Enzymatic production of allulose

Abstract

The present invention relates to an improved process for the enzymatic production of allulose using an enzyme characterized by improved expression, improved stability, and low allulose to fructose conversion activity compared to enzymes in other allulose production methods. The improved process includes converting fructose-6-phosphate to allulose-6-phosphate (A6P) using an allulose-6-phosphate epimerase and converting A6P to allulose using an allulose-6-phosphate phosphatase.

Description

[Technical Field]

[0001] Sequence Listing The Sequence Listing submitted herein is an ASCII text file via EFS-Web (2021-05-18_Sequence_Listing_ST25, created May 18, 2021, 48,103 bytes) and is incorporated herein by reference.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Application No. 63 / 026,294, filed May 18, 2020, which is incorporated herein by reference in its entirety.

[0003] The present invention relates to an improved enzymatic process for producing D-allulose. [Background technology]

[0004] D-Allulose, also known as D-psicose or simply allulose, is a low-calorie natural sweetener that is 70% as sweet as sucrose but contains only 10% of its calories. It is a naturally occurring monosaccharide hexose found in small amounts in plants such as wheat. Allulose was approved as a food additive by the Food and Drug Administration (FDA) in 2012 and is generally recognized as safe (GRAS). However, its high cost has limited its use as a sweetener. Nevertheless, in addition to having 10% of the calories of sucrose, allulose boasts many health benefits, including a low glycemic index, complete absorption in the small intestine without being metabolized, elimination in urine and feces, and inhibition of alpha-amylase, sucrase, and maltase, which help regulate blood sugar, all of which have similar food and beverage functionality to sucrose. Therefore, allulose has a variety of applications in the food and beverage industry.

[0005] Allulose is mainly produced by a method involving the enzymatic isomerization of fructose. See, for example, PCT Application Publication No. 2014 / 049373. Generally, such methods are not commercially viable due to the expensive separation of allulose from fructose and the relatively low product yields associated therewith, resulting in higher raw material costs.

[0006] Although alternative processes for producing allulose using epimerase to catalyze fructose 6-phosphate to allulose 6-phosphate, followed by a dephosphorylation step, have been described in PCT Publication Nos. 2018 / 112139, 2018 / 004308, and 2018 / 129275, these and other alternative allulose production methods do not satisfy the long-standing need for a process for producing allulose that provides higher yields using lower amounts of enzyme. With this improvement in mind, the following disclosure describes enzymes for use in the production of allulose that have higher expression, stability, and are associated with lower undesired allulose conversion activity compared to currently used allulose production methods. The above improvements meet the strong industrial and commercial interest in reducing the cost of allulose production. Summary of the Invention

[0007] The present invention provides an improved method for preparing allulose by enzymatically converting sugars, such as polysaccharides, oligosaccharides, disaccharides, sucrose, D-glucose, and D-fructose, into allulose. In one aspect, the improved process for producing allulose from sugars of the present invention comprises converting fructose-6-phosphate (F6P) to allulose-6-phosphate (A6P) using allulose-6-phosphate epimerase (A6PE), wherein A6PE comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1. In another aspect, the improved process for producing allulose from sugars of the present invention comprises converting A6P to allulose using allulose-6-phosphate phosphatase (A6PP), wherein A6PP comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2. In an embodiment of the present invention, the improved process comprises converting fructose-6-phosphate (F6P) to allulose-6-phosphate (A6P) using allulose-6-phosphate epimerase (A6PE), wherein A6PE comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:1, and converting A6P to allulose using allulose-6-phosphate phosphatase (A6PP), wherein A6PP comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:2.

[0008] The process of the invention for preparing allulose may also involve converting glucose 6-phosphate (G6P) to F6P in a step catalyzed by phosphoglucoisomerase (PGI). Other processes according to the invention may further comprise converting glucose 1-phosphate (G1P) to G6P by a reaction catalyzed by phosphoglucomutase (PGM), while other processes may further comprise converting a sugar to G1P by a reaction catalyzed by at least one other enzyme.

[0009] The sugar used in any of the methods described herein may be selected from the group consisting of starch or its derivatives, cellulose or its derivatives, and sucrose. In this regard, the starch or its derivatives may be, for example, amylose, amylopectin, soluble starch, amylodextrin, maltodextrin, maltose, maltotriose, or glucose. In some improved processes of the present invention, starch is converted to a starch derivative by enzymatic hydrolysis or by acid hydrolysis of starch. Examples of enzymatic hydrolysis of starch to obtain a starch derivative include, but are not limited to, reactions catalyzed by isoamylase, pullulanase, alpha-amylase, or a combination of two or more of these enzymes. Some processes of the present invention may further involve the addition of 4-glucan transferase (4GT).

[0010] Another process of the present invention for preparing allulose further comprises a step of converting fructose to F6P catalyzed by at least one enzyme. Another process of the present invention further comprises a step of converting sucrose to fructose in a reaction catalyzed by at least one enzyme. The G6P used in the process for preparing allulose can also be produced by converting glucose to G6P in a reaction catalyzed by at least one enzyme. Glucose can then be produced by converting sucrose to glucose catalyzed by at least one enzyme.

[0011] The process of the present invention can be carried out under a variety of reaction conditions, including at a temperature ranging from about 37°C to about 85°C, a pH ranging from about 4 to about 9, and / or for about 0.5 hours to about 48 hours, or as a continuous reaction. In some embodiments, the process steps for preparing allulose are carried out under any one or more of the aforementioned reaction conditions in a single reactor. In other embodiments, the reaction steps are carried out under the aforementioned reaction conditions using multiple bioreactors, which can be arranged in series.

[0012] In some processes of the invention, the steps for preparing allulose are carried out under conditions that are not adenosine triphosphate (ATP) or NAD(H), i.e., ATP-free or NAD(H)-free, at a phosphate concentration of about 0.1 mM to about 150 mM, phosphate is recycled, and / or the steps for converting A6P to allulose involve energetically favorable chemical reactions. [Brief explanation of the drawings]

[0013] [Figure 1] 1 is a schematic diagram showing an enzymatic pathway for converting starch or its derivative products to allulose. The following abbreviations are used: αGP, alpha-glucan phosphorylase or starch phosphorylase; PGM, phosphoglucomutase; PGI, phosphoglucoisomerase; A6PE, allulose-6-phosphate epimerase; A6PP, allulose-6-phosphate phosphatase; IA, isoamylase; PA, pullulanase; MP, maltose phosphorylase; PPGK, polyphosphate glucokinase. In the process of the present invention, A6PE comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1, and / or A6PP comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2. [Figure 2]

[0023] Figure 1 shows an enzymatic pathway for converting cellulose or a derivative thereof to allulose. CDP, cellodextrin phosphorylase; CBP, cellobiose phosphorylase; PPGK, polyphosphate glucokinase; PGM, phosphoglucomutase; PGI, phosphoglucoisomerase; A6PE, allulose-6-phosphate epimerase; and A6PP, allulose-6-phosphate phosphatase. In the process of the present invention, A6PE comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1, and / or A6PP comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2. [Figure 3]1 is a schematic diagram showing the enzymatic pathway for converting fructose to allulose. PPFK, polyphosphate fructokinase; A6PE, allulose-6-phosphate epimerase; A6PP, allulose-6-phosphate phosphatase. In the process of the present invention, A6PE comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1, and / or A6PP comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2. [Figure 4] 1 is a schematic diagram showing the enzymatic pathway for converting glucose to allulose. PPGK, polyphosphate glucokinase; PGI, phosphoglucoisomerase; A6PE, allulose-6-phosphate epimerase; A6PP, allulose-6-phosphate phosphatase. In the process of the present invention, A6PE comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1, and / or A6PP comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2. [Figure 5]

