Enzymatic production of hexose

Abstract

To provide methods of producing hexoses from saccharides by improved enzymatic processes.SOLUTION: Methods of producing hexoses from saccharides by improved enzymatic processes are disclosed herein. The improved processes utilize enzymes with higher activities than those previously reported to convert starch or a starch derivative, cellulose or a cellulose derivative, or sucrose to a glucose 6-phosphate (G6P) intermediate.SELECTED DRAWING: Figure 2

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Patent Application No. 62 / 752,061, filed Oct. 29, 2018, and U.S. Patent Application No. 62 / 857,543, filed Jun. 5, 2019, each of which is incorporated herein by reference.

[0002] The present invention relates to a method for preparing hexose monosaccharides. More specifically, the present invention provides an improved method for preparing an intermediate, glucose 6 - phosphate (G6P), using an enzyme having higher activity than previously reported.

Background Art

[0003] Hexoses are monosaccharides having six carbon atoms. Hexoses can be classified by functional groups, with aldoses having an aldehyde at the 1 - position and ketoses having a ketone at the 2 - position. Aldohexoses (or aldoses) include allose, altrose, glucose, gulose, galactose, idose, talose, and mannose. Ketohexoses (or ketoses) include psicose (allulose), fructose, tagatose, and sorbose. Inositol is a hexose that does not have an aldehyde group or a ketose group and is characterized as a cyclic carbon sugar.

[0004] International Publication No. 2018 / 169957, which is hereby incorporated by reference in its entirety, describes a method for preparing hexose from saccharides by enzymatic conversion. International Publication No. 2017 / 059278 and International Publication No. 2018 / 004310, which are hereby incorporated by reference in their entirety, describe methods for preparing tagatose from saccharides by enzymatic conversion. International Publication No. 2018 / 112139, which is hereby incorporated by reference in its entirety, describes a method for preparing allose from saccharides by enzymatic conversion. Korean Patent No. 20040098757, which is hereby incorporated by reference in its entirety, describes a method for preparing fructose 6-phosphate from saccharides by enzymatic conversion. Chinese Patent No. 106148425, which is hereby incorporated by reference in its entirety, describes a method for preparing inositol from saccharides by enzymatic conversion. In each of these methods, glucose 6-phosphate (G6P) is an intermediate in the enzymatic pathway.

[0005] Despite the development of enzymatic hexose production in high yields, there remains a need to provide further improved methods for hexose production, for example, to obtain higher yields with smaller amounts of enzymes. There is a strong industrial and commercial interest in reducing the cost of hexose production, which includes reducing the amount of enzyme used, using enzymes with higher activity, and using more effective combinations of enzymes to convert saccharides to the G6P intermediate.

Summary of the Invention

Means for Solving the Problems

[0006] The invention described herein generally relates to improved processes for preparing hexose by enzymatic conversion from various saccharide starting materials. The saccharides can be selected from starch or starch derivatives, cellulose or cellulose derivatives, or sucrose. In the improved methods of the invention, as shown, the enzymes used in the method steps have improved activity compared to the enzymes previously disclosed for the preparation of hexose.

[0007] Some improved methods of the present invention for the enzymatic production of hexoses from starch or starch derivatives, wherein the improvement comprises: a) a step of converting glucose 1-phosphate (G1P) to glucose 6-phosphate (G6P) catalyzed by phosphoglucomutase (PGM), wherein PGM comprises an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NOs: 2-8; b) a step of converting a starch derivative to G1P catalyzed by alpha-glucan phosphorylase (αGP), wherein αGP comprises an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NOs: 10-13; and a step of transglycosylating a starch derivative catalyzed by 4-alpha-glucanotransferase (4GT), wherein 4GT comprises an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NOs: 15-17, and comprising at least one of the above steps.

[0008] Some improved methods of the present invention for the enzymatic production of hexoses from cellulose or cellulose derivatives, wherein the improvement comprises a step of converting G1P to glucose 6-phosphate (G6P) catalyzed by phosphoglucomutase (PGM), wherein PGM comprises an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NOs: 2-8.

[0009] Some improved methods of the present invention for the enzymatic production of hexoses from sucrose, wherein the improvement comprises: a) a step of converting glucose 1-phosphate (G1P) to glucose 6-phosphate (G6P) catalyzed by phosphoglucomutase (PGM), wherein PGM comprises an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NOs: 2-8; and b) a step of converting sucrose to glucose 1-phosphate (G1P) using sucrose phosphorylase, wherein sucrose phosphorylase comprises an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NOs: 19-25, and comprising at least one of the above steps.

[0010] In some improved methods of the present invention, the hexose is selected from allose, mannose, galactose, fructose, altrose, talose, sorbose, gulose, idose, allulose, inositol, and tagatose. Some improved methods of the present invention further include the step of dephosphorylating hexose phosphate using hexose phosphate phosphatase.

[0011] In some improved methods of the present invention, the method steps are carried out in a single reaction vessel. In other improved methods of the present invention, the method steps are carried out in two or more reaction vessels. In some improved methods of the present invention, the method steps are carried out without ATP (ATP-free), without NAD(P)(H) (NAD(P)(H)-free), at a phosphate concentration of about 0.1 mM to about 150 mM, the phosphate is recycled, and / or the step of dephosphorylating hexose phosphate involves an energetically favorable chemical reaction. In some improved methods of the present invention, the method steps are carried out under at least one method condition of a temperature in the range of about 37°C to about 85°C, a pH in the range of about 5.0 to about 8.0, or a time of about 0.5 hours to about 48 hours. In some improved methods of the present invention, the method steps are carried out as a continuous reaction.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0031] The invention described herein provides an enzymatic pathway or method for synthesizing hexose with a high product yield while significantly reducing the product separation cost and hexose production cost. The enzymatic methods described herein generally relate to improved methods for preparing hexose monosaccharides from saccharides by enzymatic conversion. An artificial (non-natural) ATP-free enzymatic pathway for converting saccharides to hexose using a cell-free enzyme cocktail is provided. In contrast to cell-based production methods, the enzymatic methods of the present invention include cell-free preparation of hexose and have a relatively high reaction rate due to the removal of cell membranes, which often delays the transport of substrates / products in and out of cells. The method also has a final product that does not contain a nutrient-rich fermentation medium / cell metabolites. The saccharide can be selected from starch or starch derivatives, cellulose or cellulose derivatives, or sucrose. In the improved method of the present invention, as shown, the enzymes used in the method steps have improved activity compared to the enzymes previously disclosed for the preparation of hexose. In one embodiment, the present invention relates to an improved method for converting starch and its derivatives into hexoses using at least one of αGP, PGM, and 4GT having higher activity instead of the previously disclosed αGP, PGM, and 4GT. See International Publication No. WO 2018 / 169957, which discloses an alpha-glucan phosphorylase (αGP) (Uniprot ID G4FEH8) from Thermotoga maritima, a phosphoglucomutase (PGM) (Uniprot ID Q68BJ6) from Thermococcus kodakaraensis, and a 4-α-glucanoltransferase (Uniprot ID O32462) from Thermococcus litoralis. Some improved methods of the present invention for the enzymatic production of hexoses from starch or starch derivatives, wherein the improvement comprises: a) a step of converting glucose 1-phosphate (G1P) to glucose 6-phosphate (G6P) catalyzed by phosphoglucomutase (PGM), wherein PGM comprises an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NOs: 2-8; b) a step of converting a starch derivative to G1P catalyzed by alpha-glucan phosphorylase (αGP), wherein αGP comprises an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NOs: 10-13; and a step of transglycosylating a starch derivative catalyzed by 4-α-glucanotransferase (4GT), wherein 4GT comprises an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NOs: 15-17, comprising at least one of the steps.In the improved method of the present invention, the method comprises: a) a step of converting glucose 1-phosphate (G1P) to glucose 6-phosphate (G6P), which is catalyzed by phosphoglucomutase (PGM), wherein the PGM comprises an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NOs: 2 to 8; b) a step of converting a starch derivative to G1P, which is catalyzed by alpha-glucan phosphorylase (αGP), wherein the αGP comprises an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NOs: 10 to 13; and c) a step of transglycosylating a starch derivative, which is catalyzed by 4-alpha-glucanotransferase (4GT), wherein the 4GT comprises an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NOs: 15 to 17.

[0032] In another embodiment, the present invention relates to an improved method for converting cellulose and its derivatives to hexoses using a PGM having higher activity instead of PGM from Thermococcus kodakaraensis (Uniprot ID Q68BJ6). In some improved methods of the present invention for the enzymatic production of hexoses from cellulose or cellulose derivatives, the improvement comprises a step of converting G1P to glucose 6-phosphate (G6P), which is catalyzed by PGM, and the PGM comprises an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NOs: 2 to 8.

[0033] In yet another embodiment, the present invention relates to an improved method for converting sucrose to hexose using at least one of an SP having higher activity instead of the previously disclosed SP (Uniprot ID D9TT09) derived from Thermoanaerobacterium thermosaccharolyticum and a PGM having higher activity instead of the PGM (Uniprot ID Q68BJ6) derived from Thermococcus kodakaraensis. Some improved methods of the present invention for the enzymatic production of hexose from sucrose, the improvement comprising: a) a step of converting glucose 1-phosphate (G1P) to glucose 6-phosphate (G6P) catalyzed by phosphoglucomutase (PGM), wherein the PGM comprises an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NOs: 2 to 8; and b) a step of converting sucrose to glucose 1-phosphate (G1P) using sucrose phosphorylase, wherein the sucrose phosphorylase comprises an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NOs: 19 to 25, and the improvement comprises at least one of the steps. In the improved method of the present invention, the method comprises: a) a step of converting glucose 1-phosphate (G1P) to glucose 6-phosphate (G6P) catalyzed by phosphoglucomutase (PGM), wherein the PGM comprises an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NOs: 2 to 8; and b) a step of converting sucrose to glucose 1-phosphate (G1P) using sucrose phosphorylase, wherein the sucrose phosphorylase comprises an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NOs: 19 to 25.

