Enzymatic production of hexoses

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 priority to U.S. Patent Application No. 62 / 752,061, filed October 29, 2018, and U.S. Patent Application No. 62 / 857,543, filed June 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 the intermediate, glucose 6-phosphate (G6P), using an enzyme with higher activity than previously reported. [Background technology]

[0003] Hexoses are monosaccharides containing six carbon atoms. Hexoses can be classified by their functional group: aldohexoses have an aldehyde at position 1, and ketohexoses have a ketone at position 2. 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 without an aldehyde or ketose group and is characterized as a carbocyclic sugar.

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

[0005] Despite the development of high-yield enzymatic hexose production, there remains a need to provide further improved methods for producing hexoses, e.g., by which higher yields can be obtained with less enzyme. There is 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 enzyme combinations that are more effective at converting sugars to G6P intermediates. Summary of the Invention [Means for solving the problem]

[0006] The invention described herein generally relates to improved processes for preparing hexoses by enzymatic conversion from various sugar starting materials. The sugars may be selected from starch or starch derivatives, cellulose or cellulose derivatives, or sucrose. In the improved processes of the invention, as shown, the enzymes used in the process steps have improved activity over previously disclosed enzymes for the preparation of hexoses.

[0007] Some improved methods of the present invention for enzymatically producing hexose from starch or a starch derivative include at least one of the following steps: a) converting glucose 1-phosphate (G1P) to glucose 6-phosphate (G6P), catalyzed by phosphoglucomutase (PGM), where PGM comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 2-8; b) converting the starch derivative to G1P, catalyzed by alpha-glucan phosphorylase (αGP), where αGP comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 10-13; and transglycosylating the starch derivative, catalyzed by 4-α-glucan transferase (4GT), where 4GT comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 15-17.

[0008] In some improved methods of the present invention for enzymatically producing hexose from cellulose or cellulose derivatives, the improvements include converting G1P to glucose 6-phosphate (G6P) catalyzed by phosphoglucomutase (PGM), wherein PGM comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 2-8.

[0009] Some improved methods of the present invention for enzymatically producing hexose from sucrose include at least one of the following steps: a) 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 to any one of SEQ ID NOs: 2-8; and b) converting sucrose to glucose 1-phosphate (G1P) using sucrose phosphorylase, wherein sucrose phosphorylase comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 19-25.

[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 comprise the step of dephosphorylating the hexose phosphate using a hexose phosphate phosphatase.

[0011] In some improved methods of the present invention, the method steps are performed in a single reaction vessel. In other improved methods of the present invention, the method steps are performed in two or more reaction vessels. In some improved methods of the present invention, the method steps are performed in the absence of ATP (ATP-free), NAD(P)(H) (NAD(P)(H)-free), at a phosphate concentration of about 0.1 mM to about 150 mM, where phosphate is recycled, and / or the step of dephosphorylating the hexose phosphate involves an energetically favorable chemical reaction. In some improved methods of the present invention, the method steps are performed under at least one of the following process conditions: 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 for about 0.5 hours to about 48 hours. In some improved methods of the present invention, the method steps are performed as a continuous reaction. [Brief explanation of the drawings]

[0012] [Figure 1] 1 is a schematic diagram showing the enzymatic pathway for converting sucrose to G6P. The following abbreviations are used: SP, sucrose phosphate, and PGM, phosphoglucomutase. The improved methods of the invention include one or more of the following improvements: SP comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 19-25; and PGM comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 2-8.

[0013] [Figure 2]1 is a schematic diagram showing the enzymatic pathway for converting the starch derivative, maltodextrin, to G6P. The following abbreviations are used: IA, isoamylase; PA, pullulanase; αGP, α-glucan phosphorylase or starch phosphorylase; 4GT, 4-glucan transferase; and PGM, phosphoglucomutase. The improved methods of the present invention include one or more of the following improvements: PGM comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOS: 2-8; αGP comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOS: 10-13; and 4GT comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOS: 15-17.

[0014] [Figure 3] 1 is a schematic diagram showing the enzymatic pathway for converting cellulose to G6P. The following abbreviations are used: CDP, cellodextrin phosphorylase, and PGM, phosphoglucomutase. In the improved method of the present invention, PGM comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 2-8.

[0015] [Figure 4] 1 is a schematic diagram showing an enzymatic pathway for converting G6P to allose. The following abbreviations are used: PGI, phosphoglucoisomerase; P6PE, psicose-6-phosphate-3-epimerase; A6PI, allose-6-phosphate isomerase; A6PP, allose-6-phosphate phosphatase. For an enzymatic pathway for an improved method according to the present invention, this pathway is combined with the pathway of FIG. 1, FIG. 2, or FIG. 3.

[0016] [Figure 5] 1 is a schematic diagram showing an enzymatic pathway for converting G6P to mannose. The following abbreviations are used: PGI, phosphoglucoisomerase; PMI, phosphomannose isomerase; and M6PP, mannose 6-phosphate phosphatase. For an enzymatic pathway for an improved method according to the invention, combine this pathway with the pathways of FIG. 1, FIG. 2, or FIG. 3.

[0017] [Figure 6] 1 is a schematic diagram showing an enzymatic pathway for converting G6P to galactose. The following abbreviations are used: PGI, phosphoglucoisomerase; F6PE, fructose 6-phosphate isomerase; Gal6PI, galactose 6-phosphate isomerase; Gal6PP, galactose 6-phosphate phosphatase. For an enzymatic pathway for an improved method according to the invention, combine this pathway with the pathways of FIG. 1, FIG. 2, or FIG. 3.

[0018] [Figure 7] 1 is a schematic diagram showing an enzymatic pathway for converting G6P to fructose. The following abbreviations are used: PGI, phosphoglucoisomerase; F6PP, fructose-6-phosphate phosphatase. For an enzymatic pathway for an improved method according to the present invention, combine this pathway with the pathways of FIG. 1, FIG. 2, or FIG. 3.

[0019] [Figure 8] 1 is a schematic diagram showing an enzymatic pathway for converting G6P to altrose. The following abbreviations are used: PGI, phosphoglucoisomerase; P6PE, psicose-6-phosphate epimerase; Alt6PI, altrose-6-phosphate isomerase; Alt6PP, altrose-6-phosphate phosphatase. For an enzymatic pathway for an improved method according to the present invention, this pathway is combined with the pathway of FIG. 1, FIG. 2, or FIG. 3.

[0020] [Figure 9] 1 is a schematic diagram showing an enzymatic pathway for converting G6P to talose. The following abbreviations are used: PGI, phosphoglucoisomerase; F6PE, fructose-6-phosphate epimerase; Tal6PI, talose-6-phosphate isomerase; Tal6PP, talose-6-phosphate phosphatase. For an enzymatic pathway for an improved method according to the invention, combine this pathway with the pathways of FIG. 1, FIG. 2, or FIG. 3.

[0021] [Figure 10]1 is a schematic diagram showing an enzymatic pathway for converting G6P to sorbose. The following abbreviations are used: PGI, phosphoglucoisomerase; F6PE, fructose-6-phosphate epimerase; S6PE, sorbose-6-phosphate epimerase; S6PP, sorbose-6-phosphate phosphatase. For an enzymatic pathway for an improved method according to the invention, combine this pathway with the pathways of FIG. 1, FIG. 2, or FIG. 3.

