Enzymatic production of D-psicose

Abstract

The present disclosure provides a method for the enzymatic conversion of a saccharide to psicose. The invention also relates to a process for the preparation of psicose, wherein the process involves the catalytic conversion of fructose 6-phosphate (F6P) to psicose 6-phosphate (A6P) by 6-phosphate 3-isomerase (A6PE); and converting the A6P into the psicose under the catalysis of the psicose-6-phosphate phosphatase (A6PP).

Description

Technical Field

[0001] The present invention relates to the preparation of the sugar D-psicose. More specifically, the present invention relates to a method for preparing D-psicose by enzymatically converting saccharides (e.g., polysaccharides, oligosaccharides, disaccharides, sucrose, D-glucose, and D-fructose) into D-psicose. Background Art

[0002] D-psicose (also known as D-pseudofructose) (hereinafter referred to as allulose) is a low-calorie natural sweetener with 70% of the sweetness of sucrose but only 10% of the calories of sucrose. Psicose is a naturally occurring monosaccharide hexose that is only present in small amounts in wheat and other plants. In 2012, the Food and Drug Administration (FDA) approved allulose as a food additive and designated it as Generally Recognized as Safe (GRAS). However, due to the high selling price of allulose, its use as a sweetener is limited. Psicose has countless health benefits: it is low in calories (10% of sucrose); it has a very low glycemic index of 1; it is completely absorbed by the small intestine but is not metabolized, but secreted in urine and feces; it helps regulate blood sugar by inhibiting α-amylase, sucrase, and maltase; and it has similar functions to sucrose in food and beverages. Therefore, allulose obviously has a variety of applications in the food and beverage industry.

[0003] Currently, psicose is mainly produced by enzymatic isomerization of fructose (WO 2014049373). In general, the process is impaired by high raw material costs, expensive separation of psicose from fructose, and relatively low product yields.

[0004] There is a need to develop a cost-effective synthetic route for high-yield production of psicose, wherein at least one step of the process involves an energetically favorable chemical reaction. In addition, 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

[0005] A method for producing psicose that can be carried out at relatively low concentrations of phosphate, wherein the phosphate can be recycled, and / or the method does not require the use of adenosine triphosphate (ATP) as a phosphate source is needed. A psicose production pathway that does not require the use of expensive nicotinamide adenosine dinucleotide (NAD(H)) coenzyme in any reaction step is also needed. Summary of the invention

[0006] The invention described herein relates to a method for preparing psicose. In various aspects, the method relates to converting fructose 6-phosphate (F6P) into psicose 6-phosphate (A6P) catalyzed by 6-phosphate 3-epimerase (A6PE); and converting A6P into psicose catalyzed by psicose-6-phosphate phosphatase (A6PP). The present invention also relates to psicose prepared by any of the methods described herein.

[0007] In some aspects of the invention, the method for preparing psicose further involves a step of converting glucose-6-phosphate (G6P) into F6P, wherein the step is catalyzed by phosphoglucose isomerase (PGI). In other aspects, the method for synthesizing psicose further comprises a step of converting glucose 1-phosphate (G1P) into G6P, and this conversion step is catalyzed by phosphoglucose mutase (PGM).

[0008] In various aspects, a method for preparing psicose may involve converting sugars into G1P catalyzed by at least one enzyme; converting G1P into G6P catalyzed by phosphoglucomutase (PGM); converting G6P into F6P catalyzed by phosphoglucomutase (PGI); converting F6P into psicose 6-phosphate (A6P) catalyzed by A6PE; and converting the generated A6P into psicose catalyzed by A6PP.

[0009] The carbohydrate used in any of the methods may be selected from the group consisting of starch or its derivatives, cellulose or its derivatives, and sucrose. The starch or its derivatives may be amylose, amylopectin, soluble starch, amylopectin, maltodextrin, maltose, or glucose. In some aspects of the invention, the method for preparing psicose involves converting starch into a starch derivative by enzymatic hydrolysis of starch or by acid hydrolysis of starch. In other aspects, the starch derivative may be prepared by enzymatic hydrolysis of starch catalyzed by isoamylase, pullulanase, α-amylase, or a combination of two or more of these enzymes. In certain aspects, the preparation of psicose involves converting starch into a starch derivative by enzymatic hydrolysis of starch or by acid hydrolysis of starch.

[0010] The sugar method may also involve the addition of 4-glucanotransferase (4GT).

[0011] In various aspects, the method for preparing psicose may involve converting fructose into F6P catalyzed by at least one enzyme; converting F6P into psicose 6-phosphate (A6P) catalyzed by A6PE; and converting the produced A6P into psicose catalyzed by A6PP. In other embodiments, the method for producing psicose involves converting sucrose into fructose catalyzed by at least one enzyme; converting fructose into F6P catalyzed by at least one enzyme; converting F6P into psicose 6-phosphate (A6P) catalyzed by A6PE; and converting the produced A6P into psicose catalyzed by A6PP.

