Enzymatic production of tagatose

Abstract

To provide improved processes for preparing D-tagatose, capable of enzymatically converting saccharides into D-tagatose.SOLUTION: Disclosed herein are an improved process for the production of tagatose, including the step for: converting fructose-6-phosphate (F6P) to tagatose 6-phosphate (T6P), catalyzed by fructose 6-phosphate epimerase (F6PE); and converting the T6P to tagatose, catalyzed by tagatose-6-phosphate phosphatase (T6PP), using enzymes with higher activities compared to F6PE and T6PP previously used in the process to produce tagatose.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Application No. 62 / 747,877, filed Oct. 19, 2018, and U.S. Application No. 62 / 790,788, filed Jan. 10, 2019, each of which is incorporated herein by reference.

[0002] Field of the Invention The present invention relates to the field of biotechnology related to the production of D - tagatose. More specifically, the present invention provides an improved method for preparing D - tagatose that can enzymatically convert sugars (e.g., polysaccharides, oligosaccharides, disaccharides, sucrose, D - glucose, and D - fructose) to D - tagatose.

Background Art

[0003] D - tagatose (hereinafter referred to as tagatose) is a low - calorie natural sweetener that has 92% of the sweetness of sucrose but only 38% of the calories. Due to its high selling price, its use as a sweetener is limited. Tagatose boasts numerous health benefits: it is non - cariogenic; low - calorie; has a very low glycemic index of 3; attenuates the glycemic index of glucose by 20%; can lower the average blood glucose level; helps prevent cardiovascular diseases, strokes, and other vascular diseases by promoting high - density lipoprotein (HDL) cholesterol; and is a proven prebiotic and an antioxidant. Lu et al., Tagatose, a New Antidiabetic and Obesity Control Drug, Diabetes Obes. Metab. 10(2): 109 - 34 (2008). Therefore, tagatose clearly has various uses in various industries such as consumables, pharmaceuticals, biotechnology, academia, food, beverages, nutritional supplements, and the food industry.

[0004] Tagatose is primarily produced by the hydrolysis of lactose by lactase, forming D-glucose and D-galactose (see WO2011 / 150556, CN103025894, US5,002,612, US6,057,135, and US8,802,843). D-galactose is then isomerized to D-tagatose chemically by calcium hydroxide under alkaline conditions, or enzymatically by L-arabinose isomerase under neutral pH conditions. The final product is separated by a combination of filtration and ion-exchange chromatography. This method is problematic due to the cost of separating D-glucose and D-galactose and the low yield of the product. Several methods using microbial cell fermentation have been developed, but none have proven to be a practical alternative due to their reliance on expensive raw materials (such as galactitol and D-psicose), low product yields, and costly separation. Other processes for preparing tagatose have also been reported. See, for example, Lee et al., Scientific Reports | 7: 1934 | DOI:10.1038 / s41598-017-02211-3, pp. 1-8; U.S. Patent Publication No. 2018 / 0023073; International Patent Application Publication Nos. WO2014 / 196811, WO2018 / 004310, WO2018 / 021894, WO2018 / 182344, WO2018 / 182345, WO2018 / 182354, WO2018 / 182355, and WO2016 / 064146. [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] International Patent Application Publication No. WO2017 / 059278 recently described the enzymatic synthesis of tagatose in a process comprising the steps of converting fructose 6-phosphate (F6P) to tagatose 6-phosphate (T6P) catalyzed by an epimerase, fructose 6-phosphate epimerase, and converting T6P to tagatose catalyzed by a phosphatase, tagatose 6-phosphate phosphatase. However, despite improvements in enzymatic tagatose production, there remains a desire and need to provide a further improved process for producing tagatose that can provide higher yields with smaller amounts of enzymes, for example. There is a strong industrial and commercial interest in reducing the cost of tagatose production, and this reduction includes reducing the amount of enzymes used and using enzyme combinations that are more effective than those previously used. [Means for solving the problem]

[0006] Summary of the Invention The present invention provides an improved method for preparing tagatose, which can enzymatically convert sugars (e.g., polysaccharides, oligosaccharides, disaccharides, sucrose, D-glucose, and D-fructose) into tagatose. In one embodiment, the improved process of the present invention for producing tagatose from sugar comprises the step of converting fructose-6-phosphate (F6P) to tagatose-6-phosphate (T6P) using fructose-6-phosphate epimerase (F6PE), wherein F6PE comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 7. In another embodiment, the improved process of the present invention for producing tagatose from sugar comprises the step of converting T6P to tagatose using tagatose-6-phosphate phosphatase (T6PP), wherein T6PP comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. In some embodiments of the present invention, the improved process includes the step of converting fructose-6-phosphate (F6P) to tagatose-6-phosphate (T6P) using fructose-6-phosphate epimerase (F6PE), wherein F6PE comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 7, and the step of converting T6P to tagatose using tagatose-6-phosphate phosphatase (T6PP), wherein T6PP comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.

[0007] In some improved processes of the present invention, the process for preparing tagatose also includes a step of converting glucose 6-phosphate (G6P) to F6P, which is catalyzed by phosphoglucose isomerase (PGI). Some processes according to the present invention further include a step of converting glucose 1-phosphate (G1P) to G6P, which is catalyzed by phosphoglucumutase (PGM). Some processes according to the present invention further include a step of converting a sugar to G1P, which is catalyzed by at least one enzyme.

[0008] The sugar used in any process can be selected from the group consisting of starch or its derivatives, cellulose or its derivatives, and sucrose. Starch or its derivatives may be amylose, amylopectin, soluble starch, amylodextrin, maltodextrin, maltose, maltotriose, or glucose. In some improved processes of the present invention, the process for preparing tagatose involves converting starch to a starch derivative by enzymatic hydrolysis or acid hydrolysis of starch. In other processes, the starch derivative is prepared by enzymatic hydrolysis of starch catalyzed by isoamylase, pullulanase, α-amylase, or a combination of two or more of these enzymes. Some processes of the present invention may further include the addition of 4-glucantransferase (4GT).