[0022] [Figure 11] 1 is a schematic diagram showing an enzymatic pathway for converting G6P to gulose. The following abbreviations are used: PGI, phosphoglucoisomerase; F6PE, fructose-6-phosphate epimerase; S6PE, sorbose-6-phosphate epimerase; Gul6PI, gulose-6-phosphate isomerase; Gul6PP, gulose-6-phosphate phosphatase. For an enzymatic pathway for an improved method according to the invention, combine this pathway with the pathway of FIG. 1, FIG. 2, or FIG. 3.

[0023] [Figure 12] 1 is a schematic diagram showing an enzymatic pathway for converting G6P to idose. The following abbreviations are used: PGI, phosphoglucoisomerase; F6PE, fructose-6-phosphate epimerase; S6PE, sorbose-6-phosphate epimerase; 16PI, idose-6-phosphate isomerase; 16PP, idose-6-phosphate phosphatase. For an enzymatic pathway for an improved method according to the invention, combine this pathway with the pathway of FIG. 1, FIG. 2, or FIG. 3.

[0024] [Figure 13] 1 is a schematic diagram showing the enzymatic pathway for converting G6P to tagatose. The following abbreviations are used: PGI, phosphoglucoisomerase; F6PE, fructose-6-phosphate epimerase; T6PP, tagatose-6-phosphate phosphatase. For the enzymatic pathway for the improved method of the present invention, this pathway is combined with the pathway of FIG. 1, FIG. 2 or FIG. 3.

[0025] [Figure 14]1 is a schematic diagram showing an enzymatic pathway for converting G6P to inositol. The following abbreviations are used: IPS, inositol-phosphate synthase; IMP, inositol monophosphatase. For an enzymatic pathway for an improved method according to the invention, combine this pathway with the pathways of FIG. 1, FIG. 2 or FIG. 3.

[0026] [Figure 15] 1 is a schematic diagram showing an enzymatic pathway for converting G6P to psicose (allulose). The following abbreviations are used: PGI, phosphoglucoisomerase; P6PE, psicose-6-phosphate epimerase; P6PP, psicose-6-phosphate phosphatase. For an enzymatic pathway for an improved method according to the present invention, this pathway is combined with the pathway of FIG. 1, FIG. 2, or FIG. 3.

[0027] [Figure 16] FIG. 1 shows Gibbs energies of reaction between intermediates based on Gibbs energies of formation for the conversion of glucose 1-phosphate to another hexose.

[0028] [Figure 17] HPLC chromatograms showing the conversion of maltodextrins to G6P. (Dashed line) 0-hour chromatogram; (Dotted line) 30-minute method using previously disclosed αGP (Uniprot ID G4FEH8) and PGM (Uniprot ID Q68BJ6); (Solid line) 30-minute "improved" method with more active αGP (Uniprot ID D1B926) and more active PGM (Uniprot ID A0A150LLZ1). (1) void and maltodextrins, (2) G1P and G6P, (3) maltotriose, and (4) maltose.

[0029] [Figure 18]Figure 1 shows chromatograms of sucrose phosphorylase activity. The top chromatogram shows the reaction with Uniprot ID D9TT09 (reference SP) compared to a more active SP, Uniprot ID F6BJS0. The 0-hour reaction (sucrose to fructose) is shown as a dashed line, and 2-hour reactions using the same amount of SP and other enzymes are shown for both the reference (solid line) and the more active SP (dotted line). At 2 hours, the more active SP produces approximately 150% of the amount of fructose as the reference SP. The bottom chromatogram compares Uniprot ID D9TT09 (reference SP) to the more active Uniprot ID F6BJS0. The 0-hour reaction (sucrose to fructose) is shown as a dashed line, and 6-hour reactions using the same amount of SP and other enzymes are shown for both the reference SP (solid line) and the more active SP (dotted line). At 6 h (maximum yield for both reactions), the more active SP produces approximately 130% of the amount of fructose as the reference SP.

[0030] [Figure 19] Figure 1 shows a chromatogram of the reaction with Uniprot ID A0A150LLZ1 compared to a more highly active PGM. The chromatogram shows the level of fructose made from glucose 1-phosphate after incubation with limiting PGM, excess phosphoglucoisomerase, and excess fructose 6-phosphate phosphatase. Using equal amounts of all other PGMs improves fructose production, thus clearly demonstrating the enhanced PGM activity due to its role as the limiting enzyme in the enzymatic process. In the figure, Uniprot ID A0A0P6YKY9 = solid line, Uniprot ID E8N4Y6 = dashed line, Uniprot ID R7RR04 = dotted line, Uniprot ID A0A023DI95 = circle line, UniParc ID UPI0001D17AE3 = triangle line, Uniprot ID A0A150LLZ1 = square line. The activity of the much lower (see Table 1) reference PGM Uniprot ID Q68BJ6 is not shown. DETAILED DESCRIPTION OF THE INVENTION

[0031] The inventions described herein provide enzymatic pathways or methods for synthesizing hexoses with high product yields while significantly reducing product separation costs and hexose production costs. The enzymatic methods described herein generally relate to improved methods for preparing hexose monosaccharides from sugars by enzymatic conversion. An artificial (non-natural) ATP-free enzymatic route for converting sugars to hexoses using a cell-free enzyme cocktail is provided. In contrast to cell-based production methods, the enzymatic methods of the invention involve cell-free preparation of hexoses and have relatively high reaction rates due to the removal of cell membranes, which often slow the transport of substrates / products into and out of cells. The methods also have end products that are free of nutrient-rich fermentation media / cellular metabolites. The sugars may 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 over enzymes previously disclosed for the preparation of hexoses. In one embodiment, the present invention relates to an improved method for converting starch and its derivatives to hexoses by using at least one of αGP, PGM, and 4GT with higher activity instead of the previously disclosed αGP, PGM, and 4GT (see WO 2018 / 169957, which discloses α-glucan phosphorylase (αGP) from Thermotoga maritima (Uniprot ID G4FEH8), phosphoglucomutase (PGM) from Thermococcus kodakaraensis (Uniprot ID Q68BJ6), and 4-α-glucanoltransferase from Thermococcus litoralis (Uniprot ID O32462). Some improved methods of the present invention for enzymatically producing hexose from starch or a starch derivative include at least one of the following steps: a) converting glucose 1-phosphate (G1P) to glucose 6-phosphate (G6P), catalyzed by phosphoglucomutase (PGM), where PGM comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 2-8; b) converting the starch derivative to G1P, catalyzed by alpha-glucan phosphorylase (αGP), where αGP comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 10-13; and transglycosylating the starch derivative, catalyzed by 4-α-glucan transferase (4GT), where 4GT comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 15-17.In the improved method of the present invention, the method comprises the steps of: a) 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 to any one of SEQ ID NOs: 2 to 8; b) 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 to any one of SEQ ID NOs: 10 to 13; and transglycosylating the starch derivative, catalyzed by 4-α-glucan transferase (4GT), wherein 4GT comprises an amino acid sequence having at least 90% sequence identity to 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 hexose using a more active PGM instead of PGM from Thermococcus kodakaraensis (Uniprot ID Q68BJ6). In some improved methods of the present invention for enzymatically producing hexose from cellulose or cellulose derivatives, the improvement comprises converting G1P to glucose 6-phosphate (G6P) catalyzed by PGM, wherein PGM comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 2-8.