[0012] In other aspects of the present invention, G6P to be used in the method for preparing psicose can be produced by converting glucose into G6P catalyzed by at least one enzyme. Glucose can then be produced by converting sucrose into glucose catalyzed by at least one enzyme.

[0013] In other aspects of the present invention, the steps of the method for preparing psicose are performed 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 method involves an energetically favorable chemical reaction. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] These drawings illustrate certain aspects of some embodiments of the invention and should not be used to limit or define the invention.

[0015] Figure 1 is a schematic diagram illustrating the enzyme pathway for converting fructose 6-phosphate to psicose 6-phosphate and then to psicose.

[0016] Figure 2 is a schematic diagram illustrating the enzymatic pathway for converting starch or its derivatives into psicose. The following abbreviations are used: αGP, α-glucan phosphorylase or starch phosphorylase; PGM, phosphoglucomutase; PGI, phosphoglucoisomerase; IA, isoamylase; PA, pullulanase; MP, maltose phosphorylase; PPGK, polyphosphoglucokinase.

[0017] Figure 3 The enzymatic pathway for converting cellulose or its derivatives into psicose is shown. CDP, cellodextrin phosphorylase; CBP, cellobiose phosphorylase; PPGK, polyphosphodiesterase

[0018] PGM, phosphoglucomutase; PGI, phosphoglucoisomerase.

[0019] Figure 4 is a schematic diagram illustrating the enzyme pathway for converting fructose to psicose. PPFK, polyphosphofructokinase.

[0020] Figure 5 is a schematic diagram illustrating the enzyme pathway for converting glucose to psicose. PPGK, polyphosphoglucokinase; PGI, phosphoglucose isomerase.

[0021] Figure 6 The enzymatic pathway for converting sucrose or its derivatives into psicose is shown. SP, sucrose phosphorylase; PPFK, polyphosphofructokinase; PGM, phosphoglucomutase; PGI, phosphoglucoisomerase.

[0022] Figure 7 The reaction Gibbs energy between the intermediates based on the Gibbs energy formed for converting glucose 1-phosphate into psicose is shown. DETAILED DESCRIPTION

[0023] The present invention provides an enzyme pathway or method for synthesizing psicose with a high product yield while greatly reducing the product separation cost and the psicose production cost.

[0024] The present invention relates to a method for preparing psicose, wherein the method involves converting fructose 6-phosphate (F6P) into psicose 6-phosphate (A6P) catalyzed by an epimerase, and converting the generated A6P into psicose by a phosphatase (e.g., psicose 6-phosphate phosphatase, A6PP). The method is generally shown in Figure 1 In certain embodiments, the epimerase that catalyzes the conversion of F6P to A6P is psicose-6-phosphate 3-epimerase (A6PE).

[0025] An epimerase that converts F6P to A6P can be used in the methods of the present invention. An epimerase can also convert A6P to F6P. In some aspects of the present invention, an epimerase suitable for use in the method of converting F6P to A6P comprises an amino acid sequence having the following degree of identity with the amino acid sequence of SEQ ID NO: 3 or 6: 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%,

[0026] 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%. Suitable epimerases are encoded by polynucleotides comprising a nucleotide sequence having a degree of identity to the nucleotide sequence of SEQ ID NOs: 1, 2, 4 and 5 of 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%.

[0027] Examples of A6PE include, but are not limited to, the following proteins identified by UNIPROT ID numbers: D9TQJ4, A0A090IXZ8, and P32719. Among them, D9TQJ4 and A0A090IXZ8 are from thermophilic organisms. P32719 is from a mesophilic organism. P32719 has 53% identity with A0A090IXZ8 and 55% identity with D9TQJ4, and each protein catalyzes the epimerization of F6P to A6P. In addition, A0A090IXZ8 has 45% identity with D9TQJ4. In contrast, other epimerase proteins identified by UNIPROT ID numbers, A0A101D823, R1AXD6, A0A150LBU8, A0A023CQG9 and H1XWY2, which have a degree of identity of 45% or less to D9TQJ4, do not catalyze the epimerization of F6P to A6P.

[0028] In some aspects of the invention, the epimerase used in the method for converting F6P to A6P utilizes a divalent metal cofactor: preferably, but not limited to, cobalt. In other aspects of the invention, the epimerase comprises, but is not limited to, containing an (α / β)8-barrel domain for catalysis; additionally, but not limited to, containing a phosphate binding domain including Ser at the end of the 7th β-strand of the barrel, Ser at the end of the 8th β-strand of the barrel, and Gly in the active site loop; additionally, but not limited to, containing a metal binding domain including His in the 2nd and 3rd β-strands of the barrel; additionally, but not limited to, containing Asp in the 2nd and 7th β-strands of the barrel to serve as an acid / base catalyst for 1,1-proton transfer; and additionally, but not limited to, containing a His-hydrophobic residue-Asp tag in the 2nd β-strand of the barrel, wherein the His is used for metal binding and the Asp is used for acid / base catalysis. These features are known in the art, and see, for example, Chan et al., Structural Basis for Substrate Specificity in

[0029] 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 used in the method of the present invention contains a (α / β) 8-barrel domain for catalysis, Ser at the end of the 7th β-strand of the barrel, Ser at the end of the 8th β-strand of the barrel, Gly in the active site loop, His in the 2nd and 3rd β-strands of the barrel, Asp in the 2nd and 7th β-strands of the barrel, and a His-hydrophobic residue-Asp tag in the 2nd β-strand of the barrel.