[0034] In some improved methods of the present invention, the hexose is selected from allose, mannose, galactose, fructose, altrose, talose, sorbose, gulose, idose, allulose, inositol, and tagatose. Some improved methods of the present invention further include the step of dephosphorylating hexose phosphate using hexose phosphate phosphatase.

[0035] Some improved methods according to the present invention for the enzymatic production of hexose from starch or starch derivatives, cellulose or cellulose derivatives, or sucrose include the step of converting glucose 1-phosphate (G1P) to glucose 6-phosphate (G6P) catalyzed by phosphoglucomutase (PGM), where the PGM comprises an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NOs: 2-8. Preferably, the PGM comprises the amino acid sequence of any one of SEQ ID NOs: 2-8. More preferably, the PGM comprises the amino acid sequence of SEQ ID NO: 8.

[0036] Phosphoglucomutase (PGM) (EC 5.4.2.2) catalyzes the interconversion of glucose 1-phosphate and glucose 6-phosphate. In the improved method of the present invention for producing hexose from sugar, the reaction proceeds in the direction of G6P and is then further processed downstream, and the final enzymatic step of the method is an energetically favorable irreversible step of dephosphorylating hexose phosphate.

[0037] In the improved method of the present invention, the PGM has higher activity compared to the aforementioned PGM (Uniprot ID Q68BJ6) derived from Thermococcus kodakaraensis, which has the amino acid sequence set forth in SEQ ID NO: 1. Preferably, the PGM used in the method of the present invention has at least 10%, at least 100%, at least 200%, at least 500%, at least 1000%, at least 2000%, at least 2500%, at least 3000%, at least 3500%, at least 4000%, at least 4500%, at least 5000%, or at least 5500% improved enzyme activity compared to the activity of the PGM (Uniprot ID Q68BJ6) derived from Thermococcus kodakaraensis.

[0038] For example, as shown in Example 1, the PGM for use in the method of the present invention has improved activity compared to the PGM from Thermococcus kodakaraensis (Uniprot ID Q68BJ6). The PGM from Geobacillus stearothermophilus NUB3621 (Uniprot ID A0A023CRS6) has an approximately 700% improved enzyme activity. The PGM from Caldibacillus debilis (Uniprot ID A0A150LLZ1) has an approximately 1900% improved enzyme activity. The PGM from Geobacillus thermoglucosidasius (UniParc ID UPI0001D17AE3) has an approximately 2100% improved enzyme activity. The PGM from Parageobacillus caldoxylosilyticus NBRC107762 (Uniprot ID A0A023DI95) has an approximately 1980% improved enzyme activity. The PGM from Thermobrachium celere DSM8682 (Uniprot ID R7RR04) has an approximately 5100% improved enzyme activity. The PGM from Anaerolinea thermophila (Uniprot ID E8N4Y6) has an approximately 5800% improved enzyme activity. The PGM from Thermanaerothrix daxensis (Uniprot ID A0A0P6YKY9) has an approximately 6500% improved enzyme activity. The following examples provide a protocol for those skilled in the art to determine the activity of PGM as part of an enzymatic method, which includes, for example, incubating the enzyme with its substrate and then measuring the amount of reactants and products or downstream products thereof via HPLC. The measurement of the relative activity of any two enzymes is carried out under the same reaction conditions such as buffer, pH, temperature, etc.

[0039] Examples of PGMs for use in the improved method of the present invention include PGM from Geobacillus stearothermophilus NUB3621 (Uniprot ID A0A023CRS6) having the amino acid sequence set forth in SEQ ID NO: 2, PGM from Caldibacillus debilis (Uniprot ID A0A150LLZ1) having the amino acid sequence set forth in SEQ ID NO: 3, PGM from Geobacillus thermoglucosidasius (UniParc ID UPI0001D17AE3) having the amino acid sequence set forth in SEQ ID NO: 4, PGM from Parageobacillus caldoxylosilyticus NBRC107762 (Uniprot ID A0A023DI95) having the amino acid sequence set forth in SEQ ID NO: 5, PGM from Thermobrachium celere DSM8682 (Uniprot ID R7RR04) having the amino acid sequence set forth in SEQ ID NO: 6, PGM from Anaerolinea thermophila (Uniprot ID E8N4Y6) having the amino acid sequence set forth in SEQ ID NO: 7, PGM from Thermanaerothrix daxensis (Uniprot ID A0A0P6YKY9) having the amino acid sequence set forth in SEQ ID NO: 8, and proteins of PGMs comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity with any one of SEQ ID NOs: 2 to 8, but are not limited thereto.

[0040] The PGM for use in the improved method of the present invention is generally part of the α-D-phosphohexomutase superfamily (IPR005841) and contains four domains that form the active site of the enzyme. The first three domains consist of an αβ core, and the fourth domain contains a TATA box-binding protein-like fold (Mehra-Chaudhary et al., Proteins 79(4):1215-29, 2011). The first domain contributes to a conserved serine residue for phosphoryl transfer to the active site (Ser147 of SEQ ID NO: 3). The second domain contributes to conserved Mg2+-binding residues for the active site (Asp306, Asp308, and Asp310 of SEQ ID NO: 3). The third domain contributes to residues conserved for substrate specificity for the active site (Glu406 and Ser408 of SEQ ID NO: 3). The fourth domain contributes to a conserved residue for phosphate binding to the active site (Arg538 of SEQ ID NO: 3). Further, positively charged residues (Lys / Arg) are conserved and play a role in catalysis at Lys420 of SEQ ID NO: 3. The conserved residues are cited from Lee et al., FEBS J 280(11):2622-32, 2013 and Levin et al., "Protein Engineering, Design and Selection" 12(9):737-746, 1999.

[0041] Some improved methods of the present invention for producing hexose from starch or a starch derivative according to the present invention include a step of converting a starch derivative to G1P catalyzed by αGP, where αGP includes an amino acid sequence having at least 90% amino acid sequence identity with any one of SEQ ID NOs: 10 to 13. Preferably, αGP includes the amino acid sequence of any one of SEQ ID NOs: 10 to 13. More preferably, αGP includes the amino acid sequence of SEQ ID NO: 11.

[0042] α-Glucan phosphorylase or starch phosphorylase (αGP) (EC 2.4.1.1) cleaves maltooligosaccharides by phosphorolysis to yield G1P. Starch phosphorylase also catalyzes the reverse reaction involving the release of phosphate, i.e., the transfer of glucosyl units from G1P to the non-reducing end of an α-1,4-D-glucan chain. Generally, the degree of polymerization of the oligosaccharide chain is 4 or more. In the improved method of the present invention for producing hexose from a starch derivative, the reaction proceeds in the direction of G1P and is then further processed downstream, and the final enzymatic step of the method is an energetically favorable irreversible step of dephosphorylating hexose phosphate.

[0043] In the improved method of the present invention, the αGP has higher activity compared to the aforementioned alpha-glucan phosphorylase (αGP) (Uniprot ID G4FEH8) from Thermotoga maritima having the amino acid sequence set forth in SEQ ID NO: 9. Preferably, the αGP used in the method of the present invention has at least 10%, at least 50%, at least 100%, at least 150%, or at least 200% improved enzyme activity compared to the activity of the αGP from Thermotoga maritima (Uniprot ID G4FEH8). For example, as shown in Example 2, the αGP from Thermus thermophilus (Uniprot ID Q5SJ42) has about 71% improved enzyme activity compared to the αGP from Thermotoga maritima (Uniprot ID G4FEH8), the αGP from Thermus sp. CCB_US3_UF1 (Uniprot ID G8NCC0) has about 186% improved enzyme activity compared to the αGP from Thermotoga maritima (Uniprot ID G4FEH8), the αGP from Thermoanaerobacter pseudethanolicus strain ATCC 33223 (Uniprot ID B0K7V8) has about 128% improved enzyme activity compared to the αGP from Thermotoga maritima (Uniprot ID G4FEH8), and the αGP from Thermanaerovibrio acidaminovorans strain ATCC49978 (Uniprot ID D1B926) has about 111% improved enzyme activity compared to the αGP from Thermotoga maritima (Uniprot ID G4FEH8).The following examples provide those skilled in the art with a protocol for determining the activity of αGP as part of an enzymatic method, which protocol includes, for example, incubating the enzyme with its substrate and then measuring the amounts of reactants and products or subsequent downstream products via spectrophotometry and HPLC. The measurement of the relative activities of any two enzymes is performed under the same reaction conditions such as buffer, pH, temperature, etc.

[0044] Examples of αGP for use in the improved method of the present invention include αGP from Thermus thermophilus (Uniprot ID Q5SJ42) having the amino acid sequence set forth in SEQ ID NO: 10, αGP from Thermus sp CCB_US3_UF1 (Uniprot ID G8NCC0) having the amino acid sequence set forth in SEQ ID NO: 11, αGP from Thermoanaerobacter pseudethanolicus strain ATCC33223 (Uniprot ID B0K7V8) having the amino acid sequence set forth in SEQ ID NO: 12, αGP from Thermmanaerovibrio acidaminovorans strain ATCC49978 (Uniprot ID D1B926) having the amino acid sequence set forth in SEQ ID NO: 13, and proteins of αGP having at least 90%, at least 95%, at least 97%, at least 99% or 100% amino acid sequence identity with SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13, but are not limited thereto.

