sequence number: 13, sequence number: 15, sequence number: 17, sequence number: 19, sequence number: 21, sequence number: 23, sequence number: 25, sequence number: 27, sequence number: 29, sequence number: 31, sequence number: 33, sequence number: 35, sequence number: 37, sequence number: 39, sequence number: 41, sequence number: 43, sequence number: 45, sequence number: 47, sequence number: 49.

[0201] Enzyme activity test

[0202] Bacillus flavocaldarius Bacillus flavocaldarius The activity of pullulanase (Sequence No. 1) was tested using pullulan as a substrate and 3,5-dinitrosalicylic acid (DNS) for detection.

[0203] Pullulanase was incubated at 65°C with 1% (weight / volume) pullulan in 50 mM potassium phosphate buffer pH 7.0 and 5 mM MgCl2. At different time points, 24 μl of subsamples were transferred to 96 μl of DNS reagent (10 g 3,5-dinitrosalicylic acid, 300 g potassium sodium tartrate, and 16 g sodium hydroxide per liter). After heating at 95°C for 5 minutes, 100 μl was transferred to a flat-bottom microtiter plate and measured at 540 nm using a spectrophotometer (Multiskan GO, Thermo Fisher).

[0204] Myothermus Louver Meiothermus ruberThe α-glucanotransferase (SEQ No. 3) of Myothermus Ruber was tested for activity and ability to form larger oligosaccharides using maltotriose as a substrate. Detection of activity was performed using TLC (isopropanol:ethyl acetate:water (3:1:1)) and thymol spray reagent (0.5 g thymol, 95 ml ethanol, 5 ml sulfuric acid 97%). The α-glucanotransferase of Myothermus Ruber was incubated at 65°C with 50 mM maltotriose in 50 mM potassium phosphate buffer pH 7.0 and 5 mM MgCl2.

[0205] Thermomotor naphthophila ( Thermotoga naphthophila The α-glucan phosphorylase (SEQ No. 5) was tested for activity using an enzyme combination to detect the product α-D-glucose-1-phosphate. α-D-glucose-1-phosphate was converted to α-D-glucose-6-phosphate, which was subsequently oxidized to α-D-gluconate-6-phosphate by α-D-glucose-6-phosphate dehydrogenase. WST-1 was converted using 1-methoxy-5-methylphenazinium methyl sulfate (PMS) as an electron carrier with the generated NADH. The α-glucan phosphorylase was incubated at 65°C with 1% (weight / volume) soluble starch and 0.1 mM pyridoxal-5-phosphate in 50 mM potassium phosphate buffer pH 7.0 and 5 mM MgCl2. At different time points, 24 μl of subsamples were transferred to 190 μl of 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. After incubating the mixture for 30 minutes, measurements were taken at 440 nm using a spectrophotometer (Multiskan GO, Thermo Fisher).

[0206] Bifidobacterium aderenalis Bifidobacterium adolescentisThe activity of sucrose phosphorylase (SEQ No. 51) was tested using sucrose as a substrate and 3,5-dinitrosalicylic acid for detection. In the assay using 3,5-dinitrosalicylic acid for detection, only the product fructose generates a signal. 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 different time points, 24 μl of subsamples were transferred to 96 μl of DNS reagent (10 g 3,5-dinitrosalicylic acid, 300 g potassium sodium tartrate, and 16 g sodium hydroxide per liter). After heating at 95°C for 5 minutes, 100 μl was transferred to a flat-bottom micro-titer plate and measured at 540 nm using a spectrophotometer (Multiskan GO, Thermo Fisher).

[0207] Saccharobus sulfataricus ( Saccharolobus sulfataricus ) P2 (Sequence No.: 7) or Clostridium thermocellum ( Chlostridium thermocellum The activity of phosphoglucomutase (SEQ No. 9) was tested 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 product α-D-glucose-6-phosphate generates a signal. 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 different time points, 24 μl of subsamples were transferred to 96 μl of DNS reagent (10 g 3,5-dinitrosalicylic acid, 300 g potassium sodium tartrate, and 16 g sodium hydroxide per liter). After heating at 95°C for 5 minutes, 100 μl was transferred to a flat-bottom micro-titer plate and measured at 540 nm using a spectrophotometer (Multiskan GO, Thermo Fisher).