[0030] The method of the present invention uses a phosphatase to convert A6P to D-psicose. In some aspects of the present invention, the phosphatase suitable for use in the method for converting A6P to D-psicose comprises an amino acid sequence having an identity with the amino acid sequence of SEQ ID NO: 9 to the extent of 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%. Suitable epimerases are encoded by a polynucleotide comprising a nucleotide sequence having a degree of identity with the nucleotide sequence of SEQ ID NOs: 7 and 8: 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%.

[0031] Examples of A6PP include, but are not limited to, the following proteins identified by UNIPROT ID numbers: A3DC21, Q5LGR4, and Q89ZR1. A3DC21 has 46% identity with Q5LGR4 and 45% identity with Q89ZR1, and each protein catalyzes the specific dephosphorylation of A6P to psicose. In contrast, other phosphatases from the halogen acid dehydrogenase superfamily, i.e., proteins identified by UNIPROT ID numbers: H0UQ29, Q67LU4, A0A0K6IPM3, C8WSJ0, A0A151YX61, etc., which have less than 45% identity with A3DC21, do not catalyze the specific dephosphorylation of A6P to psicose.

[0032] The phosphatase for converting A6P to psicose suitable for the method of the present invention is specific for psicose 6-phosphate. As used herein, specificity for psicose-6-phosphate means having higher specific activity for psicose 6-phosphate than glucose 1-phosphate, glucose-6-phosphate or fructose-6-phosphate.

[0033] The phosphatase that converts A6P to psicose utilizes a divalent metal cofactor: preferably magnesium. In another aspect of the invention, the phosphatase contains, but is not limited to, a Rossmanoid fold domain for catalysis; additionally, but not limited to, a C1 capping domain for substrate specificity; additionally, but not limited to, a DXD tag for coordination with magnesium in the first β-strand of the Rossmanoid fold, wherein the second Asp is a general acid / base catalyst; additionally, but not limited to, a Thr or Ser at the end of the second β-strand of the Rossmanoid fold that contributes to the stability of the reaction intermediate; additionally, but not limited to, a Lys at the N-terminus of the α-helix at the C-terminus of the third β-strand of the Rossmanoid fold that contributes to the stability of the reaction intermediate; and additionally, but not limited to, a GDxxxD tag at the end of the fourth β-strand of the Rossmanoid fold that contributes to the coordination with magnesium. These features are known in the art and are described in, for example, 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 psicose used in the method of the present invention contains a Rossmanoid fold domain for catalysis, a C1 capping domain, a DxD tag in 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 the α-helix at the C-terminus of the third β-strand of the Rossmanoid fold, and a GDxxxD tag at the end of the fourth β-strand of the Rossmanoid fold.

[0034] In some embodiments, the method for preparing psicose according to the present invention further comprises a step of enzymatically converting glucose-6-phosphate (G6P) into F6P, and this step is catalyzed by phosphoglucose isomerase (PGI). In other embodiments, the method for preparing psicose further comprises a step of converting glucose 1-phosphate (G1P) into G6P, wherein the step is catalyzed by phosphoglucose mutase (PGM). In yet another embodiment, the psicose production method further comprises a step of converting sugars into G1P catalyzed by at least one enzyme.

[0035] Therefore, the method for preparing psicose according to the present invention may, for example, include the following steps: (i) converting a saccharide into glucose 1-phosphate (G1P) using one or more enzymes; (ii) converting G1P into G6P using phosphoglucomutase (PGM, EC5.4.2.2); (iii) converting G6P into F6P using phosphogluconate isomerase (PGI, EC 5.3.1.9); (iv) converting F6P into A6P via A6PE, and (v) converting A6P into psicose via A6PP. An example of a method in which the saccharide is starch is shown in Figure 2 middle.

[0036] Typically, the ratio of enzyme units used in the disclosed method is 1:1:1:1:1 (αGP:PGM:PGI:A6PE:A6PP). In order to optimize product yield, these ratios can be adjusted in any combination of numbers. For example, a ratio of 3:1:1:1:1 can be used to maximize the concentration of phosphorylated intermediates, which will result in increased activity of downstream reactions. In contrast, a ratio of 1:1:1:1:3 can be used to maintain a robust phosphate supply of αGP, which will result in more efficient phospholysis of α-1,4-glycosidic bonds. An enzyme ratio of, for example, 3:1:1:1:3 can be used to further increase the reaction rate. Therefore, the enzyme ratio (including other optional enzymes discussed below) can be changed to increase the efficiency of psicose production. For example, relative to the amount of other enzymes, a specific enzyme can be present in an amount of about 2x, 3x, 4x, 5x, etc.

