Enzymatic production of D-allulose

Abstract

The present disclosure provides methods for enzymatically converting sugars into allulose. [Solution] The present invention relates to a method for producing allulose, which method comprises converting fructose 6-phosphate (F6P) to allulose 6-phosphate (A6P) catalyzed by allulose 6-phosphate 3-epimerase (A6PE), and converting A6P to allulose catalyzed by allulose 6-phosphate phosphatase (A6PP).

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Application No. 62 / 434,033, filed December 14, 2016, which is incorporated herein by reference.

[0002] FIELD OF THE INVENTION The present invention relates to the preparation of the sugar D-allulose. More specifically, the present invention relates to a method for preparing D-allulose by enzymatically converting sugars (e.g., polysaccharides, oligosaccharides, disaccharides, sucrose, D-glucose, and D-fructose) to D-allulose. [Background technology]

[0003] Background of the Invention D-Allulose (also known as D-psicose) (hereafter referred to as allulose) is a low-calorie natural sweetener that possesses 70% of the sweetness of sucrose but only 10% of the calories. It is a naturally occurring monosaccharide hexose found in small amounts in wheat and other plants. Allulose was approved as a food additive by the Food and Drug Administration (FDA) in 2012 and is generally recognized as safe (GRAS) designated. However, its high retail price limits its use as a sweetener. Allulose offers numerous health benefits, including low calories (10% of sucrose), a very low glycemic index (GI) of 1, complete absorption in the small intestine but not metabolized (instead excreted in urine and feces), aiding in blood sugar regulation by inhibiting alpha-amylase, sucrase, and maltase, and possessing functionality similar to sucrose in foods and beverages. Thus, it is clear that allulose has numerous applications in the food and beverage industry.

[0004] Currently, allulose is mainly produced through the enzymatic isomerization of fructose (WO 2014049373). Generally, this method has problems such as high raw material costs, high separation costs from fructose to allulose, and relatively low product yields. Summary of the Invention [Means for solving the problem]

[0005] There is a need to develop a cost-effective synthetic route for producing allulose in high yields, where at least one step of the process involves an energetically favorable chemical reaction.Furthermore, there is a need for a production method in which the process steps can be carried out in a single tank or bioreactor.There is also a need for a process for producing allulose that can be carried out at a relatively low concentration of phosphate, where the phosphate can be recycled and / or the process does not require the use of adenosine triphosphate (ATP) as a phosphate source.There is also a need for an allulose production route that does not require the use of expensive nicotinamide adenosine dinucleotide (NAD(H)) coenzyme in any reaction step.

[0006] Summary of the Invention The invention described herein relates to a process for preparing allulose. In various embodiments, the process comprises converting fructose 6-phosphate (F6P) to allulose 6-phosphate (A6P) catalyzed by allulose 6-phosphate 3-epimerase (A6PE), and converting the A6P to allulose 6-phosphate phosphatase (A6PP). The invention also relates to allulose prepared by any of the processes described herein.

[0007] In some aspects of the invention, the process for preparing allulose also includes converting glucose 6-phosphate (G6P) to said F6P, which is catalyzed by phosphoglucoisomerase (PGI). In other aspects, the process for allulose synthesis also includes converting glucose 1-phosphate (G1P) to said G6P, which is catalyzed by phosphoglucomutase (PGM).

[0008] In various embodiments, the process for preparing allulose can include catalyzing at least one enzyme to convert sugars to the G1P, catalyzing phosphoglucomutase (PGM) to convert G1P to G6P, catalyzing phosphoglucoisomerase (PGI) to convert G6P to F6P, catalyzing A6PE to convert F6P to allulose 6-phosphate (A6P), and catalyzing A6PP to convert the resulting A6P to allulose.

[0009] The sugars used in any of the processes can be selected from the group consisting of starch or its derivatives, cellulose or its derivatives, and sucrose. The starch or its derivatives can be amylose, amylopectin, soluble starch, amylodextrin, maltodextrin, maltose, or glucose. In some embodiments of the present invention, the process for preparing allulose involves converting starch into a starch derivative by enzymatic or acid hydrolysis of the starch. In other embodiments, the starch derivative can be prepared by enzymatic hydrolysis of starch catalyzed by isoamylase, pullulanase, alpha-amylase, or a combination of two or more of these enzymes. In certain embodiments, the process for preparing allulose can also include the addition of 4-glucan transferase (4GT).

[0010] In various aspects, the process for preparing allulose can include converting fructose to F6P catalyzed by at least one enzyme, converting F6P to allulose 6-phosphate (A6P) catalyzed by A6PE, and converting the resulting A6P to allulose catalyzed by A6PP. In other embodiments, the allulose production process includes converting sucrose to fructose catalyzed by at least one enzyme, converting fructose to F6P catalyzed by at least one enzyme, converting F6P to allulose 6-phosphate (A6P) catalyzed by A6PE, and converting the resulting A6P to allulose catalyzed by A6PP.

[0011] In another aspect of the present invention, the G6P used in the method for preparing allulose can be produced by converting glucose to the G6P catalyzed by at least one enzyme, which in turn can be produced by converting sucrose to glucose catalyzed by at least one enzyme.

[0012] In other aspects of the invention, the steps of the process for preparing allulose are carried out at a phosphate concentration of about 0 mM to about 150 mM, in the absence of ATP, in the absence of NAD(H), the phosphate is recycled, and / or at least one step of the process involves an energetically favorable chemical reaction. The following is further disclosed in relation to the present invention. [1] A process for preparing allulose, comprising: Epimerase-catalyzed conversion of fructose 6-phosphate (F6P) to allulose 6-phosphate (A6P); and The resulting A6P is converted into allulose by phosphatase catalysis. The process comprising: [2] The process described in [1], further comprising a step of converting glucose 6-phosphate (G6P) to said F6P, said step being catalyzed by phosphoglucoisomerase (PGI). [3] The process described in [2], further comprising a step of converting glucose 1-phosphate (G1P) to said G6P, said step being catalyzed by phosphoglucomutase (PGM). [4] The process described in [3] further comprises a step of converting sugars into the G1P, the step being catalyzed by at least one enzyme, and the sugars being selected from the group consisting of starch or a derivative thereof, cellulose or a derivative thereof, and sucrose. [5] The process described in [4], wherein 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. [6] [4] The process according to [4], wherein the sugar is a starch or a derivative thereof selected from the group consisting of amylose, amylopectin, soluble starch, amylodextrin, maltodextrin, maltose, and glucose. [7] [6] The process according to [6], further comprising a step of converting starch into a starch derivative, wherein the starch derivative is prepared by enzymatic hydrolysis of starch or acid hydrolysis of starch. [8] The process according to [6], wherein 4-glucan transferase (4GT) is added to the process. [9] [7] The process according to [7], wherein the starch derivative is prepared by enzymatic hydrolysis of starch catalyzed by isoamylase, pullulanase, alpha-amylase, or a combination thereof.

[10] The process of [1] further comprises a step of converting fructose to the F6P, the step being catalyzed by at least one enzyme, and optionally a step of converting sucrose to the fructose, the step being catalyzed by at least one enzyme.

[11] [2] The process described in [2], further comprising a step of converting glucose to said G6P, said step being catalyzed by at least one enzyme, and optionally a step of converting sucrose to said glucose, said step being catalyzed by at least one enzyme.