[0208] Thermomoto is Maritima ( Thermotoga maritima The activity of phosphoglucoisomerase (SEQ No. 11) was tested using fructose-6-phosphate as a substrate and α-D-glucose-6-phosphate dehydrogenase for detection. WST-1 was converted using 1-methoxy-5-methylphenazine methyl sulfate (PMS) as an electron carrier with the generated NADH. Phosphoglucoisomerase 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 different time points, 10 μl of subsamples were transferred to 190 μl of detection solution containing 0.1 U of α-D-glucose-6-phosphate dehydrogenase, 0.1 mM WST-1, and 0.005 mM PMS. After incubating the mixture for 30 minutes, it was measured at 440 nm using a spectrophotometer (Multiskan GO, Thermo Fisher).

[0209] The activity of phosphatase of Thermomotoga naphthophila (SEQ Nos: 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49) was tested using fructose-6-phosphate and phosphate assays as substrates. The phosphate assay consists of three solutions: Solution A (12% (weight / volume) 1-ascorbic acid in 1 N HCl solution), Solution B (2% (weight / volume) Na2MoO4 x 2H2O in ddH2O), and Solution F (2% (weight / volume) citric acid and 2% (weight / volume) acetic acid in ddH2O). Solution D is a 2:1 mixture of Solution A and Solution B and is prepared fresh prior to measurement. Pipette 75 μl of Solution D into the required wells of a flat-bottom microtiter plate. In the next step, add 25 μl of the sample or a standard of the specified concentration and incubate at room temperature for 5 minutes. As a final step, stop the reaction by adding 75 μl of Solution F and incubating at room temperature for an additional 15 minutes. Measure the sample at 655 nm using a spectrophotometer (Multiskan GO, Thermo Fisher).

[0210] All tested enzymes meet the requirements for catalyzing one of the steps in the process and are stable under process conditions (65°C, 50 mM potassium phosphate buffer pH 7.0, 5 mM MgCl2 0.005 mM PLP).

[0211] 3. Conversion of starch to fructose (Example 1 of the invention; IE1)

[0212] The conversion experiment was performed on a 15 ml scale using a 50 ml Falcon tube containing 7 g of natural starch. The natural starch was pretreated with 6 ml of water and buffer and heated to a maximum of 95°C for 30 minutes to allow for near-complete expansion. Subsequently, the expanded starch was cooled to 65°C and enzymes were added. 10.5 ml of buffer, salt, and enzymes were added to 7 g of natural starch. Additionally, maltose was added at 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-glucanphosphorylase, 1.4 mg phosphoglucomutase, 1.2 mg phosphoglucoisomerase, and 14 mg phosphatase. Enzymes (phosphorylase and phosphatase) from Bacillus flavocaldarius, Myothermus ruber, and Thermothoganaphthophila, and enzymes from Clostridium thermocellum and Thermothoganamatima were used. Starting with 0-hour incubation, samples were taken periodically and filtered using a 10 kDa spin filter (VWR 82031-350). If necessary, the sample was diluted with water / acetonitrile in a 3:7 ratio for subsequent analysis.

[0213] The result of the conversion of starch to fructose according to Example 1 (IE1) of the invention (35% dry matter starch corresponds to monosaccharides at a concentration of 2153.5 mM) is presented in Table 1 as well as in Figure 8.

[0214] Example 1 of the invention Time [hour] Fructose [mM] Dextrose [mM] Remainder [mM] 0 0 0 2153.5 2 194 16 1943.5 4 312 18 1823.5 6 349 22 1782.5 8 464 40 1649.5 24 1059 186 908.5 48 1511 317 325.5

[0215] From Table 1, it can be inferred that fructose was obtained with a yield of about 70% after 48 hours.

[0216] 4. Conversion of starch to fructose in the presence of maltose (IE2 to IE5), maltotriose (IE6 to IE9), maltotetraose (IE10 to IE12), maltodextrin glusidex 12 (IE13 to IE15) or maltodextrin glusidex 19 (IE16 to IE18)

[0217] Potassium phosphate buffer 50 mM pH 7.0, MgCl2 5 mM, PLP 0.005 mM in a total volume of 5 ml, expanded natural starch 1% (w / vol), maltose (0.01 mM, 0.1 mM, 1.0 mM or 10 mM; IE2 to IE5) or maltotriose (0.01 mM, 0.1 mM, 1.0 mM or 10 mM; IE6 to IE9) or maltotetrase (0.01 mM, 0.1 mM or 1.0 mM; IE10 to IE12) or maltodextrin glusidex 12 (0.0342% (w / vol), 0.00342% (w / vol), or 0.000342% (w / vol); IE13 to IE15) or maltodextrin glusidex 19 (0.0342% (weight / volume), 0.00342% (weight / volume), or 0.000342% (weight / volume); IE16 to IE18), glucan phosphorylase (0.0025 mg / ml), alpha-glucanotransferase (0.075 mg / ml), and pullulanase (0.02 mg / ml) were incubated at 65°C for 1 hour, and partial samples were removed to confirm glucose-1-phosphate. The glucose-1-phosphate assay is described in the activity test of the enzymes above.