[0037] One of the important advantages of the method is that the method steps can be carried out in one bioreactor or reaction vessel. Alternatively, the steps can also be carried out in a plurality of bioreactors or reaction vessels arranged in series.

[0038] The phosphate ions generated by the dephosphorylation of A6P can then be recycled in the process steps for converting sugars to G1P, especially when all process steps are performed in a single bioreactor or reaction vessel. The ability to recycle phosphate in the disclosed process allows the use of non-stoichiometric amounts of phosphate, which keeps the reaction phosphate concentration low. This affects the overall pathway and overall rate of the process, but does not limit the activity of individual enzymes and allows for the overall efficiency of the psicose production process.

[0039] 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.

[0040] It is about 0.1mM, about 0.5mM, about 1mM, about 1.5mM, about 2mM, about 2.5mM, about 5mM, about 6mM, about 7mM, about 8mM, about 9mM, about 10mM, about 15mM, about 20mM, about 25mM, about 30mM, about 35mM, about 40mM, about 45mM, about 50mM, or about 55mM.

[0041] Therefore, low phosphate concentrations lead to lower production costs due to low total phosphate and therefore reduced phosphate removal costs. It also prevents inhibition of A6PP by high concentrations of free phosphate and reduces the possibility of phosphate contamination.

[0042] In addition, the method disclosed herein can be carried out without adding ATP as a phosphate source, i.e., in the absence of ATP. The method can also be carried out without having to add NAD(H), i.e., in the absence of NAD(H). Other advantages also include the fact that at least one step of the disclosed method for producing psicose involves an energetically favorable chemical reaction ( Figure 7 ).

[0043] Examples of enzymes for converting carbohydrates to G1P include α-glucan phosphorylase (αGP, EC 2.4.1.1, which also includes maltodextrin phosphorylase, starch phosphorylase, glycogen phosphorylase and other α-1,4 glycosidic bond degrading 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 enzyme combination depends on the carbohydrate used in the method.

[0044] The carbohydrate used to produce G1P can be a polysaccharide, an oligosaccharide and / or a disaccharide. For example, the carbohydrate can be starch, one or more derivatives of starch, cellulose, one or more derivatives of cellulose, sucrose, one or more derivatives of sucrose, or a combination thereof.

[0045] Starch is the most widely used energy storage compound in nature and is mainly stored in plant seeds. Natural starch contains straight-chain starch and branched amylopectin. Examples of starch derivatives include straight-chain starch, amylopectin, soluble starch, amylopectin, 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.

[0046] Derivatives of starch can be prepared by enzymatic hydrolysis of starch or by acid hydrolysis of starch. Specifically, enzymatic hydrolysis of starch can be catalyzed or enhanced by: isoamylase (IA, EC.3.2.1.68), which hydrolyzes α-1,6-glycosidic bonds; pullulanase (PA, EC.3.2.1.41), which hydrolyzes α-1,6-glycosidic bonds; 4-α-glucosyltransferase (4GT, EC.2.4.1.25), which catalyzes the transglycosylation of short maltooligosaccharides to obtain longer maltooligosaccharides; or α-amylase (EC 3.2.1.1), which cleaves α-1,4-glycosidic bonds.

[0047] Furthermore, derivatives of cellulose can be prepared by enzymatic hydrolysis of cellulose catalyzed by a cellulase enzyme mixture, by acid or by pretreatment of the biomass.

[0048] In certain embodiments, the enzyme used to convert carbohydrates into G1P contains αGP. In this step, when the carbohydrates include starch, G1P is produced from starch through αGP; when the carbohydrates contain soluble starch, starch dextrin or maltodextrin, G1P is produced from soluble starch, starch dextrin or maltodextrin through αGP.

[0049] When the sugar includes maltose and the enzyme contains maltose phosphorylase, G1P is produced from maltose by maltose phosphorylase. If the sugar includes sucrose and the enzyme contains sucrose phosphorylase, G1P is produced from sucrose by sucrose phosphorylase.

[0050] In yet another embodiment, when the carbohydrate comprises cellobiose and the enzyme comprises cellobiose phosphorylase, G1P is produced from cellobiose by the cellobiose phosphorylase.

[0051] In another embodiment, when the carbohydrate contains cellodextrins and the enzyme includes cellodextrin phosphorylase, G1P is produced from cellodextrins by the cellodextrin phosphorylase.

[0052] In an alternative embodiment of converting a carbohydrate to G1P, when the carbohydrate comprises cellulose and the enzyme comprises cellulose phosphorylase, G1P is produced from cellulose by the cellulose phosphorylase.