[12] The process according to any one of [1] to

[11] , wherein the epimerase is allulose-6-phosphate-3-epimerase.

[13] The process of

[12] , wherein the allulose-6-phosphate 3-epimerase comprises an amino acid sequence having 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% sequence identity to SEQ ID NO: 3 or 6, and the epimerase catalyzes the conversion of F6P to A6P.

[14] The allulose-6-phosphate 3-epimerase is (α / β) 8 - a barrel domain, a Ser at the end of the seventh β-strand of the barrel, a Ser at the end of the eighth β-strand of the barrel, a Gly in the active site loop, a His in the second and third β-strands of the barrel, an 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.

[15] The process according to any one of [1] to

[11] , wherein the phosphatase is allulose-6-phosphate phosphatase.

[16]

[15] The process of

[15] , wherein the allulose-6-phosphate phosphatase comprises an amino acid sequence having at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% sequence identity to SEQ ID NO: 9, and the phosphatase catalyzes the conversion of A6P to allulose.

[17] The process described in

[15] , wherein the allulose 6-phosphate phosphatase is specific for allulose 6-phosphate.

[18] The process described in

[15] , wherein the allulose 6-phosphate phosphatase comprises a rosemanoid fold domain for catalysis, a C1 capping domain, a DxD signature on the first β-strand of the rosemanoid fold, a Thr or Ser at the end of the second β-strand of the rosemanoid fold, a Lys at the N-terminus of an α-helix whose C-terminus faces the third β-strand of the rosemanoid fold, and a GDxxxD signature at the end of the fourth β-strand of the rosemanoid fold.

[19] The process according to any one of [1] to

[11] , wherein the process steps are carried out at a temperature in the range of about 40°C to about 70°C, at a pH in the range of about 5.0 to about 8.0, and / or for a period of about 8 hours to about 48 hours.

[20] The process according to any one of [1] to

[11] , wherein the process steps are carried out in one bioreactor or in multiple bioreactors arranged in series.

[21] The process according to any one of [1] to

[11] , wherein the process steps are carried out in the absence of ATP, in the absence of NAD(H), and at a phosphate concentration of about 0 mM to about 150 mM, the phosphate is recycled, and / or at least one step of the process involves an energetically favorable chemical reaction.

[22] Allulose produced by the process described in any one of [1] to

[11] . [Brief explanation of the drawings]

[0013] Brief description of the drawings These drawings illustrate some specific aspects of embodiments of the invention and should not be used to limit or define the invention.

[0014] [Figure 1] FIG. 1 is a schematic diagram showing the enzymatic pathway that converts fructose 6-phosphate to allulose 6-phosphate and then to allulose.

[0015] [Figure 2] Figure 2 is a schematic diagram showing the enzymatic pathway for converting starch or its derivatives to allulose. The following abbreviations are used: αGP, alpha-glucan phosphorylase or starch phosphorylase; PGM, phosphoglucomutase; PGI, phosphoglucoisomerase; IA, isoamylase; PA, pullulanase; MP, maltose phosphorylase; PPGK, polyphosphate glucokinase.

[0016] [Figure 3] Figure 3 shows the enzymatic pathway for converting cellulose or its derivatives to allulose. CDP, cellodextrin phosphorylase. CBP, cellobiose phosphorylase. PPGK, polyphosphate glucokinase. PGM, phosphoglucomutase. PGI, phosphoglucoisomerase.

[0017] [Figure 4] Figure 4 is a schematic diagram showing the enzymatic pathway for converting fructose to allulose. PPFK, polyphosphate fructokinase.

[0018] [Figure 5] Figure 5 is a schematic diagram showing the enzymatic pathway for converting glucose to allulose. PPGK, polyphosphate glucokinase. PGI, phosphoglucoisomerase.

[0019] [Figure 6]Figure 6 shows the enzymatic pathway for converting sucrose or its derivatives to allulose. SP, sucrose phosphorylase. PPFK, polyphosphate fructokinase. PGM, phosphoglucomutase. PGI, phosphoglucoisomerase.

[0020] [Figure 7] FIG. 7 shows the Gibbs energies of reaction between intermediates based on the Gibbs energies of formation for the conversion of glucose 1-phosphate to allulose. DETAILED DESCRIPTION OF THE INVENTION

[0021] Detailed Description of the Invention The present invention provides an enzymatic pathway or process for synthesizing allulose in high yield, while significantly reducing the cost of product isolation and allulose production.

[0022] The present invention relates to a process for preparing allulose, which comprises converting fructose 6-phosphate (F6P) to allulose 6-phosphate (A6P) catalyzed by an epimerase, and converting the resulting A6P to allulose catalyzed by a phosphatase (e.g., allulose 6-phosphate phosphatase, A6PP). A schematic of this process is shown in Figure 1. In a specific embodiment, the epimerase catalyzing the conversion of F6P to A6P is allulose 6-phosphate 3-epimerase (A6PE).

[0023] Epimerases that convert F6P to A6P can be used in the process of the present invention. Epimerases can also convert A6P to F6P. In some embodiments of the present invention, epimerases suitable for use in the process of converting F6P to A6P have a certain degree of identity to the amino acid sequence of SEQ ID NO: 3 or 6, i.e., at least 45%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, at least 91%, at least 92%, at least 93%, or at least 94%, most preferably at least 95%, and even most preferably at least 96, 97, 98, 99, or 100% identity. Suitable epimerases are encoded by polynucleotides comprising a nucleotide sequence having a degree of identity, i.e., at least 30%, preferably at least 35%, more preferably at least 40%, more preferably at least 45%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 96, 97, 98, 99, or 100% identity, to the nucleotide sequences of SEQ ID NOs: 1, 2, 4, and 5.

[0024] Examples of A6PE include, but are not limited to, proteins identified by UNIPROT IDs D9TQJ4, A0A090IXZ8, and P32719. Of these, D9TQJ4 and A0A090IXZ8 are obtained from thermophilic organisms. P32719 is obtained from mesophilic organisms. P32719 is 53% identical to A0A090IXZ8 and 55% identical to D9TQJ4, and each protein catalyzes the epimerization of F6P to A6P. Furthermore, A0A090IXZ8 is 45% identical to D9TQJ4. Conversely, other epimerase proteins identified with UNIPROT IDs of A0A101D823, R1AXD6, A0A150LBU8, A0A023CQG9, and H1XWY2 share less than 45% identity with D9TQJ4 and do not catalyze the epimerization of F6P to A6P.

[0025] In some embodiments of the present invention, suitable epimerases for use in processes for converting F6P to A6P utilize a divalent metal cofactor, preferably, but not limited to, cobalt. In a further aspect of the present invention, the epimerase includes, but is not limited to, an (α / β)8-barrel domain for catalysis; a phosphate-binding site including, but not limited to, 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; a His at the second and third β-strands of the barrel; an Asp at the second and seventh β-strands of the barrel to act as an acid / base catalyst for 1,1 proton transfer; and a His-hydrophobic Asp-signature at the second β-strand of the barrel to utilize His for metal binding and Asp for acid / base catalysis. These features are known in the art and are reported, 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. Preferably, epimerases for use in the processes of the present invention contain an (α / β)8-barrel domain for catalysis, a Ser at the end of the seventh β-strand of the barrel, a Ser at the end of the eighth β-strand of the barrel, a Gly in the active site loop, a His in the second and third β-strands of the barrel, an Asp in the second and seventh β-strands of the barrel, and a His-hydrophobic Asp signature in the second β-strand of the barrel.