[0009] Other processes of the present invention for preparing tagatose further include the step of converting fructose to F6P, catalyzed by at least one enzyme. Other processes of the present invention further include the step of converting sucrose to fructose, catalyzed by at least one enzyme. G6P used in some processes for preparing tagatose can also be produced by converting glucose to G6P, catalyzed by at least one enzyme. Glucose can then be produced by converting sucrose to glucose, catalyzed by at least one enzyme.

[0010] The method of the present invention is carried out under reaction conditions including a temperature in the range of about 37°C to about 85°C, a pH in the range of about 5.0 to about 9.0, and / or a duration of about 1 hour to about 48 hours, or as a continuous reaction. In some embodiments, the steps of the process for preparing tagatose are carried out in a single bioreactor under those reaction conditions. In other embodiments, these steps are carried out in multiple bioreactors arranged in series under those reaction conditions.

[0011] In some processes of the present invention, the steps for preparing tagatose are carried out in an ATP-free, NAD(H)-free manner, at a phosphate concentration of about 0.1 mM to about 150 mM, the phosphate is recycled, and / or the steps for converting T6P to tagatose involve energetically favorable chemical reactions. [Brief explanation of the drawing]

[0012] [Figure 1] Figure 1 is a schematic diagram showing the enzymatic pathway that converts starch or its derivatives to tagatose. The following abbreviations are used: αGP, α-glucan phosphorylase or starch phosphorylase; PGM, phosphoglucomutase; PGI, phosphoglucoisomerase; F6PE, fructose 6-phosphate epimerase; T6PP, tagatose 6-phosphate phosphatase; IA, isoamylase; PA, pullulanase; MP, maltose phosphorylase; PPGK, polyphosphate glucokinase.

[0013] [Figure 2] Figure 2 shows the enzymatic pathways that convert cellulose or its derivatives to tagatose. CDP, cellodextrin phosphorylase; CBP, cellobiose phosphorylase; PPGK, polyphosphate glucokinase; PGM, phosphoglucomutase; PGI, phosphoglucoisomerase; F6PE, fructose 6-phosphate epimerase; T6PP, tagatose 6-phosphate phosphatase.

[0014] [Figure 3] Figure 3 is a schematic diagram showing the enzymatic pathway that converts fructose to tagatose. PPFK, polyphosphate fructokinase; F6PE, fructose 6-phosphate epimerase; T6PP, tagatose 6-phosphate phosphatase.

[0015] [Figure 4]Figure 4 is a schematic diagram showing an enzymatic pathway for converting glucose to tagatose. PPGK, polyphosphate glucokinase; PGI, phosphoglucose isomerase; F6PE, fructose 6-phosphate epimerase; T6PP, tagatose 6-phosphate phosphatase.

[0016] [Figure 5] Figure 5 shows an enzymatic pathway for converting sucrose or its derivative products to tagatose. SP, sucrose phosphorylase; PPFK, polyphosphate fructokinase; PGM, phosphoglucocomutase; PGI, phosphoglucose isomerase; F6PE, fructose 6-phosphate epimerase; T6PP, tagatose 6-phosphate phosphatase.

[0017] [Figure 6] Figure 6 shows the reaction Gibbs energy between intermediates based on the formation Gibbs energy for converting glucose 1-phosphate to tagatose.

[0018] [Figure 7] Figure 7 shows the conversion of F6P to tagatose described in Example 3 in an HPLC chromatogram. (Solid line) 0-hour chromatogram; (dashed line) 2 hours with F6PE (Uniprot ID B5YBD7) and T6PP (Uniprot ID O29805); and (dotted line) 2 hours with an improved process using F6PE (Uniprot ID A0A0P6XN50) and T6PP (Uniprot ID D6YBK5). The peaks shown are (1) void, (2) F6P and T6P, (3) free phosphate ion (free phosphate), (4) tagatose, and (5) fructose.

Mode for Carrying Out the Invention

[0019] Detailed Description The present invention generally relates to an improved enzymatic process for converting sugars to tagatose. For example, the present invention relates to an improved process for converting sugars and their derived products, such as starch, cellulose, sucrose, glucose, and fructose, to tagatose using a cell-free enzyme cocktail. In contrast to cell-based production methods, the present invention involves a cell-free preparation of tagatose, which has a relatively high reaction rate due to the removal of cell membranes, which often slows the transport of substrates / products into and out of cells. The process of the present invention also results in a final product that does not contain nutrient-rich fermentation media / cell metabolites.

[0020] In one embodiment, the present invention relates to an improved process for producing tagatose, comprising the steps of: converting F6P to T6P catalyzed by F6PE; and converting T6P to tagatose catalyzed by T6PP using an enzyme having improved activity compared to F6PE and / or T6PP previously used in processes for producing tagatose. For example, F6PE and T6PP species: F6PE from Anaerolinea thermophila UNI-1 (Uniprot ID E8N0N6); F6PE from Caldicellulosiruptor kronotskyensis (Uniprot ID E4SEH3); F6PE from Caldilinea aerophila (Uniprot ID I0I507); F6PE from Caldihrix abyssi (Uniprot ID H1XRG1); and F6PE from Dictyoglomus thermophilum (Uniprot ID B5YBD7); T6PP from Archaeoglomus fulgidus (Uniprot ID O29805); T6PP from Archaeoglomus profundus (Uniprot ID D2RHV2_ARCPA); T6PP from Archaeoglomus veneficus (Uniprot ID See International Patent Application Publication WO2017 / 059278 disclosing F2KMK2_ARCVS). Using enzymes with higher activity allows for the use of smaller amounts of enzyme, thus reducing the overall process cost.