[0053] According to the present invention, psicose can also be produced from fructose. An example of the method is shown in Figure 4 For example, the method involves producing F6P from fructose and polyphosphate catalyzed by polyphosphofructokinase (PPFK); converting F6P to A6P catalyzed by A6PE; and converting A6P to psicose catalyzed by A6PP. Fructose can be produced, for example, by enzymatic conversion of sucrose.

[0054] In other embodiments, psicose can be produced from sucrose. An example of such a method is shown in Figure 6 The method provides an in vitro synthetic pathway, which includes the following enzymatic steps: production of G1P from sucrose and free phosphate catalyzed by sucrose phosphorylase (SP); conversion of G1P to G6P catalyzed by PGM; conversion of G6P to F6P catalyzed by PGI; conversion of F6P to A6P catalyzed by A6PE; and conversion of A6P to psicose catalyzed by A6PP.

[0055] The phosphate ions generated when A6P is converted to psicose can then be recycled in the step of converting sucrose to G1P. Figure 6 As shown, PPFK and polyphosphate can be used to increase the psicose yield by producing F6P from fructose produced by the phospholysis of sucrose by SP.

[0056] In some embodiments, the method for preparing psicose comprises the steps of producing glucose from polysaccharides and oligosaccharides by enzymatic hydrolysis or acid hydrolysis, converting glucose into G6P by at least one enzyme, producing fructose from polysaccharides and oligosaccharides by enzymatic hydrolysis or acid hydrolysis, and converting fructose into G6P by at least one enzyme. Examples of polysaccharides and oligosaccharides are listed above.

[0057] In other embodiments, G6P is produced from glucose and sodium polyphosphate by polyphosphoglucokinase.

[0058] The present disclosure provides methods for converting sugars (such as polysaccharides and oligosaccharides in starch, cellulose, sucrose, and their derivatives) into psicose. In certain embodiments, an artificial (non-natural) ATP-free enzyme pathway is provided to convert starch, cellulose, sucrose, and their derivatives into psicose using a cell-free enzyme mixture.

[0059] As shown above, several enzymes can be used to hydrolyze starch to increase the G1P yield. Such enzymes include isoamylase, pullulanase, and α-amylase. Corn starch contains many branches that hinder the action of αGP. Isoamylase can be used to debranch the starch, resulting in linear starch dextrins. Starch pretreated with isoamylase can result in a higher concentration of F6P in the final product. Isoamylase and pullulanase cleave α-1,6-glycosidic bonds, which allows α-glucan phosphorylase to degrade starch more completely. α-amylase cleaves α-1,4-glycosidic bonds, so α-amylase is used to degrade starch into fragments for faster conversion to psicose.

[0060] like Figure 2As shown, maltose phosphorylase (MP) can be used to increase the yield of psicose by cleaving the degradation product maltose phosphate into G1P and glucose. Alternatively, 4-glucanotransferase (4GT) can be used to increase the yield of psicose by recycling the degradation products glucose, maltose and maltotriose into longer maltooligosaccharides; the longer maltooligosaccharides can be cleaved by αGP phosphate to obtain G1P.

[0061] In addition, cellulose is the most abundant biological resource and is the main component of plant cell walls. Non-edible lignocellulosic biomass contains cellulose, hemicellulose and lignin and other trace components. Pure cellulose can be prepared by a series of treatments, including Avicel (microcrystalline cellulose), regenerated amorphous cellulose, bacterial cellulose, filter paper, etc. The partially hydrolyzed cellulose substrate includes water-insoluble cellodextrins with a degree of polymerization greater than 7, water-soluble cellodextrins with a degree of polymerization of 3-6, cellobiose, glucose and fructose.

[0062] In certain embodiments, cellulose and its derivative products can be converted to psicose through a series of steps. An example of such a method is shown in Figure 3 The method provides an in vitro synthetic pathway involving the following steps: generating G1P from cellodextrin and cellobiose and free phosphate, catalyzed by cellodextrin phosphorylase (CDP) and cellobiose phosphorylase (CBP), respectively; 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 psicose, catalyzed by A6PP. In this method, phosphate ions can be recycled through the steps of converting cellodextrin and cellobiose to G1P.

[0063] Several enzymes can be used to hydrolyze solid cellulose into water-soluble cellodextrins and cellobiose. Such enzymes include endoglucanases and cellobiohydrolases, but do not include β-glucosidases (cellobiases).

[0064] Prior to cellulose hydrolysis and G1P production, cellulose and biomass can be pretreated to increase their reactivity and reduce the degree of polymerization of the cellulose chains. Cellulose and biomass pretreatment methods include dilute acid pretreatment, cellulose solvent-based lignocellulose fractionation, ammonia fiber swelling, ammonia soaking, ionic liquid treatment, and partial hydrolysis using concentrated acids (including hydrochloric acid, sulfuric acid, phosphoric acid, and combinations thereof).