[0026] The process of the present invention uses a phosphatase that converts A6P to allulose (D-allulose). In some embodiments of the present invention, the phosphatase suitable for use in the process of converting A6P to allulose has a degree of identity to the amino acid sequence of SEQ ID NO: 9, i.e., at least 45%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, at least 91%, at least 92%, at least 93%, or at least 94%, and even most preferably at least 96, 97, 98, 99, or 100% identity. Suitable phosphatases are encoded by polynucleotides comprising a nucleotide sequence having a degree of identity, i.e., at least 30%, preferably at least 35%, more preferably at least 40%, more preferably at least 45%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 96, 97, 98, 99 or 100% identity, to the nucleotide sequences of SEQ ID NOs: 7 and 8.

[0027] Examples of A6PP include, but are not limited to, proteins identified by UNIPROT IDs A3DC21, Q5LGR4, and Q89ZR1. A3DC21 is 46% identical to Q5LGR4 and 45% identical to Q89ZR1, and each protein catalyzes the specific dephosphorylation of A6P to allulose. Conversely, other phosphatases from the haloacid dehydrogenase superfamily, such as proteins identified by UNIPROT IDs H0UQ29, Q67LU4, A0A0K6IPM3, C8WSJ0, and A0A151YX61, and other proteins, share less than 45% identity with A3DC21 and do not catalyze the specific dephosphorylation of A6P to allulose.

[0028] Phosphatases suitable for use in the process of the present invention for converting A6P to allulose are specific for allulose 6-phosphate. As used herein, specific for allulose 6-phosphate refers to a higher specific activity for allulose 6-phosphate than for glucose 1-phosphate, glucose 6-phosphate, or fructose 6-phosphate.

[0029] The phosphatase for converting A6P to allulose utilizes a divalent metal cofactor, preferably magnesium. In a further embodiment of the present invention, the phosphatase includes, but is not limited to, a Rossmanoid fold domain for catalysis, a C1 capping domain for substrate specificity, a DxD signature in the first β-strand of the Rossmanoid fold for coordinating magnesium, a general acid / base catalyst, a Thr or Ser at the end of the second β-strand of the Rossmanoid fold to stabilize reaction intermediates, a Lys at the N-terminus of the α-helix C-terminal to the third β-strand of the Rossmanoid fold to stabilize reaction intermediates, and a GDxxxD signature at the end of the fourth β-strand of the Rossmanoid fold for coordinating magnesium. These characteristics are known in the art and are reported, for example, in 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.Preferably, the phosphatase used in the process of the present invention for converting A6P to allulose comprises a rosemanoid fold domain for catalysis, a C1 capping domain, a DxD signature on the first β-strand of the rosemanoid fold, a Thr or Ser at the end of the second β-strand of the rosemanoid fold, a Lys at the N-terminus of the α-helix whose C-terminus points toward the third β-strand of the rosemanoid fold, and a GDxxxD signature at the end of the fourth β-strand of the rosemanoid fold.

[0030] In some embodiments, the process for preparing allulose of the present invention comprises the enzymatic conversion of glucose 6-phosphate (G6P) to F6P, which is catalyzed by phosphoglucose isomerase (PGI). In other embodiments, the process for preparing allulose further comprises the conversion of glucose 1-phosphate (G1P) to G6P, which is catalyzed by phosphoglucomutase (PGM). In yet further embodiments, the allulose production process also comprises the conversion of sugars to G1P, catalyzed by at least one enzyme.

[0031] Therefore, the process for preparing allulose of the present invention can include, for example, 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 using A6PE; and (v) converting A6P to allulose using A6PP. An example of the process for converting sugars to starch is shown in Figure 2.

[0032] Typically, the ratio of enzyme units used in the disclosed process is 1:1:1:1:1 (αGP:PGM:PGI:A6PE:A6PP). These ratios can be adjusted in any number of combinations to optimize product yield. For example, a ratio of 3:1:1:1:1 can be used to maximize the concentration of phosphorylated intermediates, thereby increasing the activity of downstream reactions. Conversely, a ratio of 1:1:1:1:3 can be used to maintain a steady supply of phosphate for αGP, thereby efficiently phosphorolytically cleaving the alpha-1,4-glycosidic bond. For example, a 3:1:1:1:3 enzyme ratio can be used to further increase the reaction rate. Therefore, varying the enzyme ratio, including any other enzymes described below, can increase the efficiency of allulose production. For example, a particular enzyme can be present in an amount that is about 2-fold, 3-fold, 4-fold, 5-fold, etc., compared to the amount of other enzymes.

[0033] One of the key advantages of this process is that the process steps can be carried out in one bioreactor or reaction vessel, or alternatively, the steps can be carried out in multiple bioreactors or reaction vessels arranged in series.

[0034] In particular, when all process steps are carried out in a single bioreactor or reaction vessel, the phosphate ions produced in the dephosphorylation of A6P can then be reused in the process step that converts sugars to G1P. The ability to reuse phosphate in the disclosed process allows for the use of non-stoichiometric amounts of phosphate, thereby keeping the reaction phosphate concentration low. This allows for the overall efficiency of the process to obtain allulose without limiting the activity of individual enzymes, which would affect the overall pathway and overall rate of the process.

[0035] For example, the reaction phosphate concentration may be in the range of about 0 mM to about 300 mM, about 0 mM to about 150 mM, about 1 mM to about 50 mM, preferably about 5 mM to about 50 mM, or more preferably about 10 mM to about 50 mM. For example, the reaction phosphate concentration may be about 0.1 mM, about 0.5 mM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, or about 55 mM.

[0036] Therefore, a low concentration of phosphate reduces the cost of phosphate removal and therefore production costs because the total amount of phosphate is low, which also prevents inhibition of A6PP by high concentrations of free phosphate and reduces the possibility of phosphate contamination.

[0037] Furthermore, the processes disclosed herein can be performed without added ATP as a phosphate source, i.e., ATP-free. These processes can also be performed without the need for added NAD(H), i.e., NAD(H)-free. Other advantages include the fact that at least one step of the disclosed processes for producing allulose involves an energetically favorable chemical reaction (Figure 7).

[0038] Examples of enzymes used to convert sugars to G1P include alpha-glucan phosphorylase (αGP, EC 2.4.1.1, including maltodextrin phosphorylase, starch phosphorylase, glycogen phosphorylase, and other α-1,4 glycosidic bond-cleaving phosphorylases), maltose phosphorylase (MP, EC 2.4.1.8), cellodextrin phosphorylase (CDP, EC 2.4.1.49), cellobiose phosphorylase (CBP, EC 2.4.1.20), cellulose phosphorylase, sucrose phosphorylase (SP, EC 2.4.1.7), and combinations thereof. The choice of enzyme or combination of enzymes depends on the sugars used in the process.

[0039] The sugars used to produce G1P can be polysaccharides, oligosaccharides, and / or disaccharides. For example, the sugars can be starch, one or more starch derivatives, cellulose, one or more cellulose derivatives, sucrose, one or more sucrose derivatives, or combinations thereof.

[0040] 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.