[0033] In yet another embodiment, the present invention relates to an improved method for converting sucrose to hexose using at least one of a SP having higher activity in place of the previously disclosed SP from Thermoanaerobacterium thermosaccharolyticum (Uniprot ID D9TT09) and a PGM having higher activity in place of PGM from Thermococcus kodakaraensis (Uniprot ID Q68BJ6). Some improved methods of the present invention for enzymatically producing hexose from sucrose include at least one of the following steps: a) 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 to any one of SEQ ID NOs: 2-8; and b) converting sucrose to glucose 1-phosphate (G1P) using sucrose phosphorylase, wherein sucrose phosphorylase comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 19-25. In the improved method of the present invention, the method comprises: a) 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 to any one of SEQ ID NOs: 2 to 8; and b) 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 to 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 comprise the step of dephosphorylating the hexose phosphate using a hexose phosphate phosphatase.

[0035] Some improved methods according to the present invention for enzymatically producing hexose from starch or a starch derivative, cellulose or a cellulose derivative, or sucrose include 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 to any one of SEQ ID NOs: 2 to 8. Preferably, PGM comprises the amino acid sequence of any one of SEQ ID NOs: 2 to 8. More preferably, 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 hexoses from sugars, the reaction proceeds in the direction of G6P, which is then further processed downstream, and the final enzymatic step of the method is an energetically favorable irreversible step that dephosphorylates the hexose phosphate.

[0037] In the improved methods of the invention, the PGM has higher activity than the aforementioned PGM (Uniprot ID Q68BJ6) from Thermococcus kodakaraensis, which has the amino acid sequence set forth in SEQ ID NO: 1. Preferably, the PGM used in the methods of the invention has an enzymatic activity that is 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 over the activity of PGM (Uniprot ID Q68BJ6) from Thermococcus kodakaraensis.

[0038] For example, as shown in Example 1, PGM for use in the methods of the invention has improved activity over PGM from Thermococcus kodakaraensis (Uniprot ID Q68BJ6), PGM from Geobacillus stearothermophilus NUB3621 (Uniprot ID A0A023CRS6) has about 700% improved enzymatic activity, PGM from Caldibacillus debilis (Uniprot ID A0A150LLZ1) has about 1900% improved enzymatic activity, and PGM from Geobacillus thermoglucosidasius (Uniprot ID A0A150LLZ1) has about 1900% improved enzymatic activity. PGM from Parageobacillus caldoxylosilyticus NBRC107762 (Uniprot ID A0A023DI95) had approximately 1980% improved enzyme activity, PGM from Thermobrachium celere DSM8682 (Uniprot ID R7RR04) had approximately 5100% improved enzyme activity, PGM from Anaerolinea thermophila (Uniprot ID E8N4Y6) had approximately 5800% improved enzyme activity, and PGM from Thermanaerothrix daxensis (Uniprot ID A0A0P6YKY9) has an approximately 6500% improved enzymatic activity. The following example provides one skilled in the art with a protocol for determining the activity of a PGM as part of an enzymatic method, which may involve, for example, incubating the enzyme with its substrate and then measuring the amount of reactant and product or downstream product via HPLC. Measurements of the relative activity of any two enzymes are performed under identical reaction conditions, such as buffer, pH, temperature, etc.

[0039] Examples of PGMs for use in the improved methods 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 (Uniprot ID UPI0001D17AE3) having the amino acid sequence set forth in SEQ ID NO:4, PGM from Parageobacillus caldoxylosilyticus NBRC107762 (Uniprot ID UPI0001D17AE3) having the amino acid sequence set forth in SEQ ID NO:5, and PGM from Parageobacillus caldoxylosilyticus NBRC107762 (Uniprot ID UPI0001D17AE3) having the amino acid sequence set forth in SEQ ID NO:6. A0A023DI95), PGM from Thermobrachium celere DSM8682 having the amino acid sequence set forth in SEQ ID NO:6 (Uniprot ID R7RR04), PGM from Anaerolinea thermophila having the amino acid sequence set forth in SEQ ID NO:7 (Uniprot ID E8N4Y6), PGM from Thermanaerothrix daxensis having the amino acid sequence set forth in SEQ ID NO:8 (Uniprot ID A0A0P6YKY9), and proteins of PGM comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to any one of SEQ ID NOs:2-8.

[0040] PGMs for use in the improved methods of the present invention are generally part of the α-D-phosphohexomutase superfamily (IPR005841) and contain four domains that form the enzyme's active site. The first three domains comprise the 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 a conserved serine residue for phosphoryl transfer to the active site (Ser147 in SEQ ID NO:3). The second domain contributes conserved Mg2+-binding residues to the active site (Asp306, Asp308, and Asp310 in SEQ ID NO:3). The third domain contributes conserved residues for substrate specificity to the active site (Glu406 and Ser408 in SEQ ID NO:3). The fourth domain contributes a conserved residue for phosphate binding to the active site (Arg538 in SEQ ID NO:3). Additionally, a positively charged residue (Lys / Arg) is conserved that plays a role in catalysis at Lys420 in SEQ ID NO: 3. Conserved residues are taken 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 include converting a starch derivative to G1P, catalyzed by αGP, wherein αGP comprises an amino acid sequence having at least 90% amino acid sequence identity with any one of SEQ ID NOs: 10 to 13. Preferably, αGP comprises the amino acid sequence of any one of SEQ ID NOs: 10 to 13. More preferably, αGP comprises the amino acid sequence of SEQ ID NO: 11.

[0042] Alpha-glucan phosphorylase or starch phosphorylase (αGP) (EC 2.4.1.1) phosphorolytically cleaves maltooligosaccharides to produce G1P. Starch phosphorylase also catalyzes the reverse reaction, i.e., the transfer of a glucosyl unit from G1P to the non-reducing end of an α-1,4-D-glucan chain, with the release of phosphate. Generally, the degree of polymerization of the oligosaccharide chain is 4 or greater. In the improved method of the present invention for producing hexoses from starch derivatives, the reaction proceeds toward G1P, which is then further processed downstream, and the final enzymatic step of the method is an energetically favorable irreversible step that dephosphorylates the hexose phosphate.

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

[0044] Examples of αGP for use in the improved methods of the invention include αGP from Thermus thermophilus having the amino acid sequence set forth in SEQ ID NO: 10 (Uniprot ID Q5SJ42), αGP from Thermus sp. CCB_US3_UF1 having the amino acid sequence set forth in SEQ ID NO: 11 (Uniprot ID G8NCC0), αGP from Thermoanaerobacter pseudethanolicus strain ATCC 33223 having the amino acid sequence set forth in SEQ ID NO: 12 (Uniprot ID B0K7V8), αGP from Thermanaerobacter acidaminovorans strain ATCC 49978 having the amino acid sequence set forth in SEQ ID NO: 13 (Uniprot ID B0K7V9), and αGP from Thermanaerovibrio acidaminovorans strain ATCC 49978 having the amino acid sequence set forth in SEQ ID NO: 14 (Uniprot ID B0K7V9). D1B926), and αGP proteins having at least 90%, at least 95%, at least 97%, at least 99% or 100% amino acid sequence identity to SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 or SEQ ID NO:13.

[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 conserved residues 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 a starch derivative, the improvement comprises transglycosylating the starch derivative using 4-α-glucantransferase (4GT), wherein 4GT comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 15-17. Preferably, 4GT comprises the amino acid sequence of any one of SEQ ID NOs: 15-17. More preferably, αGP comprises the amino acid sequence of SEQ ID NO: 17.