[0021] In the improved process of the present invention, F6PE has higher activity compared to the previously disclosed thermophilic F6PE (Uniprot ID B5YBD7) from Dictyoglomus thermophilum. See International Patent Application Publication WO2017 / 059278. Preferably, the F6PE used in the process of the present invention has enzyme activity improved by at least 10%, at least 30%, at least 80%, at least 100%, at least 150%, at least 180%, or at least 200% compared to the thermophilic F6PE (Uniprot ID B5YBD7) from Dictyoglomus thermophilum. For example, as shown in Example 1, the thermophilic F6PE from Thermonaerothrix daxensis (Uniprot A0A0P6XN50) showed approximately 150% higher enzyme activity compared to the thermophilic F6PE from Dictyoglomus thermophilum (Uniprot ID:B5YBD7), the thermophilic F6PE from Candidatus Thermofonsia Clade 3 (Uniprot ID:A0A2M8QBR9) showed approximately 120% higher enzyme activity compared to the thermophilic F6PE from Dictyoglomus thermophilum (Uniprot ID:B5YBD7), and the thermophilic F6PE from Thermoanaerobacterium thermosaccharolyticum (Uniprot A0A223HVJ3) showed approximately 178% higher enzyme activity compared to the thermophilic F6PE from Dictyoglomus thermophilum (Uniprot ID:B5YBD7). The following example provides a protocol for determining the activity of F6PE, comprising incubating the enzyme with its substrate and then measuring the amounts of reactants and products via HPLC. The measurement of the relative activity of any two enzymes is performed under identical reaction conditions, such as buffer, pH, and temperature.

[0022] The F6PE used in the process of the present invention is specific to F6P and T6P. The epimerization catalyzed by F6PE is a reversible reaction. Specific means that it has higher activity for F6P / T6P than other phosphorylated monosaccharides present in the reaction. For example, F6PE exhibits higher epimerization activity for F6P / T6P than, for example, G6P.

[0023] Examples of F6PEs for use in the improved processes of the present invention include, but are not limited to, the following proteins: thermophilic F6PE from Thermonaerothrix daxensis having the amino acid sequence described in SEQ ID NO: 1 (Uniprot ID A0A0P6XN50); thermophilic F6PE from Thermoanaerobacterium thermosaccharolyticum having the amino acid sequence described in SEQ ID NO: 2 (Uniprot ID A0A223HVJ3); thermophilic F6PE from Candidatus Thermofonsia Clade 3 having the amino acid sequence described in SEQ ID NO: 7 (Uniprot ID A0A2M8QBR9); and F6PEs having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 7. An improved process for producing tagatose from sugar according to the present invention comprises the step of converting fructose-6-phosphate (F6P) using F6PE to tagatose-6-phosphate (T6P), wherein F6PE comprises an amino acid sequence having at least 90% amino acid sequence identity with SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 7.

[0024] The F6PE used in the process of the present invention is an epimerase capable of converting F6P to T6P. F6PE utilizes a divalent metal cofactor such as magnesium, manganese, cobalt, or zinc, preferably magnesium. The F6PE for use in the process of the present invention comprises an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 7, and preferably comprises an aldolase-type TIM barrel. See Wichelecki et al., (2015) J. Biol. Chem., v290, pp. 28963-76.

[0025] In the improved process of the present invention, T6PP has higher activity compared to the previously disclosed T6PP from Archaeoglobus fulgidus (Uniprot ID O29805). See International Patent Application Publication WO2017 / 059278. Preferably, the T6PP used in the process of the present invention has enzyme activity that is at least 10%, at least 30%, at least 80%, at least 100%, at least 150%, at least 300%, at least 500%, at least 600%, at least 900%, at least 1200%, at least 1500%, at least 1800%, or at least 2100% improved compared to the activity of T6PP from Archaeoglobus fulgidus (Uniprot ID O29805). For example, as shown in Example 2, T6PP from Methanosarcina thermophila CHTI-55 (Uniprot ID A0A0E3NCH4) showed approximately 614% higher enzyme activity compared to T6PP from Archaeoglobus fulgidus (Uniprot ID O29805), T6PP from Thermobispora bispora strain ATCC19993 (Uniprot D6YBK5) showed approximately 1328% higher enzyme activity compared to T6PP from Archaeoglobus fulgidus (Uniprot ID O29805), T6PP from Spirochaeta thermophila ATCC 49972 (Uniprot ID E0RT70) showed approximately 2075% higher enzyme activity compared to T6PP from Archaeoglobus fulgidus (Uniprot ID DO29805), and Sphaerobacter thermophilus DSM T6PP from 20745 (Uniprot ID D1C7G9) exhibits approximately 814% higher enzyme activity compared to T6PP from Archaeoglobus fulgidus (Uniprot ID O29805). The following example provides to those skilled in the art a protocol for determining the activity of T6PP, which includes incubating the enzyme with its substrate and then measuring the amounts of reactants and products via HPLC.The relative activity of any two enzymes is measured under identical reaction conditions, including buffer, pH, and temperature.

[0026] The T6PP used in the process of the present invention is specific to T6P. In the case of T6PP, specific means that it has higher dephosphorylation activity towards T6P than other phosphorylated monosaccharides in the process. For example, T6PP has higher dephosphorylation activity towards T6P than, for example, G1P, G6P, and F6P.

[0027] Examples of T6PPs for use in the process of the present invention include, but are not limited to, the following proteins: T6PP from Methanosarcina thermofila CHTI-55 having the amino acid sequence described in SEQ ID NO: 3 (Uniprot ID A0A0E3NCH4); T6PP from Thermobispora bispora strain ATCC19993 having the amino acid sequence described in SEQ ID NO: 4 (Uniprot ID D6YBK5); T6PP from Spirochaeta thermophila strain ATCC49972 having the amino acid sequence described in SEQ ID NO: 5 (Uniprot ID E0RT70); T6PP from Sphaerobacter thermophilus strain DSM20745 having the amino acid sequence described in SEQ ID NO: 6 (Uniprot ID D1C7G9); and T6PPs having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. The present invention for producing tagatose from sugar comprises converting fructose-6-phosphate (F6P) to tagatose-6-phosphate (T6P) using fructose-6-phosphate epimerase (F6PE), and converting the resulting T6P to tagatose using tagatose-6-phosphate phosphatase (T6PP), wherein T6PP comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.