[0041] Starch derivatives can be prepared by enzymatic or acid hydrolysis of starch, which can be catalyzed or facilitated by isoamylase (IA, EC.3.2.1.68), which hydrolyzes α-1,6-glucosidic bonds; pullulanase (PA, EC.3.2.1.41), which hydrolyzes α-1,6-glucosidic bonds; 4-α-glucanotransferase (4GT, EC.2.4.1.25), which catalyzes the transglycosylation of short maltooligosaccharides to give longer maltooligosaccharides; or α-amylase (EC.3.2.1.1), which cleaves α-1,4-glucosidic bonds.

[0042] Additionally, derivatives of cellulose can be prepared by enzymatic hydrolysis of cellulose catalyzed by cellulase mixtures, by acid, or by pretreatment of biomass.

[0043] In certain embodiments, the enzyme used to convert sugars to G1P comprises αGP, wherein in this step, if the sugars include starch, the G1P is produced from the starch by αGP, and if the sugars include soluble starch, amylodextrin, or maltodextrin, the G1P is produced from the soluble starch, amylodextrin, or maltodextrin by αGP.

[0044] When the sugars include maltose and the enzyme contains maltose phosphorylase, the G1P is produced from maltose by maltose phosphorylase.When the sugars include sucrose and the enzyme contains sucrose phosphorylase, the G1P is produced from sucrose by sucrose phosphorylase.

[0045] In yet another embodiment, when the sugars include cellobiose and the enzyme contains cellobiose phosphorylase, the G1P is produced from cellobiose by cellobiose phosphorylase.

[0046] In a further embodiment, when the sugars include cellodextrin and the enzyme includes cellodextrin phosphorylase, the G1P is produced from cellodextrin by cellodextrin phosphorylase.

[0047] In another embodiment of converting sugars to G1P, when the sugars include cellulose and the enzyme contains cellulose phosphorylase, the G1P is produced from cellulose by the cellulose phosphorylase.

[0048] In the present invention, allulose can also be produced from fructose. An example of this process is shown in Figure 4. For example, this process involves producing F6P from fructose and polyphosphate using polyphosphate fructokinase (PPFK) catalysis, converting F6P to A6P using A6PE catalysis, and converting A6P to allulose using A6PP catalysis. This fructose can be produced, for example, by enzymatic conversion of sucrose.

[0049] In other embodiments, allulose can be produced from sucrose. An example of such a process is shown in Figure 6. This process provides an in vitro synthesis pathway that includes the following enzymatic steps: sucrose phosphorylase (SP)-catalyzed production of G1P from sucrose and free phosphate, PGM-catalyzed conversion of G1P to G6P, PGI-catalyzed conversion of G6P to F6P, A6PE-catalyzed conversion of F6P to A6P, and A6PP-catalyzed conversion of A6P to allulose.

[0050] The phosphate ions produced during the conversion of A6P to allulose can then be reused in the conversion of sucrose to G1P. Additionally, as shown in Figure 6, PPFK and polyphosphate can be used to produce F6P from fructose produced by the phosphorolytic cleavage of sucrose by SP, thereby increasing the yield of allulose.

[0051] In some embodiments, the process for preparing allulose comprises the steps of: producing glucose from polysaccharides and oligosaccharides by enzymatic or acid hydrolysis, converting the glucose to G6P by at least one enzyme-catalyzed conversion, producing fructose from polysaccharides and oligosaccharides by enzymatic or acid hydrolysis, and converting the fructose to G6P by at least one enzyme-catalyzed conversion. Examples of polysaccharides and oligosaccharides are described above.

[0052] In other embodiments, G6P is generated from glucose and sodium polyphosphate by polyphosphate glucokinase.

[0053] The present disclosure provides a process for converting sugars, such as polysaccharides and oligosaccharides comprising starch, cellulose, sucrose, and their derivatives, to allulose. In certain embodiments, a cell-free enzyme cocktail is used to provide an artificial (non-naturally occurring), ATP-free enzymatic pathway for the conversion of starch, cellulose, sucrose, and their derivatives to allulose.

[0054] As mentioned above, several enzymes can be used to hydrolyze starch and increase G1P yield. These enzymes include isoamylase, pullulanase, and alpha-amylase. Corn starch contains numerous branches that interfere with the action of alpha-GP. Isoamylase can be used to debranch starch and produce linear amylodextrin. Pre-treating starch with isoamylase can result in a higher concentration of F6P in the final product. Isoamylase and pullulanase cleave alpha-1,6-glycosidic bonds, allowing alpha-glucan phosphorylase to more completely degrade starch. Because alpha-amylase cleaves alpha-1,4-glycosidic bonds, alpha-amylase can be used to break down starch into fragments for more rapid conversion to allulose.

[0055] As shown in Figure 2, allulose yield can be increased by using maltose phosphorylase (MP) to phosphorolytically cleave the degradation product maltose to G1P and glucose. Alternatively, allulose yield can be increased by using 4-glucan transferase (4GT) to recycle the degradation products glucose, maltose, and maltotriose into long maltooligosaccharides, which can then be phosphorolytically cleaved by αGP to produce G1P.

[0056] Additionally, 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, such as Avicel (microcrystalline cellulose), regenerated amorphous cellulose, bacterial cellulose, and filter paper, can be prepared through a series of processes. Partially hydrolyzed cellulosic substrates contain water-insoluble cellodextrins with degrees of polymerization greater than 7, water-soluble cellodextrins with degrees of polymerization between 3 and 6, cellobiose, glucose, and fructose.

[0057] In certain embodiments, cellulose and its derivatives can be converted into allulose through a series of steps. An example of such a process is shown in Figure 3. This process provides an in vitro synthesis pathway including the following steps: producing G1P from cellodextrin, cellobiose, and free phosphate, respectively, catalyzed by cellodextrin phosphorylase (CDP) and cellobiose phosphorylase (CBP); converting G1P to G6P, catalyzed by PGM; converting G6P to F6P, catalyzed by PGI; converting F6P to A6P, catalyzed by A6PE; and converting A6P to allulose, catalyzed by A6PP. In this process, phosphate ions can be recycled by converting cellodextrin and cellobiose to G1P.

[0058] Several enzymes can be used to hydrolyze solid cellulose into water-soluble cellodextrins and cellobiose, including endoglucanases and cellobiohydrolases, but not beta-glucosidase (cellobiase).

[0059] Prior to cellulose hydrolysis and G1P production, cellulose and biomass can be pretreated to increase their reactivity and suppress the degree of polymerization of the cellulose chains. Pretreatment processes 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, phosphoric acid, and combinations thereof.

[0060] In some embodiments, as shown in FIG. 3, polyphosphate and polyphosphate glucokinase (PPGK) can be added to the process, thus increasing the yield of allulose by phosphorylating the degradation product glucose to G6P.

[0061] In other embodiments, allulose can be produced from glucose. An example of such a process is shown in Figure 5. This process includes the steps of producing G6P from glucose and polyphosphate catalyzed by polyphosphate glucokinase (PPGK), converting G6P to F6P catalyzed by PGI, converting F6P to A6P catalyzed by enzymes, and converting A6P to allulose catalyzed by A6PP.

[0062] Any suitable physiological buffer known in the art, such as HEPES, PBS, BIS-TRIS, MOPS, DIPSO, or Trizma, can be used in the process of the present invention. The reaction buffer for all embodiments can have a pH ranging from 5.0 to 8.0. More preferably, the pH of the reaction buffer can range from about 6.0 to about 7.3. For example, the pH of the reaction buffer can be 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, or 7.3.