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

[0048] Some methods of the present invention for producing hexose include an energetically favorable dephosphorylation step to convert hexose phosphates to hexose. In such methods, particularly when carried out in a single reaction vessel, 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 toward amylodextrins with a degree of polymerization (DP) less than 4. In such methods according to the present invention, 4GT is added to the reaction to improve the overall yield. 4GT transglycosylates amylodextrins with a DP less than 4, such as maltotriose, so that they are converted to long-chain amylodextrins that can be degraded by αGP. Without 4GT, maltotriose accumulates as the end product of the process rather than being recycled into long-chain amylodextrins that can be re-entered into the process to increase yield. 4GT increases yield when amylodextrins are sufficiently degraded by αGP to produce significant amounts of maltotriose. This occurs due to an energetically favorable dephosphorylation step and phosphate recycling.

[0049] In the improved methods of the present invention, 4GT has higher activity than the aforementioned 4-α-glucantransferase (4GT) from Thermococcus litoralis (Uniprot ID O32462), which has the amino acid sequence set forth in SEQ ID NO: 14. Preferably, the 4GT used in the methods of the present invention has an enzymatic activity that is at least 10%, at least 50%, at least 100%, at least 150%, or at least 200% improved over the activity of 4GT from Thermococcus litoralis (Uniprot ID O32462). For example, as shown in Example 3, 4GT from Oceanithermus profundus DSM14977 (Uniprot ID E4U8S9) has an approximately 128% improved enzymatic activity over 4GT from Thermococcus litoralis (Uniprot ID O32462), 4GT from Meiothermus silvanus strain ATCC700542 (Uniprot ID D7BF07) has an approximately 212% improved enzymatic activity over 4GT from Thermococcus litoralis (Uniprot ID O32462), and 4GT from Anaerolinea thermophila strain DSM14523 (Uniprot ID D7BF08) has an approximately 212% improved enzymatic activity over 4GT from Thermococcus litoralis (Uniprot ID O32462). The enzyme 4GT (E8MXP8) from Thermococcus litoralis (Uniprot ID O32462) has an enzymatic activity that is approximately 184% improved. The following example provides a protocol for determining the activity of 4GT as part of an enzymatic method, which involves, for example, incubating the enzyme with its substrate and then measuring the amount of glucose by spectrophotometry. Measurement of the relative activity of any two enzymes is performed under identical reaction conditions, such as buffer, pH, and temperature.

[0050] Examples of 4GT for use in the improved methods of the present invention include, but are not limited to, 4GT from Oceanithermus profundus DSM 14977 having the amino acid sequence set forth in SEQ ID NO: 15 (Uniprot ID E4U8S9), 4GT from Meiothermus silvanus strain ATCC 700542 having the amino acid sequence set forth in SEQ ID NO: 16 (Uniprot ID D7BF07), 4GT from Anaerolinea thermophila strain DSM 14523 having the amino acid sequence set forth in SEQ ID NO: 17 (Uniprot ID E8MXP8), and 4GT proteins 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.

[0051] The 4-glucantransferase for use in the improved methods of the present invention is genetically part of the glycoside transferase superfamily (IPR017853), more specifically, glycoside hydrolase family 77 (IPR003385). Generally, 4-glucantransferases contain 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). 4GT for use in the improved methods of the present invention also contains conserved residues involved in 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 some improved methods for enzymatically producing hexose from sucrose, the improvement includes converting sucrose to glucose 1-phosphate (G1P) catalyzed by sucrose phosphorylase, wherein the sucrose phosphorylase comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 19 to 25. Preferably, the SP comprises the amino acid sequence of any one of SEQ ID NOs: 19 to 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 hexose from sucrose, the reaction proceeds in the direction of G1P, which is then further processed downstream, and the final enzymatic step of the method is an energetically favorable irreversible step that dephosphorylates the hexose phosphate.

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

[0055] The following examples provide those skilled in the art with protocols for determining the activity of SPs as part of an enzymatic method, including, for example, incubating the enzyme with its substrate and then measuring the amount of reactant and product or subsequent downstream product via HPLC. Measurements of the relative activity of any two enzymes are performed under identical reaction conditions, such as buffer, pH, temperature, etc.

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

[0057] The SPs for use in the improved methods of the present invention are generally part of the glycoside hydrolase superfamily (IPR017853), more specifically the sucrose phosphorylase family (IPR022527), and contain a glycosyl hydrolase, family 13, catalytic domain (IPR006047). The glycosyl hydrolase domain consists of a (β / α)8 barrel, which is the catalytic domain. The entire sucrose phosphorylase consists of four domains: the N-terminal domain, the glycosyl hydrolase domain, the B domain (formed by a large loop within the glycosyl domain), and the 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). Sequence alignment to the well-studied sucrose phosphorylase from Leuconostoc mesenteroides (Uniprot Q59495) reveals conservation of the catalytic residue Asp196 (Schwarz A, Nidetzky B (July 2006) "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, mostly stored in plant seeds. Native starch includes 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 or acid hydrolysis of starch. Specifically, enzymatic hydrolysis of starch can be catalyzed or enhanced by isoamylase (EC 3.2.1.68), which hydrolyzes α-1,6-glucosidic bonds; pullulanase (EC 3.2.1.41), which hydrolyzes α-1,6-glucosidic bonds; or α-amylase (EC 3.2.1.1), which cleaves α-1,4-glucosidic bonds. Cornstarch contains many branches that interfere with the action of αGP. Isoamylase can be used to debranch starch, resulting in linear amylodextrin. Pre-treated starch with isoamylase can 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 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 the major component of plant cell walls. Non-food lignocellulosic biomass contains cellulose, hemicellulose, and lignin, as well as other minor components. Pure cellulose, including Avicel (microcrystalline cellulose), regenerated amorphous cellulose, bacterial cellulose, and filter paper, can be prepared through a series of processes. Partially hydrolyzed cellulosic substrates include water-insoluble cellodextrins with a degree of polymerization greater than 7, water-soluble cellodextrins with a degree of polymerization between 3 and 6, cellobiose, glucose, and fructose. Cellulose derivatives include pretreated biomass, regenerated amorphous cellulose, cellodextrins, cellobiose, fructose, and glucose. Furthermore, cellulose derivatives can be prepared by enzymatic hydrolysis of cellulose catalyzed by a cellulase mixture, acid, or biomass pretreatment. In some methods of the present invention, G1P is produced from cellulose using cellulose phosphorylase. In some methods, G1P is produced 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. Hexoses are monosaccharides with six carbon atoms. Hexoses can be classified by functional group, with aldohexoses having an aldehyde at position 1 and ketohexoses having a ketone at position 2. 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 carbocyclic hexose. The improved methods of the present invention can be used to convert starch and its derivatives into hexoses selected from allose, mannose, galactose, fructose, altrose, talose, sorbose, gulose, tagatose, allulose, inositol, and idose. Accordingly, some embodiments of the present invention relate to improved methods for producing these individual hexoses.