[0023] Figure 1 shows an enzymatic pathway for converting sucrose or a derivative thereof to allulose. SP, sucrose phosphorylase; PPFK, polyphosphate fructokinase; PGM, phosphoglucomutase; PGI, phosphoglucoisomerase; A6PE, allulose-6-phosphate epimerase; and A6PP, allulose-6-phosphate phosphatase. In the process of the present invention, A6PE comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1, and / or A6PP comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2. [Figure 6]The conversion of maltodextrin to allulose, as measured by HPLC, was performed using the following enzymes: αGP (Uniprot ID G8NCC0), PGM (Uniprot ID A0A150LLZ1), PGI (Uniprot ID Q5SLL6), A6PP (Uniprot ID A0A0E3NCH4), and 4GT (Uniprot ID E8MXP8). A6PE (Uniprot ID A0A090IXZ8) and (Uniprot ID UPI000411882A) were compared to the A6PE used in the improved process of the present invention (A0A223HZI7). [Figure 7] The conversion of G1P to allulose as measured by HPLC is shown. The following enzymes were used: PGM (Uniprot ID A0A150LLZ1), PGI (Uniprot ID Q5SLL6), and A6PE (Uniprot ID D9TQJ4). A6PP (Uniprot ID A3DC21) was compared to A6PP (Uniprot ID A0A0E3NCH4) used in the improved process of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention described herein relates to an improved enzymatic process for converting sugars into allulose. More specifically, the present invention relates to an improved cell-free enzymatic process for converting sugars into allulose. Examples of sugars that can be converted to allulose by the process of the present invention include, but are not limited to, starch, cellulose, sucrose, glucose, fructose, and products derived from any of the aforementioned sugars. The enzymes used in the process according to the present invention can be combined into a single cell-free enzyme cocktail. Indeed, compared to cell-based production methods for producing allulose, the process offers a higher reaction rate, at least in part due to the absence of cell membranes, which can slow the transport of substrates, products, or both, in and out of the cells used in the process. The process of the present invention also results in a final allulose product that is free of fermentation medium and nutrient-rich metabolic products associated with the cells used in the cell-based process.

[0015] Some processes of the present invention for producing allulose improve the step of converting fructose-6-phosphate (F6P) to allulose-6-phosphate (A6P) by a reaction catalyzed by allulose-6-phosphate epimerase (A6PE), which has one or more improved properties compared to any one of the A6PE enzymes used in the previous allulose production methods of the present invention. Other processes of the present invention improve the step of converting A6P to allulose by a reaction catalyzed by allulose-6-phosphate phosphatase (A6PP), which has one or more improved properties compared to any one of the A6PP enzymes used in the previous allulose production methods of the present invention. In some processes of the present invention, the process for producing allulose may include the steps of converting (F6P to A6P) and (A6P to allulose), and improved A6PE and improved A6PP are used in the steps of converting (F6P to A6P) and (A6P to allulose), respectively. Other processes of the present invention for producing allulose may use unimproved A6PP for the A6P-to-allulose conversion step while using improved A6PE for the F6P-to-A6P conversion step. Conversely, processes of the present invention may use unimproved A6PE for the F6P-to-A6P conversion step and improved A6PP for the A6P-to-allulose conversion step. For examples of A6PE and A6PP, including A6PE from Thermoanaerobacterium thermosaccharolyticum (UniProt ID D9TQJ4), A6PE from Bacillus thermoamylovorans (UniProt ID A0A090IXZ8), and A6PP from Clostridium thermocellum (UniProt ID A3DC21), see PCT Publication No. 2018 / 112139, which is incorporated herein by reference in its entirety.

[0016] In a process for producing allulose, the use of an enzyme with higher activity and better properties, such as improved stability, allows for the use of less enzyme, thereby reducing the overall cost of the process. As discussed above, the conversion of F6P to A6P in the improved process of the present invention is catalyzed by A6PE with improved properties, contributing to improvements in the allulose production process, including one or more of the following improved properties: higher expression yield, thermostability, and lower undesired allulose to fructose epimerization activity, compared to the thermophilic A6PE from Thermoanaerobacterium thermosaccharolyticum (UniProt ID D9TQJ4).

[0017] In some processes of the present invention, the expression yield of A6PE in the improved process has an expression yield that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, or at least 400% higher than the expression yield of A6PE in an unimproved process, such as, but not limited to, A6PE having the amino acid sequence of UniProt ID D9TQJ4 (Thermoanaerobacterium thermosaccharolyticum). For example, in the process of the present invention for producing allulose, the step of converting F6P to A6P uses A6PE having the amino acid sequence of UniProt ID A0A223HZI7 (Clostridium thermosaccharolyticum), which has an expression yield that is approximately 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, or 400% higher than A6PE having the amino acid sequence of UniProt ID D9TQJ4.

[0018] In some processes of the present invention, A6PE in the improved processes is more stable than A6PE in the unimproved processes. More specifically, A6PE in the improved processes of the present invention may be more thermostable than A6PE in the unimproved processes. Indeed, A6PE in some processes of the present invention may remain 50% to 60% soluble, 60% to 70% soluble, 70% to 80% soluble, 80% to 90% soluble, 90% to 100% soluble, or 100% soluble after 30 minutes at 50°C to 60°C. In processes of the invention where the conversion of F6P to A6P uses A6PE in an improved process having the amino acid sequence of UniProt ID A0A223HZI7, the A6PE may remain about 80% soluble after 30 minutes at about 60°C, and therefore the A6PE in the improved process may be at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, or 85% soluble after 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 minutes at 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, or 66°C.

[0019] The conversion of allulose to fructose by A6PE is undesirable in the process of producing allulose.In some improved processes of the present invention, the A6PE-dependent allulose conversion activity to fructose is lower than that of the unimproved process for producing allulose.In the improved processes of the present invention, for example, 1% or less, 0.9% or less, 0.8% or less, 0.7% or less, 0.6% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, 0.1% or less, or 0% of the allulose produced by the process of the present invention is converted to fructose.

[0020] The A6PE enzyme used in the improved process of the present invention is specific for F6P / A6P, and epimerization catalyzed by A6PE is reversible. As used herein, the term "specific" means that the F6 / A6P epimerization activity of A6PE in the improved process of the present invention is higher than that for other phosphorylated monosaccharides present in the reaction. For example, the F6P / A6P epimerization activity of A6PE in the improved process of the present invention is higher than its epimerization activity for G6P.

[0021] The conversion of F6P to A6P in the improved processes of the invention may utilize a divalent metal A6PE cofactor, such as magnesium, manganese, cobalt, or zinc. In some processes of the invention, for example, cobalt is a cofactor for A6PE in the F6P to A6P conversion reaction step.

[0022] As described above, exemplary properties of the A6PE enzyme in the improved processes of the present invention include, but are not limited to, increased expression yield, increased stability, and reduced conversion of allulose to fructose. In some improved processes of the present invention, the amino acid sequence of A6PE is thermophilic. More specifically, in certain improved processes of the present invention, A6PE has an amino acid sequence that shares 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 99%, or 100% sequence identity with A6PE derived from Clostridium thermosaccharolyticum. Thus, in some improved processes of the invention, the amino acid sequence of A6PE shares 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 99%, or 100% sequence identity with the amino acid sequence of a C. thermosaccharolyticum thermophilic epimerase having the sequence of UniPROt ID A0A223HZI7.

[0023] Another structural feature of A6PE in some improved processes of the present invention is an (α / β)8 barrel domain for catalysis. In some of these improved processes of the present invention, the (α / β)8 barrel domain contains a phosphate-binding domain with Ser at the end of the seventh β-strand of the barrel, Ser at the end of the eighth β-strand of the barrel, and Gly in the active site loop. In other improved processes of the present invention, A6PE contains a metal-binding domain with His in the second and third β-strands of the barrel. In other improved processes of the present invention, A6PE contains Asp in the second and seventh β-strands of the barrel, functioning as an acid / base catalyst for 1,1 proton transfer. In other improved processes of the present invention, A6PE contains a His hydrophobic residue-Asp signature in the second β-strand of the barrel, where His is utilized by Asp for metal binding and acid / base catalysis. In other improved processes of the present invention, A6PE is a member of the ribulose-phosphate 3-epimerase family (Pfam PF00834). In still other improved processes of the present invention, A6PE contains two or more of any of the above (α / β)8 barrel domain structural features. Thus, in some improved processes of the present invention, A6PE contains an (α / β)8 barrel domain for catalysis, Ser at the end of the seventh β-strand of the barrel, Ser at the end of the eighth β-strand of the barrel, Gly in the active site loop, His in the second and third β-strands of the barrel, Asp in the second and seventh β-strands of the barrel, and a His-hydrophobic residue-Asp signature in the second β-strand of the barrel, and is a member of the ribulose-phosphate 3-epimerase family (Pfam PF00834). These characteristics of (α / β)8 barrel domains are known in the art and are referenced, for example, in Chan et al. Structural Basis for Substrate Specificity in Phosphate Binding (beta / alpha)8-Barrels: D-Allulose 6-Phosphate 3-Epimerase from Escherichia coli K-12. Biochemistry 2008;47(36):9608-9617.