[0063] The reaction buffer may also contain important metal cations. Examples of metal ions include Mg 2+ , Co 2+ , and Zn 2+ There is.

[0064] The reaction temperature at which the process steps are carried out can range from 37 to 85°C. More preferably, the steps are carried out at a temperature in the range of about 40 to about 70°C. This 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.

[0065] The reaction time of the disclosed process can be adjusted as needed and can range from about 8 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.

[0066] The process of the present invention is capable of achieving high yields due to the highly favorable equilibrium constant throughout the reaction, and theoretically, yields of up to 99% can be achieved if the starting material is completely converted to the intermediate.

[0067] The process of the present invention uses inexpensive starting materials and reduces costs associated with separating raw materials and products, thereby reducing production costs. Starch, cellulose, sucrose, and some of their derivatives are cheaper raw materials than, for example, fructose. When allulose is produced from fructose, the yield is lower than that in the present invention, and allulose must be separated from fructose via chromatography, which increases production costs.

[0068] Furthermore, according to the present invention, the step of converting A6P to allulose is an irreversible phosphatase reaction regardless of the raw material, and therefore allulose is produced in very high yields while effectively minimizing the cost of subsequent product isolation.

[0069] In contrast to cell-based manufacturing processes, the present invention involves a cell-free preparation of allulose, which has a relatively high reaction rate due to the elimination of cell membranes, which often slows the transport of substrates / products into and out of cells, and which has a final product that is free of nutrient-rich fermentation media / cellular metabolic products.

[0070] Example Materials and Methods chemicals

[0071] All chemicals, including corn starch, soluble starch, maltodextrin, maltose, glucose, and filter paper, were reagent grade or higher unless otherwise noted and were purchased from Sigma-Aldrich (St. Louis, MO, USA) or Fisher Scientific (Pittsburgh, PA, USA). Restriction enzymes, T4 ligase, and Phusion DNA polymerase were purchased from New England Biolabs (Ipswich, MA, USA). Oligonucleotides were synthesized at either Integrated DNA Technologies (Coralville, IA, USA) or Eurofins MWG Operon (Huntsville, AL, USA). The nucleotide sequence, SEQ ID NO: 1, encodes the thermophilic A6PE (UNIPROT ID D9TQJ4) from Thermoanaerobacterium thermosaccharolyticum. SEQ ID NO: 2 is a codon-optimized version of the nucleotide sequence. SEQ ID NO: 3 is the amino acid sequence of the enzyme. The nucleotide sequence SEQ ID NO:4 encodes thermophilic A6PE (UNIPROT ID A0A090IXZ8) from Bacillus thermoamylovorans. SEQ ID NO:5 is a codon-optimized version of the nucleotide sequence. SEQ ID NO:6 is the amino acid sequence of the enzyme. The nucleotide sequence SEQ ID NO:7 encodes thermophilic A6PP (UNIPROT ID A3DC21) from Clostridium thermocellum. SEQ ID NO:8 is a codon-optimized version of the nucleotide sequence. SEQ ID NO:9 is the amino acid sequence corresponding to the enzyme.The regenerated amorphous cellulose used for enzyme purification was prepared from Avicel PH105 (FMC BioPolymer, Philadelphia, PA, USA) through its dissolution and regeneration as described by Ye et al., "Fusion of a family 9 cellulose-binding module improves the catalytic potential of Clostridium thermocellum cellodextrin phosphorylase on insoluble cellulose." Appl. Microbiol. Biotechnol. 2011;92:551-560. Escherichia coli Sig10 (Sigma-Aldrich, St. Louis, MO, USA) was used as the host cell for DNA manipulation, and E. coli BL21(DE3) (Sigma-Aldrich, St. Louis, MO, USA) was used as the host cell for recombinant protein expression. ZYM-5052 medium containing either 100 mg / L ampicillin or 50 mg / L kanamycin was used for E. coli cell growth and recombinant protein expression. Cellulase (catalog number: C2730) and pullulanase (catalog number: P1067) from Trichoderma reesei were purchased from Sigma-Aldrich (St. Louis, MO, USA) and manufactured by Novozymes (Franklinton, NC, USA). Maltose phosphorylase (catalog number: M8284) was purchased from Sigma-Aldrich.

[0072] Recombinant enzyme production and purification

[0073] E. coli BL21(DE3) strains harboring protein expression plasmids were inoculated into 1 L Erlenmeyer flasks containing 100 mL of ZYM-5052 medium containing either 100 mg / L ampicillin or 50 mg / L kanamycin. Cells were grown for 16–24 h at 37°C with rotary shaking at 220 rpm. The 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 (heat-precipitated and cellulose-binding modules) or 20 mM HEPES (pH 7.5) containing 300 mM NaCl and 5 mM imidazole (Ni-purified). The cell pellets were resuspended in the same buffer and lysed by sonication (Fisher Scientific Sonic Dismembrator Model 500; 5 sec pulse on and 10 sec off, 50% amplitude, total 21 min). After centrifugation, the target protein was purified in the supernatant.

[0074] Various recombinant proteins were purified using three approaches. His-tagged proteins were purified with Profinity IMAC Ni-Charged Resin (Bio-Rad, Hercules, CA, USA). Fusion proteins containing a cellulose-binding module (CBM) and a self-cleaving intein were purified via high-affinity adsorption to regenerated amorphous cellulose with a large surface area. Hyperthermostable enzymes were purified using heat precipitation at 70–95°C for 5–30 min. The purity of the recombinant proteins was examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). A6PE was purified with 80 μM CoCl2 present in the growth medium, elution buffer, dialysis buffer, and protein storage buffer.

[0075] Enzymes used and their activity assays

[0076] Alpha-glucan phosphorylase (αGP) from Thermotoga maritima (UNIPROT ID G4FEH8) was used. Activity was assayed at 50°C in 50 mM sodium phosphate buffer (pH 7.2) containing 1 mM MgCl2, 5 mM DTT, and 30 mM maltodextrin. The reaction was terminated by filtering the enzyme using a Vivaspin 2 concentrator (10,000 molecular weight cutoff) (Vivaproducts, Inc., Littleton, MA, USA). Glucose 1-phosphate (G1P) was measured using a glucose hexokinase / G6PDH assay kit (Sigma Aldrich, catalog number GAHK20-1KT) supplemented with 25 U / mL phosphoglucomutase. Units (U) are expressed as μmol / min.

[0077] Phosphoglucomutase (PGM) from Thermococcus kodakaraensis (UNIPROT ID Q68BJ6) was used. Activity was measured at 50°C in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCl2 and 5 mM G1P. The reaction was terminated by filtering the enzyme using a Vivaspin 2 concentrator (10,000 molecular weight cutoff). The product, glucose 6-phosphate (G6P), was determined using a hexokinase / G6PDH assay kit (Sigma-Aldrich, catalog number GAHK20-1KT).

[0078] Two different sources of phosphoglucoisomerase (PGI) were used: Clostridium thermocellum (UNIPROT ID A3DBX9) and Thermus thermophilus (UNIPROT ID Q5SLL6). Activity was measured at 50°C in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCl2 and 10 mM G6P. The reaction was terminated by enzyme filtration using Vivaspin 2 concentrators (10,000 molecular weight cutoff). The product, fructose 6-phosphate (F6P), was determined using a fructose 6-phosphate kinase (F6PK) / pyruvate dehydrogenase (PK) / lactate dehydrogenase (LD) coupled enzyme assay, in which a decrease in absorbance at 340 nm indicates the production of F6P. The 200 μL reaction contained 50 mM HEPES (pH 7.2), 5 mM MgCl2, 10 mM G6P, 1.5 mM ATP, 1.5 mM phosphoenolpyruvate, 200 μM NADH, 0.1 U PGI, 5 U PK, and 5 U LD.