[0045] The α-glucan phosphorylase for use in the improved method of the present invention is generally part of glycosyltransferase, family 35 (IPR000811) and contains an "α-glucan phosphorylase" domain (IPR011834). Some αGPs have residues conserved for PLP binding, PLP stabilization, and phosphate binding. For example, in Uniprot ID D1B926 (SEQ ID NO: 13), Lys585 is conserved for PLP binding, Arg484 and Thr581 are conserved for PLP stabilization, and Gly110, Arg485, and Lys490 are conserved for phosphate binding (the conserved residues are cited from Watson et al., EMBO Journal Vol.16 No.1 pp.1-14, 1997).

[0046] In some improved methods according to the present invention for producing hexose from starch derivatives, the improvement includes the step of transglycosylating a starch derivative using a 4-α-glucanotransferase (4GT), where the 4GT includes an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NOs: 15 to 17. Preferably, the 4GT includes an amino acid sequence of any one of SEQ ID NOs: 15 to 17. More preferably, the αGP includes the amino acid sequence of SEQ ID NO: 17.

[0047] 4-α-Glucanotransferase (4GT) (EC 2.4.1.25) catalyzes the transglycosylation of maltooligosaccharides, yielding maltooligosaccharides and glucose of various lengths. Maltose and glucose can be used as acceptors in the transfer reaction.

[0048] Some methods of the present invention for producing hexoses include an energetically favorable dephosphorylation step of converting hexose phosphate to hexose. In such methods, particularly when carried out in a single reaction vessel, the phosphate is recycled and used upstream in the process, for example in a reaction catalyzed by αGP converting amylodextrin to G1P. However, αGP does not react with high activity towards amylodextrins having a degree of polymerization (DP) of less than 4. In such methods according to the present invention, 4GT is added to the reaction to improve the overall yield. 4GT transglycosylates amylodextrin such that amylodextrins with a DP smaller than 4, such as maltotriose, are converted to longer-chain amylodextrins that can be degraded by αGP. Without 4GT, maltotriose is not recycled to longer-chain amylodextrins that can participate again in the process to increase the yield, but accumulates as the final product of the process. 4GT increases the yield when amylodextrin is sufficiently degraded by αGP to produce a significant amount of maltotriose. This occurs due to the energetically favorable dephosphorylation step and the recycling of phosphate.

[0049] In the improved method of the present invention, the 4GT has higher activity compared to the aforementioned 4-α-glucanotransferase (4GT) (Uniprot ID O32462) derived from Thermococcus litoralis having the amino acid sequence set forth in SEQ ID NO: 14. Preferably, the 4GT used in the method of the present invention has at least 10%, at least 50%, at least 100%, at least 150%, or at least 200% improved enzyme activity compared to the activity of the 4GT (Uniprot ID O32462) derived from Thermococcus litoralis. For example, as shown in Example 3, the 4GT (Uniprot ID E4U8S9) of Oceanithermus profundus DSM14977 has approximately 128% improved enzyme activity compared to the 4GT (Uniprot ID O32462) of Thermococcus litoralis, the 4GT (Uniprot ID D7BF07) derived from Meiothermus silvanus strain ATCC700542 has approximately 212% improved enzyme activity compared to the 4GT (Uniprot ID O32462) derived from Thermococcus litoralis, and the 4GT (Uniprot ID E8MXP8) derived from Anaerolinea thermophila strain DSM14523 has approximately 184% improved enzyme activity compared to the 4GT (Uniprot ID O32462) derived from Thermococcus litoralis. The following examples provide a protocol for determining the activity of 4GT as part of an enzymatic method, which protocol includes, for example, incubating the enzyme with its substrate and then measuring the amount of glucose by spectrophotometry. The measurement of the relative activity of any two enzymes is performed under the same reaction conditions such as buffer, pH, temperature, etc.

[0050] Examples of 4GTs for use in the improved methods of the present invention include 4GT from Oceanithermus profundus DSM14977 (Uniprot ID E4U8S9) having the amino acid sequence set forth in SEQ ID NO: 15, 4GT from Meiothermus silvanus strain ATCC700542 (Uniprot ID D7BF07) having the amino acid sequence set forth in SEQ ID NO: 16, 4GT from Anaerolinea thermophila strain DSM14523 (Uniprot ID E8MXP8) having the amino acid sequence set forth in SEQ ID NO: 17, and proteins of 4GT having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17, but are not limited thereto.

[0051] The 4-glucanotransferase for use in the improved method of the present invention is genetically part of the glycoside transferase superfamily (IPR017853), more specifically family 77 of glycoside hydrolases (IPR003385). Generally, 4-glucanotransferase contains a (β / α)8 barrel catalytic domain with the active site at the C-terminus of the barrel β-strand. The active site contains a conserved catalytic triad. In SEQ ID NO: 17, these conserved residues correspond to Asp298 (nucleophile), Glu345 (proton donor), and Asp398 (transition state stabilizer). The 4GT for use in the improved method of the present invention also contains conserved residues related to substrate binding. For example, in SEQ ID NO: 17, these conserved residues correspond to Tyr60, Asp218, Arg296, and His397. See Przylas et al., Journal of Molecular Biology 296(3):873-886, 2000.

[0052] In several improved methods for the enzymatic production of hexoses from sucrose, the improvement includes the step of converting sucrose to glucose 1-phosphate (G1P) catalyzed by a sucrose phosphorylase, where the sucrose phosphorylase comprises an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NOs: 19-25. Preferably, the SP comprises the amino acid sequence of any one of SEQ ID NOs: 19-25. More preferably, the SP comprises the amino acid sequence of SEQ ID NO: 21.

[0053] Sucrose phosphorylase (EC: 2.4.1.7) catalyzes the conversion of sucrose and inorganic phosphate to fructose and G1P. In the improved method of the present invention for producing hexoses from sucrose, the reaction proceeds in the direction of G1P and is then further processed downstream, and the final enzymatic step of the method is an energetically favorable irreversible step of dephosphorylating hexose phosphate.

[0054] In the improved method of the present invention, the SP has higher activity compared to the aforementioned SP (Uniprot ID D9TT09) derived from Thermoanaerobacterium thermosaccharolyticum having the amino acid sequence set forth in SEQ ID NO: 18. Preferably, the SP used in the method of the present invention has an enzyme activity improved by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 100% or at least 200% compared to the activity of the SP (Uniprot ID D9TT09) derived from Thermoanaerobacterium thermosaccharolyticum. In some embodiments, the SP used in the method of the present invention exhibits an improved maximum yield of hexose, particularly fructose. For example, some SPs with higher activity are shown in Example 4. The SP (Uniprot ID A0A1X2FWC2) derived from Thermoanaerobacterium sp PSU-2 has an enzyme activity improved by 11% compared to the activity of the SP (Uniprot ID D9TT09) derived from Thermoanaerobacterium thermosaccharolyticum, and a maximum fructose yield 9% higher. The SP (Uniprot ID L0IL15) derived from Thermoanaerobacterium thermosaccharolyticum has an enzyme activity improved by 50% compared to the SP (Uniprot ID D9TT09) derived from Thermoanaerobacterium thermosaccharolyticum, and a maximum fructose yield 1% higher.The SP (Uniprot ID F6BJS0) derived from Thermoanaerobacterium xylanolyticum has an enzyme activity 49% higher than that of the SP (Uniprot ID D9TT09) derived from Thermoanaerobacterium thermosaccharolyticum, and a maximum fructose yield 32% higher. The SP (Uniprot ID A0A1Y3Q6Q6) derived from Thermobacillus sp ZCTH02-B1 has an enzyme activity 50% improved compared to that of the SP (Uniprot ID D9TT09) derived from Thermoanaerobacterium thermosaccharolyticum, and a maximum fructose yield 3% higher. The SP (Uniprot ID Q84HQ2) derived from Bifidobacterium adolescentis has an enzyme activity 31% improved compared to that of the SP (Uniprot ID D9TT09) derived from Thermoanaerobacterium thermosaccharolyticum, and a maximum fructose yield 37% higher. The SP (Uniprot ID A0A388NK91) derived from Paenibacillus thermophilus has an enzyme activity 22% improved compared to that of the SP (Uniprot ID D9TT09) from Thermoanaerobacterium thermosaccharolyticum, and a maximum fructose yield 19% higher.The SP (Uniprot ID A0A135L6L9) derived from Tepidibacillus decaturensis has an enzyme activity improved by 44% compared to the activity of the SP (Uniprot ID D9TT09) derived from Thermoanaerobacterium thermosaccharolyticum, and a maximum yield of fructose that is 9% higher. The increase in the maximum yield is important in commercial processes and is involved in the achievable maximum yield of fructose by each SP. Presumably, the contributing factors include product inhibition of sucrose phosphorylase by fructose, the rate of the reverse reaction (G1P + fructose ⇔ sucrose + P. i ) and, more broadly, the balance between fructose formation and fructose degradation in the later stages of the reaction.

[0055] The following examples provide a protocol for a person skilled in the art to determine the activity of an SP as part of an enzymatic method, the protocol including, for example, incubating the enzyme with its substrate and then measuring the amounts of reactants and products or subsequent downstream products via HPLC. The measurement of the relative activity of any two enzymes is carried out under the same reaction conditions such as buffer, pH, temperature.

[0056] Examples of SPs for use in the improved method of the present invention include an SP (Uniprot ID A0A1X2FWC2) derived from Thermoanaerobacterium sp PSU-2 having the amino acid sequence set forth in SEQ ID NO: 19, an SP (Uniprot ID L0IL15) derived from Thermoanaerobacterium thermosaccharolyticum having the amino acid sequence set forth in SEQ ID NO: 20, an SP (Uniprot ID F6BJS0) derived from Thermoanaerobacterium xylanolyticum having the amino acid sequence set forth in SEQ ID NO: 21, an SP (Uniprot ID A0A1Y3Q6Q6) derived from Thermobacillus sp ZCTH02-B1 having the amino acid sequence set forth in SEQ ID NO: 22, an SP (Uniprot ID Q84HQ2) derived from Bifidobacterium adolescentis having the amino acid sequence set forth in SEQ ID NO: 23, an SP (Uniprot ID A0A388NK91) derived from Paenibacillus thermophilus having the amino acid sequence set forth in SEQ ID NO: 24, an SP (Uniprot ID A0A135L6L9) derived from Tepidibacillus decaturensis having the amino acid sequence set forth in SEQ ID NO: 25, and proteins of SPs having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity with any one of SEQ ID NOs: 19 to 25, but are not limited thereto.

