Enzymatic production of D-allulose

Abstract

The present disclosure provides a method for enzymatically converting sugars into allulose. The present invention also relates to a method for producing allulose, comprising converting fructose 6-phosphate (F6P) to allulose 6-phosphate (A6P) catalyzed by allulose 6-phosphate 3-epimerase (A6PE), and converting A6P to allulose 6-phosphate phosphatase (A6PP). [Selected Figure] Figure 1

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 THEINVENTION The present invention relates to the preparation of the saccharide D-allulose. More specifically, the present invention relates to a method for preparing D-allulose by enzymatically converting saccharides (e.g., polysaccharides, oligosaccharides, disaccharides, sucrose, D-glucose, and D-fructose) to D-allulose. [Background technology]

[0003] 2. Background of the Invention D-Allulose (also known as D-psicose) (hereafter allulose) is a low-calorie natural sweetener that has 70% of the sweetness of sucrose but only 10% of the calories. It is a naturally occurring monosaccharide hexose that is present 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 designated as Generally Recognized as Safe (GRAS). However, its high selling price limits its use as a sweetener. Allulose has numerous health benefits, including low calories (10% of sucrose), a very low glycemic index of 1, is completely absorbed in the small intestine but is not metabolized, instead being secreted into the urine and feces, aids in blood sugar regulation by inhibiting alpha-amylase, sucrase, and maltase, and has similar functionality to sucrose in foods and beverages. Thus, it is clear that allulose has a variety of uses 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 yield, in which 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 one 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, which can be recycled and / or which does not require the use of adenosine triphosphate (ATP) as a source of phosphate.There is also a need for an allulose production route that does not require the use of expensive nicotinamide adenosine dinucleotide (NAD(H)) coenzymes 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 catalyzed by 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 a step of converting glucose 6-phosphate (G6P) to said F6P, which is catalyzed by phosphoglucoisomerase (PGI). In other aspects, the process for synthesizing allulose also includes a step of converting glucose 1-phosphate (G1P) to said G6P, which is catalyzed by phosphoglucomutase (PGM).

[0008] In various aspects, 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 aspects of the invention, the process for preparing allulose includes converting starch into a starch derivative by enzymatic or acid hydrolysis of starch. In other aspects, 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. The process for preparing allulose can also include adding 4-glucan transferase (4GT) in certain aspects.

[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 the 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 embodiment of the present invention, the G6P used in the method for preparing allulose can be produced by converting glucose to said G6P, catalyzed by at least one enzyme. Glucose can then be produced by converting sucrose to glucose, catalyzed by at least one enzyme.

[0012] In other aspects of the invention, the process steps 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. In relation to the present invention, the following is further disclosed: [1] A process for preparing allulose, comprising: converting fructose 6-phosphate (F6P) to allulose 6-phosphate (A6P) catalyzed by epimerase; and The produced A6P is converted to 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 of [3], further comprising 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] 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] 7. The process according to claim 6, further comprising a step of converting starch into a starch derivative, the starch derivative being prepared by enzymatic hydrolysis of starch or by acid hydrolysis of starch. [8] The process according to [6], wherein 4-glucantransferase (4GT) is added to the process. [9] 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 described in [1], further comprising 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] The process described in [2], further comprising a step of converting glucose to the G6P, the step being catalyzed by at least one enzyme, and optionally a step of converting sucrose to the glucose, the 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 capable of catalyzing (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] 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 of

[15] , wherein the allulose 6-phosphate phosphatase comprises a rosemanoid fold domain for catalysis, a C1 capping domain, a DxD signature at 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 description 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] [Diagram 2] Figure 2 is a schematic diagram showing the enzymatic pathway for the conversion of 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] [Diagram 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] [Diagram 5] Figure 5 is a schematic diagram showing the enzymatic pathway for the conversion of glucose to allulose. PPGK, polyphosphate glucokinase. PGI, phosphoglucoisomerase.

[0019] [Figure 6]Figure 6 shows the enzymatic pathway that converts 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 PREFERRED EMBODIMENTS

[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 the cost of producing allulose.