[0065] In some embodiments, polyphosphate and polyphosphoglucokinase (PPGK) can be added to the method to increase the yield of psicose by phosphorylating the degradation product glucose to G6P, such as Figure 3 shown.

[0066] In other embodiments, psicose can be produced from glucose. An example of this method is shown in Figure 5 The method involves the following steps: generating G6P from glucose and polyphosphate catalyzed by polyphosphoglucokinase (PPGK); converting G6P into F6P catalyzed by PGI; converting F6P into A6P catalyzed by enzyme; and converting A6P into psicose catalyzed by A6PP.

[0067] Any suitable biological buffer known in the art can be used in the method of the present invention, such as HEPES, PBS, BIS-TRIS, MOPS, DIPSO, Trizma, etc. The reaction buffer of all embodiments can have a pH range of 5.0-8.0. More preferably, the reaction buffer pH can be in the range of about 6.0 to about 7.3. For example, the reaction buffer pH can be 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2 or 7.3.

[0068] The reaction buffer may also contain key metal cations. Examples of such metal ions include Mg 2+ 、Co 2+ and Zn 2+ .

[0069] The reaction temperature for carrying out the reacting step may be in the range of 37° C. to 85° C. More preferably, the step may be carried out in the range of about 40° C. to about 70° C. The temperature may 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.

[0070] The reaction time of the disclosed method can be adjusted as needed, and can be within the range of 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.

[0071] Due to the very favorable equilibrium constant of the overall reaction, the process according to the invention can be realized

[0072] Theoretically, if the starting material is completely converted into the intermediate, a yield of up to 99% can be achieved.

[0073] The method of the present invention uses low-cost starting materials and reduces production costs by reducing costs associated with separation of raw materials and products. Starch, cellulose, sucrose and some of their derivatives are cheaper raw materials than fructose. When psicose is produced from fructose, the yield is lower than the present invention, and psicose must be separated from fructose by chromatography, which results in higher production costs.

[0074] Moreover, the step of converting A6P to psicose according to the present invention is an irreversible phosphatase reaction, which is independent of the raw materials. Therefore, psicose is produced in a very high yield while effectively minimizing the subsequent product separation cost.

[0075] In contrast to cell-based manufacturing methods, the present invention relates to cell-free preparation of psicose, which has a relatively high reaction rate due to the elimination of cell membranes, which usually slow down the transport of substrates / products in and out of cells. It also has a final product that is free of nutrient-rich fermentation medium / cellular metabolites.

[0076] Example

[0077] Materials and methods

[0078] Chemicals

[0079] Unless otherwise specified, all chemicals, including corn starch, soluble starch, maltodextrin, maltose, glucose, filter paper were reagent grade or higher and 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 by 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 a thermophilic A6PE from Bacillus thermoamylovorans (UNIPROT ID A0A090IXZ8). 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 a thermophilic A6PP from Clostridium thermocellum (UNIPROT ID A3DC21). 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. Regenerated amorphous cellulose for enzyme purification was prepared from Avicel PH105 (FMC BioPolymer, Philadelphia, PA, USA) by its dissolution and regeneration as described by 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 a host cell for DNA manipulation, and Escherichia coli BL21 (DE3) (Sigma-Aldrich, St. Louis, MO, USA) was used as a host cell for recombinant protein expression. 100 mg L was contained. -1 Ampicillin or 50 mg L -1 Kanamycin-containing ZYM-5052 medium was used for E. coli cell growth and recombinant protein expression. Cellulase (Catalog No.: C2730) and pullulanase (Catalog No.: P1067) from Trichoderma reesei were purchased from Sigma-Aldrich (St. Louis, MO, USA) and produced by Novozymes (Franklinton, NC, USA). Maltose phosphorylase (Catalog No.: M8284) was purchased from Sigma-Aldrich.

[0080] Production and purification of recombinant enzymes

[0081] The Escherichia coli BL21 (DE3) strain carrying the protein expression plasmid was cultured in a 1 L Erlenmeyer flask with 100 mg L -1 Ampicillin or 50 mg L -1 100mL ZYM-5052 culture medium of kanamycin is incubated together.Cells are cultured at 30 ℃ with 220rpm rotary shaking for 16-24 hours.Cells are harvested by centrifugation at 12 ℃, and washed once with 20mM HEPES (pH 7.5) (heat precipitation and cellulose binding module) containing 50mM NaCl and 5mM MgCl2 or 20mM HEPES (pH 7.5) (Ni purification) containing 300mM NaCl and 5mM imidazole.Cell pellets are resuspended in the same buffer and cracked by ultrasonic treatment (Fisher Scientific ultrasonic crusher model 500; 5s pulse on and 10s off, 21 minutes in total under 50% amplitude).After centrifugation, the target protein in the purification supernatant is obtained.