[0024] In the improved process of the present invention, allulose-6-phosphate phosphatase (A6PP) is a phosphatase that specifically converts A6P to allulose. Therefore, the A6PP in the improved process of the present invention is specific to A6P. The term "specific" as used herein means that the A6P dephosphorylation activity of A6PP is higher than that of other phosphorylated monosaccharides in the process. For example, the A6PP in the improved process of the present invention has higher dephosphorylation activity for A6P than for G1P, G6P, and F6P. The A6PP in the improved process of the present invention may also utilize a divalent metal cofactor such as zinc, manganese, cobalt, or magnesium, preferably magnesium.

[0025] The conversion of A6P to allulose in the improved processes of the present invention may use A6PP with increased activity compared to any of the A6PP enzymes used in previous allulose-producing processes of the present invention. In some improved processes of the present invention, for example, A6PP converts A6P to allulose with greater activity than A6PP from Clostridium thermocellum (UniProt ID A3DC21). See International Patent Applications 2018 / 112139 and 2018 / 129275, which are incorporated herein by reference in their entireties. More specifically, in the improved process of the present invention, the A6PP has the amino acid sequence of A6PP from Methanosarcina CHTI-55, which has A6P activity that is at least 10%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, or 300% improved compared to the activity of A6PP from Clostridium thermocellum, which has the amino acid sequence of UNIPROT ID A3DC21. More specifically, in some improved processes of the present invention, the A6PP has the amino acid sequence of Uniprot ID A0A0E3NCH4 (SEQ ID NO: 2) and has A6P activity that is at least 10%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, or 300% improved compared to the activity of A6PP from Clostridium thermocellum having the amino acid sequence of Uniprot ID A3DC21.

[0026] In some improved processes of the invention, A6PP has an amino acid sequence that shares 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 99%, or 100% sequence identity with A6PP from Methanosarcina thermophila CHTI-55. Thus, in some improved processes of the invention, the amino acid sequence of A6PP shares 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 99%, or 100% sequence identity with the amino acid sequence of Methanosarcina thermophila CHTI-55 phosphatase, which has the sequence of UniProt ID A0A0E3NCH4 (SEQ ID NO: 2).

[0027] Another structural feature of A6PE in some improved processes of the present invention is a Rossmanoid-fold domain for catalysis. In some of these improved processes of the present invention, A6PP contains one or more of the following features: a C1 capping domain for substrate specificity, a DxD signature in the first β-strand of the Rossmanoid-fold for the second Asp to coordinate magnesium, a general acid / base catalyst, a Thr or Ser at the end of the second β-strand of the Rossmanoid-fold to aid in the stability of reaction intermediates, a Lys at the N-terminal end of the alpha-helix C-terminal to the third β-strand of the Rossmanoid-fold to aid in the stability of reaction intermediates, and an E(D / N) signature at the end of the fourth β-strand of the Rossmanoid-fold to coordinate divalent metal cations such as magnesium. See, e.g., Burroughs et al., Evolutionary Genomics of the HAD Superfamily: Understanding the Structural Adaptations and Catalytic Diversity in a Superfamily of Phosphoesterases and Allied Enzymes. J. Mol. Biol. 2006;361;1003-1034.

[0028] As established herein, the improved process of the present invention includes converting F6P to A6P using A6PE and converting A6P to allulose using A6PP. The improved process of the present invention may also include additional upstream steps. For example, some processes of the present invention use phosphoglucose isomerase (PGI) to produce allulose from sugars and convert glucose 6-phosphate (G6P) to F6P. Exemplary PGIs that can be used include those disclosed in International Patent Application Publication No. 2017 / 059278, PGI from Clostridium thermocellum (Uniprot ID A3DBX9), and PGI from Thermus thermophilus (Uniprot ID Q5SLL6).

[0029] Some improved processes of the present invention also include converting glucose 1-phosphate (G1P) to G6P using phosphoglucomutase (PGM). Examples of PGM include PGM from Thermococcus kodakaraensis (Uniprot ID Q68BJ6), disclosed in International Patent Application Publication No. 2017 / 059278, or PGM from Caldibacilus devilis (Uniprot A0A150LLZ1), disclosed in PCT Application Publication No. 2020 / 092315.

[0030] Some improved processes of the present invention also include a step of converting sugars to G1P using at least one enzyme. For example, the improved process may convert a sugar selected from starch or a derivative thereof described in FIG. 1, cellulose or a derivative thereof described in FIG. 2, fructose described in FIG. 3, glucose described in FIG. 4, or sucrose described in FIG. 5. The enzyme used in the step of converting sugars to G1P in such improved processes of the present invention may be, for example, alpha-glucan phosphorylase (αGP), maltose phosphorylase, sucrose phosphorylase, cellodextrin phosphorylase, cellobiose phosphorylase, and / or cellulose phosphorylase, and mixtures thereof. The selection of an enzyme or combination of enzymes to arrive at F6P depends on the sugar used in the process.

[0031] 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 cellulose 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.

[0032] In some improved processes of the present invention, cellulose and its derivatives can be converted into allulose by a series of enzymatic steps including the production of free phosphate from G1P derived from cellodextrin and cellobiose, and cellodextrin phosphorylase (CDP) and cellobiose phosphorylase (CBP), respectively, the conversion of G1P to G6P catalyzed by PGM, the conversion of G6P to F6P catalyzed by PGI, and the conversion of F6P to allulose as described above, and the phosphate ions can be recycled by the step of converting cellodextrin and cellobiose to G1P.

[0033] Several enzymes can be used to hydrolyze solid cellulose into water-soluble cellodextrins and cellobiose. Such enzymes include endoglucanases and cellobiohydrolases, but not beta-glucosidases (cellobiases). Prior to cellulose hydrolysis and G1P production, cellulose and biomass can be pretreated to increase their reactivity and reduce the degree of polymerization of the cellulose chains. Pretreatment methods for cellulose and biomass include dilute acid pretreatment, cellulose solvent-based lignocellulosic fractionation, ammonia fiber swelling, aqueous ammonia soaking, ionic liquid treatment, and partial hydrolysis using concentrated acids, including hydrochloric acid, sulfuric acid, and phosphoric acid, as well as combinations thereof.

[0034] When the sugar contains cellobiose and the enzyme contains cellobiose phosphorylase, G1P is produced from cellobiose and phosphate by cellobiose phosphorylase. When the sugar contains cellodextrin and the enzyme contains cellodextrin phosphorylase, G1P is produced from cellodextrin and phosphate by cellodextrin phosphorylase. When the sugar contains cellulose and the enzyme contains cellulose phosphorylase, G1P is produced from cellulose and phosphate by cellulose phosphorylase.

[0035] When the sugar contains maltose and the enzyme contains maltose phosphorylase, G1P is produced from maltose and phosphate by maltose phosphorylase. When the sugar contains sucrose and the enzyme contains sucrose phosphorylase, G1P is produced from sucrose and phosphate by sucrose phosphorylase.

[0036] When the sugar is starch or a starch derivative, the derivative may be selected from the group consisting of amylose, amylopectin, soluble starch, amylodextrin, maltodextrin, maltose, maltotriose, and glucose, and mixtures thereof. In a specific process of the present invention, the enzyme used to convert sugar to G1P contains αGP. In this step, when the sugar contains starch, G1P is produced by αGP from starch and phosphate; when the sugar contains soluble starch, amylodextrin, or maltodextrin, G1P is produced by αGP from soluble starch and phosphate, amylodextrin and phosphate, or maltodextrin and phosphate. Examples of αGP are αGP from Thermotoga maritima (Uniprot ID G4FEH8) disclosed in International Patent Application Publication No. 2017 / 059278, or αGP from Thermus sp. CCB_US3_UF1 (Uniprot G8NCC0) disclosed in International Patent Application Publication No. 2020 / 092315.