[0218] Comparative Examples 1 to 5 (CE1 to CE5) were carried out by adding only 1 mM of maltose, maltotriose, maltotetraose, glusidex 12, or glusidex 19, respectively, without adding starch. Comparative Example 6 was carried out without adding additional sugars such as maltose, maltotriose, maltotetraose, glusidex 12, or glusidex 19.

[0219] The test results regarding the effect of additional sugars, such as short oligosaccharides like maltose, maltotriose, maltotetraose, Glusidex 12 or Glusidex 19, on the conversion rate to glucose-1-phosphate are presented in Table 2. Each diagram is illustrated in Fig. 9.

[0220] Examples (sugar residues) Absorbance at 440 nm (detection of G1P) At 0 [minutes] In 15 minutes In 30 minutes IE 2 (maltose 10 mM) 0.147 1.351 1.966 IE 3 (maltose 1 mM) 0.125 1.138 1.857 IE 4 (maltose 0.1 mM) 0.096 0.694 1.288 IE 5 (maltose 0.01 mM) 0.095 0.678 1.286 IE 6 (maltotriose 10 mM) 0.134 1.222 1.793 IE 7 (maltotriose 1 mM) 0.120 1.148 1.748 IE 8 (maltotriose 0.1 mM) 0.087 0.681 1.323 IE 9 (maltotriose 0.01 mM) 0.094 0.713 1.352 IE 10 (maltotetraose 1 mM) 0.101 0.832 1.369 IE 11 (maltotetraose 0.1 mM) 0.097 0.784 1.383 IE 12 (maltotetraose 0.01 mM) 0.097 0.629 1.184 IE 13(Glucidex 12 0.0342%(weight / volume)) 0.092 0.689 1.226 IE 14(Glucidex 12 0.00342%(weight / volume)) 0.093 0.652 1.042 IE 15(Glucidex 12 0.000342%(weight / volume)) 0.094 0.679 1.159 IE 16 (Glucidex 19 0.0342% (weight / volume)) 0.099 0.720 1.254 IE 17(Glucidex 19 0.00342%(weight / volume)) 0.093 0.595 1.106 IE 18(Glucidex 19 0.000342%(weight / volume)) 0.095 0.643 1.212 CE 1 (Maltose alone (1 mM)) 0.053 0.144 0.228 CE 2 (maltotriose alone (1 mM)) 0.068 0.364 0.569 CE 3 (maltotetraose alone (1 mM)) 0.079 0.446 0.728 CE 4 (Glucidex alone 12 0.0342% (weight / volume)) 0.063 0.237 0.413 CE 5 (Glucidex alone 19 0.0342% (weight / volume)) 0.065 0.255 0.418 CE 6 (natural starch) 0.092 0.500 0.905

[0221] The conversion of large polysaccharides such as starch or similar substrates such as cellulose within 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 release glucose-1-phosphate by catalyzing the cleavage of the α-1,4 bond using an inorganic phosphate between the terminal glucose residue of the polymer and the remainder. The combined action of pullulanase, α-glucanotransferase, and short oligosaccharides, such as maltose (IE2 to IE5), maltotriose (IE6 to IE9), maltotetraose (IE10 to IE12), maltodextrin glusidex 12 (IE13 to IE15) or maltodextrin glusidex 19 (IE16 to IE18) (see Table 2), shows that the conversion rate is increased by the addition of short oligosaccharides. This leads to higher glucose-1-phosphate production and thus allows for faster conversion of starch to fructose within the process of the invention.

[0222] The conversion rates for all Comparative Examples 1 to 6 are slower compared to Examples 2 to 18 of the invention.

[0223] 5. Phosphate Test

[0224] Phosphate assays were performed to monitor the different activities of variants for two sugar-phosphate intermediates generated in different phosphatases and processes.

[0225] 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, and subsamples were removed at regular intervals to test for released phosphate. The phosphate assay is described in more detail above.