[0082] Three methods were used to purify various recombinant proteins. His-tagged proteins were purified by Profinity IMAC Ni packed resin (Bio-Rad, Hercules, CA, USA). Fusion proteins containing a cellulose binding module (CBM) and a self-cleaving intein were purified by high affinity adsorption on large surface area regenerated amorphous cellulose. Heat precipitation at 70-95°C for 5-30 minutes was used to purify ultrathermostable enzymes. The purity of the recombinant proteins was detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). A6PE was purified with 80 μM CoCl2, elution buffer, dialysis buffer, and protein storage buffer present in the growth medium.

[0083] Enzymes used and their activity assays

[0084] α-glucan phosphorylase (αGP) (UNIPROT ID G4FEH8) from Thermotoga maritima was used. Activity was measured at 50°C in 50mM sodium phosphate buffer (pH 7.2) containing 1mM MgCl2, 5mM DTT and 30mM maltodextrin. The reaction was terminated by filtering the enzyme with a Vivaspin 2 concentrator (10,000MWCO) (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 25U / mL phosphoglucomutase. Units (U) are described as μmol / minute.

[0085] Use the phosphoglucomutase (PGM) (UNIPROT IDQ68BJ6) from Thermococcus kodakaraensis. Activity was measured at 50°C in 50mM HEPES buffer (pH 7.2) containing 5mM MgCl and 5mM G1P. The reaction was terminated by filtering the enzyme with a Vivaspin 2 concentrator (10,000MWCO). The product glucose-6-phosphate (G6P) was measured using a hexokinase / G6PDH assay kit (Sigma Aldrich, catalog number GAHK20-1KT).

[0086] Two different sources of phosphorus were used, from Clostridium thermocellum (UNIPROT ID A3DBX9) and Thermus thermophilus (UNIPROT ID Q5SLL6).

[0087] Acid glucose isomerase (PGI). Activity was measured at 50°C in 50mM HEPES buffer (pH 7.2) containing 5mM MgCl and 10mM G6P. The reaction was terminated by filtering the enzyme with a Vivaspin 2 concentrator (10,000MWCO). The product fructose 6-phosphate (F6P) was determined using a fructose 6-phosphate kinase (F6PK) / pyruvate dehydrogenase (PK) / lactate dehydrogenase (LD) coupled enzyme assay, wherein the reduction in absorbance at 340nm indicated the production of F6P. This 200 μL reaction contained 50mM HEPES (pH 7.2), 5mM MgCl, 10mM G6P, 1.5mM ATP, 1.5mM phosphoenolpyruvate, 200 μM NADH, 0.1U PGI, 5UPK and 5ULD.

[0088] The psicose-6-phosphate 3-epimerase (A6PE) from Thermoanaerobacterium thermosaccharolyticum (UNIPROTID 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 with a Vivaspin 2 concentrator (10,000 MWCO). The product psicose 6-phosphate (A6P) was determined using psicose-6-phosphate phosphatase and detecting free phosphate release. To detect free phosphate release, 500 μL of a solution (pH 5) containing 0.1 M zinc acetate and 2 mM ammonium molybdate was added to a 50 μL reaction. It was mixed, and then 125 μL of 5% ascorbic acid (pH 5) was added. The solution was mixed and then incubated for 20 minutes at 30° C. The absorbance at 850 nm was read to determine free phosphate release.

[0089] The psicose-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 terminated by filtering the enzyme with a Vivaspin 2 concentrator (10,000 MWCO). The product psicose was determined by detecting free phosphate release as described for A6PE.

[0090] Recombinant cellodextrin phosphorylase and cellobiose phosphorylase from Clostridium thermocellum are described in Ye et al. Spontaneous high-yield production of hydrogen from cellular materials and water catalyzed by enzyme cocktails. ChemSusChem 2009; 2: 149-152. Their activities were determined as described.

[0092] 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. Its activity was determined as described.

[0093] The 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. Its activity was determined as described.

[0094] The recombinant 4-α-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. Its activity was measured as described.

[0095] 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 organophosphate. Glucose 1-phosphate (G1P) was measured using a glucose hexokinase / G6PDH assay kit supplemented with 25 U / mL phosphoglucomutase, as was α-glucan phosphorylase.

[0096] The enzyme units used in each of the following examples can be increased or decreased to adjust the reaction time as needed. For example, if one wants to perform Example 9 in 8 hours instead of 24 hours, the enzyme units will increase by about 3 times. On the contrary, if one wants to perform Example 9 in 48 hours instead of 24 hours, the enzyme units can be reduced by about 2 times. These examples illustrate how the amount of enzyme units can be used to increase or decrease the reaction time while maintaining a constant productivity.