[0057] The SP for use in the improved method of the present invention generally belongs to a part of the glycoside hydrolase superfamily (IPR017853), more specifically the sucrose phosphorylase family (IPR022527), and includes the glycosyl hydrolase, family 13, catalytic domain (IPR006047). The glycosyl hydrolase domain consists of a (β / α)8 barrel and is the catalytic domain. The entire sucrose phosphorylase consists of four domains, namely, an N-terminal domain, a glycosyl hydrolase domain, a B domain (formed from a large loop within the glycosyl domain), and a C-terminal domain (Sprogoe D, van den Broek LA, Mirza O, Kastrup JS, Voragen AG, Gajhede M, Skov LK (February 2004) "Crystal structure of sucrose phosphorylase from Bifidobacterium adolescentis" Biochemistry. 43(5):1156-62.doi:10.1021 / bi0356395.PMID 14756551). Through sequence alignment to the well-studied sucrose phosphorylase from Leuconostoc mesenteroides (Uniprot Q59495), conservation of the catalytic residue Asp196 can be seen (Schwarz A, Nidetzky B (July 2006) "The Asp-196-->Ala mutant of Leuconostoc mesenteroides sucrose phosphorylase exhibits altered stereochemical course and kinetic mechanism of glucosyl transfer to and from phosphate" FEBS Letters.580(16):3905 - 10.doi:10.1016 / j.febslet.2006.06.020.PMID 16797542.), Glu237 (Schwarz A, Brecker L, Nidetzky B (May 2007) "Acid - base catalysis in Leuconostoc mesenteroides sucrose phosphorylase probed by site - directed mutagenesis and detailed kinetic comparison of wild - type and Glu237->Gln mutant enzymes". The Biochemical Journal. 403(3):441 - 9.doi:10.1042 / BJ20070042.PMC 1876375.PMID 17233628.), and Asp 295 (Mueller M, Nidetzky B (April 2007) "The role of Asp - 295 in the catalytic mechanism of Leuconostoc mesenteroides sucrose phosphorylase probed with site - directed mutagenesis". FEBS Letters. 581(7):1403 - 8.doi:10.1016 / j.febslet.2007.02.060.PMID 17350620).

[0058] Starch is the most widely used energy storage compound in nature, and most of it is stored in plant seeds. Natural starch contains linear amylose and branched amylopectin. Examples of starch derivatives include amylose, amylopectin, soluble starch, amylodextrin, maltodextrin, maltose, fructose, and glucose. Starch derivatives can be prepared by enzymatic hydrolysis or acid hydrolysis of starch. Specifically, enzymatic hydrolysis of starch can be catalyzed or enhanced by isoamylase (IA, EC.3.2.1.68) that hydrolyzes α-1,6-glucoside bonds, pullulanase (PA, EC.3.2.1.41) that hydrolyzes α-1,6-glucoside bonds, or α-amylase (EC 3.2.1.1) that cleaves α-1,4-glucoside bonds. Corn starch contains many branches that interfere with the action of αGP. Isoamylase can be used to debranch starch to obtain linear amylodextrin. Starch pretreated with isoamylase can result in a higher F6P concentration in the final product. Isoamylase and pullulanase cleave α-1,6-glycoside bonds, enabling more complete degradation of starch by α-glucan phosphorylase. Since α-amylase cleaves α-1,4-glycoside bonds, α-amylase is used to break down starch into fragments for more rapid conversion to hexoses and improved solubility.

[0059] Cellulose is the most abundant biological resource and a major component of plant cell walls. Non-food lignocellulosic biomass contains cellulose, hemicellulose, lignin, and other minor components. Pure cellulose, including Avicel (microcrystalline cellulose), regenerated amorphous cellulose, bacterial cellulose, filter paper, etc., can be prepared by a series of treatments. Examples of partially hydrolyzed cellulose-based substrates include water-insoluble cellodextrins with a degree of polymerization exceeding 7, water-soluble cellodextrins with a degree of polymerization of 3 - 6, cellobiose, glucose, fructose, etc. Examples of cellulose derivatives include pretreated biomass, regenerated amorphous cellulose, cellodextrin, cellobiose, fructose, glucose, etc. Furthermore, cellulose derivatives can be prepared by enzymatic hydrolysis of cellulose catalyzed by a cellulase mixture, by acids, or by pretreatment of biomass. In some methods of the present invention, G1P is produced from cellulose by cellulose phosphorylase. In some methods, G1P is generated from cellodextrin and cellobiose and free phosphate catalyzed by cellodextrin phosphorylase (CDP) and cellobiose phosphorylase (CBP).

[0060] In some improved methods of the present invention, the hexose is selected from allose, mannose, galactose, fructose, altrose, talose, sorbose, gulose, idose, allulose, inositol, and tagatose. A hexose is a monosaccharide having six carbon atoms. Hexoses can be classified by functional groups, where an aldohexose has an aldehyde at the 1-position and a ketohexose has its ketone at the 2-position. Aldohexoses (or aldoses) include allose, altrose, glucose, gulose, galactose, idose, talose, and mannose. Ketohexoses (or ketoses) include psicose (allulose), fructose, tagatose, and sorbose. Inositol has neither an aldehyde group nor a ketose group and is characterized as a cyclic carbon hexose. Using the improved method of the present invention, starch and its derivatives can be converted into hexoses selected from allose, mannose, galactose, fructose, altrose, talose, sorbose, gulose, tagatose, allulose, inositol, and idose. Accordingly, some of the embodiments of the present invention relate to improved methods for producing these individual hexoses.

[0061] The improved method of the present invention for producing hexose includes an additional step of dephosphorylating hexose phosphate using hexose phosphate phosphatase. In some improved methods of the present invention for producing hexose, the method steps are carried out in a single reaction vessel. In other improved methods of the present invention, the method steps are carried out in two or more single reaction vessels. In some improved methods, the method steps are carried out at a phosphate concentration of about 0.1 mM to about 150 mM without ATP and without NAD(P)(H), the phosphate is recycled, and / or the step of dephosphorylating hexose phosphate involves an energetically favorable chemical reaction. In some improved methods of the present invention, the method steps are carried out under at least one method condition of a temperature in the range of about 37 °C to about 85 °C, a pH in the range of about 5.0 to about 8.0, or a time of about 0.5 hour to about 48 hours. In some improved methods of the present invention, the method steps are carried out as a continuous reaction.

[0062] Some of the methods of the present invention for preparing hexoses include an additional step of dephosphorylating hexose phosphate using hexose phosphate phosphatase. The phosphatase used in the method of the present invention is specific for hexose phosphate. For example, allose 6-phosphate is converted to allose by allose 6-phosphate phosphatase, mannose 6-phosphate is converted to mannose by mannose 6-phosphate phosphatase, galactose 6-phosphate is converted to galactose by galactose 6-phosphate phosphatase, fructose 6-phosphate is converted to fructose by fructose 6-phosphate phosphatase, altrose 6-phosphate is converted to altrose by altrose 6-phosphate phosphatase, talose 6-phosphate is converted to talose by talose 6-phosphate phosphatase, sorbose 6-phosphate is converted to sorbose by sorbose 6-phosphate phosphatase, glucosone 6-phosphate is converted to glucosone by glucosone 6-phosphate phosphatase, tagatose 6-phosphate is converted to tagatose by tagatose 6-phosphate phosphatase, psicose 6-phosphate is converted to psicose by psicose 6-phosphate phosphatase, inositol 3-phosphate is converted to inositol by inositol monophosphatase, and idose 6-phosphate is converted to idose by idose 6-phosphate phosphatase. As used herein, specific means having a higher specific activity for the indicated hexose than for other hexoses. For example, allose 6-phosphate phosphatase has a higher specific activity for allose 6-phosphate than, for example, sorbose 6-phosphate or talose 6-phosphate. In the method of the present invention, the hexose phosphate phosphatase has a higher activity for the indicated hexose phosphate compared to other hexose phosphate intermediates in the method. As an illustrative example, in a method for converting maltodextrin to allose, allose 6-phosphate phosphatase has a higher activity for allose 6-phosphate compared to other hexose phosphate intermediates in the method, such as G1P, G6P, F6P, and psicose 6-phosphate.Please refer to FIGS. 2 and 4.

[0063] Sugar alcohols can be produced from various hexose sugars produced by the improved methods of the present invention. For example, mannitol and sorbitol can be produced, which are currently of commercial interest to the medical and food industries. The ketose or aldose products of these improved enzymatic methods can be reduced to the sugar alcohol form using a reducing agent such as hydrogen gas or sodium borohydride. Preferably, hydrogen gas is used as the reducing agent as already described in U.S. Pat. No. 6,570,043, U.S. Pat. No. 8,816,068, or U.S. Pat. No. 5,466,795.