[0061] Improved methods of the invention for producing hexose include the additional step of dephosphorylating hexose phosphates using hexose phosphate phosphatase. In some improved methods of the invention for producing hexose, the method steps are performed in a single reaction vessel. In other improved methods of the invention, the method steps are performed in two or more single reaction vessels. In some improved methods, the method steps are performed without ATP, without NAD(P)(H), at a phosphate concentration of about 0.1 mM to about 150 mM, phosphate is recycled, and / or the step of dephosphorylating hexose phosphates involves an energetically favorable chemical reaction. In some improved methods of the invention, the method steps are performed under at least one of the following process conditions: 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 period of about 0.5 hours to about 48 hours. In some improved methods of the invention, the method steps are performed as a continuous reaction.

[0062] Some methods of the present invention for preparing hexose include the additional step of dephosphorylating hexose phosphates using hexose phosphate phosphatase. The phosphatase used in the methods of the present invention is specific for hexose phosphates. 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, and talose 6-phosphate is converted to talose by talose 6-phosphate phosphatase. , sorbose 6-phosphate is converted to sorbose by sorbose 6-phosphate phosphatase, gulose 6-phosphate is converted to gulose by gulose 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 sorbose 6-phosphate or talose 6-phosphate. In the methods of the invention, the hexose phosphate phosphatase has higher activity on 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, the allose 6-phosphate phosphatase has higher activity on allose 6-phosphate compared to other hexose phosphate intermediates in the method, such as G1P, G6P, F6P, and psicose 6-phosphate.Please refer to Figures 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 previously described in U.S. Pat. Nos. 6,570,043, 8,816,068, or 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: a) converting G1P to G6P, catalyzed by a PGM having at least 90% sequence identity to any one of SEQ ID NOS: 2-8; b) converting the starch derivative to G1P, catalyzed by αGP, having at least 90% sequence identity to any one of SEQ ID NOS: 10-13; and c) transglycosylating the starch derivative, catalyzed by 4GT, having at least 90% sequence identity to any one of SEQ ID NOS: 15-17. When cellulose or a cellulose derivative is the starting material, the improvement is converting G1P to G6P, catalyzed by a PGM having at least 90% sequence identity to any one of SEQ ID NOS: 2-8. When sucrose is the starting material, the improvement is selected from one or both of: a) converting G1P to G6P, catalyzed by a PGM having at least 90% sequence identity to any one of SEQ ID NOs: 2 to 8, and b) converting sucrose to G1P, catalyzed by an SP having at least 90% sequence identity to any one of SEQ ID NOs: 19 to 25. The method further includes converting G6P to fructose 6-phosphate (F6P), catalyzed by phosphoglucoisomerase (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) converting G1P to G6P, catalyzed by a PGM having at least 90% sequence identity to any one of SEQ ID NOS: 2-8; b) converting the starch derivative to G1P, catalyzed by αGP, having at least 90% sequence identity to any one of SEQ ID NOS: 10-13; and c) transglycosylating the starch derivative, catalyzed by 4GT, having at least 90% sequence identity to any one of SEQ ID NOS: 15-17. When cellulose or a cellulose derivative is the starting material, the improvement is converting G1P to G6P, catalyzed by a PGM having at least 90% sequence identity to any one of SEQ ID NOS: 2-8. When sucrose is the starting material, the improvement is selected from one or both of: a) converting G1P to G6P, catalyzed by a PGM having at least 90% sequence identity to any one of SEQ ID NOs: 2 to 8, and b) converting sucrose to G1P, catalyzed by a SP having at least 90% sequence identity to any one of SEQ ID NOs: 19 to 25. The method further includes converting G6P to fructose 6-phosphate (F6P), catalyzed by phosphoglucoisomerase (PGI), converting F6P to mannose 6-phosphate (M6P), catalyzed by mannose 6-phosphate isomerase (M6PI) or phosphoglucose / phosphomannose isomerase (PGPMI), and 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) converting G1P to G6P, catalyzed by a PGM having at least 90% sequence identity to any one of SEQ ID NOS: 2-8; b) converting the starch derivative to G1P, catalyzed by αGP, having at least 90% sequence identity to any one of SEQ ID NOS: 10-13; and c) transglycosylating the starch derivative, catalyzed by 4GT, having at least 90% sequence identity to any one of SEQ ID NOS: 15-17. When cellulose or a cellulose derivative is the starting material, the improvement is converting G1P to G6P, catalyzed by a PGM having at least 90% sequence identity to any one of SEQ ID NOS: 2-8. When sucrose is the starting material, the improvement is selected from one or both of: a) converting G1P to G6P, catalyzed by a PGM having at least 90% sequence identity to any one of SEQ ID NOs: 2 to 8, and b) converting sucrose to G1P, catalyzed by an SP having at least 90% sequence identity to any one of SEQ ID NOs: 19 to 25. The method further includes converting G6P to fructose 6-phosphate (F6P), catalyzed by phosphoglucoisomerase (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: a) converting G1P to G6P, catalyzed by a PGM having at least 90% sequence identity to any one of SEQ ID NOS: 2-8; b) converting the starch derivative to G1P, catalyzed by αGP, having at least 90% sequence identity to any one of SEQ ID NOS: 10-13; and c) transglycosylating the starch derivative, catalyzed by 4GT, having at least 90% sequence identity to any one of SEQ ID NOS: 15-17. When cellulose or a cellulose derivative is the starting material, the improvement is converting G1P to G6P, catalyzed by a PGM having at least 90% sequence identity to any one of SEQ ID NOS: 2-8. When sucrose is the starting material, the improvement is selected from one or both of: a) converting G1P to G6P, catalyzed by a PGM having at least 90% sequence identity to any one of SEQ ID NOs: 2 to 8, and b) converting sucrose to G1P, catalyzed by a SP having at least 90% sequence identity to any one of SEQ ID NOs: 19 to 25. The method further includes converting G6P to fructose 6-phosphate (F6P), catalyzed by phosphoglucoisomerase (PGI), and 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 altrose. When starch or a starch derivative is the starting material, the improvement is selected from one or more of: a) converting G1P to G6P, catalyzed by a PGM having at least 90% sequence identity to any one of SEQ ID NOS: 2-8; b) converting the starch derivative to G1P, catalyzed by αGP, having at least 90% sequence identity to any one of SEQ ID NOS: 10-13; and c) transglycosylating the starch derivative, catalyzed by 4GT, having at least 90% sequence identity to any one of SEQ ID NOS: 15-17. When cellulose or a cellulose derivative is the starting material, the improvement is converting G1P to G6P, catalyzed by a PGM having at least 90% sequence identity to any one of SEQ ID NOS: 2-8. When sucrose is the starting material, the improvement is selected from one or both of a) converting G1P to G6P, catalyzed by a PGM having at least 90% sequence identity to any one of SEQ ID NOs: 2 to 8, and b) converting sucrose to G1P, catalyzed by a SP having at least 90% sequence identity to any one of SEQ ID NOs: 19 to 25. The method further includes converting G6P to fructose 6-phosphate (F6P), catalyzed by phosphoglucoisomerase (PGI), converting F6P to P6P, catalyzed by P6PE, converting P6P to altrose 6-phosphate (Alt6P), catalyzed by altrose 6-phosphate isomerase (Alt6PI), and converting the generated Alt6P to altrose, catalyzed by altrose 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: a) converting G1P to G6P, catalyzed by a PGM having at least 90% sequence