[0037] Some processes according to the present invention may further include a step of converting starch into a starch derivative, which is prepared by enzymatic or acid hydrolysis of starch. In certain processes of the present invention, maltose phosphorylase (MP) can be used to increase allulose yield by phosphorolytically cleaving the degradation product maltose into G1P and glucose. Alternatively, 4-glucan transferase (4GT) can be used to increase allulose yield by recycling the degradation products glucose, maltose, and maltotriose into longer maltooligosaccharides, which can be phosphorolytically cleaved by αGP to obtain G1P. Examples of 4GT are 4GT from Thermococcus litoralis (Uniprot ID O32462), disclosed in International Patent Application Publication No. WO 2017 / 059278, or 4GT from Anaerolinea thermophila strain DSM 14523 (Uniprot E8MXP8), disclosed in International Patent Application Publication No. WO 2020 / 092315. In some processes of the invention, polyphosphate and polyphosphate glucokinase (PPGK) can be added to the process, thus increasing the yield of allulose by phosphorylating the breakdown product glucose to G6P.

[0038] Starch is the most widely used energy storage compound in nature, and is mostly stored in plant seeds.Natural starch contains linear amylose and branched amylopectin.Examples of starch derivatives include amylose, amylopectin, soluble starch, amylodextrin, maltodextrin, maltose, fructose, and glucose.Examples of cellulose derivatives include pretreated biomass, regenerated amorphous cellulose, cellodextrin, cellobiose, fructose, and glucose.Sucrose derivatives include fructose and glucose.

[0039] When the process uses a starch derivative, the starch derivative can be prepared by enzymatic hydrolysis of starch catalyzed by isoamylase, pullulanase, alpha-amylase, or a combination thereof. Corn starch contains many branches that interfere with the action of αGP. Isoamylase and pullulanase can be used to debranch starch, resulting in linear amylodextrin. Isoamylase and pullulanase pretreatment of starch can result in a higher F6P concentration in the final product. Isoamylase and pullulanase cleave alpha-1,6-glycosidic bonds, allowing for more complete degradation of starch by alpha-glucan phosphorylase. Alpha-amylase cleaves alpha-1,4-glycosidic bonds, and therefore alpha-amylase is used to break down starch into fragments for faster conversion to allulose.

[0040] Allulose can also be produced from fructose. See Figure 3. The process according to the present invention can also include a step of converting fructose to F6P, which is catalyzed by at least one enzyme, and optionally a step of converting sucrose to fructose, which is catalyzed by at least one enzyme. For example, this process involves producing F6P from fructose and polyphosphate catalyzed by polyphosphate fructokinase (PPFK). The conversion of F6P to allulose is as described above. Fructose can be produced, for example, by the enzymatic conversion of sucrose. The phosphate ions produced when A6P is converted to allulose can then be recycled to the step of converting sucrose to G1P.

[0041] Allulose can also be produced from glucose. See Figure 4. The process according to the present invention can also include a step of converting glucose to G6P catalyzed by at least one enzyme, and optionally a step of converting sucrose to fructose, which step is also catalyzed by at least one enzyme. For example, this process involves producing G6P from glucose and polyphosphate catalyzed by polyphosphate glucokinase (PPGK). Glucose can be produced, for example, by 4-glucan transferase recycling of maltotriose to longer-chain maltodextrins.

[0042] In some methods of the present invention, the phosphate ions produced when A6P is converted to allulose are recycled to the step of converting starch derivatives to G1P (see, e.g., FIG. 1), cellulose derivatives to G1P (see, e.g., FIG. 2), or sucrose to G1P (see, FIG. 5), particularly when the process is carried out in a single reaction vessel. Additionally, PPFK and polyphosphates can be used to increase allulose yield by generating F6P from fructose produced by phosphorolytic cleavage of sucrose by SP.

[0043] For example, a process for preparing allulose from sugars includes the following steps: (i) converting the sugars to glucose 1-phosphate (G1P) using one or more enzymes, (ii) converting G1P to G6P using phosphoglucomutase (PGM, EC 5.4.2.2), (iii) converting G6P to F6P using phosphoglucoisomerase (PGI, EC 5.3.1.9), (iv) converting F6P to A6P via allulose 6-phosphate epimerase (A6PE), and (v) converting A6P to allulose via allulose 6-phosphate phosphatase (A6PP). In the improved process of the present invention, A6PE comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1, and / or A6PP comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2. In such a process, for example, the enzyme in step (i) is αGP. Typically, the ratio of enzyme units used in the process is 1:1:1:1:1 (αGP:PGM:PGI:A6PE:A6PP). An enzyme unit is the amount of enzyme required to convert 1 μmol of substrate to product in 1 minute. Therefore, an enzyme with higher activity will have a lower amount of enzyme, in terms of mg of enzyme per enzyme unit, compared to an enzyme with lower activity catalyzing the same reaction. These ratios can be adjusted in any number of combinations to optimize product yield. For example, a particular enzyme may be present in approximately 2x, 3x, 4x, 5x, etc. amounts relative to the amount of another enzyme.

[0044] The method for preparing allulose according to the present invention may include the following additional steps: producing glucose from polysaccharides and oligosaccharides by enzymatic or acid hydrolysis, converting glucose to G6P catalyzed by at least one enzyme, producing fructose from polysaccharides and oligosaccharides by enzymatic or acid hydrolysis, and converting fructose to G6P catalyzed by at least one enzyme. Examples of polysaccharides and oligosaccharides are listed above.

[0045] The process for preparing allulose according to the present invention can be carried out in a single bioreactor or reaction vessel. Alternatively, these steps can also be carried out in multiple bioreactors or reaction vessels arranged in series. In a preferred process, the enzymatic production of allulose is carried out in a single reaction vessel.

[0046] Enzymes used in the present invention may take the form of soluble, immobilized, structured, or aggregated proteins. These enzymes may be adsorbed to insoluble organic or inorganic supports, commonly used to enhance functionality, as is known in the art. These include polymeric supports such as agarose, methacrylate, polystyrene, phenol formaldehyde, or dextran, as well as inorganic supports such as glass, metal, or carbon-based materials. These materials are often produced with high surface-to-volume ratios and specialized surfaces that facilitate the attachment and activity of immobilized enzymes. Enzymes may be attached to these solid supports through covalent, ionic, or hydrophobic interactions. Enzymes may also be attached 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 may be attached 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 on a combination of two supports (e.g., two enzymes per support, then mixing the supports). These variations can be mixed homogeneously or in defined layers to optimize turnover in a continuous reactor. For example, the beginning of the reactor may have a layer of aGP to ensure a high initial G1P increase. The enzymes can be all immobilized on one support, on individual supports, or in groups. The enzymes can be mixed homogeneously or in defined layers or zones to optimize turnover.

[0047] Any suitable biological buffer known in the art can be used in the improved process of the present invention, such as HEPES, PBS, BIS-TRIS, MOPS, DIPSO, Trizma, or phosphate buffer. The reaction buffer of all embodiments can have a pH ranging from 4.0 to 9.0. More preferably, the pH of the reaction buffer can range from about 6.0 to about 7.8. For example, the pH of the reaction buffer can be 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, or 7.8.

[0048] In some improved processes of the present invention, the reaction buffer contains divalent metal cations. Examples include Mn 2+ , Co 2+ , Mg 2+ and Zn 2+ Preferably, Co 2+ and Mg 2+ The concentration of the divalent metal cation can be in the range of about 0 mM to about 150 mM, about 0 mM to about 100 mM, about 1 mM to about 50 mM, preferably about 5 mM to about 50 mM, or more preferably about 10 mM to about 50 mM. For example, the concentration of the divalent metal cation 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.

[0049] The reaction temperature at which the improved process steps are carried out can range from 37 to 85° C. More preferably, the steps are carried out at a temperature ranging from about 37 to about 85° C. The temperature can be, for example, about 40° C., about 45° C., about 50° C., about 55° C., or about 60° C. Preferably, the reaction temperature is about 50° C. In some improved processes of the present invention, the reaction temperature is constant and is not changed during the process.