[0022] The present invention relates to a process for preparing allulose, which comprises the conversion of fructose 6-phosphate (F6P) to allulose 6-phosphate (A6P) catalyzed by an epimerase, and the conversion of the resulting A6P to allulose catalyzed by a phosphatase (e.g., allulose 6-phosphate phosphatase, A6PP). The process is illustrated in FIG. 1. In a particular embodiment, the epimerase that catalyzes the conversion of F6P to A6P is allulose 6-phosphate 3-epimerase (A6PE).

[0023] The epimerase that converts F6P to A6P can be used in the process of the present invention. The epimerase can also convert A6P to F6P. In some aspects of the present invention, the epimerase suitable for use in the process of converting F6P to A6P has 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, where D9TQJ4 and A0A090IXZ8 are from thermophilic organisms, and P32719 is from mesophilic organisms. P32719 is 53% identical to A0A090IXZ8 and 55% identical to D9TQJ4, and each protein catalyzes the epimerization of F6P to A6P. Additionally, 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] Some embodiments of the present invention utilize a divalent metal cofactor, preferably, but not limited to, cobalt, as an epimerase suitable for use in a process for converting F6P to A6P. In further aspects of the 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, 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, and a 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 have been 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, the epimerase for use in the process of the invention contains an (alpha / beta)8-barrel domain for catalysis, a Ser at the end of the seventh beta-strand of the barrel, a Ser at the end of the eighth beta-strand of the barrel, a Gly in the active site loop, a His in the second and third beta-strands of the barrel, an Asp in the second and seventh beta-strands of the barrel, a His in the second beta-strand of the barrel, and a hydrophobic residue Asp-signature.

[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, the 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. In contrast, other phosphatases from the haloacid dehydrogenase superfamily, such as the proteins identified by UNIPROT IDs H0UQ29, Q67LU4, A0A0K6IPM3, C8WSJ0, A0A151YX61, and other proteins, have less than 45% identity to A3DC21 and do not catalyze the specific dephosphorylation of A6P to allulose.

[0028] The phosphatase suitable for use in the process of the present invention for converting A6P to allulose is specific for allulose 6-phosphate. As used herein, specific for allulose 6-phosphate refers to a higher specific activity for allulose 6-phosphate than 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 at 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-terminus of the α-helix with its C-terminus toward the third β-strand of the Rossmanoid fold to aid in the stability of reaction intermediates, and a GDxxxD signature at the end of the fourth β-strand of the Rossmanoid fold to coordinate magnesium, but is not limited to, a GDxxxD signature at the end of the fourth β-strand of the Rossmanoid fold to coordinate magnesium. These features 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 for converting A6P to allulose used in the process of the invention comprises a lossmanoid fold domain for catalysis, a C1 capping domain, a DxD signature at the first β-strand of the lossmanoid fold, a Thr or Ser at the end of the second β-strand of the lossmanoid fold, a Lys at the N-terminus of an α-helix whose C-terminus points towards the third β-strand of the lossmanoid fold, and a GDxxxD signature at the end of the fourth β-strand of the lossmanoid fold.

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

[0031] Thus, the process of 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 by A6PE; and (v) converting A6P to allulose by A6PP. An example of the process of converting sugars to starch is shown in FIG. 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 alpha-1,4-glycosidic bonds. For example, a ratio of 3:1:1:1:3 enzymes can be used to further increase the reaction rate. Thus, varying the enzyme ratios, including any other enzymes described below, can increase the efficiency of allulose production. For example, a particular enzyme can be present in an amount of 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 generated in the dephosphorylation of A6P can then be recycled to the process step that converts sugars to G1P. The ability to recycle phosphate in the disclosed process allows for the use of non-stoichiometric amounts of phosphate, which keeps the reaction phosphate concentration low. This 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] Thus, a low concentration of phosphate reduces the cost of phosphate removal and therefore production costs because the total amount of phosphate is less, which also prevents inhibition of A6PP by high concentrations of free phosphate and reduces the possibility of phosphate contamination.