[0064] In one embodiment, the improved method of the present invention relates to the production of allose. When starch or a starch derivative is the starting material, the improvement is selected from one or more of the following steps: a) converting G1P to G6P, catalyzed by a PGM having at least 90% sequence identity with any one of SEQ ID NOs: 2-8; b) converting a starch derivative to G1P, catalyzed by an αGP having at least 90% sequence identity with any one of SEQ ID NOs: 10-13; and c) transglycosylating a starch derivative, catalyzed by a 4GT having at least 90% sequence identity with any one of SEQ ID NOs: 15-17. When cellulose or a cellulose derivative is the starting material, the improvement is the step of converting G1P to G6P, catalyzed by a PGM having at least 90% sequence identity with any one of SEQ ID NOs: 2-8. When sucrose is the starting material, the improvement is selected from one or both of the following steps: a) converting G1P to G6P, catalyzed by a PGM having at least 90% sequence identity with any one of SEQ ID NOs: 2-8; and b) converting sucrose to G1P, catalyzed by an SP having at least 90% sequence identity with any one of SEQ ID NOs: 19-25. The method further includes the steps of converting G6P to fructose 6-phosphate (F6P), catalyzed by phosphoglucose isomerase (PGI); converting F6P to psicose 6-phosphate (P6P), catalyzed by psicose 6-phosphate 3-epimerase (P6PE); converting P6P to allose 6-phosphate (A6P), catalyzed by allose 6-phosphate isomerase (A6PI); and converting A6P to allose, catalyzed by allose 6-phosphate phosphatase (A6PP).

[0065] In one embodiment, the improved method of the present invention relates to the production of mannose. When starch or a starch derivative is the starting material, the improvement is selected from one or more of: a) a step of converting G1P to G6P catalyzed by a PGM having at least 90% sequence identity with any one of SEQ ID NOs: 2 to 8; b) a step of converting a starch derivative to G1P catalyzed by an αGP having at least 90% sequence identity with any one of SEQ ID NOs: 10 to 13; and c) a step of transglycosylating a starch derivative catalyzed by a 4GT having at least 90% sequence identity with any one of SEQ ID NOs: 15 to 17. When cellulose or a cellulose derivative is the starting material, the improvement is a step of converting G1P to G6P catalyzed by a PGM having at least 90% sequence identity with any one of SEQ ID NOs: 2 to 8. When sucrose is the starting material, the improvement is selected from one or both of: a) a step of converting G1P to G6P catalyzed by a PGM having at least 90% sequence identity with any one of SEQ ID NOs: 2 to 8; and b) a step of converting sucrose to G1P catalyzed by an SP having at least 90% sequence identity with any one of SEQ ID NOs: 19 to 25. The method further includes a step of converting G6P to fructose 6-phosphate (F6P) catalyzed by phosphoglucose isomerase (PGI), a step of converting F6P to mannose 6-phosphate (M6P) catalyzed by mannose 6-phosphate isomerase (M6PI) or phosphoglucose / phosphomannose isomerase (PGPMI), and a step of converting M6P to mannose catalyzed by mannose 6-phosphate phosphatase (M6PP).

[0066] In one embodiment, the improved method of the present invention relates to the production of galactose. When starch or a starch derivative is the starting material, the improvement is selected from one or more of: a) the step of converting G1P to G6P catalyzed by a PGM having at least 90% sequence identity with any one of SEQ ID NOs: 2-8; b) the step of converting a starch derivative to G1P catalyzed by an αGP having at least 90% sequence identity with any one of SEQ ID NOs: 10-13; and c) the step of transglycosylating a starch derivative catalyzed by a 4GT having at least 90% sequence identity with any one of SEQ ID NOs: 15-17. When cellulose or a cellulose derivative is the starting material, the improvement is the step of converting G1P to G6P catalyzed by a PGM having at least 90% sequence identity with any one of SEQ ID NOs: 2-8. When sucrose is the starting material, the improvement is selected from one or both of: a) the step of converting G1P to G6P catalyzed by a PGM having at least 90% sequence identity with any one of SEQ ID NOs: 2-8; and b) the step of converting sucrose to G1P catalyzed by an SP having at least 90% sequence identity with any one of SEQ ID NOs: 19-25. The method further includes the steps of converting G6P to fructose 6-phosphate (F6P) catalyzed by phosphoglucose isomerase (PGI), converting F6P to tagatose 6-phosphate (T6P) catalyzed by fructose 6-phosphate 4-epimerase (F6PE), converting T6P to galactose 6-phosphate (Gal6P) catalyzed by galactose 6-phosphate isomerase (Gal6PI), and converting Gal6P to galactose catalyzed by galactose 6-phosphate phosphatase (Gal6PP).

[0067] In one embodiment, the improved method of the present invention relates to the production of fructose. When starch or a starch derivative is the starting material, the improvement is selected from one or more of the following steps: a) converting G1P to G6P, catalyzed by a PGM having at least 90% sequence identity with any one of SEQ ID NOs: 2-8; b) converting a starch derivative to G1P, catalyzed by an αGP having at least 90% sequence identity with any one of SEQ ID NOs: 10-13; and c) transglycosylating a starch derivative, catalyzed by a 4GT having at least 90% sequence identity with any one of SEQ ID NOs: 15-17. When cellulose or a cellulose derivative is the starting material, the improvement is the step of converting G1P to G6P, catalyzed by a PGM having at least 90% sequence identity with any one of SEQ ID NOs: 2-8. When sucrose is the starting material, the improvement is selected from one or both of the following steps: a) converting G1P to G6P, catalyzed by a PGM having at least 90% sequence identity with any one of SEQ ID NOs: 2-8; and b) converting sucrose to G1P, catalyzed by an SP having at least 90% sequence identity with any one of SEQ ID NOs: 19-25. The method further includes a step of converting G6P to fructose 6-phosphate (F6P), catalyzed by phosphoglucose isomerase (PGI), and a step of converting F6P to fructose, catalyzed by fructose 6-phosphate phosphatase (F6PP).

[0068] In one embodiment, the improved method of the present invention relates to the production of allose. When starch or a starch derivative is the starting material, the improvement is selected from one or more of: a) a step of converting G1P to G6P catalyzed by a PGM having at least 90% sequence identity with any one of SEQ ID NOs: 2 to 8; b) a step of converting a starch derivative to G1P catalyzed by an αGP having at least 90% sequence identity with any one of SEQ ID NOs: 10 to 13; and c) a step of transglycosylating a starch derivative catalyzed by a 4GT having at least 90% sequence identity with any one of SEQ ID NOs: 15 to 17. When cellulose or a cellulose derivative is the starting material, the improvement is a step of converting G1P to G6P catalyzed by a PGM having at least 90% sequence identity with any one of SEQ ID NOs: 2 to 8. When sucrose is the starting material, the improvement is selected from one or both of: a) a step of converting G1P to G6P catalyzed by a PGM having at least 90% sequence identity with any one of SEQ ID NOs: 2 to 8; and b) a step of converting sucrose to G1P catalyzed by an SP having at least 90% sequence identity with any one of SEQ ID NOs: 19 to 25. The method further includes a step of converting G6P to fructose 6-phosphate (F6P) catalyzed by phosphoglucose isomerase (PGI), a step of converting F6P to P6P catalyzed by P6PE, a step of converting P6P to allose 6-phosphate (Alt6P) catalyzed by allose 6-phosphate isomerase (Alt6PI), and a step of converting the generated Alt6P to allose catalyzed by allose 6-phosphate phosphatase (Alt6PP).

[0069] In one embodiment, the improved method of the present invention relates to the production of talose. When starch or a starch derivative is the starting material, the improvement is selected from one or more of the following steps: a) converting G1P to G6P, catalyzed by a PGM having at least 90% sequence identity with any one of SEQ ID NOs: 2-8; b) converting a starch derivative to G1P, catalyzed by an αGP having at least 90% sequence identity with any one of SEQ ID NOs: 10-13; and c) transglycosylating a starch derivative, catalyzed by a 4GT having at least 90% sequence identity with any one of SEQ ID NOs: 15-17. When cellulose or a cellulose derivative is the starting material, the improvement is the step of converting G1P to G6P, catalyzed by a PGM having at least 90% sequence identity with any one of SEQ ID NOs: 2-8. When sucrose is the starting material, the improvement is selected from one or both of the following steps: a) converting G1P to G6P, catalyzed by a PGM having at least 90% sequence identity with any one of SEQ ID NOs: 2-8; and b) converting sucrose to G1P, catalyzed by an SP having at least 90% sequence identity with any one of SEQ ID NOs: 19-25. The method further includes the steps of converting G6P to fructose 6-phosphate (F6P), catalyzed by phosphoglucose isomerase (PGI); converting F6P to T6P, catalyzed by F6PE; converting T6P to talose 6-phosphate (Tal6P), catalyzed by talose 6-phosphate isomerase (Tal6PI); and converting Tal6P to talose, catalyzed by talose 6-phosphate phosphatase (Tal6PP).

[0070] In one embodiment, the improved method of the present invention relates to the production of sorbose. When starch or a starch derivative is the starting material, the improvement is selected from one or more of the following steps: a) converting G1P to G6P catalyzed by a PGM having at least 90% sequence identity with any one of SEQ ID NOs: 2-8; b) converting a starch derivative to G1P catalyzed by an αGP having at least 90% sequence identity with any one of SEQ ID NOs: 10-13; and c) transglycosylating a starch derivative catalyzed by a 4GT having at least 90% sequence identity with any one of SEQ ID NOs: 15-17. When cellulose or a cellulose derivative is the starting material, the improvement is the step of converting G1P to G6P catalyzed by a PGM having at least 90% sequence identity with any one of SEQ ID NOs: 2-8. When sucrose is the starting material, the improvement is selected from one or both of the following steps: a) converting G1P to G6P catalyzed by a PGM having at least 90% sequence identity with any one of SEQ ID NOs: 2-8; and b) converting sucrose to G1P catalyzed by an SP having at least 90% sequence identity with any one of SEQ ID NOs: 19-25. The method further includes the steps of converting G6P to fructose 6-phosphate (F6P) catalyzed by phosphoglucose isomerase (PGI), converting F6P to trehalose 6-phosphate (T6P) catalyzed by F6PE, converting T6P to sorbose 6-phosphate (S6P) catalyzed by sorbose 6-phosphate epimerase (S6PE), and converting S6P to sorbose catalyzed by sorbose 6-phosphate phosphatase (S6PP).