[0050] The reaction time of the disclosed process 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 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.

[0051] The steps in the improved process of the present invention can be carried out in a batch or continuous process using a packed-bed reactor or similar equipment. In a continuous process, solution maltodextrin is pumped through a bed of immobilized enzyme at a rate such that the conversion to allulose is complete when the solution leaves the column 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 allulose yield is achieved when the maltodextrin leaves the column. This methodology provides higher volumetric productivity than batch methods. This limits the time our product spends interacting with the column and reaction conditions, reducing the possibility of product degradation (e.g., potential hydroxymethylfurfural formation). Whether in batch or continuous mode, various steps of the process of the present invention can be carried out using the same reaction conditions as the other steps. For example, in certain processes of the present invention using a single bioreactor or reaction vessel, reaction conditions such as pH and temperature, as well as reaction buffers, are kept constant throughout all steps of the process.

[0052] The phosphate ions generated by A6PP dephosphorylation of A6P can then be recycled to the process step that converts sugars to G1P, especially when all process steps are carried out in a single bioreactor or reaction vessel. The ability to recycle phosphate in the disclosed process allows for the use of non-stoichiometric amounts of phosphate, thereby 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 allulose production process.

[0053] For example, the reaction phosphate concentration can be in the range of about 0.1 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 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.

[0054] A low phosphate concentration reduces production costs due to a lower total phosphate concentration, thus reducing the cost of phosphate removal, and it also prevents high concentrations of free phosphate from inhibiting the A6PP processing enzymes, reducing the possibility of phosphate contamination.

[0055] Furthermore, the process disclosed herein can be carried out without adding ATP as a source of phosphate, i.e., ATP-free. The process can be carried out without the need to add NAD(H), i.e., NAD(H)-free. Other advantages also include the fact that at least one step of the disclosed process for producing allulose involves an energetically favorable chemical reaction. While the use of enzymes with higher activity does not affect the overall energy, the ability to use less enzyme in the improved process is advantageous. An advantage is the reduction in the overall cost of enzymes in the total production cost of the product.

[0056] The process according to the present invention can achieve high yields due to the very favorable equilibrium constant of the entire reaction.Theoretically, when the starting material is completely converted into the intermediate, a yield of up to 99% can be achieved.In addition, the step of converting A6P into allulose according to the present invention is an irreversible phosphatase reaction regardless of the raw material.Therefore, allulose is produced in a very high yield.

[0057] The process of the present invention uses low-cost starting materials and reduces the costs associated with raw material and product separation, thereby reducing product costs.Starch and its derivatives, cellulose and its derivatives, and sucrose are, for example, cheaper raw materials than crystalline fructose.When allulose is produced from fructose, the yield is only about 28% (WO2016 / 160573).Fructose and allulose are then separated by chromatography, which at the same time leads to higher production costs than the disclosed method.

[0058] The process according to the present invention allows for easy recovery of allulose and minimizes separation costs. Preferably, in the process of the present invention, allulose is not recovered via chromatographic separation. After allulose is produced in a continuous reaction, the product is instead subjected to ultrafiltration, ion exchange (cation, then anion, not mixed bed), concentration, crystallization, crystal isolation, and drying. Because the yield of allulose is high, only the crystallization step is required to purify allulose. To further purify allulose before crystallization, ultrafiltration can be used to co-crystallize it with allulose, eliminating the risk of enzymes present in the crystallization process and nanofiltration, or ultrafiltration can be used to remove any unconverted dextrins that may limit the recyclability of the mother liquor (maltodextrin, maltotetraose, maltotriose, maltose, etc.).

[0059] The improved process for preparing allulose according to the present invention comprises the following steps: (i) converting sugars to glucose 1-phosphate (G1P) using one or more enzymes, (ii) converting G1P to G6P using phosphoglucomutase (PGM, EC 5.4.2.2), (iii) converting G6P to F6P using phosphoglucoisomerase (PGI, EC 5.3.1.9), (iv) converting F6P to A6P via allulose 6-phosphate epimerase (A6PE), and (v) converting A6P to allulose via allulose 6-phosphate phosphatase (A6PP), wherein A6PE comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1 and / or A6PP comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2. This process is preferably carried out in a single bioreactor or reaction vessel.

[0060] Preferably, the improved process for preparing allulose according to the present invention comprises the following steps: (i) converting sugars into glucose 1-phosphate (G1P) using αGP, wherein the sugars are selected from the group consisting of starch, one or more derivatives of starch, or a combination thereof, (ii) converting G1P to G6P using phosphoglucomutase (PGM, EC 5.4.2.2), (iii) converting G6P to F6P using phosphoglucoisomerase (PGI, EC 5.3.1.9), (iv) converting F6P to A6P via allulose 6-phosphate epimerase (A6PE), and (v) converting A6P to allulose via allulose 6-phosphate phosphatase (A6PP), wherein A6PE comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1 and / or A6PP comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2. The process is preferably carried out in a single reactor vessel and may incorporate one or more of the various process conditions discussed above. [Example]

[0061] Materials and Methods: All chemicals, including glucose 1-phosphate, magnesium chloride, and sodium phosphate (monobasic and dibasic), were reagent grade or higher and purchased from Sigma-Aldrich (St. Louis, MO, USA) or Fisher Scientific (Pittsburgh, PA, USA) unless otherwise noted. 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 amplification and recombinant protein expression.

[0062] Production and purification of recombinant enzyme. E. coli BL21(DE3) strain containing the protein expression plasmid (pET28a) was cultured at 50 mg L -1The cells were incubated in a 1 L Erlenmeyer flask with 100 mL of ZYM-5052 medium containing 100 mM kanamycin. Cells were grown at 37°C with rotary shaking at 220 rpm for 16–24 h. Cells were harvested by centrifugation at 12°C and washed once with either 20 mM HEPES (pH 7.5) containing 50 mM NaCl and 5 mM MgCl2 for heat precipitation or 20 mM HEPES (pH 7.5) containing 300 mM NaCl and 5 mM imidazole for 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. His-tagged proteins were purified using Profinity IMAC Ni-Charged Resin (Bio-Rad, Hercules, CA, USA). The amount of purified protein was quantified by absorbance at 280 nm and used to calculate the relative expression yields in Table 1.

[0063] E. coli BL21(DE3) strain containing the thermostable protein expression plasmid (pET28a) was cultured at 50 mg L -1 The cells were incubated in a 1-L Erlenmeyer flask with 100 mL of ZYM-5052 medium containing 100 mM kanamycin. Cells were grown at 37°C with rotary shaking at 220 rpm for 16–24 hours. Cells were harvested by centrifugation at 12°C and washed once with either 20 mM HEPES (pH 7.5) containing 50 mM NaCl and 5 mM MgCl2. The cell pellet was resuspended in the same buffer and lysed by sonication. After centrifugation, the target protein in the supernatant was tested for thermal stability at 50–80°C for 30 minutes. The stability of the recombinant protein was examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and recorded by visual inspection, as described in Table 1. [Table 1]

[0064] Example 1. Evaluating the relative activity of allulose 6-phosphate epimerase (A6PE)-dependent conversion of G1P to allulose in improved processes. The conversion of G1P to allulose by an enzymatic process involving A6PE having the amino acid sequence of UniProt ID A0A223HZI7 ("A0A223HZI7 A6PE") was compared to G1P to allulose conversion processes that differed only in the A6PE used in each process. More specifically, the conversion of G1P to allulose was compared using processes including PGM having the amino acid sequence of UniProt ID A0A150LLZ1, PGI having the amino acid sequence of UniProt ID Q5SLL6, A6PE or A6PE having the amino acid sequence of UniProt ID D9TQJ4, UniProt ID A0A090IXZ8, UniProt ID A8UV28, UniProt ID G7M2I3, UniProt ID A0A094WLM1, Uniprot ID UPI000411882A, or Uniprot P32719 (the P32719 enzyme is unstable at 50°C), and A6PP having the amino acid sequence of Uniprot ID A0A0E3NCH4. Each process was carried out with a 0.20 mL reaction mixture containing 38.5 mM G1P, 50 mM HEPES (pH 7.2), 15 mM MgCl, 0.5 mM CoCl, 0.05 g / L PGM, 0.05 g / L PGI, 0.025 g / L A6PE, and 0.15 g / L A6PP. The reaction was incubated at 50°C for 3 hours. A6PE was the rate-limiting enzyme in the conversion of G1P to allulose. The reaction was terminated by enzyme filtration using a Vivaspin® 2 concentrator (10,000 MWCO) and analyzed by HPLC (Agilent 1100 series) using an Agilent Hi-Plex H column and a refractive index detector. Sample runs were performed at 65°C for 15.5 minutes at 0.6 mL / min in 5 mM H2SO4.