[0028] In the process of the present invention, T6PP is a phosphatase that converts T6P to tagatose. T6PP utilizes a divalent metal cofactor such as zinc, manganese, cobalt, or magnesium, preferably magnesium. In the process of the present invention, T6PP has at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6; and preferably a rosmanoid fold domain for catalytic activity, and further, a C1 capping domain for substrate specificity; a DxD signature on the first β-strand of the rosmanoid fold for coordinating magnesium, where the second Asp is a general acid / base catalyst; Thr or Ser at the end of the second β-strand of the rosmanoid fold to aid in the stability of the reaction intermediate; Lys at the N-terminus of the α-helix C-terminus to the third β-strand of the rosmanoid fold to aid in the stability of the reaction intermediate; and an E(D / N) signature at the end of the fourth β-strand of the rosmanoid fold for coordinating divalent metal cations such as magnesium. For example, Burroughs et al. See Evolutionary Genomics of the HAD Superfamily: Understanding the Structural Adaptations and Catalytic Diversity in a Superfamily of Phosphoesterases and Allied Enzymes J. Mol. Biol, 2006; 361; 1003-1034.

[0029] A preferred enzyme process according to the present invention includes the step of converting fructose-6-phosphate (F6P) to tagatose-6-phosphate (T6P) using fructose-6-phosphate epimerase (F6PE), wherein F6PE comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 7, and the step of converting T6P to tagatose using tagatose-6-phosphate phosphatase (T6PP), wherein T6PP comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. More preferably, F6PE comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2, and T6PP comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 5. Most preferably, F6PE has the amino acid sequence described in SEQ ID NO: 2, and T6PP has the amino acid sequence described in SEQ ID NO: 5.

[0030] The process for preparing tagatose from sugars according to the present invention also includes the step of enzymatically converting glucose 6-phosphate (G6P) to F6P, which is catalyzed by phosphoglucose isomerase (PGI). Exemplary PGIs that can be used include those disclosed in International Patent Application Publication WO2017 / 059278: PGI from Clostridium thermocellum (Uniprot ID A3DBX9) and PGI from Thermus thermophilus (Uniprot ID Q5SLL6).

[0031] The process for preparing tagatose according to the present invention further comprises the step of converting glucose 1-phosphate (G1P) to G6P, which is catalyzed by phosphoglucomutase (PGM). An example of PGM is PGM from Thermococcus kodakaraensis (Uniprot ID Q68BJ6), disclosed in International Patent Application Publication WO2017 / 059278.

[0032] Furthermore, the process according to the present invention may further include a step of converting a sugar to G1P, which is catalyzed by at least one enzyme, and the sugar is selected from the group consisting of starch or its derivatives (Figure 1), cellulose or its derivatives (Figure 2), fructose (Figure 3), glucose (Figure 4), and sucrose (Figure 5). The enzyme(s) used in the step of converting the sugar to G1P in the process according to the present invention may be alpha-glucan phosphorylase (αGP), maltose phosphorylase, sucrose phosphorylase, cellodextrin phosphorylase, cellobiose phosphorylase, and / or cellulose phosphorylase and mixtures thereof. The selection of enzymes or combinations of enzymes to reach F6P depends on the sugar used in the process.

[0033] Cellulose is the most abundant biological resource and the main component of plant cell walls. Non-food lignocellulosic biomass contains cellulose, hemicellulose, lignin, and other trace components. Pure cellulose, including Avicel (microcrystalline cellulose), recycled amorphous cellulose, bacterial cellulose, and filter paper, can be prepared through a series of processes. Partially hydrolyzed cellulose substrates include water-insoluble cellodextrins with a degree of polymerization greater than 7, water-soluble cellodextrins with a degree of polymerization of 3-6, cellobiose, glucose, and fructose.

[0034] In a specific process according to the present invention, cellulose and its derivative products can be converted to tagatose through a series of steps. See Figure 2. For example, the process according to the present invention provides a pathway comprising the following steps: generating G1P from cellodextrin and cellobiose catalyzed by cellodextrin phosphorylase (CDP) and cellobiose phosphorylase (CBP), respectively, and free phosphate ions; converting G1P to G6P catalyzed by PGM; converting G6P to F6P catalyzed by PGI; converting F6P to tagatose as described above; the phosphate ions can be recycled in the step of converting cellodextrin and cellobiose to G1P.

[0035] Solid cellulose can be hydrolyzed into water-soluble cellodextrin and cellobiose using several enzymes. Such enzymes include endoglucanase and cellobiohydrolase, but not beta-glucosidase (cellobiase). Before cellulose hydrolysis and G1P production, cellulose and biomass can be pretreated to increase their reactivity and reduce the degree of polymerization of the cellulose chains. Pretreatment methods for cellulose and biomass include dilute acid pretreatment, cellulose solvent-based lignocellulose fractionation, ammonia fiber swelling, ammonia aqueous immersion, and ionic liquid treatment, and partial hydrolysis using concentrated acids including hydrochloric acid, sulfuric acid, phosphoric acid, and combinations thereof.

[0036] If the sugar contains cellobiose and the enzyme contains cellobiose phosphorylase, G1P is produced from cellobiose by cellobiose phosphorylase. If the sugar contains cellodextrin and the enzyme contains cellodextrin phosphorylase, G1P is produced from cellodextrin by cellodextrin phosphorylase. If the sugar contains cellulose and the enzyme contains cellulose phosphorylase, G1P is produced from cellulose by cellulose phosphorylase.

[0037] If the sugar contains maltose and the enzyme contains maltose phosphorylase, G1P is produced from maltose by maltose phosphorylase. If the sugar contains sucrose and the enzyme contains sucrose phosphorylase, G1P is produced from sucrose by sucrose phosphorylase.

[0038] When the sugar is starch or a starch derivative, the derivative may be selected from the group consisting of amylose, amylopectin, soluble starch, amylodextrin, maltodextrin, maltose, maltotriose, and glucose, and mixtures thereof. In a particular process of the present invention, the enzyme used to convert the sugar to G1P includes αGP. In this step, if the sugar is starch, G1P is produced from starch by αGP. If the sugar is soluble starch, amylodextrin, or maltodextrin, G1P is produced from soluble starch, amylodextrin, or maltodextrin by αGP. An example of αGP is αGP from Thermotoga maritima (Uniprot ID G4FEH8), disclosed in International Patent Application Publication WO2017 / 059278.