[0079] Allulose 6-phosphate 3-epimerase (A6PE) from Thermoanaerobacterium thermosaccharolyticum (UNIPROT ID D9TQJ4, SEQ ID NO: 3) was used. Activity was measured at 50°C in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCl2, 80 μM CoCl2, 1 U / mL A6PP, and 10 mM F6P. The reaction was terminated by filtering the enzyme using a Vivaspin 2 concentrator (10,000 molecular weight cutoff). The product, allulose 6-phosphate (A6P), was confirmed using allulose 6-phosphate phosphatase and detecting the release of free phosphate. To detect the release of free phosphate, 500 μl of a solution containing 0.1 M zinc acetate and 2 mM ammonium molybdate (pH 5) was added to 50 μl of the reaction mixture. This was mixed, followed by the addition of 125 μl of 5% ascorbic acid (pH 5). The solution was mixed and then incubated at 30° C. for 20 minutes. The absorbance was read at 850 nm to confirm the release of free phosphate.

[0080] Allulose 6-phosphate phosphatase (A6PP) from Clostridium thermocellum (UNIPROT ID A3DC21, SEQ ID NO: 9) was used. Activity was measured at 50°C in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCl, 80 μM CoCl, 1 U / mL A6PE, and 10 mM F6P. The reaction was terminated by filtering the enzyme using a Vivaspin 2 concentrator (10,000 molecular weight cutoff). The product, allulose, was confirmed by detecting the release of free phosphate as described for A6PE.

[0081] Recombinant cellodextrin phosphorylase and cellobiose phosphorylase from C. thermocellum are described in Ye et al., Spontaneous high-yield production of hydrogen from cellulosic materials and water catalyzed by enzyme cocktails. ChemSusChem 2009;2:149-152. Their activities were assayed as described.

[0082] Recombinant polyphosphate glucokinase from Thermobifida fusca YX was prepared as described in Liao et al., "One-step purification and immobilization of thermophilic polyphosphate glucokinase from Thermobifida fusca YX: glucose-6-phosphate generation without ATP." Appl. Microbiol. Biotechnol. 2012;93:1109-1117. Their activities were assayed as described.

[0083] Recombinant isoamylase from Sulfolobus tokodaii is described in Cheng et al., "Doubling power output of starch biobattery treated by the most thermostable isoamylase from an archaeon, Sulfolobus tokodaii." Scientific Reports 2015;5:13184. Their activities were assayed as described.

[0084] Recombinant 4-alpha-glucanotransferase from Thermococcus litoralis is described in Jeon et al. 4-α-Glucanotransferase from the Hyperthermophilic Archaeon Thermococcus Litoralis. Eur. J. Biochem. 1997;248:171-178. Their activity was assayed as described.

[0085] Sucrose phosphorylase (UNIPROT H1XT50) from Caldithrix abyssi was used. Its activity was measured in 50 mM HEPES buffer (pH 7.5) containing 10 mM sucrose and 12 mM organic phosphates. Glucose 1-phosphate (G1P) was measured similarly to alpha-glucan phosphorylase using a glucose hexokinase / G6PDH assay kit supplemented with 25 U / mL phosphoglucomutase.

[0086] The reaction time can be adjusted by increasing or decreasing the enzyme units used in each of the following examples as needed. For example, if Example 9 is performed for 8 hours instead of 24 hours, the enzyme units should be increased by approximately three times. Conversely, if Example 9 is performed for 48 hours instead of 24 hours, the enzyme units should be reduced by approximately half. These examples illustrate how the amount of enzyme units can be used to adjust the reaction time while maintaining constant productivity.

[0087] Example 1

[0088] To verify the technical feasibility of enzymatic biosynthesis of fructose 6-phosphate from starch, three enzymes were recombinantly expressed: alpha-glucan phosphorylase from T. maritima (UNIPROT ID G4FEH8), phosphoglucomutase from Thermococcus kodakaraensis (UNIPROT ID Q68BJ6), and phosphoisomerase from Clostridium thermocellum (UNIPROT ID A3DBX9). These recombinant proteins were overexpressed in E. coli BL21(DE3) and purified as described above.

[0089] A 0.20 mL reaction mixture containing 10 g / L soluble starch, 50 mM phosphate-buffered saline (pH 7.2), 5 mM MgCl2, 0.5 mM ZnCl2, 0.01 U αGP, 0.01 U PGM, and 0.01 U PGI was incubated at 50°C for 24 hours. The reaction was terminated by enzyme filtration using a Vivaspin 2 concentrator (10,000 molecular weight cutoff). The product, fructose 6-phosphate (F6P), was determined using a fructose 6-phosphate kinase (F6PK) / pyruvate dehydrogenase (PK) / lactate dehydrogenase (LD) coupled enzyme assay, as described above, in which a decrease in absorbance at 340 nm indicates F6P production. The final F6P concentration after 24 hours was 3.6 g / L.

[0090] Example 2

[0091] The same test as in Example 1 (except for the reaction temperature) was carried out at 40-80°C. After 40 hours of reaction, it was observed that 10 g / L of soluble starch produced 0.9 g / L of F6P at 40°C and 3.6 g / L of F6P at 80°C. These results suggest that increasing the reaction temperature for this enzyme set will increase the F6P yield, although too high a temperature may impair enzyme activity.

[0092] Example 3

[0093] At 80°C, an enzyme unit ratio of approximately 1:1:1 of αGP:PGM:PGI was found to rapidly produce F6P. It was noted that the enzyme ratio did not significantly affect the final F6P concentration if the reaction time was long enough. However, the enzyme ratio did affect the reaction rate and the overall cost of enzymes used in the system.

[0094] Example 4

[0095] A 0.20 mL reaction mixture containing 10 g / L maltodextrin, 50 mM phosphate-buffered saline (pH 7.2), 5 mM MgCl2, 0.5 mM ZnCl2, 0.01 U αGP, 0.01 U PGM, and 0.01 U PGI was incubated at 50°C for 24 hours. The reaction was terminated by filtration of the enzyme using a Vivaspin 2 concentrator (10,000 molecular weight cutoff). The product, fructose 6-phosphate (F6P), was determined using a fructose 6-phosphate kinase (F6PK) / pyruvate dehydrogenase (PK) / lactate dehydrogenase (LD) coupled enzyme assay, as described above, in which a decrease in absorbance at 340 nm indicates the production of F6P. The final concentration of F6P after 24 hours was 3.6 g / L.