[0065] The results showed no significant A6PE-dependent differences in activity, except that A6PE with the UniProt A8UV28 amino acid sequence was not active under the conditions tested.

[0066] Example 2. Evaluation of lower allulose-6-phosphate epimerase (A6PE)-dependent allulose-to-fructose conversion activity. The conversion of allulose to fructose by an enzymatic process including A0A223HZI7 A6PE was compared with allulose-to-fructose conversion processes that differed only in the A6PE used in each process. More specifically, the conversion of allulose to fructose was compared using a process including A0A223HZI7 A6PE or A6PE having the amino acid sequence of UniProt ID D9TQJ4, UniProt ID A0A090IXZ8, UniProt ID G7M2I3, UniProt ID A0A094WLM1, UniProt ID A0A223HZI7, or UniParc ID UPI000411882A (A6PEs having the amino acid sequences of UniProt ID P32719 and UniProt ID A8UV28 were omitted due to incompatibility with the pathway). Each process was carried out in a 0.20 mL reaction mixture containing 200 g / L allulose, 10 mM HEPES (pH 7.2), 5 mM MgCl, 0.5 mM CoCl, and 0.025 g / L or 0.25 g / L A6PE. The reaction was incubated at 50°C for 6 hours. The reaction was terminated by filtration of the enzyme through a Vivaspin 2 concentrator (10,000 MWCO) and analyzed by HPLC (Agilent 1100 series) using a SupelCogel Pb column and a refractive index detector. Samples were run in ultrapure water at 80°C for 40 minutes at 0.6 mL / min.

[0067] The results summarized in Table 2 showed that the tested A6PEs produced relatively little fructose from allulose, except for the enzymes with the amino acid sequences of UniProt ID A0A090IXZ8 and UniParc ID UPI000411882A. Notably, no conversion of allulose to fructose was observed for A0A223HZI7 A6PE at 0.025 g / L of the enzyme composition. [Table 2]

[0068] Example 3. Evaluation of the relative activity of allulose-6-phosphate epimerases (A6PEs) with improved properties in the conversion of maltodextrin to allulose. The complete cascade conversion of maltodextrin to allulose by an enzymatic process including A0A223HZI7 A6PE (which did not demonstrate fructose-to-allulose activity) was compared to maltodextrin-to-allulose conversion processes that differed only in the A6PE used in each process to determine how F6PE, which has different fructose-to-allulose activity, affected allulose yield. More specifically, the conversion of maltodextrin to allulose was compared using processes including αGP having the amino acid sequence of UniProt ID G8NCC0, PGM having the amino acid sequence of UniProt ID A0A150LLZ1, PGI having the amino acid sequence of UniProt ID Q5SLL6, A6PE or A6PE having UniProt ID A0A223HZI7 (which exhibited high fructose to allulose activity) or UniProt ID UPI000411882A (which exhibited moderate fructose to allulose activity), A6PP having the amino acid sequence of UniProt ID A0A0E3NCH4, and 4GT having the amino acid sequence of UniProt ID E8MXP8. Each process was carried out with a 0.20 mL reaction mixture containing 100 g / L debranched Sigma-Aldrich maltodextrin DE 4-7, 25 mM sodium phosphate (pH 7.2), 15 mM MgCl, 0.5 mM CoCl, 0.3 g / L αGP, 0.075 g / L PGM, 0.075 g / L PGI, 0.1 g / L A6PE, 0.1 g / L A6PP, and 0.04 g / L 4GT. Reactions that were not completely complete at the time of analysis were incubated at 50°C for 18 hours. The reactions were terminated by filtration of the enzyme through a Vivaspin® 2 concentrator (10,000 MWCO) and analyzed by HPLC (Agilent® 1100 series) using a SupelCogel® Pb column and a refractive index detector.Sample runs were in ultrapure water at 80° C. for 40 minutes at 0.6 mL / min.

[0069] The results, shown in Figure 6, indicated that the process involving A6PE, having the amino acid sequence of UniProt ID UPI000411882A, produced substantially more fructose than the process involving A0A223HZI7A6PE. As expected, the process involving A6PE, having the amino acid sequence of UniProt ID A0A090IXZ8, produced intermediate amounts of fructose. The process involving A0A223HZI7 A6PE produced the most allulose at 18 hours. This resulted in the highest allulose yield pathway, more efficient than any previously disclosed route for producing allulose using A6PE. The relative affinity of F6P / A6P to allulose for a given A6PE enzyme is demonstrated by the observed difference between the fructose-to-allulose activity and the allulose-to-maltrodextrin cascade. For example, A6PE having the amino acid sequence of UniProt ID A0A090IXZ8 produced higher amounts of fructose from allulose alone compared to A6PE having the amino acid sequence of Uniparc ID UPI000411882A, but in the complete cascade reaction, A6PE having the amino acid sequence of Uniprot ID A0A090IXZ8 performed better because F6P / A6P could compete with allulose for this enzyme more efficiently than A6PE having the amino acid sequence of Uniparc ID UPI000411882A.

[0070] Example 4. Evaluation of the relative activity of allulose 6-phosphate phosphatase (A6PP) with improved properties in the conversion of G1P to allulose. The conversion of G1P to allulose by an enzymatic process using A6PP having the amino acid sequence of UniProt ID A0A0E3NCH4 ("A0A0E3NCH4 A6PP") was compared with G1P for allulose conversion processes that differed only in terms of the A6PP used in each process. More specifically, the conversion of G1P to allulose was compared using processes containing either A0A0E3NCH4 A6PP or A6PP having the amino acid sequence of UniProt ID A0A150LLZ1, PGI having the amino acid sequence of UniProt ID Q5SLL6, A6PE having the amino acid sequence of UniProt ID D9TQJ4, and UniProt ID A3DC21. Previously disclosed A6PP, with the amino acid sequence of either Uniprot ID Q5LGR4 or Uniprot ID Q89ZR1, could not be included in the comparison due to the instability of these enzymes at 50°C. Each process was carried out in a 0.20 mL reaction mixture containing 38.5 mM G1P, 50 mM HEPES (pH 7.2), 0.5 mM CoCl, 0.05 g / L PGM, 0.05 g / L PGI, 0.025 g / L A6PE, and 0.05 g / L A6PP. The reaction was incubated at 50°C for 3 hours. A6PP was the rate-limiting enzyme in the conversion of G1P to allulose. The reaction was terminated by filtration of the enzyme using a Vivaspin® 2 concentrator (10,000 MWCO) and analyzed by HPLC (Agilent 1100 series) using an Agilent Hi-Plex® H column and a refractive index detector. The sample run was in 5 mM H2SO4 at 0.6 mL / min for 15.5 minutes at 65°C. The results, summarized in Table 3, showed a 2.2-fold improvement in allulose production using A0A0E3NCH4A6PP compared to the previously disclosed process using A6PP, which has the amino acid sequence of UniProt ID A3DC21. [Table 3]