[0037] Additionally, the processes disclosed herein can be carried out without the addition of ATP as a phosphate source, i.e., ATP-free. These processes can also be carried out without the need for the addition of 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 decomposition 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 the 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 promoted by isoamylase (IA, EC.3.2.1.68), which hydrolyzes α-1,6-glucosidic bonds, pullulanases (PA, EC.3.2.1.41), which hydrolyze 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 alpha-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, in which, if the sugars comprise starch, the G1P is produced from the starch by αGP, and if the sugars comprise 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 generated from maltose by maltose phosphorylase.When the sugars include sucrose and the enzyme contains sucrose phosphorylase, the G1P is generated from sucrose by sucrose phosphorylase.

[0045] In yet another embodiment, when the sugar comprises cellobiose and the enzyme comprises 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 generated 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 cellulose phosphorylase.

[0048] In the present invention, allulose can also be produced from fructose. An example of the process is shown in Figure 4. For example, the process includes the steps of polyphosphate fructokinase (PPFK) catalyzing the production of F6P from fructose and polyphosphate, A6PE catalyzing the conversion of F6P to A6P, and A6PP catalyzing the conversion of A6P to allulose. The fructose can be produced, for example, by the 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 route that includes the following enzymatic steps: sucrose phosphorylase (SP) catalyzes the production of G1P from sucrose and free phosphate, PGM catalyzes the conversion of G1P to G6P, PGI catalyzes the conversion of G6P to F6P, A6PE catalyzes the conversion of F6P to A6P, and A6PP catalyzes the conversion of A6P to allulose.

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

[0051] In some embodiments, the process for preparing allulose includes the following steps: producing glucose from polysaccharides and oligosaccharides by enzymatic or acid hydrolysis, converting glucose to G6P by catalysis of at least one enzyme, producing fructose from polysaccharides and oligosaccharides by enzymatic or acid hydrolysis, and converting fructose to G6P by catalysis of at least one enzyme. 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, such as those constituting starch, cellulose, sucrose, and their derivatives, into allulose. In certain embodiments, a cell-free enzyme cocktail is used to provide an artificial (non-natural) ATP-free enzymatic pathway for the conversion of starch, cellulose, sucrose, and their derivatives into allulose.

[0054] As mentioned above, several enzymes can be used to hydrolyze starch and increase the G1P yield. These enzymes include isoamylase, pullulanase, and alpha-amylase. Corn starch contains numerous branches that prevent αGP action. Isoamylase is used to debranch starch and produce linear amylodextrin. Starch pretreated with isoamylase can have a high 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. Alpha-amylase cleaves alpha-1,4-glycosidic bonds, so alpha-amylase is used to break down starch into fragments for more rapid conversion to allulose.

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

[0056] In addition, cellulose is the most abundant biological resource and is also 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 route that includes the following steps: cellodextrin phosphorylase (CDP) and cellobiose phosphorylase (CBP) catalyze cellodextrin, cellobiose, and free phosphate to produce G1P, PGM catalyze G1P to G6P, PGI catalyze G6P to F6P, A6PE catalyze F6P to A6P, and A6PP catalyze A6P to allulose. 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 including 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. Cellulose and biomass pretreatment processes 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 enzyme, and converting A6P to allulose catalyzed by A6PP.

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

[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 can be carried out at a temperature in the range of about 40° C. to about 70° 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.

[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 constants 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 the costs associated with the separation of raw materials and products are reduced, so production costs are reduced. 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 smaller 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 separation.

[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 in and out of the cell, and which has an end product that does not contain nutrient-rich fermentation medium / cell metabolic products.

[0070] Working Example Materials and Methods Chemicals

[0071] All chemicals, including corn starch, soluble starch, maltodextrin, maltose, glucose, and filter paper, were of reagent grade or better 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 the 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 the 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 in Ye et al., Fusion of a family 9 cellulose-binding module improves 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 carrying protein expression plasmids were incubated in 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 hours 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 supernatant contained the purified target protein.