[0096] Example 5

[0097] To test for F6P production from Avicel, cellulose was hydrolyzed at 50°C using Sigma cellulase. To remove beta-glucosidase from commercial cellulase, 10 filter paper activity / mL of cellulase was mixed with 10 g / L of Avicel in an ice bath for 10 minutes. After centrifugation at 4°C, the supernatant containing beta-glucosidase was decanted. The Avicel combined with cellulase containing endoglucanase and cellobiohydrolase was resuspended in citrate buffer (pH 4.8) at 50°C for 3 days for hydrolysis. The cellulose hydrolysate was mixed with 5 U / mL cellodextrin phosphorylase, 5 U / mL cellobiose phosphorylase, 5 U / mL αGP, 5 U / mL PGM, and 5 U / mL PGI in 100 mM HEPES buffer (pH 7.2) containing 10 mM phosphate, 5 mM MgCl2, and 0.5 mM ZnCl2. The reaction was carried out at 60°C for 72 hours, and high concentrations of F6P were observed (only small amounts of glucose and no cellobiose were present). F6P was detected using the coupled enzyme assay described above. Glucose was detected using a hexokinase / G6PDH assay kit as described above.

[0098] Example 6

[0099] To increase the F6P yield from Avicel, Avicel was pretreated with concentrated phosphoric acid to produce amorphous cellulose (RAC) as described by Zhang et al. (2006;7:644-648). To remove beta-glucosidase from commercial cellulase, 10 g / L of cellulase containing 10 filter paper degradation activity / mL was mixed with 10 g / L of RAC for 5 minutes in an ice bath. After centrifugation at 4°C, the supernatant containing beta-glucosidase was decanted. The RAC bound to the cellulase containing endoglucanase and cellobiohydrolase was resuspended in citrate buffer (pH 4.8) at 50°C for 12 hours for hydrolysis. The RAC hydrolysate was mixed with 5 U / mL cellodextrin phosphorylase, 5 U / mL cellobiose phosphorylase, 5 U / mL αGP, 5 U / mL PGM, and 5 U / mL PGI in 100 mM HEPES buffer (pH 7.2) containing 10 mM phosphate, 5 mM MgCl2, and 0.5 mM ZnCl2. The reaction was carried out at 60°C for 72 hours. Because no enzymes were added to convert glucose to F6P, high concentrations of F6P and glucose were recovered. F6P was detected using the coupled enzyme assay described above. Glucose was detected using a hexokinase / G6PDH assay kit as described above.

[0100] Example 7

[0101] To further enhance the F6P yield from RAC, polyphosphate glucokinase and polyphosphate were added. To remove beta-glucosidase from commercial cellulase, cellulase with 10 filter paper degradation activity / mL was mixed with 10 g / L RAC for 5 minutes in an ice bath. After centrifugation at 4°C, the supernatant containing beta-glucosidase was decanted. The RAC bound to cellulase containing endoglucanase and cellobiohydrolase was resuspended in citrate buffer (pH 4.8) at 50°C and incubated for 12 hours in citrate buffer (pH 4.8) for hydrolysis. The RAC hydrolysate was mixed with 5 U / mL polyphosphate glucokinase, 5 U / mL cellodextrin phosphorylase, 5 U / mL cellobiose phosphorylase, 5 U / mL αGP, 5 U / mL PGM, and 5 U / mL PGI in 100 mM HEPES buffer (pH 7.2) containing 50 mM polyphosphate, 10 mM phosphate, 5 mM MgCl2, and 0.5 mM ZnCl2. The reaction was carried out at 50°C for 72 hours. F6P was found at high concentrations, while glucose was present only in small amounts. F6P was detected using the coupled enzyme assay described above. Glucose was detected using a hexokinase / G6PDH assay kit as described above.

[0102] Example 8

[0103] To verify the production of allulose from F6P, 2 g / L F6P was mixed with 1 U / ml A6PE and 1 U / ml A6PP in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCl2 and 80 μM CoCl2. The reaction mixture was incubated at 50°C for 6 hours. A 99% conversion of F6P to allulose was observed using an HPLC (Agilent 1100 series) equipped with an Agilent Hi-Plex H-column and a refractive index detector. The sample was run at 0.6 mL / min in 5 mM H2SO4.

[0104] Example 9

[0105] To verify the production of allulose from maltodextrin, a 0.20 mL reaction mixture containing 20 g / L maltodextrin, 50 mM phosphate-buffered saline (pH 7.2), 5 mM MgCl, 80 μM CoCl, 0.05 U αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U A6PE, and 0.05 U A6PP was incubated at 50°C for 24 hours. The reaction was terminated by filtering the enzyme using a Vivaspin 2 concentrator (10,000 molecular weight cutoff). Allulose was detected and quantified using an Agilent 1100 Series HPLC equipped with a refractive index detector and an Agilent Hi-Plex H-column. The mobile phase was 5 mM HSO, flowing at 0.6 mL / min. The yield was quantified using standards of various concentrations of allulose.

[0106] Example 10 A reaction mixture containing 200 g / L maltodextrin, 10 mM acetate buffer (pH 5.5), 5 mM MgCl, 80 μM CoCl, and 0.1 g / L isoamylase is incubated at 80° C. for 24 hours. This is used to generate another reaction mixture containing 20 g / L isoamylase-treated maltodextrin, 50 mM phosphate-buffered saline (pH 7.2), 5 mM MgCl, 0.05 U αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U A6PE, and 0.05 U A6PP, and incubated at 50° C. for 24 hours. Allulose production is quantified as described in Example 9.

[0107] Example 11 A reaction mixture containing 200 g / L maltodextrin, 10 mM acetate buffer (pH 4.5), 5 mM MgCl, and a 1:200 dilution of Novozymes D6 pullulanase is incubated at 50° C. for 4 hours. This is used to generate another reaction mixture containing 20 g / L pullulanase-treated maltodextrin, 50 mM phosphate-buffered saline pH 7.2, 5 mM MgCl, 80 μM CoCl, 0.05 U αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U A6PE, and 0.05 U A6PP, and incubated at 50° C. for 24 hours. Allulose production is quantified as described in Example 9.

[0108] Example 12

[0109] To further increase the allulose yield from maltodextrin, 0.05 U of 4-glucan transferase (4GT) is added to the reaction described in Example 9.

[0110] A 0.2 mL reaction mixture containing 20 g / L isoamylase-treated maltodextrin (see Example 9), 50 mM phosphate buffered saline pH 7.2, 5 mM MgCl, 80 μM CoCl, 0.05 U αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U A6PE, 0.05 U A6PP, and 0.05 U 4GT is incubated for 24 hours at 50° C. Allulose production is quantified as described in Example 9.

[0111] Example 13

[0112] To determine the concentration range of phosphate-buffered saline (PBS), a 0.20 mL reaction mixture containing 50 g / L maltodextrin, 6.25 mM, 12.5 mM, 25 mM, 37.5 mM, or 50 mM phosphate-buffered saline pH 7.2, 5 mM MgCl2, 0.1 U aGP, 0.1 U PGM, and 0.1 U PGI was incubated at 50°C for 6 hours. Shorter durations ensured that the reaction did not go to completion, thus clearly indicating differences in efficiency. F6P production was determined using a fructose 6-phosphate kinase (F6PK) / pyruvate dehydrogenase (PK) / lactate dehydrogenase (LD) coupled enzyme assay, in which a decrease in absorbance at 340 nm indicates F6P production. Reactions containing 6.25 mM, 12.5 mM, 25 mM, 37.5 mM, or 50 mM phosphate-buffered saline (pH 7.2) yielded F6P yields of 4.5 g / L, 5.1 g / L, 5.6 g / L, 4.8 g / L, or 4.9 g / L, respectively (Table 1). These results indicate that 25 mM PBS pH 7.2 is ideal for these specific reaction conditions. It is important to note that even the use of 6.25 mM PBS at pH 7.2 resulted in significant turnover due to phosphate recycling. This indicates that the disclosed phosphate recycling method can maintain industrial-level volumetric productivity (e.g., 200–300 g / L of maltodextrin) even at low levels of phosphate. [Table 1]