[0226] The results of the phosphate assay are presented in Table 3 and Figure 10. The amounts of G6P and F6P were detected, and the F6P / G6P ratio was measured. The F6P / G6P ratio indicates the selectivity of the tested phosphatase.

[0227] Examples (phosphatase) Hours [minutes] Released phosphate G6P[nmol] F6P[nmol] F6P / G6P ratio CE7(WT) 10 12.165 194.647 16 20 20.276 347.584 17.143 30 25.490 476.191 18.682 IE19(T) 10 12.745 358.591 28.136 20 31.862 652.879 20.491 30 50.400 958.753 19.023 IE20(TY) 10 6.952 216.082 31.083 20 15.641 400.301 25.593 30 23.752 590.314 24.854 IE21(TYL) 10 5.793 363.226 62.700 20 11.296 547.735 48.487 30 15.062 717.182 47.615 IE22(TYI) 10 8.110 151.199 18.643 20 9.848 300.660 30.529 30 17.959 442.011 24.613 IE23(HTY) 10 15.641 387.557 24.778 20 31.862 729.348 22.891 30 48.082 1027.691 21.373 IE24(HTYL) 10 17.959 714.865 39.806 20 37.945 1357.896 35.786 30 62.565 1993.975 31.870 IE25(HTYI) 10 12.745 379.446 29.773 20 25.490 832.464 32.659 30 39.393 1256.517 31.897 IE26(STYL) 10 5.214 213.185 40.889 20 9.848 426.949 43.353 30 13.324 637.238 47.826 IE27(STYI) 10 7.531 155.834 20.692 20 6.372 313.405 49.182 30 13.324 448.963 33.696

[0228] As can be seen in Table 3 as well as Figure 10, the introduction of the Thermomotoga naphthophila phosphatase mutant P46T (IE19(T)) increases the overall activity of the enzyme and decreases the activity toward glucose-6-phosphate (G6P). This allows for better process economies in the context of reduced enzyme levels and better overall fructose production. P46TE47Y (IE20(TY)) has slightly improved activity toward F6P compared to the wild type (CE7(WT)) and nearly identical activity toward G6P, which reduces unwanted byproducts such as glucose. P46TE47YG50L (IE21(TYL)) has improved activity toward F6P and decreased activity toward G6P, which also reduces unwanted byproducts such as glucose, compared to the wild type (CE7(WT)). Y23HP46TE47Y(IE23(HTY)) also possesses improved activity toward F6P and slightly increased activity toward G6P compared to CE7(WT). However, regarding both activities, this variant also demonstrates improved performance for the process of the invention, allowing for an improved process that reduces unwanted byproducts such as glucose. P46TE47YG50I(IE22(TYI)) possesses improved selectivity toward F6P. Y23HP46TE47YG50L(IE24(HTYL)) possesses improved activity toward F6P and decreased activity toward G6P compared to CE7(WT) in the context of overall increased activity. Y23HP46TE47YG50I(IE25(HTYI)) possesses improved activity toward F6P and decreased activity toward G6P in the context of overall increased activity. Y23SP46TE47YG50L (IE26(STYL)) exhibits improved activity toward F6P and reduced activity toward G6P compared to CE7(WT) in the context of increased overall activity. Y23SP46TE47YG50I (IE27(STYI)) exhibits improved selectivity toward F6P.

[0229] The phosphatase of the invention (see Table 3 and Fig. 10) exhibits increased activity, increased selectivity, or both.

[0230] The results of additional phosphate assays testing additional phosphatases compared to wild-type phosphatase are presented in Table 4 and Figure 11. The time interval between measurements was shortened because the phosphatase used showed improved activity.

[0231] Examples (phosphatase) Hours [minutes] Released phosphate G6P[nmol] F6P[nmol] F6P / G6P ratio CE8(WT) 6 1.30 30.34 23.33 12 5.75 58.71 10.19 18 4.36 76.96 17.63 IE28(HTYL) 6 1.08 86.67 80.05 12 31.15 298.15 9.57 18 22.02 341.79 15.51 IE29(Y47T) 6 1.29 194.18 149.64 12 22.42 526.27 23.46 18 37.10 659.17 17.76 IE30(Y47F) 6 2.27 354.07 155.77 12 31.94 893.24 27.96 18 43.05 1129.69 26.23 IE31(V45M) 6 1.97 335.33 170.03 12 17.76 735.26 41.38 18 30.47 1064.51 34.92 IE32(V45Q) 6 1.97 267.81 135.80 12 23.72 602.21 25.38 18 40.80 881.02 21.59 IE33(V45R) 6 17.46 792.46 45.38 12 55.09 1415.99 25.69 18 83.29 1778.20 21.34