[0074] Three approaches were used to purify various recombinant proteins. 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 on regenerated amorphous cellulose with a large surface area. Hyperthermostable enzymes were purified using heat precipitation at 70–95°C for 5–30 min. Purity of 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 stopped by filtering the enzyme using Vivaspin 2 concentrators (10,000 molecular weight cut-off) (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 stopped 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) from Clostridium thermocellum (UNIPROT ID A3DBX9) and PGI from Thermus thermophilus (UNIPROT ID Q5SLL6) were used. 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 stopped by filtering 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 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) (UNIPROT ID D9TQJ4), SEQ ID NO: 3, from Thermoanaerobacterium thermosaccharolyticum 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 stopped 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. This was mixed followed by the addition of 125 μl of 5% ascorbic acid (pH 5). The solution was mixed and then incubated for 20 minutes at 30° C. 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 MgCl2, 80 μM CoCl2, 1 U / mL A6PE, and 10 mM F6P. The reaction was stopped by filtering the enzyme using Vivaspin 2 concentrators (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 is 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 activities were assayed as described.

[0085] Sucrose phosphorylase from Caldithrix abyssi (UNIPROT H1XT50) was used. Its activity was measured in 50 mM HEPES buffer (pH 7.5) containing 10 mM sucrose and 12 mM organic phosphate. 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 enzyme units used in each of the following examples can be increased or decreased as necessary to adjust the reaction time. For example, if Example 9 is performed for 8 hours instead of 24 hours, the enzyme units are increased by about three times. Conversely, if Example 9 is performed for 48 hours instead of 24 hours, the enzyme units are reduced by about half. These examples illustrate how the amount of enzyme units can be used to increase or decrease 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 stopped by filtering 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.

[0090] Example 2

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

[0092] Example 3

[0093] At 80° C., an enzyme unit ratio of about 1:1:1 αGP:PGM:PGI was found to rapidly generate 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 stopped by filtering 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 cellulase was mixed with 10 g / L 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 / L 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). F6P was detected using the coupled enzyme assay described above. Glucose was detected using the 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. A transition from cellulose swelling to cellulose dissolution by o-phosphoric acid: evidence from enzymatic hydrolysis and supramolecular structure. Biomacromolecules 2006;7:644-648. To remove beta-glucosidase from commercial cellulase, 10 filter paper decomposition activity / mL cellulase was mixed with 10 g / L RAC in an ice bath for 5 minutes. After centrifugation at 4°C, the supernatant containing beta-glucosidase was decanted. The RAC combined with 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 / L 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. High concentrations of F6P and glucose were recovered because no enzyme was added to convert glucose to F6P. F6P was detected using the coupled enzyme assay described above. Glucose was detected using the hexokinase / G6PDH assay kit as described above.

[0100] Example 7

[0101] To further increase the F6P yield from RAC, polyphosphate glucokinase and polyphosphate were added. To remove beta-glucosidase from commercial cellulase, cellulase with 10 filter paper decomposition activity / mL was mixed with 10 g / L RAC in an ice bath for 5 min. After centrifugation at 4°C, the supernatant containing beta-glucosidase was decanted. The RAC combined with cellulase containing endoglucanase and cellobiohydrolase was resuspended in citrate buffer (pH 4.8) at 50°C for hydrolysis and incubated in citrate buffer (pH 4.8) at 50°C for 12 h for hydrolysis. The RAC hydrolysate was mixed with 5 U / mL polyphosphate glucokinase, 5 U / mL cellodextrin phosphorylase, 5 U / L 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 in high concentrations, while glucose was present in small amounts. F6P was detected using the coupled enzyme assay described above. Glucose was detected using the 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 was incubated at 50°C for 6 h. 99% conversion of F6P to allulose was observed on an HPLC (Agilent 1100 series) equipped with an Agilent Hi-Plex H-column and a refractive index detector. Samples were run in 5 mM H2SO4 at 0.6 mL / min.