identity to any one of SEQ ID NOS: 2-8; b) converting the starch derivative to G1P, catalyzed by αGP, having at least 90% sequence identity to any one of SEQ ID NOS: 10-13; and c) transglycosylating the starch derivative, catalyzed by 4GT, having at least 90% sequence identity to any one of SEQ ID NOS: 15-17. When cellulose or a cellulose derivative is the starting material, the improvement is converting G1P to G6P, catalyzed by a PGM having at least 90% sequence identity to any one of SEQ ID NOS: 2-8. When sucrose is the starting material, the improvement is selected from one or both of: a) converting G1P to G6P, catalyzed by a PGM having at least 90% sequence identity to any one of SEQ ID NOs: 2 to 8, and b) converting sucrose to G1P, catalyzed by a SP having at least 90% sequence identity to any one of SEQ ID NOs: 19 to 25. The method further includes converting G6P to fructose 6-phosphate (F6P), catalyzed by phosphoglucoisomerase (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: a) converting G1P to G6P, catalyzed by a PGM having at least 90% sequence identity to any one of SEQ ID NOS: 2-8; b) converting the starch derivative to G1P, catalyzed by αGP, having at least 90% sequence identity to any one of SEQ ID NOS: 10-13; and c) transglycosylating the starch derivative, catalyzed by 4GT, having at least 90% sequence identity to any one of SEQ ID NOS: 15-17. When cellulose or a cellulose derivative is the starting material, the improvement is converting G1P to G6P, catalyzed by a PGM having at least 90% sequence identity to any one of SEQ ID NOS: 2-8. When sucrose is the starting material, the improvement is selected from one or both of: a) converting G1P to G6P, catalyzed by a PGM having at least 90% sequence identity to any one of SEQ ID NOs: 2 to 8, and b) converting sucrose to G1P, catalyzed by an SP having at least 90% sequence identity to any one of SEQ ID NOs: 19 to 25. The method further includes converting G6P to fructose 6-phosphate (F6P), catalyzed by phosphoglucoisomerase (PGI), converting F6P to 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 glose. When starch or a starch derivative is the starting material, the improvement is selected from one or more of: a) converting G1P to G6P, catalyzed by a PGM having at least 90% sequence identity to any one of SEQ ID NOS: 2-8; b) converting the starch derivative to G1P, catalyzed by αGP, having at least 90% sequence identity to any one of SEQ ID NOS: 10-13; and c) transglycosylating the starch derivative, catalyzed by 4GT, having at least 90% sequence identity to any one of SEQ ID NOS: 15-17. When cellulose or a cellulose derivative is the starting material, the improvement is converting G1P to G6P, catalyzed by a PGM having at least 90% sequence identity to any one of SEQ ID NOS: 2-8. When sucrose is the starting material, the improvement is selected from one or both of: a) converting G1P to G6P, catalyzed by a PGM having at least 90% sequence identity to any one of SEQ ID NOs: 2 to 8, and b) converting sucrose to G1P, catalyzed by an SP having at least 90% sequence identity to any one of SEQ ID NOs: 19 to 25. The method further includes converting G6P to fructose 6-phosphate (F6P), catalyzed by phosphoglucoisomerase (PGI), converting F6P to T6P, catalyzed by F6PE, converting S6P to gulose 6-phosphate (Gul6P), catalyzed by gulose 6-phosphate isomerase (Gul6PI), and 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 is selected from one or more of: a) converting G1P to G6P, catalyzed by a PGM having at least 90% sequence identity to any one of SEQ ID NOS: 2-8; b) converting the starch derivative to G1P, catalyzed by αGP, having at least 90% sequence identity to any one of SEQ ID NOS: 10-13; and c) transglycosylating the starch derivative, catalyzed by 4GT, having at least 90% sequence identity to any one of SEQ ID NOS: 15-17. When cellulose or a cellulose derivative is the starting material, the improvement is converting G1P to G6P, catalyzed by a PGM having at least 90% sequence identity to any one of SEQ ID NOS: 2-8. When sucrose is the starting material, the improvement is selected from one or both of: a) converting G1P to G6P, catalyzed by a PGM having at least 90% sequence identity to any one of SEQ ID NOs: 2 to 8; and b) converting sucrose to G1P, catalyzed by a SP having at least 90% sequence identity to any one of SEQ ID NOs: 19 to 25. The method further includes converting G6P to fructose 6-phosphate (F6P) catalyzed by phosphoglucoisomerase (PGI), converting F6P to T6P catalyzed by F6PE, converting T6P to sorbose 6-phosphate (S6P) catalyzed by sorbose 6-phosphate epimerase (S6PE), converting S6P to idose 6-phosphate (I6P) catalyzed by idose 6-phosphate isomerase (I6PI), and 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 to any one of SEQ ID NOS: 2-8; b) converting the starch derivative to G1P, catalyzed by αGP, having at least 90% sequence identity to any one of SEQ ID NOS: 10-13; and c) transglycosylating the starch derivative, catalyzed by 4GT, having at least 90% sequence identity to any one of SEQ ID NOS: 15-17. When cellulose or a cellulose derivative is the starting material, the improvement is converting G1P to G6P, catalyzed by a PGM having at least 90% sequence identity to any one of SEQ ID NOS: 2-8. When sucrose is the starting material, the improvement is selected from one or both of: a) 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) converting sucrose to G1P, catalyzed by a SP having at least 90% sequence identity with any one of SEQ ID NOs: 19 to 25. The method further includes converting G6P to fructose 6-phosphate (F6P), catalyzed by phosphoglucoisomerase (PGI), converting F6P to tagatose 6-phosphate (T6P), catalyzed by fructose 6-phosphate epimerase (F6PE), and 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: a) converting G1P to G6P, catalyzed by a PGM having at least 90% sequence identity to any one of SEQ ID NOS: 2-8; b) converting the starch derivative to G1P, catalyzed by αGP, having at least 90% sequence identity to any one of SEQ ID NOS: 10-13; and c) transglycosylating the starch derivative, catalyzed by 4GT, having at least 90% sequence identity to any one of SEQ ID NOS: 15-17. When cellulose or a cellulose derivative is the starting material, the improvement is converting G1P to G6P, catalyzed by a PGM having at least 90% sequence identity to any one of SEQ ID NOS: 2-8. When sucrose is the starting material, the improvement is selected from one or both of: a) converting G1P to G6P, catalyzed by a PGM having at least 90% sequence identity to any one of SEQ ID NOs: 2 to 8, and b) converting sucrose to G1P, catalyzed by a SP having at least 90% sequence identity to any one of SEQ ID NOs: 19 to 25. The method further includes converting G6P to fructose 6-phosphate (F6P), catalyzed by phosphoglucoisomerase (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 is selected from one or more of: a) converting G1P to G6P, catalyzed by a PGM having at least 90% sequence identity to any one of SEQ ID NOS: 2-8; b) converting the starch derivative to G1P, catalyzed by αGP, having at least 90% sequence identity to any one of SEQ ID NOS: 10-13; and c) transglycosylating the starch derivative, catalyzed by 4GT, having at least 90% sequence identity to any one of SEQ ID NOS: 15-17. When cellulose or a cellulose derivative is the starting material, the improvement is converting G1P to G6P, catalyzed by a PGM having at least 90% sequence identity to any one of SEQ ID NOS: 2-8. When sucrose is the starting material, the improvement is selected from one or both of: a) converting G1P to G6P, catalyzed by a PGM having at least 90% sequence identity to any one of SEQ ID NOS: 2-8; and b) converting sucrose to G1P, catalyzed by a SP having at least 90% sequence identity to any one of SEQ ID NOS: 19-25. The method further comprises converting G6P to inositol 3-phosphate (I3P) using an inositol phosphate synthase and converting I3P to inositol using an inositol monophosphatase. In another embodiment, the improved method of the present invention relates to the production of inositol from sucrose.