[0071] In one embodiment, the improved method of the present invention relates to the production of growth. When starch or a starch derivative is the starting material, the improvement is selected from one or more of: a) the step of converting G1P to G6P catalyzed by a PGM having at least 90% sequence identity with any one of SEQ ID NOs: 2-8; b) the step of converting a starch derivative to G1P catalyzed by an αGP having at least 90% sequence identity with any one of SEQ ID NOs: 10-13; and c) the step of transglycosylating a starch derivative catalyzed by a 4GT having at least 90% sequence identity with any one of SEQ ID NOs: 15-17. When cellulose or a cellulose derivative is the starting material, the improvement is the step of converting G1P to G6P catalyzed by a PGM having at least 90% sequence identity with any one of SEQ ID NOs: 2-8. When sucrose is the starting material, the improvement is selected from one or both of: a) the step of converting G1P to G6P catalyzed by a PGM having at least 90% sequence identity with any one of SEQ ID NOs: 2-8; and b) the step of converting sucrose to G1P catalyzed by an SP having at least 90% sequence identity with any one of SEQ ID NOs: 19-25. The method further includes the step of converting G6P to fructose 6-phosphate (F6P) catalyzed by phosphoglucose isomerase (PGI), the step of converting F6P to T6P catalyzed by F6PE, the step of converting S6P to gulose 6-phosphate (Gul6P) catalyzed by gulose 6-phosphate isomerase (Gul6PI), and the step of converting Gul6P to gulose catalyzed by gulose 6-phosphate phosphatase (Gul6PP).

[0072] In one embodiment, the improved method of the present invention relates to the production of idose. When starch or a starch derivative is the starting material, the improvement comprises one or more selected from: a) a step of converting G1P to G6P catalyzed by a PGM having at least 90% sequence identity with any one of SEQ ID NOs: 2-8; b) a step of converting a starch derivative to G1P catalyzed by an αGP having at least 90% sequence identity with any one of SEQ ID NOs: 10-13; and c) a step of transglycosylating a starch derivative catalyzed by a 4GT having at least 90% sequence identity with any one of SEQ ID NOs: 15-17. When cellulose or a cellulose derivative is the starting material, the improvement is a step of converting G1P to G6P catalyzed by a PGM having at least 90% sequence identity with any one of SEQ ID NOs: 2-8. When sucrose is the starting material, the improvement is selected from one or both of: a) a step of converting G1P to G6P catalyzed by a PGM having at least 90% sequence identity with any one of SEQ ID NOs: 2-8; and b) a step of converting sucrose to G1P catalyzed by an SP having at least 90% sequence identity with any one of SEQ ID NOs: 19-25. The method further comprises a step of converting G6P to fructose 6-phosphate (F6P) catalyzed by phosphoglucose isomerase (PGI), a step of converting F6P to T6P catalyzed by F6PE, a step of converting T6P to sorbose 6-phosphate (S6P) catalyzed by sorbose 6-phosphate epimerase (S6PE), a step of converting S6P to idose 6-phosphate (I6P) catalyzed by idose 6-phosphate isomerase (I6PI), and a step of converting I6P to idose catalyzed by idose 6-phosphate phosphatase (I6PP).

[0073] In one embodiment, the improved method of the present invention relates to the production of tagatose. When starch or a starch derivative is the starting material, the improvement is selected from one or more of the following steps: a) converting G1P to G6P, catalyzed by a PGM having at least 90% sequence identity with any one of SEQ ID NOs: 2-8; b) converting a starch derivative to G1P, catalyzed by an αGP having at least 90% sequence identity with any one of SEQ ID NOs: 10-13; and c) transglycosylating a starch derivative, catalyzed by a 4GT having at least 90% sequence identity with any one of SEQ ID NOs: 15-17. When cellulose or a cellulose derivative is the starting material, the improvement is the step of converting G1P to G6P, catalyzed by a PGM having at least 90% sequence identity with any one of SEQ ID NOs: 2-8. When sucrose is the starting material, the improvement is selected from one or both of the following steps: a) converting G1P to G6P, catalyzed by a PGM having at least 90% sequence identity with any one of SEQ ID NOs: 2-8; and b) converting sucrose to G1P, catalyzed by an SP having at least 90% sequence identity with any one of SEQ ID NOs: 19-25. The method further includes a step of converting G6P to fructose 6-phosphate (F6P), catalyzed by phosphoglucose isomerase (PGI); a step of converting F6P to tagatose 6-phosphate (T6P), catalyzed by fructose 6-phosphate epimerase (F6PE); and a step of converting T6P to tagatose, catalyzed by tagatose 6-phosphate phosphatase (T6PP).

[0074] In one embodiment, the improved method of the present invention relates to the production of psicose. When starch or a starch derivative is the starting material, the improvement is selected from one or more of the following steps: a) converting G1P to G6P, catalyzed by a PGM having at least 90% sequence identity with any one of SEQ ID NOs: 2-8; b) converting a starch derivative to G1P, catalyzed by an αGP having at least 90% sequence identity with any one of SEQ ID NOs: 10-13; and c) transglycosylating a starch derivative, catalyzed by a 4GT having at least 90% sequence identity with any one of SEQ ID NOs: 15-17. When cellulose or a cellulose derivative is the starting material, the improvement is the step of converting G1P to G6P, catalyzed by a PGM having at least 90% sequence identity with any one of SEQ ID NOs: 2-8. When sucrose is the starting material, the improvement is selected from one or both of the following steps: a) converting G1P to G6P, catalyzed by a PGM having at least 90% sequence identity with any one of SEQ ID NOs: 2-8; and b) converting sucrose to G1P, catalyzed by an SP having at least 90% sequence identity with any one of SEQ ID NOs: 19-25. The method further includes the steps of converting G6P to fructose 6-phosphate (F6P), catalyzed by phosphoglucose isomerase (PGI); converting F6P to psicose 6-phosphate (P6P), catalyzed by psicose 6-phosphate epimerase (P6PE); and converting P6P to psicose, catalyzed by psicose 6-phosphate phosphatase (P6PP).

[0075] In one embodiment, the improved method of the present invention relates to the production of inositol. When starch or a starch derivative is the starting material, the improvement comprises one or more selected from: a) the step of converting G1P to G6P, catalyzed by a PGM having at least 90% sequence identity with any one of SEQ ID NOs: 2-8; b) the step of converting a starch derivative to G1P, catalyzed by an αGP having at least 90% sequence identity with any one of SEQ ID NOs: 10-13; and c) the step of transglycosylating a starch derivative, catalyzed by a 4GT having at least 90% sequence identity with any one of SEQ ID NOs: 15-17. When cellulose or a cellulose derivative is the starting material, the improvement is the step of converting G1P to G6P, catalyzed by a PGM having at least 90% sequence identity with any one of SEQ ID NOs: 2-8. When sucrose is the starting material, the improvement is selected from one or both of: a) the step of converting G1P to G6P, catalyzed by a PGM having at least 90% sequence identity with any one of SEQ ID NOs: 2-8; and b) the step of converting sucrose to G1P, catalyzed by an SP having at least 90% sequence identity with any one of SEQ ID NOs: 19-25. The method further comprises the steps of converting G6P to inositol 3-phosphate (I3P) using inositol phosphate synthase and converting I3P to inositol using inositol monophosphatase. In another embodiment, the improved method of the present invention relates to the production of inositol from sucrose.

[0076] Figure 1 shows the enzymatic pathway for converting sucrose into the G6P intermediate. The improved method of the present invention includes one or more of the following improvements: SP comprises an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NOs: 19-25; and PGM comprises an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NOs: 2-8. In a preferred method of the present invention, SP comprises an amino acid sequence of any one of SEQ ID NOs: 19-25, and PGM comprises an amino acid sequence of any one of SEQ ID NOs: 2-8. In a more preferred method, SP comprises the amino acid sequence of SEQ ID NO: 21, and PGM comprises the amino acid sequence of SEQ ID NO: 8.

[0077] Figure 2 is a schematic diagram showing the enzymatic pathway for converting the starch derivative, maltodextrin, into the G6P intermediate. The following abbreviations are used: IA, isoamylase; PA, pullulanase; αGP, α-glucan phosphorylase or starch phosphorylase; 4GT, 4-glucanotransferase; and PGM, phosphoglucomutase. The improved method of the present invention includes one or more of the following improvements: PGM comprises an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NOs: 2-8; αGP comprises an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NOs: 10-13; and 4GT comprises an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NOs: 15-17. In a preferred method of the present invention, PGM comprises an amino acid sequence of any one of SEQ ID NOs: 2-8, αGP comprises an amino acid sequence of any one of SEQ ID NOs: 10-13, and 4GT comprises an amino acid sequence of any one of SEQ ID NOs: 15-17. In a more preferred method of the present invention, PGM comprises the amino acid sequence of SEQ ID NO: 8, αGP comprises the amino acid sequence of SEQ ID NO: 11, and 4GT comprises the amino acid sequence of SEQ ID NO: 17.

[0078] Figure 3 shows the enzymatic pathway for converting cellulose to the G6P intermediate. In the improved method of the present invention, PGM comprises an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NOs: 2-8. Preferably, PGM comprises the amino acid sequence of any one of SEQ ID NOs: 2-8. More preferably, PGM comprises the amino acid sequence of SEQ ID NO: 8. In some improved methods of the present invention for producing hexose, one or more of the method steps are carried out in a single reaction vessel. In other improved methods of the present invention, the method steps are carried out in two or more reaction vessels. Subsequently, the phosphate ions generated by the dephosphorylation of hexose phosphate can be reused in the method step of converting the starch derivative to G1P, particularly when all the method steps are carried out in a single bioreactor or reaction vessel. The ability to reuse phosphate allows the use of a non-stoichiometric amount of phosphate and keeps the reaction phosphate concentration low. This affects the overall pathway and the overall rate of the method, but does not limit the activity of the individual enzymes and enables the overall efficiency of the hexose production method.