[0104] Example 9

[0105] To verify the production of allulose from maltodextrin, 0.20 mL of reaction mixture containing 20 g / L maltodextrin, 50 mM phosphate buffered saline pH 7.2, 5 mM MgCl2, 80 μM CoCl2, 0.05 U αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U A6PE, and 0.05 U A6PP is incubated at 50° C. for 24 hours. The reaction is stopped by filtering the enzyme using a Vivaspin 2 concentrator (10,000 molecular weight cutoff). Allulose is 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 is 5 mM H2SO4 and is run at 0.6 mL / min. The yield is 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 MgCl2, 80 μM CoCl2, 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 MgCl2, 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 MgCl2, and a 1:200 dilution of Novozymes D6 pullulanase is incubated for 4 hours at 50° C. 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 MgCl2, 80 μM CoCl2, 0.05 U αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U A6PE, and 0.05 U A6PP, and incubated for 24 hours at 50° C. Allulose production is quantified as described in Example 9.

[0108] Example 12

[0109] To further increase the yield of allulose 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 MgCl2, 80 μM CoCl2, 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), 0.20 mL reaction mixtures 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 are incubated at 50° C. for 6 hours. The shorter duration ensures that it has not gone to completion, and therefore allows a clear indication of efficiency differences. The production of F6P is 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. Reactions containing 6.25 mM, 12.5 mM, 25 mM, 37.5 mM, or 50 mM phosphate buffered saline (pH 7.2) yielded F6P at 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 a concentration of 25 mM PBS pH 7.2 is ideal for these particular reaction conditions. It is important to note that even the use of 6.25 mM PBS, pH 7.2, results in significant turnover due to phosphate recycling. This indicates that the disclosed phosphate recycling method can maintain industrial volumetric productivity (e.g., 200-300 g / L 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 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 is incubated at 50° C. for 16 hours. The units are reduced to ensure that the reaction did not go to completion, allowing differences in efficiency to be clearly shown. F6P production is 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 the system will work over a wide pH range, but that 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 MgCl2, 80 μM CoCl2, 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 yield of allulose 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 MgCl2, 80 μM CoCl2, 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 MgCl2, 80 μM CoCl2, 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 MgCl2, 80 μM CoCl2, 0.05 U sucrose polyphosphate, 0.05 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 from sugars, comprising: (i) converting sugars to glucose 1-phosphate (G1P) catalyzed by at least one enzyme; (ii) converting the produced G1P into glucose 6-phosphate (G6P) by catalysis with phosphoglucomutase (PGM); (iii) converting the produced G6P into fructose 6-phosphate (F6P) by catalysis with phosphoglucoisomerase (PGI); (iv) converting the F6P to allulose 6-phosphate (A6P) by catalysis with allulose 6-phosphate 3-epimerase (A6PE); and (v) converting the produced A6P to allulose by catalysis with allulose 6-phosphate phosphatase (A6PP) specific for allulose 6-phosphate; The process comprising the steps of:

2. 2. The process of claim 1, wherein the sugar is selected from the group consisting of starch or a derivative thereof, cellulose or a derivative thereof, and sucrose.

3. 2. The process of claim 1, 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.

4. 3. The process of claim 2, 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.

5. 5. The process of claim 4, 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.

6. 2. The process of claim 1, wherein 4-glucan transferase (4GT) is added to the process.

7. 3. The process of claim 2, wherein the sugars are starch derivatives, and the starch derivatives are prepared by enzymatic hydrolysis of starch catalyzed by isoamylase, pullulanase, alpha-amylase, or combinations thereof.

8. 8. The process of any one of claims 1 to 7, 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.

9. The A6PE is an (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.

10. The process of any one of claims 1 to 7, 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.

11. 8. The process of any one of claims 1 to 7, wherein the A6PP 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 points towards the third β-strand of the rosemanoid fold, and a GDxxxD signature at the end of the fourth β-strand of the rosemanoid fold.

12. 8. The process of any one of claims 1 to 7, 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 hours to 48 hours.

13. 8. The process according to any one of claims 1 to 7, wherein the process steps are carried out in one bioreactor or in multiple bioreactors arranged in series.

14. 8. The process of any one of claims 1 to 7, wherein the process steps are carried out in the absence of ATP, in the absence of NAD(H), at a phosphate concentration of >0 mM to <150 mM, the phosphate is recycled, and / or at least one step of the process involves an energetically favorable chemical reaction.

15. The process of any one of claims 1 to 7, wherein the process step is cell-free.

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