[0076] 1 shows an enzymatic pathway for converting sucrose to a G6P intermediate. The improved methods of the present invention include one or more of the following improvements: SP comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOS: 19-25; and PGM comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOS: 2-8. In a preferred method of the present invention, SP comprises the amino acid sequence of any one of SEQ ID NOS: 19-25, and PGM comprises the 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 NOS: 21, and PGM comprises the amino acid sequence of SEQ ID NOS: 8.

[0077] Figure 2 is a schematic diagram showing the enzymatic pathway for converting the starch derivative, maltodextrin, to a G6P intermediate. The following abbreviations are used: IA, isoamylase; PA, pullulanase; αGP, α-glucan phosphorylase or starch phosphorylase; 4GT, 4-glucan transferase; and PGM, phosphoglucomutase. The improved methods of the present invention include 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 the amino acid sequence of any one of SEQ ID NOS: 2-8, αGP comprises the amino acid sequence of any one of SEQ ID NOS: 10-13, and 4GT comprises the amino acid sequence of any one of SEQ ID NOS: 15-17. In a more preferred method of the 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 an enzymatic pathway for converting cellulose to a G6P intermediate. In the improved methods of the present invention, PGM comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOS: 2-8. Preferably, PGM comprises any one of SEQ ID NOS: 2-8. More preferably, PGM comprises the amino acid sequence of SEQ ID NOS: 8. In some improved methods of the present invention for producing hexose, one or more of the process steps are carried out in a single reaction vessel. In other improved methods of the present invention, the process steps are carried out in two or more reaction vessels. The phosphate ions generated by dephosphorylation of the hexose phosphates can then be reused in the process step converting the starch derivative to G1P, especially when all process steps are carried out in a single bioreactor or reaction vessel. The ability to recycle phosphate allows for the use of non-stoichiometric amounts of phosphate, keeping 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, allowing for the overall efficiency of the hexose production process.

[0079] In the improved methods of the present invention for producing hexoses, the method steps are carried out without ATP, without NAD(P)(H), at a phosphate concentration of about 0.1 mM to about 150 mM, and the phosphate is recycled and / or the step of dephosphorylating the hexose phosphate involves an energetically favorable chemical reaction. In the improved methods of the present invention, the method steps are carried out under at least one of the following process conditions: 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, for about 0.5 hours to about 48 hours, or in a continuous process.

[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. A low phosphate concentration reduces production costs due to low total phosphate, and reduces phosphate removal costs. It also prevents inhibition of phosphatases or other enzymes in the process by high concentrations of free phosphate, reducing the possibility of phosphorus contamination.

[0081] Additionally, each of the methods disclosed herein can be performed without the addition of ATP as a phosphate source, i.e., without the inclusion of ATP. Each method can also be performed without the need for the addition of NAD(P)(H), i.e., without the inclusion of NAD(P)(H).

[0082] Any suitable biologically compatible buffer known in the art, such as HEPES, PBS, BIS-TRIS, MOPS, DIPSO, or Trizma, can be used in the methods of the present invention. Reaction buffers for the methods of the present invention can have a pH ranging from 5.0 to 8.0. More preferably, the reaction buffer pH can range from 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 may also contain a divalent metal cation. In some methods, the step includes adding Mg 2+ , Zn 2+ , Ca 2+ , Co 2+ , Mn 2+and combinations thereof. As is known in the art, the desired metal cation can be introduced using an appropriate salt.

[0084] In each method of the present invention, the reaction temperature at which the method steps are carried out can range from 37 to 85°C. More preferably, the steps 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 range from 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 process 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 when 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 (e.g., maintained at 50°C) so that maximum tagatose yield is achieved when the maltodextrin leaves the column. This methodology offers greater volumetric productivity than batch processes. This limits the time the product is in contact with the column and reaction conditions, reducing the possibility of product degradation (e.g., potential hydroxymethylfurfural formation). It also facilitates automation of production, thereby reducing operating costs.

[0087] The enzymes used in the steps of the present invention can take the form of soluble, immobilized, aggregated, entrapped, or aggregated proteins. These enzymes can be adsorbed to insoluble organic or inorganic supports, commonly used to improve functionality, as 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 high surface-to-volume ratio and specialized surfaces that promote the attachment and activity of immobilized enzymes. Enzymes can be immobilized to these solid supports through covalent bonds, ionic, or hydrophobic interactions. Enzymes can also be immobilized through engineered interactions, such as covalent fusion to another protein or peptide sequence with affinity for the solid support, most often a polyhistidine sequence. Enzymes can be immobilized directly to a surface or surface coating, or to other proteins already present on the surface or surface coating. The enzymes can be immobilized all on one support, on individual supports, or a combination of the two (e.g., two enzymes per support are immobilized and then the supports are mixed). These variations can be mixed homogeneously or in defined layers to optimize turnover in a continuous process. For example, at the start of the reactor, there can be a layer of αGP 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 homogeneously or in defined layers or zones to optimize turnover.

[0088] Each of the methods according to the invention can achieve high yields due to the highly favorable equilibrium constant of the overall reaction: theoretically, yields of up to 99% can be achieved if the starting material is completely converted to the phosphorylated intermediate.

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

[0090] The methods of the present invention allow for easy recovery of hexoses, minimizing separation costs. In some preferred methods of the present invention, recovery of the desired hexose is achieved without chromatographic separation. After producing hexoses in a continuous reaction, the product is passed through microfiltration, ion exchange (cation, then anion, with possible mixed bed for polishing), concentration, crystallization, crystal isolation, and drying. Due to the high yield of hexoses, only a crystallization step is required to purify the hexoses. To further purify the hexoses prior to crystallization, nanofiltration can be used to eliminate the risk of enzymes being present in the crystallization process and to remove unconverted dextrins (such as maltodextrins, maltotetraose, maltotriose, and maltose) that may co-crystallize with the hexoses or limit the reusability of the mother liquor.

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

[0092] example The following examples describe improved methods using enzymes 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 noted. 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 kanamycin was used for E. coli cell growth and recombinant protein expression.

[0095] Recombinant enzyme production and purification

[0096] E. coli BL21(DE3) strain harboring the protein expression plasmid (pET28a) was incubated in a 1 L Erlenmeyer flask containing 100 mL of ZYM-5052 medium containing 50 mg L-1 kanamycin. Cells were grown for 16–24 h at 30°C with rotary shaking at 220 rpm. Cells were harvested by centrifugation at 12°C and washed once with either 20 mM HEPES (pH 7.5) containing 300 mM NaCl and 5 mM imidazole (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 Profinity IMAC Ni-Charged Resin (Bio-Rad, Hercules, CA) using an increasing imidazole gradient in the previously described buffer. The purity of the recombinant protein was examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

[0097] Example 1 Improved Process with Higher Activity PGM

[0098] Various phosphoglucomutases (PGMs) were isolated and assayed for the conversion of G1P to G6P in a multistep enzymatic reaction. The relative activity of each PGM was measured as follows: Reactions were prepared in 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 and incubated at 50°C for 30 min. The reaction was terminated by filtration of the enzyme using a Vivaspin 2 concentrator (10,000 MWCO). The product fructose was assayed using a Hi-Plex H+ column and a refractive index detector. Samples were assayed in 5 mM H2SO4 at 0.6 mL / min for 15.5 min at 65°C. The amount of fructose produced in 30 min was used to determine the relative efficiency of each PGM in the enzymatic process. The average increase in fructose peak area and peak height was used to determine the increase in activity.