[0039] Some processes according to the present invention may further include a step of converting starch to a starch derivative, where the starch derivative is prepared by enzymatic hydrolysis or acid hydrolysis of starch. In certain processes of the present invention, the yield of tagatose can be increased by using maltose phosphorylase (MP) to phosphorylated cleave the degradation product maltose into G1P and glucose. Alternatively, the yield of tagatose can be increased by using 4-glucantransferase (4GT) to recycle the degradation products glucose, maltose, and maltotriose into longer maltooligosaccharides, which can be phosphorylated cleaved by αGP to produce G1P. An example of 4GT is 4GT from Thermococcus litoralis (Uniprot ID O32462), disclosed in International Patent Application Publication WO2017 / 059278. In some processes of the present invention, polyphosphate and polyphosphate glucokinase (PPGK) can be added to the process, and thus the yield of tagatose can be increased by phosphorylating the degradation product glucose to G6P.

[0040] Starch is the most widely used energy storage compound in nature, mostly stored in plant seeds. Natural starches include linear amylose and branched amylopectin. Examples of starch derivatives include amylose, amylopectin, soluble starch, amylodextrin, maltodextrin, maltose, fructose, and glucose. Examples of cellulose derivatives include pre-treated biomass, regenerated amorphous cellulose, cellodextrin, cellobiose, fructose, and glucose. Sucrose derivatives include fructose and glucose.

[0041] When the process uses starch derivatives, these can be prepared by enzymatic hydrolysis of starch catalyzed by isoamylase, pullulanase, α-amylase, or a combination thereof. Corn starch contains many branches that hinder the action of αGP. Starch can be debranched using isoamylase to produce linear amylodextrin. Starch pretreated with isoamylase may result in a higher F6P concentration in the final product. Isoamylase and pullulanase cleave α-1,6-glycosidic bonds, allowing for more complete degradation of starch by α-glucan phosphorylase. α-amylase cleaves α-1,4-glycosidic bonds, so α-amylase can be used to break down starch into fragments and rapidly convert them to tagatose.

[0042] Tagatose can also be produced from fructose. See Figure 3. The process according to the present invention may also include a step of converting fructose to F6P, which is catalyzed by at least one enzyme, and optionally a step of converting sucrose to fructose, which is catalyzed by at least one enzyme. For example, this process includes producing F6P from fructose and polyphosphate catalyzed by polyphosphate fructokinase (PPFK). The conversion of F6P to tagatose is as described above. Fructose can be produced, for example, by the enzymatic conversion of sucrose. The phosphate ions produced when T6P is converted to tagatose can then be recycled in the step of converting sucrose to G1P.

[0043] Tagatose can also be produced from glucose. See Figure 4. The process according to the present invention may also include the step of converting glucose to G6P catalyzed by at least one enzyme, and optionally the step of converting sucrose to fructose, the latter of which is catalyzed by at least one enzyme. For example, this process may include producing G6P from glucose and polyphosphate catalyzed by polyphosphate glucokinase (PPGK). Glucose can be produced, for example, by the enzymatic conversion of sucrose. See Figure 5.

[0044] In some methods of the present invention, the phosphate ions produced when T6P is converted to tagatose are recycled in the step of converting a starch derivative to G1P (see, for example, Figure 1), the step of converting a cellulose derivative to G1P (see, for example, Figure 2), or, particularly when the process is carried out in a single reaction vessel, in the step of converting sucrose to G1P (see Figure 5). Furthermore, the yield of tagatose can be increased by producing F6P from fructose produced by the phosphate decomposition cleavage of sucrose by SP using PPFK and polyphosphate.

[0045] For example, a process for preparing tagatose from sugar includes the following steps: (i) converting the sugar to glucose 1-phosphate (G1P) using one or more enzymes; (ii) converting G1P to G6P using phosphoglucocomutase (PGM, EC 5.4.2.2); (iii) converting G6P to F6P using phosphoglucoisomerase (PGI, EC 5.3.1.9); (iv) converting F6P to T6P via fructose 6-phosphate epimerase (F6PE); and (v) converting T6P to tagatose via tagatose 6-phosphate phosphatase (T6PP). In the improved process of the present invention, F6PE comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 7, and / or T6PP comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. In such a process, for example, the enzyme in step (i) is αGP. Typically, the ratio of enzyme units used in the process is 1:1:1:1:1 (αGP:PGM:PGI:F6PE:T6PP). An enzyme unit is the amount of enzyme required to convert 1 μmol of substrate into the product per minute. Therefore, an enzyme with higher activity will require less enzyme per enzyme unit in mg of enzyme compared to an enzyme with lower activity catalyzing the same reaction. To optimize the product yield, these ratios can be adjusted in any number of combinations. For example, a particular enzyme may be present in amounts approximately 2, 3, 4, or 5 times greater than the amounts of other enzymes.

[0046] The process for preparing tagatose according to the present invention may include the following additional steps: producing glucose from polysaccharides and oligosaccharides by enzymatic or acid hydrolysis; converting glucose to G6P catalyzed by at least one enzyme; producing fructose from polysaccharides and oligosaccharides by enzymatic or acid hydrolysis; and converting fructose to G6P catalyzed by at least one enzyme. Examples of polysaccharides and oligosaccharides are listed above.

[0047] The process for preparing tagatose according to the present invention can be carried out in a single bioreactor or reaction vessel. Alternatively, the steps can be carried out in multiple bioreactors or reaction vessels arranged in series. In the preferred process, the enzymatic production of tagatose is carried out in a single reaction vessel.

[0048] The enzymes used in this invention may take the form of soluble, immobilized, aggregated, or condensed proteins. These enzymes may be adsorbed onto insoluble organic or inorganic supports, as is known in the art, which are commonly used to improve functionality. These include polymeric supports such as agarose, methacrylate, polystyrene, or dextran, as well as inorganic supports such as glass, metal, or carbon-based materials. These materials are often manufactured with a large surface-to-volume ratio and special surfaces to facilitate the attachment and activity of immobilized enzymes. Enzymes may be attached to these solid supports via covalent, ionic, or hydrophobic interactions. Enzymes may also be immobilized via genetically engineered interactions, such as covalent fusion to another protein or peptide sequence, most often a polyhistidine sequence, that has affinity for the solid support. Enzymes may attach directly to the surface or surface coating, or they may attach to other proteins already present on the surface or surface coating. All enzymes can be immobilized on one support, individual supports, or a combination of two (for example, mixing two enzymes per support and then mixing those supports). These variations can be mixed uniformly or in defined layers to optimize turnover in a continuous reactor. For example, the reactor start may have a layer of aGP to ensure a high initial G1P increase. The enzymes can be immobilized all on one support, on individual supports, or in groups. These enzymes can be mixed uniformly or in defined layers or zones to optimize turnover.