[0071] Sequence Listing SEQ ID NO: 1, A6PE, UniProt ID A0A223HZI7, Thermoanaerobacterium thermosaccharolyticum MKPMFAPSLMCANFLDLKNQIEILNERADIYHIDIMDGHYVKNFALSPYLMEQLKTIAKIPMDAHLMVENPADFLECIAKSGATYISPHAETINKDAFRIMRTIKALGCKTG IVLNPATPVEYIKYYIGMLDKITILTVDAGFAGQTFINEMLDKIAEIKSLRDQNGYSYLIEVDGSCNEKTFKQLAEAGTDVFVVGSSGLFNLDTDLKVAWDKMMDTFTRCTSN SEQ ID NO:2, A6PP, UniProt ID A0A0E3NCH4, Methanosarcina thermophila MLKALIFDMDGVLVDSMPFHAAAWKKAFFEMGMEIQDSDIFAIEGSNPRNGLPLLIRKARKEPEAFDFEAITSIYRQEFKRVFEPKAFEGMKECLEVLKKRFLLSVVSGS DHVIVHSIINRLFPGIFDIVVTGDDIINSKPHPDPFLKAVELLNVRREECVVIENAILGVEAAKNARIYCIGVPTYVEPSHLDKADLVVEDHRQLMQHLLSLEPANGFRQ SEQ ID NO: 3, A6PE, UniProt ID D9TQJ4, Thermoanaerobacterium thermosaccharolyticum MKYLFSPSLMCMNLIKLNEQISVLNSKADFLHVDIMDGHFVKNITLSPFFIEQIKSYVNIPIDAHLMVENPGDYIEICEKSGASFITIHAETINREAFRIIDRIKSHGLM VGIALNPATPISEIKHYINKIDKITIMTVDPGFAGQPFIPEVLEKIRDLKRLKDDNNYNYLIEADGSCNKNTFQVLKDAGCKVFVLGSSGLFNLSDDLGKAWEIMIGNFNG SEQ ID NO: 4, A6PE, UniProt ID A0A090IXZ8, Bacillus thermoamylovorans MSNKIEFSPSLMTMDLDKFKEQITFLNNHVGSYHIDIMDGHYVPNITLSPWFVQEVRKISDVPMSAHLMVTNPSFWVQQLIDIKCEWICMHVETLDGLAFRLIDQIHDAGLKAGV VLNPETSVDAIRPYIDLVDKVTIMTVDPGFAGQRFIDSTLEKIVELRKLREEHGYKYVIEMDGSSNRKSFKKIYEAGPDIYIIGRSGLFGLHEDIEKAWEIMCKDFEEMTGEKVL SEQ ID NO: 5, A6PE, UniProt ID P32719, E. coli MKISPSLMCMDLLKFKEQIEFIDSHADYFHIDIMDGHFVPNLTLSPFFVSQVKKLATKPLDCHLMVTRPQDYIAQLARAGADFITLHPETINGQAFRLIDEIRRHDMKVGLILNP ETPVEAMKYYIHKADKITVMTVDPGFAGQPFIPEMLDKLAELKAWREREGLEYEIEVDGSCNQATYEKLMAAGADVFIVGTSGLFNHAENIDEAWRIMTAQILAAKSEVQPHAKTA SEQ ID NO: 6, A6PE, UniProt ID A8UV28, Hydrogenivirga sp. 128-5-R1-1 MEKLLAPSILAGDWWNIGEQIEATLRGGADIIHFDVMDGHFVPNITVGPEILTSISRRVNVPVDAHLMIENPDRYIPSFVEAGAKWISVHIENVPHIHRTLTLIRELGAKA GVVLNPGTPLSAVEEAIHYADYVLLMSVNPGFSGQRFIERSLERLSLLRDMRDRLNPDCLIEVDGGVKEDNVVEVVRAGADVVVVGSGIFSAKDVEAQTRKLKDLISSAVAV SEQ ID NO: 7, A6PE, UniParc ID UPI000411882A, Brevibacillus thermolvar MGFKFSPSLMCMNLDIQHQIEVMNRRADLVHIDIMDGHYVKNLTLSPFFIEQLKESLHVPMDVHLMVENPTDFIERVKEAGASIISPHAETINTDAFRIIDKVKSLGCQMGIV LNPATPIAYIQHYIHLVDKITIMTVDPGYAGQKFIPEMLEKIRQAKRLKEERGYRYLIEVDGSCNVGTFKRLAEAGAEVFIVGSSGLFNLHPDLEVAWDMMMDNFQREVGETTA SEQ ID NO: 8, A6PE, UniProt ID G7M2I3, Clostridium sp. DL-VIII MKPMFAPSLMCANFLDLKNQIEILNERADIFHVDIMDGHYVKNFSLSPAMMEQLKTITKIPMDAHLMVENPADFLEGIAKAGATYISPHAETINKDAFRIMRTIKALGCKTG VVLNPATPVEYIKHYLGMLDKITILTVDAGFAGQTFIEEMLDKIEEVKRLREENGYSYLIEVDGSCNEKTFKKLAEAGTEVFIVGSSGLFNLDADLKVSWDKMMNMFNKCINN SEQ ID NO: 9, A6PE, UniProt ID A0A094WLM1, Bacillus alkalophilus MYKFSPSLMCMDLSRFKEQVEVLNDKADFYHVDIMDGHFVKNITLSPFFIQELKKITDVPIDAHLMVTNPADFVEMTIDAGADYISLHAETINGNAFRLINQIKEKGKKFGVVL NPATPLESIRHYIQHVDKLTIMTVDPGFAGQKFVEEMIGKIKEAKELKERNGYKYLITIDGSCNKNTFKKLVEAGAEVLIVGSSGLFGLDEDVNIAWDKMMDTFHLEVKDISQV SEQ ID NO: 10, A6PP, UniProt ID A3DC21, Hungateiclostridium thermocellum MIKYKAVFFDFDYTLADSSKAVIECINYALQKMGYPESSPESICRTIGLTLAEAFKILSGDTSDSNADLFRQYFKERADLVMCDRTVMYSTVECVLKKLKKADVKTGI VSTKYRYRIEDILKRDKLLQYFDVIVGGEDVAAHKPDPEGLLKAISMVGCQKEEVLFVGDSTVDARTAKNAGVDFVAVLTGTTGANEFSEYNPGAVIEDLSGLLDMFML SEQ ID NO: 11, A6PP, UniProt ID Q5LGR4, Bacteroides fragilis MKYTVYLFDFDYTLADSSRGIVTCFRSVLERHGYTGITDDMIKRTIGKTLEESFSILTGITDADQLESFRQEYSKEADIYMNANTILFPDTLPTLTHLKKQGIRIGIISTKYRFRILSFLRNNHMPDDWFDIIIGGEDVTHHKPDPEGLLLAIDRLKACPEEVLYIGDSTVDAGTAAAAGVSFTGVTSGMTTAQEFQAYPYDRIISTLGQLISVPEDKSGCPL SEQ ID NO: 12, A6PP, UniProt ID Q89ZR1, Bacteroides thetaiotaomicron MNYKTYLFDFDYTLADSSRGIVTCFRNVLNRHQYTNVTDEAIKRTIGKTLEESFSILTGVTDWEQLTAFRQEYRLEADVHMNVNTRLFPDTLSTLKELKERGARIGIISTKYRFRILSFLDEYLPENFLDIVVGGEDVQAAKPSPEGIKFALEHLGRTPQETLYIGDSTVDAETAQNAGVDFAGVLNGMTTADELRAYPHRFIMENLSGLLYI SEQ ID NO: 13, PGM, UniProt ID A0A150LLZ1, Caldibacillus devilis MEWKQRAERWLRFENLDPELKKQLEEMAKDEKKLEDLFYKYLEFGTGGMRGEIGPGTNRINIYTVRKASEGLARFLLASGGEEKAKQGVVIAYDSRRKSREFALETAKTVGKHGIKAYVFESLRPTPELSFAVRYLHAAAGVVITASHNPPEYNGYKVYGEDGGQLTPKAADELIRYVYEVEDELSLTVPGEQELIDRGLLQYIGENIDLAYIEKLKTIQLNRDVILNGGKDLKIVFTPLHGTAGQLVQTGLREFGFQNVYVVKEQEQPDPDFSTVKSPNPEEHEAFEIAIRYGKKYDADLIMGTDPDSDRLGIVVKNGQGDYVVLTGNQTGAILLYYLLSQKKEKGMLVRNSAVLKTIVTSELGRAIASDFGVETIDTLTGFKFIGEKIKEFKETGSHVFQFGYEESYGYLIGDFVRDKDAIQAALFAAEAAAYYKAQGKSLYDVLMEIYKKYGFYKESLRSITLKGKDGAEKIRAIMDAFRQNPPEEVSGIPVAITEDYLTQKRVDKAAGQTTPIHLPKSNVLKYYLADESWFCIRPSGTEPKCKFYFAVRGDSEAQSEARLRQLETNVMAMVEKILQK Sequence number 14, PGI, UniProt ID Q5SLL6, Thermus thermophilus MLRLDTRFLPGFPEALSRHGPLLEEARRRLLAKRGEPGSMLGWMDLPEDTETLREVRRYREANPWVEDFVLIGIGGSALGPKALEAAFNESGVRFHYLDHVEPEPILRLLRTLDPRKTLVNAVSKSGSTAETLAGLAVFLKWLKAHLGEDWRRHLVVTTDPKEGPLRAFAEREGLKAFAIPKEVGGRFSALSPVGLLPLAFAGADLD ALLMGARKANETALAPLEESLPLKTALLLHLHRHLPVHVFMVYSERLSHLPSWFVQLHDESLGKVDRQGQRVGTTAVPALGPKDQHAQVQLFREGPLDKLLALV IPEAPLEDVEIPEVEGLEAASYLFGKTLFQLLKAEAEATYEALAEAGQRVYALFLPEVSPYAVGWLMQHLMWQTAFLGELWEVNAFDQPGVELGKVLTRKRLAG SEQ ID NO: 15, 4GT, UniProt ID E8MXP8, Anaerolinea thermophila MSLFKRASGILLHPTSLPGPDGIGDLPGEAYRWVNFLAESGCSLWQILPLGPTGFGDSPYQCFSAFAGNPYLVSPALLLDEGLLTSEDLADRPEFPASRVDYGPVIQWKLTLLDRAYVRFKRSTSQK RKAAFEFKEEQRAWLLDFSLFMAIKEAHGGASWDYWPEPLRKRDPEALNAFHRAHEVDVERHSFRQFLFFRQWQALRQYAHEKGVQIIGDVPIFVAYDSADVWSHPDLFYLDETGKPTVVAGVPPDY FSATGQLWGNPLYRWDYHRETGFAWWLERLKATFAMVDIVRLDHFRGFAGYWEVPYGMPTAEKGRWVPGPGIALFEAIRNALGGLPIIAEDLGEITPDVIELREQLGLPGMKIFQFAFASDADDPFLP HNYVQNCVAYTGTHDNDTAIGWYNSAPEKERDFVRRYLARSGEDIAWDMIRAVWSSVAMFAIAPLQDFLKLGPEARMNYPGRPAGNWGWRYEAFMLDDGLKNRIKEINYLYGRLPEHMKPPKVVKKWT Accession number 16, αGP, UniProt ID G8NCC0, Thermus species CCB_US3_UF1 MPLLPEPLSGLKELAYNLWWSWNPEAAELFQEIDPSLWKRFRGNPVKLLLEADPGRLEGLAATSYPARVGAVVEALRAYLREREEKQGPLVAYFSAEYGFHSSLPIYSGGLGVLAGDHVKAASDLGLNLVGVGIFYHEGYFHQRLSPEGVQVEVYETLHPEELPLYPVQDREGRPLRVGVEFPGRTLWLSAYRVQVGAVPVYLLTANLPENTPEDRAITARLYAPGLEMRIQQELVLGLGGVRLLRALGLAPEVFHMNEGHSAFLGLERVRELVAEGHPFPVALELARAGALFTTHTPVPAGHDAFPLELVERYLGGFWERMGTDRETFLSLGLEEKPWGKVFSMSNLALRTSAQANGVSRLHGEVSREMFHHLWPGFLREEVPIGHVTNGVHTWTFLHPRLRRHYAEVFGPEWRKRPEDPETWKVEALGEEFWQIHKDLRAELVREVRTRLYEQRRRNGESPSRLREAEKVLDPEALTIGFARRFATYKRAVLLFKDPERLRRLLHGHYPIQFVFAGKAHPKDEPGKAYLQELFAKIREYGLEDRMVVLEDYDMYLARVLVHGSDVWLNTPRRPMEASGTSGMKAALNGALNLSVLDGWWAEAYNGKNGFAIGDERVYESEEAQDMADAQALYDVLEFEVLPLFYAKGPEGYSSGWLSMVHESLRTVGPRYSAARMVGDYLEIYRRGGAWAEAARAGQEALAAFHQALPALQGVTLRAQVPGDLTLNGVPMRVRAFLEGEVPEALRPFLEVQLVVRRSSGHLEVVPMRPGPDGYEVAYRPSRPGSYAYGVRLALRHPITGHVAWVRWA