[0113] Example 14

[0114] To determine the pH range of the cascade reaction, a 0.20 mL reaction mixture containing 50 g / L maltodextrin, phosphate-buffered saline (PBS) at pH 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, or 7.3, 5 mM MgCl2, 0.02 U αGP, 0.02 U PGM, and 0.02 U PGI was incubated at 50°C for 16 hours. Differences in efficiency were clearly demonstrated by reducing the units to ensure non-completion. F6P production was quantified as described in Example 12. Reactions containing 50 mM phosphate buffered saline at pH 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, or 7.3 gave F6P yields of 4.0 g / L, 4.1 g / L, 4.2 g / L, 4.1 g / L, 4.4 g / L, 4.1 g / L, 3.8 g / L, or 4.0 g / L, respectively (Table 2). These results indicate that this system works over a wide pH range, but a pH of 6.8 is ideal for these particular reaction conditions. [Table 2]

[0115] Example 15

[0116] To study scale-up, a 20 mL reaction mixture containing 50 g / L isoamylase-treated maltodextrin (see Example 10), 50 mM phosphate buffered saline pH 7.2, 5 mM MgCl, 80 μM CoCl, 10 U αGP, 10 U PGM, 10 U PGI, 10 U A6PE, and 10 U A6PP is incubated for 24 hours at 50° C. Allulose production is quantified as described in Example 9.

[0117] Example 16

[0118] To further increase the allulose yield from maltodextrin, 0.05 U of maltose phosphorylase is added to the reaction described in Example 9.

[0119] Example 17

[0120] To further increase the allulose yield from maltodextrin, 0.05 U of polyphosphate glucokinase and 75 mM polyphosphate are added to the reaction described in Example 9.

[0121] Example 18

[0122] To produce allulose from fructose, a reaction mixture containing 10 g / L fructose, 50 mM Tris buffer pH 7.0, 75 mM polyphosphate, 5 mM MgCl, 80 μM CoCl, 0.05 U fructose polyphosphate kinase, 0.05 U A6PE, and 0.05 U A6PP is incubated for 24 hours at 50° C. Allulose production is quantified as described in Example 9.

[0123] Example 19

[0124] To produce allulose from glucose, a reaction mixture containing 10 g / L glucose, 50 mM Tris buffer pH 7.0, 75 mM polyphosphate, 5 mM MgCl, 80 μM CoCl, 0.05 U glucose polyphosphate kinase, 0.05 U PGI, and 0.05 U A6PE is incubated for 24 hours at 50° C. Allulose production is quantified as described in Example 9.

[0125] Example 20

[0126] To produce allulose from sucrose, a reaction mixture containing 10 g / L sucrose, 50 mM Tris-buffered saline pH 7.0, 5 mM MgCl, 80 μM CoCl, 0.05 U sucrose polyphosphate, 0.05 U PGM, 0.05 U PGI, 0.05 U A6PE, and 0.05 U A6PP is incubated for 24 hours at 50° C. Allulose production is quantified as described in Example 9.

[0127] Example 21

[0128] To further increase the yield of allulose from sucrose, 75 mM polyphosphate and 0.05 mM polyphosphate fructokinase are added to the reaction described in Example 20. Allulose production is quantified as described in Example 9. [Sequence List Free Text]

[0129] SEQ ID NO:2: <223> Codon-optimized thermoanaerobacterium thermosaccharolyticum A6PE SEQ ID NO:5: <223> Thermophilic A6PE from codon-optimized Bacillus thermoamylovorans SEQ ID NO:8: <223> Codon-optimized A6PP from Clostridium thermocellum

Claims

1. A process for preparing allulose, comprising: converting fructose 6-phosphate (F6P) to allulose 6-phosphate (A6P) catalyzed by allulose 6-phosphate 3-epimerase (A6PE); and The produced A6P is converted into allulose by catalysis with allulose 6-phosphate phosphatase (A6PP) which is specific for allulose 6-phosphate. including the steps Moreover, the process steps are carried out in a single bioreactor under the same process conditions.

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

3. 3. The process of claim 2, further comprising converting glucose 1-phosphate (G1P) to said G6P, said step being catalyzed by phosphoglucomutase (PGM).

4. 4. The process of claim 3, further comprising a step of converting sugars into said G1P, said step being catalyzed by at least one enzyme, and wherein said sugars are selected from the group consisting of starch or a derivative thereof, cellulose or a derivative thereof, and sucrose.

5. 5. The process of claim 4, 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.

6. 5. The process of claim 4, wherein the sugar is a starch or derivative thereof selected from the group consisting of amylose, amylopectin, soluble starch, amylodextrin, maltodextrin, maltose, and glucose.

7. 7. The process of claim 6, further comprising the step of converting the starch into a starch derivative, wherein the starch derivative is prepared by enzymatic hydrolysis of the starch or by acid hydrolysis of the starch.

8. 7. The process of claim 6, wherein 4-glucan transferase (4GT) is added to the process.

9. 8. The process of claim 7, wherein the starch derivative is prepared by enzymatic hydrolysis of starch catalyzed by isoamylase, pullulanase, alpha-amylase, or a combination thereof.

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

11. 11. The process of claim 10, further comprising converting sucrose to said fructose, said step catalyzed by at least one enzyme.

12. 3. The process of claim 2, further comprising converting glucose to said G6P, said step catalyzed by at least one enzyme.

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

14. 13. The process of any one of claims 1 to 12, wherein the A6PE comprises an amino acid sequence having at least 90%, at least 95%, or at least 100% sequence identity to SEQ ID NO: 3 or 6.

15. The A6PE has (α / β) ８ - a barrel domain, a Ser at the end of the seventh β-strand of the barrel, a Ser at the end of the eighth β-strand of the barrel, a Gly in the active site loop, a His in the second and third β-strands of the barrel, an Asp in the second and seventh β-strands of the barrel, and a His-hydrophobic Asp-signature in the second β-strand of the barrel.

16. 13. The process of any one of claims 1 to 12, wherein the A6PP comprises an amino acid sequence having at least 90%, at least 95%, or at least 100% sequence identity to SEQ ID NO:

9.

17. 13. The process of any one of claims 1 to 12, wherein the A6PP comprises a Rossmanoid fold domain for catalysis, a C1 capping domain, a DxD signature on the first β-strand of the Rossmanoid fold, a Thr or Ser at the end of the second β-strand of the Rossmanoid fold, a Lys at the N-terminus of an α-helix whose C-terminus points toward the third β-strand of the Rossmanoid fold, and a GDxxxD signature at the end of the fourth β-strand of the Rossmanoid fold.

18. 13. The process of any one of claims 1 to 12, wherein the process steps are carried out at a temperature in the range of 37°C to 85°C, at a pH in the range of 5.0 to 8.0, and / or for a time period of 8 to 48 hours.

19. 13. The process of any one of claims 1 to 12, wherein the process steps are carried out in the absence of ATP, in the absence of NAD(H), at a phosphate concentration of greater than 0 mM to 150 mM, wherein the phosphate is recycled, and / or at least one step of the process involves an energetically favorable chemical reaction.

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