[0099] Figure 19 is a chromatogram showing the activity of various PGMs. The chromatogram shows the level of fructose made from glucose 1-phosphate after incubation with various PGMs, excess phosphoglucoisomerase, and excess fructose 6-phosphate phosphatase. PGMs are cascade restriction enzymes. Table 1 shows that PGMs for use in the improved methods of the invention have improved activity compared to a previously disclosed PGM from Thermococcus kodakaraensis (Uniprot ID Q68BJ6). See 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: 200 μL reaction mixtures were prepared in 25 mM sodium phosphate pH 7.2, 5 mM MgCl, 20 g / L maltodextrin DE4-7, 9 μg αGP, and 0.1 g / L PGM and incubated at 50°C. Samples were added at various times to 1.5 mM NAD + and 3 U / mL glucose 6-phosphate dehydrogenase. The absorbance at 340 nm at each time point was used to obtain the reaction rate. This rate was used to determine the specific activity of each αGP and was used for relative activity comparison.

[0102] Table 2 shows that αGP for use in the improved methods of the invention has improved activity compared to the previously disclosed αGP from Thermotoga maritima (Uniprot ID G4FEH8), see WO 2017 / 059278. [Table 2]

[0103] Example 3 Improved Process with Higher Activity 4GT

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

[0105] Table 3 shows that 4GT for use in the improved methods of the invention has improved activity compared to the previously disclosed 4GT from Thermococcus litoralis (Uniprot ID O32462). See WO 2018 / 169957. [Table 3]

[0106] Example 4 Improved Method with Higher Activity SP

[0107] Various sucrose phosphorylases predicted by homology to 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 the conversion of sucrose to glucose 6-phosphate.

[0108] The relative efficiency of each SP was determined as follows: Reaction mixtures were prepared in 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 and incubated at 50°C. Samples were taken at 0, 2, 6, and 8 hours. The reaction was stopped by enzyme filtration using a Vivaspin 2 concentrator (10,000 MWCO). The product fructose was assayed using a Supel Cogel Pb column and a refractive index detector. Samples were assayed in ultrapure water at 0.6 mL / min for 25 minutes at 80°C. The amount of fructose produced at 2 hours was used to determine the relative activity of each SP. The amount of fructose produced at 6 hours (confirmed complete at 8 hours) indicates the difference in the maximum achievable yield of each SP.

[0109] The effect on the complete conversion of sucrose to fructose was also examined. Of the eight enzymes tested compared to the reference (table below), seven showed improved activity over Uniprot ID D9TT09, and one showed an unexpected advantage in producing fructose from sucrose. Figure 18 shows chromatograms of sucrose phosphorylase activity, comparing Uniprot ID D9TT09 (reference SP) with Uniprot ID F6BJS0. At 2 hours, the more active SP produces approximately 150% of the amount of fructose as the reference SP. The bottom chromatogram compares Uniprot ID D9TT09 (reference SP) with Uniprot ID F6BJS0 for maximum yield. At 6 hours (maximum yield for both reactions), the more active SP produces approximately 130% of the amount of fructose as the reference SP. Interestingly, relative yield does not directly correlate with relative activity. Possible 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 equilibrium between fructose formation and fructose degradation in the later stages of the reaction. A comparative SP from Thermanaerothrix daxensis (Uniprot ID A0A0N8GPZ6) having the amino acid sequence set forth 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 improved enzyme activity, the conversion of maltodextrin to G6P was performed using αGP (Uniprot ID G4FEH8) and PGM (Uniprot ID Q68BJ6) as previously described and compared with a method using αGP (Uniprot ID D1B926) and PGM (Uniprot ID A0A150LLZ1), which have 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 min. The reaction was terminated 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. Samples were evaluated in 5 mM H2SO4 at 0.6 mL / min for 15.5 minutes at 65° C. Results were not quantified (Figure 17) because the void, maltodextrin, and G6P peaks were too close to reliably quantify the individual components, but clearly more G6P is produced using the enzymes αGP (Uniprot ID D1B926) and PGM (Uniprot ID A0A150LLZ1). [Sequence List Free Text]

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

Claims

1. 1. An improved process for the enzymatic production of hexoses from starch or starch derivatives, the improvement comprising: a) 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; b) converting a starch derivative to G1P catalyzed by α-glucan phosphorylase (aGP), wherein the aGP comprises an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NOs: 10 to 13; and c) transglycosylating a starch derivative catalyzed by 4-α-glucan transferase (4GT), wherein the 4GT comprises an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NOs: 15-17; The improved method includes at least one of the following:

2. An improved method for enzymatically producing hexose from cellulose or a cellulose derivative, the improvement comprising the 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 NOs: 2-8.

3. 1. An improved process for the enzymatic production of hexose from sucrose, the improvement comprising: a) 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) converting sucrose into 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; The improved method includes at least one of the following:

4. 4. The improved method according to any one of claims 1 to 3, wherein the hexose is selected from the group consisting of allose, mannose, galactose, fructose, altrose, talose, sorbose, gulose, idose, allulose, inositol and tagatose.

5. 10. The improved process of claim 1, wherein the starch derivative is selected from the group consisting of amylose, amylopectin, soluble starch, amylodextrin, maltotriose, maltose, and maltodextrin.

6. 5. The improved method of any one of claims 1 to 4, further comprising the step of dephosphorylating the hexose phosphates using a hexose phosphate phosphatase.

7. 7. The improved method of claim 6, wherein the method steps are carried out in a single reaction vessel.

8. 8. The improved method of claim 7, wherein the method steps are carried out ATP-free, NAD(P)(H)-free, at a phosphate concentration of about 0.1 mM to about 150 mM, phosphate is recycled, and / or the step of dephosphorylating the hexose phosphate involves an energetically favorable chemical reaction.

9. The method steps are: 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 For about 0.5 hours to about 48 hours, or as a continuous reaction, under at least one of the following process conditions: The improved method according to any one of claims 1 to 8.

10. 5. The improved method of claim 4, further comprising the step of reducing the hexose to its sugar alcohol.

11. 5. The improved method of claim 4, wherein the PGM has an activity that is at least 10% higher than the activity of PGM from Thermococcus kodakaraensis (Uniprot ID Q68BJ6).

12. 2. The improved method of claim 1, wherein the aGP has an activity that is at least 10% higher than the activity of aGP from Thermotoga maritima (Uniprot ID G4FEH8).

13. 2. The improved method of claim 1, wherein the 4GT has an activity that is at least 10% higher than the activity of 4GT from Thermococcus litoralis (Uniprot ID O32462).

14. 4. The improved method of claim 3, wherein the SP has at least 10% greater activity than SP from Thermoanaerobacterium thermosaccharolyticum (Uniprot ID D9TT09).

15. 4. The improved method of claim 3, wherein the SP has at least a 10% higher achievable yield compared to SP from Thermoanaerobacterium thermosaccharolyticum (Uniprot ID D9TT09).

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