Claims

1. 1. An improved process for the production of allulose from sugars, comprising: converting fructose-6-phosphate (F6P) to allulose-6-phosphate (A6P) using allulose-6-phosphate epimerase (A6PE); converting A6P to allulose using allulose-6-phosphate phosphatase (A6PP); wherein said A6PP comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

2.

2. The process described in claim 1, wherein the A6PE comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

1.

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

4. 4. The process of claim 3, further comprising converting glucose 1-phosphate (G1P) to said G6P, said step being catalyzed by phosphoglucomutase (PGM).

5. 5. The process of claim 4, further comprising a step of converting sugars into said G1P, said step being catalyzed by at least one enzyme, and said sugars being selected from the group consisting of starch or a derivative thereof, cellulose or a derivative thereof, and sucrose.

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

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

8. 8. The process of claim 7, further comprising the step of converting starch into a starch derivative, said starch derivative being prepared by enzymatic hydrolysis of starch or by acid hydrolysis of starch.

9. 9. The process of claim 8, wherein 4-glucan transferase (4GT) is added to the process.

10. 9. The process of claim 8, wherein the starch derivative is prepared by enzymatic hydrolysis of starch catalyzed by isoamylase, pullulanase, alpha-amylase, or a combination thereof.

11. 3. The process of claim 1 or 2, further comprising a step of converting fructose to said F6P catalyzed by at least one enzyme.

12. The process of claim 11, further comprising a step of converting sucrose to said fructose catalyzed by at least one enzyme.

13. 4. The process of claim 3, further comprising converting glucose to said G6P catalyzed by at least one enzyme.

14. The process of claim 13, further comprising a step of converting sucrose to said glucose catalyzed by at least one enzyme.

15. (i) converting sugars into glucose 1-phosphate (G1P) using alpha-glucan phosphorylase or starch phosphorylase, wherein the sugars are selected from the group consisting of starch, one or more derivatives of starch, or a combination thereof; (ii) converting said G1P to glucose 6-phosphate (G6P) using phosphoglucomutase (PGM); (iii) converting the G6P to fructose 6-phosphate (F6P) using phosphoglucoisomerase (PGI); 3. The process of claim 1 or 2, further comprising:

16. The process steps are performed under the following conditions: (a) a temperature in the range of 37°C to 85°C; (b) a pH in the range of 5.0 to 9.0; or (c) 1 hour to 48 hours 16. The process of claim 15, wherein the process is carried out under at least one of the following conditions:

17. The process steps are performed under the following conditions: (a) no adenosine triphosphate (ATP) as a source of phosphate; (b) no nicotinamide adenosine dinucleotide; (c) a phosphate concentration of 0.1 mM to 150 mM; (d) 0.1 mM to 50 mM Mg ２＋ concentration, (e) 0.1 mM to 50 mM Co ２＋ concentration, (f) the phosphate is recycled; (g) at least one step of the process involves an energetically favorable chemical reaction; 17. The process according to claim 15 or 16, wherein the process is carried out under at least one of the following conditions:

18. 18. The process of claim 17, wherein phosphate is recycled and phosphate ions generated by dephosphorylation of A6P by A6PP are used in the step of converting the sugar to G1P.

19. 18. The process of claim 17, wherein the step of converting A6P to allulose is an energetically favorable, irreversible reaction.

20. The process according to any one of claims 15 to 19, further comprising a step of separating and recovering the produced allulose, wherein the separation and recovery is not by chromatographic separation.

21. 16. The process of claim 15, wherein the derivative of starch is selected from the group consisting of amylose, amylopectin, soluble starch, amylodextrin, maltodextrin, maltotriose, maltose, and glucose.

22. Executing the process of claim 1 or 2; Adding the produced allulose; A method for manufacturing a consumable product, comprising:

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