[0079] In the improved method of the present invention for producing hexose, the method steps are carried out at a phosphate concentration of about 0.1 mM to about 150 mM without ATP and without NAD(P)(H), the phosphate is reused, and / or the step of dephosphorylating hexose phosphate involves an energetically favorable chemical reaction. In the improved method of the present invention, the method steps are carried out under at least one method condition of a temperature in the range of about 37 °C to about 85 °C, a pH in the range of about 5.0 to about 8.0, or a time of about 0.5 hours to about 48 hours, or a continuous method.

[0080] For example, the reaction phosphate concentration in each method can be in the range of about 0 mM to about 300 mM, about 0.1 mM to about 150 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 reaction phosphate concentration in each method can 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. When the phosphate concentration is low, the total phosphate is low, so the manufacturing cost is reduced and the phosphate removal cost is reduced. Also, inhibition of phosphatases or other enzymes during the method by high concentrations of free phosphate is prevented, and the possibility of phosphorus contamination is reduced.

[0081] Furthermore, each of the methods disclosed herein can be carried out without adding ATP as a source of phosphate, i.e., without containing ATP. Each method can also be carried out without the need to add NAD(P)(H), i.e., without containing NAD(P)(H).

[0082] Any suitable biologically compatible buffer known in the art, such as HEPES, PBS, BIS-TRIS, MOPS, DIPSO, Trizma, etc., can be used in each method of the present invention. The reaction buffer for the method according to the present invention can have a pH in the range of 5.0 to 8.0. More preferably, the reaction buffer pH can be in the range of about 6.0 to about 7.3. For example, the reaction buffer pH can be 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2 or 7.3.

[0083] The reaction buffer can also contain divalent metal cations. In some methods, the step is Mg 2+ , Zn 2+ , Ca 2+ , Co 2+ , Mn 2+It is carried out in the presence of a divalent metal cation selected from the group consisting of and combinations thereof. As is known in the art, appropriate salts can be used to introduce the desired metal cation.

[0084] In each method of the present invention, the reaction temperature at which the method step is carried out can be in the range of 37 to 85 °C. More preferably, the step can be carried out at a temperature in the range of about 40 °C to about 80 °C. The temperature can be, for example, about 40 °C, about 45 °C, about 50 °C, about 55 °C, about 60 °C, about 65 °C, about 70 °C, about 75 °C, about 80 °C, or about 85 °C. Preferably, the reaction temperature is about 40 °C, about 45 °C, about 50 °C, about 55 °C, about 60 °C, or about 65 °C. More preferably, the reaction temperature is in the range of about 50 °C to about 55 °C.

[0085] For some of the improved methods of the present invention, the reaction time can be adjusted as needed and can be in the range of about 0.5 hours to about 48 hours. For example, the reaction time can be about 1 hour, about 2 hours, about 4 hours, about 8 hours, about 12 hours, 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. More preferably, the reaction time is about 24 hours.

[0086] This method can also be carried out in one or more reactors as a continuous process without a set time limit. In a continuous process, for example, a maltodextrin solution is pumped through a bed of immobilized enzyme at a rate such that the conversion to tagatose is complete by the time the solution leaves one or more columns for downstream processing. For example, 200 g / L of maltodextrin can be pumped through a column packed with immobilized enzyme (maintained at, for example, 50 °C) such that maximum tagatose yield is achieved by the time the maltodextrin leaves the column. This methodology provides greater volumetric productivity than a batch process. This limits the time the product is in contact with the column and reaction conditions, reducing the potential for product degradation (e.g., potential hydroxymethylfurfural formation). Also, since automation of production is facilitated, operating costs are reduced.

[0087] The enzymes used in the steps of the present invention can take the form of soluble, immobilized, aggregated, captured, or aggregated proteins. These enzymes can be adsorbed onto insoluble organic or inorganic supports commonly used to improve functionality, as is known in the art. These supports 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 a specialized surface that promotes the attachment and activity of the immobilized enzyme. The enzyme can be immobilized on these solid supports via covalent, ionic, or hydrophobic interactions. The enzyme can also be immobilized by genetically engineered interactions, such as a covalent fusion to another protein or peptide sequence that has an affinity for the solid support, most often a polyhistidine sequence. The enzyme can be immobilized directly on the surface or surface coating, or on other proteins already present on the surface or surface coating. The enzymes can be immobilized all on one carrier, on individual carriers, or in two combinations (e.g., two enzymes are immobilized per carrier and then those carriers are mixed). These variations can be mixed in a uniform or defined layer to optimize the turnover in a continuous process. For example, at the start of the reactor, a layer of αGP can be present to ensure a high initial G1P increase. The enzymes can be immobilized all on one carrier, on individual carriers, or in groups. These enzymes can be mixed in a uniform or defined layer or zone to optimize the turnover.

[0088] Each of the methods according to the present invention can achieve a high yield due to a very favorable equilibrium constant for the overall reaction. Theoretically, a maximum yield of 99% can be achieved if the starting material is completely converted to the phosphorylated intermediate.

[0089] The method of the present invention reduces the manufacturing cost by using low-cost starting materials and reducing the costs associated with raw material and product separation. Starch and its derivatives are raw materials that are less expensive than, for example, lactose. When producing hexose from lactose, the manufacturing cost becomes high because glucose and other hexoses are separated via chromatography. Also, the step of dephosphorylating hexose by phosphatase is an irreversible phosphatase reaction regardless of the raw material. Therefore, hexose is produced in a very high yield while effectively minimizing subsequent product separation costs.

[0090] The method according to the present invention enables easy recovery of hexose and minimizes separation costs. In some preferred methods of the present invention, the recovery of the desired hexose is performed without chromatography separation. After generating hexose in a continuous reaction, the product is passed through microfiltration, ion exchange (cation, then anion, possible mixed bed for polishing), concentration, crystallization, crystal isolation, and drying. Because of the high yield of hexose, all that is necessary to purify hexose is the crystallization step. To further purify hexose before crystallization, nanofiltration can be used to eliminate the risk of enzymes being present in the crystallization process and remove untransformed dextrins (such as maltodextrin, maltotetraose, maltotriose, maltose, etc.) that can co-crystallize with hexose or limit the reusability of the mother liquor.

[0091] The improved method of the present invention also includes the production of G6P from sucrose, starch or starch derivatives, or cellulose or cellulose derivatives. G6P produced from the method of the present invention can be isolated and purified by techniques known in the art such as, for example, chromatography.

[0092] Example The following example describes an improved method using an enzyme with higher activity.

[0093] Materials and Methods

[0094] All chemicals including glucose 1-phosphate, magnesium chloride, maltodextrin DE4-7, and sodium phosphate (monobasic and dibasic) were of reagent grade or higher and were purchased from Sigma-Aldrich (St. Louis, MO, USA) or Fisher Scientific (Pittsburgh, PA, USA) unless otherwise specified. Maltotriose was purchased from Carbosynth (Berkshire, UK). Escherichia coli (E. coli) BL21(DE3) (Sigma-Aldrich, St. Louis, MO, USA) was used as the host cell for recombinant protein expression. ZYM-5052 medium containing 50 mg L-1 kanamycin was used for E. coli cell growth and recombinant protein expression.

[0095] Production and Purification of Recombinant Enzyme

[0096] E. coli BL21(DE3) strain carrying the protein expression plasmid (pET28a) was incubated in a 1 L Erlenmeyer flask containing 100 mL of ZYM-5052 medium supplemented with 50 mg L-1 kanamycin. Cells were grown at 30 °C for 16 - 24 h with rotary shaking at 220 rpm. Cells were harvested by centrifugation at 12 °C and washed once with either 300 mM NaCl and 5 mM imidazole in 20 mM HEPES (pH 7.5) or 20 mM HEPES (pH 7.5) (Ni purification). The cell pellet was resuspended in the same buffer and lysed by sonication. After centrifugation, the target protein in the supernatant was purified by standard methods. His-tagged proteins were purified using a gradient of increasing imidazole in the buffer described above with Profinity IMAC Ni-Charged Resin (Bio-Rad, Hercules, CA). The purity of the recombinant proteins was examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

[0097] Example 1 Improved Method with Higher Activity PGM

[0098] Various phosphoglucomutases (PGMs) were isolated and assayed for the conversion of G1P to G6P in a multi-step enzymatic reaction. The relative activity of each PGM was measured as follows. A reaction mixture of 50 mM HEPES pH 7.2, 5 mM MgCl2, 50 mM glucose 1-phosphate, 0.02 g / L PGM, 0.1 g / L PGI, and 0.1 g / L F6PP was prepared and incubated at 50 °C for 30 minutes. The reaction was stopped by filtration of the enzyme using a Vivaspin 2 concentrator (10,000 MWCO). The product, fructose, was evaluated using a Hi-Plex H+ column and a refractive index detector. Samples were evaluated in 5 mM H2SO4 at 0.6 mL / min at 65 °C for 15.5 minutes. The relative efficiency of each PGM in the enzymatic method was determined using the amount of fructose produced in 30 minutes. The increase in activity was determined using the average of the increase in the peak area and peak height of fructose.

[0099] Figure 19 is a chromatogram showing the activities of various PGMs. The chromatogram shows the levels of fructose made from glucose 1-phosphate after incubation with various PGMs, excess phosphoglucose isomerase, and excess fructose 6-phosphate phosphatase. PGM is the rate-limiting enzyme of the cascade. Table 1 shows that the PGMs for use in the improved method of the present invention have improved activity compared to the previously disclosed PGM from Thermococcus kodakaraensis (Uniprot ID Q68BJ6). See International Publication No. WO 2017 / 059278.