[0049] Any suitable biological buffer known in the art, such as HEPES, PBS, BIS-TRIS, MOPS, DIPSO, Trizma, etc., can be used in the process of the present invention. The reaction buffer of all embodiments may have a pH in the range of 5.0 to 9.0. More preferably, the pH of the reaction buffer may be in the range of about 6.0 to about 7.3. For example, the pH of the reaction buffer may be 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, or 7.3.

[0050] In some improved processes of the present invention, the reaction buffer contains a divalent metal cation. An example is Mn 2+ Co 2+ Mg 2+ and Zn 2+ For example, preferably Mg 2+ It is included. The concentration of divalent metal cations may be in the range of about 0 mM to about 150 mM, about 0 mM to about 100 mM, about 1 mM to about 50 mM, preferably about 5 mM to about 50 mM, or more preferably about 10 mM to about 50 mM. For example, the concentration of divalent metal cations may be about 0.1 mM, about 0.5 mM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, or about 55 mM.

[0051] The reaction temperature at which the process steps are carried out may be in the range of 37 to 85°C. More preferably, the steps can be carried out at a temperature in the range of about 37°C to about 85°C. The temperature may be, for example, about 40°C, about 45°C, about 50°C, about 55°C, or about 60°C. Preferably, the reaction temperature is about 50°C. In some processes of the present invention, the reaction temperature is constant and does not change during the process.

[0052] The reaction time of the disclosed process can be adjusted as needed and may range from about 1 hour to about 48 hours. For example, the reaction time may be about 16 hours, about 18 hours, about 20 hours, about 22 hours, about 24 hours, about 26 hours, about 28 hours, about 30 hours, about 32 hours, about 34 hours, about 36 hours, about 38 hours, about 40 hours, about 42 hours, about 44 hours, about 46 hours, or about 48 hours. Preferably, the reaction time is about 24 hours.

[0053] The reaction can be carried out in batch or continuous process using a packed-bed reactor or similar apparatus. In a continuous process, the maltodextrin solution is pumped through a bed of immobilized enzymes at a rate such that the conversion to tagatose is completed when the solution leaves the column for downstream processing. For example, 200 g / L of maltodextrin can be pumped into a column packed with immobilized enzymes (e.g., maintained at 50°C) so that the maximum tagatose yield is achieved when the maltodextrin leaves the column. This methodology offers superior volume productivity compared to batch methods. This limits the time the product is in contact with the column and reaction conditions, reducing the possibility of product degradation (such as the potential formation of hydroxymethylfurfural). Whether in batch or continuous mode, various steps of the process of the present invention can be carried out using the same reaction conditions as the other steps. For example, in a specific process of the present invention using a single bioreactor or reaction vessel, reaction conditions such as pH and temperature, as well as the reaction buffer, are kept constant throughout all steps of the process.

[0054] Next, the phosphate ions generated by the T6PP dephosphorylation of T6P can be recycled in the process step that converts the sugar to G1P, especially if all process steps are carried out in a single bioreactor or reaction vessel. The ability to recycle phosphate in the disclosed process allows for the use of non-stoichiometric amounts of phosphate, which keeps the reaction phosphate concentration low. This affects the overall pathway and the overall rate of the process, but does not limit the activity of individual enzymes, enabling the overall efficiency of the tagatose production process.

[0055] For example, the reaction phosphate concentration is in the range of about 0 mM to about 300 mM, about 0 mM to about 150 mM, about 1 mM to about 50 mM, preferably about 5 mM to about 50 mM, and more preferably about 10 mM to about 50 mM. For example, the reaction phosphoric acid concentration may be about 0.1 mM, about 0.5 mM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, or about 55 mM.

[0056] Therefore, lower phosphate concentrations reduce manufacturing costs due to lower total phosphate levels, and consequently, the cost of phosphate removal. Furthermore, it prevents inhibition of T6PP by high concentrations of free phosphate ions (free phosphate), reducing the possibility of phosphate contamination.

[0057] Furthermore, the process disclosed herein can be carried out without adding ATP as a source of phosphate, i.e., without ATP. The process can also be carried out without the need to add NAD(H), i.e., without NAD(H). Other advantages include the fact that at least one step of the disclosed process for producing tagatose involves an energetically favorable chemical reaction (Figure 6). While the use of enzymes with higher activity does not affect the overall energy, the ability to use smaller amounts of enzyme in the improved process is advantageous. The benefit is the reduction in the total cost of enzymes in the total production cost of the product.

[0058] The process according to the present invention can achieve high yields due to a very favorable equilibrium constant for the entire reaction. Theoretically, a yield of up to 99% can be achieved if the starting materials are completely converted to the intermediate. Furthermore, the step of converting T6P to tagatose according to the present invention is an irreversible phosphatase reaction regardless of the feedstock. Therefore, tagatose is produced in very high yields.

[0059] The process of the present invention reduces manufacturing costs by using low-cost starting materials and reducing costs associated with the separation of raw materials and products. Starch, cellulose, sucrose, and their derivatives are, for example, cheaper raw materials than lactose. When tagatose is produced from lactose, the production cost is high because glucose, galactose, and tagatose are separated by chromatography.

[0060] The process according to the present invention enables easy recovery of tagatose and minimizes separation costs. Preferably, in the process of the present invention, the recovery of tagatose is not by chromatographic separation. After generating tagatose in a continuous reaction, the product instead undergoes microfiltration, ion exchange (cations, then anions, not a mixed bed), concentration, crystallization, crystal separation, and drying. Due to the high yield of tagatose, only the crystallization step is required to purify tagatose. To further purify tagatose before crystallization, nanofiltration can be used to eliminate the risk of enzymes being present in the crystallization process and to remove unconverted dextrins that may co-crystallize with tagatose, or to limit the recyclability of the mother liquor (maltodextrin, maltotetraose, maltotriose, maltose, etc.).