[0232] Y23HP46TE47YG50L(HTYL) has improved activity toward F6P and reduced activity toward G6P compared to the wild type in the context of increased overall activity. For this reason, the enzyme allows for an improved process that reduces unwanted byproducts such as glucose. The same applies to variants Y23HP46TE47TG50L(Y47T), Y23HP46TE47TG50L(Y47F), Y23HV45MP46TE47TG50L(V45M), Y23HV45QP46TE47TG50L(V45Q), and Y23HV45RP46TE47TG50L(V45R).

[0233] The results of another phosphate assay testing additional phosphatases are presented in Table 5 and Figure 12. Since the phosphatase used showed improved activity, the time interval between measurements was also shortened.

[0234] Examples (phosphatase) Hours [minutes] Released phosphate G6P[nmol] F6P[nmol] F6P / G6P ratio IE34(V45R) 6 19.71 666.26 33.79 12 95.16 1339.62 14.07 18 119.81 1928.61 16.09 IE35(HTFL) 6 6.58 324.47 49.27 12 21.98 648.58 29.49 18 36.96 985.68 26.66 IE36(HTTLV45R) 6 33.29 639.46 19.20 12 119.11 1278.51 10.73 18 161.27 1811.74 11.23 IE 37(HTFLV45R) 6 32.58 743.82 22.82 12 120.89 1441.85 11.92 18 190.58 2056.57 10.79 IE38(HTFLV45M) 6 10.78 483.30 44.82 12 28.63 945.95 33.03 18 49.90 1414.95 28.35 IE39(HTFLV45Q) 6 1.68 129.95 77.05 12 9.04 281.23 31.09 18 13.87 436.05 31.43

[0235] Variants Y23HV45RP46TE47TG50L(V45R), Y23HP46TE47FG50L(HTFL), Y23HV45RP46TE47TG50L(HTTLV45R), Y23HV45MP46TE47FG50L(V45M), and Y23HV45QP46TE47FG50L(V45Q) all have improved activity toward F6P and reduced activity toward G6P in the context of overall increased activity. For this reason, the enzyme allows for an improved process that reduces unwanted byproducts such as glucose.

[0236] 6. Conversion of sucrose to fructose using an improved phosphatase enzyme

[0237] A total volume of 10 ml of potassium phosphate buffer 50 mM pH 7.0, 25 mM MgCl, 1000 mM sucrose, sucrose phosphorylase (0.25 mg / ml), phosphoglucomutase (0.04 mg / ml), phosphoglucoisomerase (0.075 mg / ml), and phosphatase (0.18 mg / ml) was incubated at 50°C for 48 hours, and glucose and fructose concentrations were determined by removing partial samples. Enzymes from Bifidobacterium aderenalis (sucrose phosphorylase), Thermotoga nafthophila (phosphatase), Clostridium thermocellum (phosphoglucomutase), and Thermotoga maritima (phosphoglucoisomerase) were used. Fructose was measured as described in the methods.

[0238] Table 6 and Figure 13 present the results of the conversion of sucrose to fructose using three different phosphatase enzymes.

[0239] Time [hour] Fructose concentration [mM] CE9(WT) IE40(TY) IE41(HTYL) 0 88.325 88.325 85.077 1 119.436 126.615 160.462 2 157.385 169.692 241.145 3 230.034 262.855 344.393 20 984.103 1127.692 1484.957 48 1317.436 1628.547 1741.368

[0240] As can be seen from Table 6, the conversion rates of the phosphatase TY (IE40) and HTYL (IE41) of the invention are higher compared to wild-type phosphatase WT (CE9).