Table 1

[0100] Example 2 Improved method with higher activity αGP

[0101] The relative activity of each αGP was measured as follows. A 200 μL reaction solution containing 25 mM sodium phosphate pH 7.2, 5 mM MgCl2, 20 g / L maltodextrin DE4-7, 9 μg αGP, and 0.1 g / L PGM was prepared and incubated at 50 °C. Samples were mixed with 1.5 mM NAD + and 3 U / mL glucose 6-phosphate dehydrogenase at various time points. The reaction rate was obtained using the absorbance at 340 nm at each time point. This rate was used to confirm the specific activity of each αGP and was used for relative activity comparison.

[0102] Table 2 shows that the αGP for use in the improved method of the present invention has improved activity compared to the previously disclosed αGP (Uniprot ID G4FEH8) from Thermotoga maritima. See International Publication No. WO 2017 / 059278.

Table 2

[0103] Example 3 Improved method with 4GT having higher activity

[0104] The relative activity of each 4GT was measured as follows. A 200 μL reaction solution containing 50 mM sodium phosphate pH 7.2, 5 mM MgCl2, 20 g / L maltotriose, and 9 μg 4GT was prepared and incubated at 50 °C. At various time points, the samples were mixed with 1.5 mM NAD+, 1 mM ATP, 1 U / mL hexokinase, and 1 U / mL glucose 6-phosphate dehydrogenase. The reaction rate was obtained using the absorbance at 340 nm at each time point. This rate was used to confirm the specific activity of each 4GT and was used for relative activity comparison.

[0105] Table 3 shows that the 4GT for use in the improved method of the present invention has improved activity compared to the previously disclosed 4GT from Thermococcus litoralis (Uniprot ID O32462). See International Publication No. WO 2018 / 169957.

Table 3

[0106] Example 4 Improved method with SP having higher activity

[0107] Various sucrose phosphorylases predicted by homology with Uniprot ID D9TT09 (Verhaeghe et al., "The quest for a thermostable sucrose phosphorylase reveals sucrose 6’-phosphate phosphorylase as a novel specificity", Appl Microbiol Biotechnol. 2014 Aug;98(16):7027-37) were isolated and assayed for conversion from sucrose to glucose 6-phosphate.

[0108] The relative efficiency of each SP was measured as follows. Reactants of 25 mM sodium phosphate pH 7.2, 5 mM MgCl2, 200 g / L sucrose, 0.15 g / L SP, 0.1 g / L PGM, 0.1 g / L PGI, and 0.3 g / L F6PP were prepared and incubated at 50 °C. Samples were taken at 0 h, 2 h, 6 h, and 8 h. The reaction was stopped by filtration of the enzyme using a Vivaspin 2 concentrator (10,000 MWCO). The product, fructose, was evaluated using a Supel Cogel Pb column and a refractive index detector. Samples were evaluated in ultrapure water at 0.6 mL / min at 80 °C for 25 min. The relative activity of each SP was determined using the amount of fructose made at 2 h. The amount of fructose made at 6 h (confirmed complete at 8 h) indicates the difference in the maximum achievable yield of each SP.

[0109] The effect on the complete conversion of sucrose to fructose was also investigated. Of the eight enzymes tested compared to the benchmark (table below), seven showed improved activity over Uniprot ID D9TT09 and one showed an unexpected advantage in producing fructose from sucrose. Figure 18 shows a chromatogram of sucrose phosphorylase activity comparing Uniprot ID D9TT09 (benchmark SP) to Uniprot ID F6BJS0. At 2 h, the higher activity SP produces approximately 150% of the amount of fructose as the benchmark SP. The lower chromatogram compares Uniprot ID D9TT09 (benchmark SP) to Uniprot ID F6BJS0 with respect to maximum yield. At 6 h (the maximum yield for both reactions), the higher activity SP produces approximately 130% of the amount of fructose as the benchmark SP. Interestingly, the relative yield does not directly correlate with the relative activity. Presumably, contributing factors include product inhibition of sucrose phosphorylase by fructose, the rate of the reverse reaction (G1P + fructose ⇔ sucrose + P i) More broadly, it includes the balance between the formation of fructose and the decomposition of fructose in the later stages of the reaction. The comparative SP (Uniprot ID A0A0N8GPZ6) from Thermanaerothrix daxensis having the amino acid sequence shown in SEQ ID NO: 26 showed lower relative activity and a lower maximum yield of fructose compared to the previously disclosed SP. [Table 4]

[0110] Example 5 Improved Enzymatic Production of G6P

[0111] To visualize the improvement in enzyme activity, the conversion of maltodextrin to G6P was carried out using the previously disclosed αGP (Uniprot ID G4FEH8) and PGM (Uniprot ID Q68BJ6), and the method was compared with the method using αGP (Uniprot ID D1B926) and PGM (Uniprot ID A0A150LLZ1) having higher activity. A 200 μL reaction mixture containing 20 g / L maltodextrin DE5, 50 mM phosphate buffer pH 7.2, 5 mM MgCl2, 0.05 g / L αGP, and 0.005 g / L PGM was incubated at 50 °C for 30 minutes. The reaction was stopped by filtering the enzyme through a Vivaspin 2 concentrator (30,000 MWCO) and analyzed by HPLC (Agilent 1100 series) using an Agilent Hi-Plex H-column and a refractive index detector. The samples were evaluated in 5 mM H2SO4 at 0.6 mL / min at 65 °C for 15.5 minutes. The results were not quantified because the peaks of void, maltodextrin, and G6P were too close to reliably quantify the individual components (Figure 17), but clearly more G6P was produced using the enzymes αGP (Uniprot ID D1B926) and PGM (Uniprot ID A0A150LLZ1). [Sequence Listing Free-Text]

[0112] Sequence Listing 10 <223>Xaa can be any naturally occurring amino acid

Claims

1. An improved method for the enzymatic production of hexoses from starch or starch derivatives, the improved method comprising: a) a step of converting glucose 1-phosphate (G1P) to glucose 6-phosphate (G6P) catalyzed by phosphoglucomutase (PGM), wherein the PGM comprises an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NO: 8 or SEQ ID NOs: 2-7, the PGM having an activity that is at least 10% higher compared to the activity of PGM from Thermococcus kodakarensis (UniProt ID Q68BJ6) in the improved method, said step being included in the improved method.

2. The improved method according to claim 1, wherein the improved method further comprises: b) a step of converting a starch derivative to G1P catalyzed by α-glucan phosphorylase (aGP).

3. The improved method according to claim 2, wherein the improved method further comprises: c) a step of transglycosylating a starch derivative catalyzed by 4-α-glucanotransferase (4GT).

4. An improved method for the enzymatic production of hexoses from cellulose or cellulose derivatives, the improved method comprising: a step of converting G1P to glucose 6-phosphate (G6P) catalyzed by phosphoglucomutase (PGM), wherein the PGM comprises an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NO: 8 or SEQ ID NOs: 2-7, the PGM having an activity that is at least 10% higher compared to the activity of PGM from Thermococcus kodakarensis (UniProt ID Q68BJ6) in the improved method, said step being included in the improved method.

5. An improved method for the enzymatic production of hexoses from sucrose, the improved method comprising: a) a step of converting glucose 1-phosphate (G1P) to glucose 6-phosphate (G6P) catalyzed by phosphoglucomutase (PGM), wherein the PGM comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 8 or any one of SEQ ID NOs: 2-7, The PGM has an activity that is at least 10% higher compared to the activity of PGM (UniProt ID Q68BJ6) from Thermococcus kodakaraensis in the improved method. The above steps, and b) converting sucrose to glucose 1-phosphate (G1P) using sucrose phosphorylase (SP). An improved method.

6. The improved method according to any one of claims 1 to 5, wherein the hexose is selected from the group consisting of allose, mannose, galactose, fructose, altrose, talose, sorbose, gulose, idose, allulose, inositol and tagatose.

7. The improved method according to claim 1, wherein the starch derivative is selected from the group consisting of amylose, amylopectin, soluble starch, amylodextrin, maltotriose, maltose and maltodextrin.

8. The improved method according to any one of claims 1 to 7, further comprising the step of dephosphorylating hexose phosphate using hexose phosphate phosphatase.

9. The improved method according to claim 8, wherein the steps of the method are carried out in a single reaction vessel.

10. The steps of the method are carried out at a phosphate concentration of 0.1 mM to 150 mM without ATP (ATP-free) and without NAD(P)(H) (NAD(P)(H)-free), the phosphate is reused, and / or the step of dephosphorylating hexose phosphate involves an energetically favorable chemical reaction.

11. The steps of the method are at a temperature in the range of 37°C to 85°C, at a pH in the range of 5.0 to 8.0, or for 0.5 hours to 48 hours, or as a continuous reaction, under at least one of the method conditions. The improved method according to any one of claims 1 to 10.

12. The improved method according to claim 6, further comprising the step of reducing the hexose to its sugar alcohol.

13. The improved method according to claim 2, wherein the aGP comprises an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NOs: 11, 10, 12, and 13, and has an activity that is at least 10% higher compared to the activity of aGP (UniProt ID G4FEH8) derived from Thermotoga maritima.

14. The improved method according to claim 3, wherein the 4GT comprises an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NOs: 17, 15, and 16, and has an activity that is at least 10% higher compared to the activity of 4GT (UniProt ID O32462) derived from Thermococcus litoralis.

15. The improved method according to claim 5, wherein the SP comprises an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NOs: 23, 19 - 22, and 24 - 25, and has an activity that is at least 10% higher compared to the SP (UniProt ID D9TT09) derived from Thermoanaerobacterium thermosaccharolyticum.

16. The improved method according to claim 5, wherein the SP comprises an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NOs: 23, 19 - 22, and 24 - 25, and has an achievable yield that is at least 10% higher compared to the SP (UniProt ID D9TT09) derived from Thermoanaerobacterium thermosaccharolyticum.

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