[0061] An improved process for preparing tagatose according to the present invention comprises the following steps: (i) converting a sugar to glucose-1-phosphate (G1P) using one or more enzymes; (ii) converting G1P to G6P using phosphoglucocomutase (PGM, EC 5.4.2.2); (iii) converting G6P to F6P using phosphoglucoisomerase (PGI, EC 5.3.1.9); (iv) converting F6P to T6P via fructose 6-phosphate epimerase (F6PE); and (v) converting T6P to tagatose via tagatose 6-phosphate phosphatase (T6PP), where F6PE comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 7, and / or T6PP comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. This process is preferably carried out in a single bioreactor or reaction vessel.

[0062] Preferably, an improved process for preparing tagatose according to the present invention comprises the following steps: (i) converting a sugar to glucose 1-phosphate (G1P) using αGP, where the sugar is selected from the group consisting of starch, one or more starch derivatives, or combinations thereof; (ii) phosphoglucomutase (PGM, EC) (iii) a step of converting G1P to G6P using 5.4.2.2); (iv) a step of converting G6P to F6P using phosphoglucoisomerase (PGI, EC5.3.1.9); (iv) a step of converting F6P to T6P via fructose 6-phosphate epimerase (F6PE); and (v) a step of converting T6P to tagatose via tagatose 6-phosphate phosphatase (T6PP), where F6PE comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 7, and / or T6PP comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. This process is preferably carried out in a single reactor vessel and can incorporate one or more of the various process conditions described above. [Examples]

[0063] example

[0064] Materials and methods

[0065] All chemicals, including glucose-1-phosphate, magnesium chloride, and sodium phosphate (monobasic and dibasic), are reagent grade or higher unless otherwise specified and are purchased from Sigma-Aldrich (St. Louis, Missouri, USA) or Fisher Scientific (Pittsburgh, Pennsylvania, USA). Escherichia coli BL21(DE3) (Sigma-Aldrich, St. Louis, Missouri, USA) was used as the host cell for recombinant protein expression. ZYM-5052 medium containing 50 mg of L-1 kanamycin was used for E. coli cell growth and recombinant protein expression.

[0066] Production and purification of recombinant enzymes

[0067] A 50 mg L of E. coli BL21 (DE3) strain containing the protein expression plasmid (pET28a) was added to a 1 L Ehrenmeier flask. ‐1 Cells were incubated with 100 mL of ZYM-5052 medium containing kanamycin. Cells were grown at 37°C at 220 rpm for 16–24 hours with rotational shaking. Cells were harvested by centrifugation at 12°C and washed once with either 20 mM HEPES (pH 7.5) containing 50 mM NaCl and 5 mM MgCl2 (thermal precipitate) or 20 mM HEPES (pH 7.5) containing 300 mM NaCl and 5 mM imidazole (Ni purified). The cell pellet was resuspended in the same buffer and lysed by sonication. After centrifugation, the target protein in the supernatant was purified. His-tagged proteins were purified using Profinity IMAC Ni-Charged Resin (Bio-Rad, Hercules, CA, USA). Thermal precipitation at 50–80°C for 5–30 minutes was used to purify thermostable enzymes. The purity of the recombinant protein was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

[0068] Example 1: F6PE with higher activity

[0069] The relative activity of different F6PEs was measured at 50°C for 1 hour in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCl2, 0.5 mM MnCl2, 38.5 mM G1P, 0.05 g / L PGM, 0.05 g / L PGI, 0.05 g / L F6PE, and 0.075 g / L T6PP (Uniprot ID A0A0E3NCH4) from Methanosarcina thermophila CHTI-55. The reaction was stopped by filtering the enzyme through a Vivaspin 2 concentrator (10,000 MWCO). The tagatose of the product was evaluated using an Agilent Hi-Plex H column and refractive index detector. Samples were analyzed in 5 mM H2SO4 at 0.6 mL / min for 15 minutes at 65°C. Table 1 shows that the F6PE used in the improved process of the present invention has higher activity compared to the previously disclosed F6PE from Dictyoglomus thermophilum (Uniprot ID B5YBD7). See International Patent Application Publication WO2017 / 059278. [Table 1]

[0070] Example 2: T6PP with higher activity

[0071] The relative activity of T6PP was measured at 50°C for 1 hour in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCl2, 0.5 mM MnCl2, 38.5 mM G1P, 0.05 g / L PGM, 0.05 g / L PGI, 0.25 g / L F6PE from Thermanaerothrix daxensis (Uniprot ID A0A0P6XN50), and 0.05 g / L T6PP. The reaction was stopped by filtering the enzyme through a Vivaspin 2 concentrator (10,000 MWCO). The tagatose of the product was evaluated using an Agilent Hi-Plex H column and refractive index detector. Samples were analyzed with 5 mM H2SO4 at 0.6 mL / min for 15 minutes at 65°C. Table 2 shows that the T6PP used in the improved process of the present invention has higher activity compared to the previously disclosed T6PP from Archaeoglobus fulgidus (Uniprot ID O29805). See International Patent Application Publication WO2017 / 059278. [Table 2]

[0072] Example 3: Improved Tagathos manufacturing from F6P

[0073] The conversion of F6P to tagatose using previously disclosed enzymes, F6PE (Uniprot ID B5YBD7) and T6PP (Uniprot ID O29805), was compared with the conversion of F6P to tagatose using enzymes useful in the improved process of the present invention, F6PE (Uniprot ID A0A0P6XN50) and T6PP (Uniprot ID D6YBK5). A 0.20 mL reaction mixture containing 38.5 mM F6P, 50 mM HEPES pH 7.2, 5 mM MgCl2, 0.5 mM MnCl2, 0.1 g / L F6PE, and 0.033 g / L T6PP was incubated at 50°C for 2 hours.