Claims

Claim 1 A method for converting at least one of an oligosaccharide and a polysaccharide into fructose, comprising: a) adding at least six enzymes to a composition comprising water, phosphate, and at least one of an oligosaccharide and a polysaccharide; and b) subsequently enzymatically converting at least one of an oligosaccharide and a polysaccharide into fructose in the presence of 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 decasaccharides, decasaccharides, dodisaccharides, decasaccharides, quinasaccharides, octasaccharides, heptasaccharides, hexasaccharides, pentasaccharides, tetrasaccharides, trisaccharides, disaccharides, and combinations thereof; the at least one additional sugar has a glycosidic bond at least partially identical to that of at least one of an oligosaccharide and a polysaccharide; and in step a), the at least six enzymes are phosphatase, transferase, phosphorylase, A method selected from the group consisting of mutases, isomerases, and hydrolases; wherein at least one enzyme in step a) is a phosphatase having an amino acid sequence that is at least 98% identical to the sequence according to SEQ ID NO: 41; and at least one of the oligosaccharides and polysaccharides is selected from at least one of glucose-based oligosaccharides and glucose-based polysaccharides. Claim 2 A method according to claim 1, wherein at least one additional sugar in step a) is selected from the group consisting of tetrasaccharides, trisaccharides, disaccharides, and combinations thereof; or in step a) the composition comprises at least one of oligosaccharides and polysaccharides in an amount of 35% or less by dry weight; or at least one of oligosaccharides and polysaccharides is selected from the group consisting of starch and its derivatives, hemicellulose and its derivatives, cellulose and its derivatives, and combinations thereof. Claim 3 A method according to claim 1, wherein in step b), the sugar phosphate is generated in the middle; or in step b), the enzymatic conversion is a one-pot reaction; or the phosphatase comprises an amino acid sequence that is at least 98.5% identical to the sequence according to SEQ ID NO: 41; or the phosphatase has an amino acid sequence according to 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. Claim 4 A method according to claim 1, wherein at least one transferase is added in step a); or at least one phosphorylase is added in step a); or at least one mutase is added in step a); or at least one isomerase is added in step a); or at least one hydrolase is added in step a). Claim 5 A method according to claim 1, wherein step b) is performed at room temperature (25℃) for at least 24 hours. Claim 6 A method according to claim 1, wherein at least 50% of the sugars present in the composition of step a) are converted into fructose after 24 hours of enzymatic conversion; or at least 70% of the sugars present in the composition of step a) are converted into fructose after 48 hours of enzymatic conversion. Claim 7 A method according to claim 1, wherein in step b), the temperature is 10 to 100°C; or the pH of the composition is 3 to 12. Claim 8 A composition for converting at least one of an oligosaccharide and a polysaccharide into fructose, comprising: - water; - phosphate; - at least six enzymes, wherein at least one enzyme is a phosphatase having an amino acid sequence at least 98% identical to the sequence according to SEQ ID NO: 41; - at least one of an oligosaccharide and a polysaccharide; wherein at least six enzymes are selected from the group consisting of phosphatase, transferase, phosphorylase, mutase, isomerase, and hydrolase; the composition further comprises at least one additional sugar, wherein at least one additional sugar is selected from the group consisting of decasaccharides, decasaccharides, dodisaccharides, decasaccharides, quinasaccharides, octasaccharides, heptasaccharides, hexasaccharides, pentasaccharides, tetrasaccharides, trisaccharides, disaccharides, and combinations thereof; and at least one additional sugar has a glycosidic bond at least partially identical to that of at least one of an oligosaccharide and a polysaccharide; A composition in which at least one of the oligosaccharides and polysaccharides is selected from at least one of glucose-based oligosaccharides and glucose-based polysaccharides. Claim 9 A composition according to claim 8, wherein the phosphatase comprises an amino acid sequence that is at least 98.5% identical to the sequence according to SEQ ID NO: 41; or the phosphatase has an amino acid sequence according to 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. Claim 10 An aqueous composition comprising: - at least 50% fructose based on dry weight; - 0.001 to 25% glucose based on dry weight; - 0.01 to 22% phosphate based on dry weight; and - at least 6 enzymes based on dry weight in an amount of 0.001 to 2%, wherein the at least 6 enzymes are selected from the group consisting of phosphatase, transferase, phosphorylase, mutase, isomerase, and hydrolase; at least one enzyme is phosphatase; and the phosphatase comprises an amino acid sequence that is at least 98% identical to the sequence according to SEQ ID NO:

41. Claim 11 An aqueous composition according to claim 10, wherein the phosphatase comprises an amino acid sequence that is at least 98.5% identical to the sequence according to SEQ ID NO: 41; or the phosphatase has an amino acid sequence according to 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. Claim 12 An aqueous composition according to claim 10, which can be obtained by a method according to any one of claims 1 to 7. Claim 13 Phosphatase comprising an amino acid sequence that is at least 98% identical to the sequence according to SEQ ID NO:

41. Claim 14 In claim 13, a phosphatase having an amino acid sequence according to 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. Claim 15 delete

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