[0074] The reaction was stopped by enzyme filtration in a Vivaspin 2 concentrator (10,000 MWCO) and analyzed by HPLC (Agilent 1100 series) using an Agilent Hi-Plex H column and refractive index detector. Samples were analyzed in 5 mM H2SO4 at 0.6 mL / min for 15.5 minutes at 65°C. The results show a 4.4-fold improvement in tagatose production with the improved process enzyme compared to previously disclosed enzymes (Figure 7). The reaction with the improved process enzyme contains a small amount of fructose (see Figure 7, shoulder, 5, peak 4). This may be an artifact due to T6PP initially detecting a large amount of F6P compared to T6P in the reaction here, and is not a problem in the overall pathway.

[0075] Sequence List α-glucan phosphorylase Thermotoga maritima (Uniprot ID G4FEH8) - Sequence ID 8 [ka]

[0076] Phosphoglucomutase Thermococcus kodakaraensis (Uniprot ID Q68BJ6) - Sequence ID 9 [ka]

[0077] Fructose 6-phosphate 4-epimerase (Comparison) Dictyoglomus thermophilum (Uniprot ID B5YBD7) - SEQ ID NO: 10 [ka]

[0078] Thermanaerothrix daxensis (Uniprot ID A0A0P6XN50) - Sequence ID 1 [ka]

[0079] Thermoanaerobacterium thermosaccharolyticum (Uniprot ID A0A223HVJ3) - Sequence ID 2 [ka]

[0080] Candidatus Thermofonsia Clade 3 bacterium (Uniprot ID A0A2M8QBR9) - Sequence ID 7 [ka]

[0081] Tagatose 6-phosphate phosphatase (Comparison) Archaeoglobus fugidis (Uniprot ID O29805) - Sequence ID 11 [ka]

[0082] Methanosarcina thermophila CHTI-55 (Uniprot ID A0A0E3NCH4) - Sequence ID 3 [ka]

[0083] Thermobispora bispora strain ATCC 19993 (Uniprot ID D6YBK5) - Sequence ID No. 4 [ka]

[0084] Spirochaeta thermophila ATCC 49972 (Uniprot ID E0RT70) - Sequence ID No. 5 [ka]

[0085] Sphaerobacter thermophilus DSM 20745 (Uniprot ID D1C7G9) - Sequence ID 6 [ka]

[0086] 4-glucantransferase Thermococcus litoralis (Uniprot ID O32462) - Sequence ID 12 [ka]

Claims

1. An improved method for producing tagatose from sugar, and this improved method Converting fructose-6-phosphate (F6P) to tagatose-6-phosphate (T6P) using fructose-6-phosphate epimerase (F6PE), and Converting T6P to tagatose using tagatose-6-phosphate phosphatase (T6PP). This includes, where T6PP contains an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 5, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO:

6. The above method.

2. The method according to claim 1, wherein F6PE comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO:

7.

3. The method according to claim 1 or 2, further comprising the step of converting glucose 6-phosphate (G6P) to F6P, wherein the step is catalyzed by phosphoglucose isomerase (PGI).

4. The method according to claim 3, further comprising the step of converting glucose 1-phosphate (G1P) to G6P, wherein this step is catalyzed by phosphoglucomutase (PGM).

5. The method according to claim 4, further comprising the step of converting a sugar to G1P, wherein this step is catalyzed by at least one enzyme, and the sugar is selected from the group consisting of starch or derivatives thereof, cellulose or derivatives thereof, and sucrose.

6. The method according to claim 5, wherein at least one enzyme is selected from the group consisting of α-glucan phosphorylase (αGP), maltose phosphorylase, sucrose phosphorylase, cellodextrin phosphorylase, cellobiose phosphorylase, and cellulose phosphorylase.

7. The method according to claim 5, wherein the sugar is a starch or derivative thereof selected from the group consisting of amylose, amylopectin, soluble starch, amylodextrin, maltodextrin, maltose, maltotriose, and glucose.

8. The method according to claim 7, further comprising the step of converting starch to a starch derivative, wherein the starch derivative is prepared by enzymatic hydrolysis of starch or acid hydrolysis of starch.

9. The method according to claim 8, wherein 4-glucantransferase (4GT) is added when converting a starch derivative to glucose 1-phosphate.

10. The method according to claim 8, wherein the starch derivative is prepared by enzymatic hydrolysis of starch using isoamylase, pullulanase, α-amylase, or a combination thereof.

11. A step of converting fructose catalyzed by at least one enzyme to F6P, and The method according to claim 1 or 2, further comprising the optional step of converting sucrose to fructose catalyzed by at least one enzyme.

12. A step of converting glucose to G6P catalyzed by at least one enzyme, and The method according to claim 3, further comprising the optional step of converting sucrose to glucose catalyzed by at least one enzyme.

13. The method according to claim 1 or 2, wherein the steps of the method are carried out under at least one of the following conditions of the method: In a single reaction vessel At temperatures ranging from approximately 37°C to approximately 85°C, pH ranges from approximately 5.0 to approximately 9.

0. In approximately 1 hour to approximately 48 hours, Any combination of the above.

14. The method according to claim 1 or 2, wherein the steps of the method are carried out under at least one of the following conditions of the method: Without adenosine triphosphate (ATP) as a phosphate source, Without nicotinamide adenosine dinucleotide, Phosphate concentrations ranging from approximately 0.1 mM to approximately 150 mM, Mg from approximately 0.1 mM to 50 mM ２＋ In terms of concentration, The phosphate ions produced by the T6PP dephosphorylation of T6P are used in the steps of the method for converting sugar to G1P. At least one step of the method involves an energetically favorable chemical reaction, or Any combination of the above.

15. The method according to claim 1 or 2, further comprising the step of separating and recovering the generated tagatose, wherein the separation and recovery is not via chromatographic separation.

16. The method according to claim 2, wherein F6PE has higher activity in an improved method compared to the activity of F6PE (SEQ ID NO: 10) from Dictyoglomus thermofilm.

17. The method according to claim 1, wherein T6PP has higher activity in an improved method compared to the activity of T6PP (SEQ ID NO: 11) from Archaeoglobus fulgidus.

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