[0097] Example 1

[0098] To verify the technical feasibility of enzymatic biosynthesis of fructose-6-phosphate from starch, three enzymes were recombinantly expressed: α-glucan phosphorylase from Thermotoga maritima (UNIPROT ID G4FEH8), phosphoglucose mutase from Thermococcuskodakaraensis (UNIPROT ID Q68BJ6), and phosphoglucose isomerase from Clostridium thermocellum (UNIPROT ID A3DBX9). The recombinant proteins were overexpressed in Escherichia coli BL21 (DE3) and purified as described above.

[0099] 0.20mL reaction mixture containing 10g / L soluble starch, 50mM phosphate buffered saline pH 7.2, 5mM MgCl2, 0.5mM ZnCl2, 0.01U αGP, 0.01U PGM and 0.01U PGI was incubated at 50°C for 24 hours. The reaction was terminated by filtering the enzyme with a Vivaspin 2 concentrator (10,000MWCO). The product fructose 6-phosphate (F6P) was determined using a fructose 6-phosphate kinase (F6PK) / pyruvate dehydrogenase (PK) / lactate dehydrogenase (LD) coupled enzyme assay, wherein the decrease in absorbance at 340nm indicates the production of F6P, as described above. The final concentration of F6P after 24 hours was 3.6g / L.

[0100] Example 2

[0101] The same test as in Example 1 was performed at 40°C to 80°C (except for the different reaction temperatures). We found that after 40 hours of reaction, 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 indicate that increasing the reaction temperature for this set of enzymes increases the F6P yield, but too high a temperature may impair some enzyme activity.

[0102] Example 3

[0103] We found that at 80°C, an approximately 1:1:1 ratio of αGP:PGM:PGI enzyme units resulted in rapid production of F6P. We note that if the reaction time is long enough, the enzyme ratio does not greatly affect the final F6P concentration. However, the enzyme ratio affects the reaction rate and the overall cost of the enzymes used in the system.

[0104] Example 4

[0105] The mixture contained 10 g / L maltodextrin, 50 mM phosphate buffered saline, pH 7.2, 5 mM

[0106] MgCl2, 0.5mM ZnCl2, 0.01U αGP, 0.01U PGM and 0.01U PGI 0.20mL reaction mixture was incubated at 50°C for 24 hours. The reaction was terminated by filtering the enzyme with a Vivaspin 2 concentrator (10,000MWCO). The product fructose 6-phosphate (F6P) was determined using a fructose 6-phosphate kinase (F6PK) / pyruvate dehydrogenase (PK) / lactate dehydrogenase (LD) coupled enzyme assay, wherein a decrease in absorbance at 340nm indicated the production of F6P, as described above. The final concentration of F6P after 24 hours was 3.6g / L.

[0107] Example 5

[0108] In order to test the production of F6P by Avicel, Sigma cellulase was used to hydrolyze cellulose at 50°C. In order to remove β-glucosidase from commercial cellulase, 10 filter paper units / mL of cellulase were mixed with 10g / L Avicel in an ice-water bath for 10 minutes. After centrifugation at 4°C, the supernatant containing β-glucosidase was decanted. Avicel combined with cellulase containing endoglucanase and cellobiohydrolase was resuspended in citrate buffer (pH 4.8) and hydrolyzed for 3 days at 50°C. Cellulose hydrolysate was mixed with 5U / mL cellodextrin phosphorylase, 5U / L cellobiose phosphorylase, 5U / mL αGP, 5U / mL PGM and 5U / mL PGI in 100mM HEPES buffer (pH 7.2) containing 10mM phosphate, 5mM MgCl and 0.5mM ZnCl. The reaction was run at 60°C for 72 hours and high concentrations of F6P were found (little 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.

[0109] Example 6

[0110] To increase the F6P yield of Avicel, Avicel was pretreated with concentrated phosphoric acid to produce amorphous cellulose (RAC) as described in 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 β-glucosidase from commercial cellulase, 10 filter paper units / mL of cellulase were mixed with 10 g / L RAC in an ice-water bath for 5 minutes. After centrifugation at 4°C, the supernatant containing β-glucosidase was decanted. RAC combined with cellulase containing endoglucanase and cellobiohydrolase was resuspended in citrate buffer (pH 4.8) for hydrolysis at 50°C for 12 hours. The RAC hydrolysate was mixed with 5U / mL cellodextrin phosphorylase, 5U / L cellobiose phosphorylase, 5U / mL αGP, 5U / mL PGM and 5U / mL PGI in 100mM HEPES buffer (pH 7.2) containing 10mM phosphate, 5mM MgCl2 and 0.5mM ZnCl2. The reaction was carried out at 60°C for 72 hours. Because no enzyme was added to convert glucose to F6P, high concentrations of F6P and glucose were recovered. F6P was detected using the above-mentioned coupled enzyme assay. Glucose was detected using the hexokinase / G6PDH assay kit as described above.

[0111] Example 7

[0112] In order to further improve the F6P yield of RAC, polyphosphoglucokinase and polyphosphate were added. In order to remove β-glucosidase from commercial cellulase, 10 filter paper units / mL of cellulase were mixed with 10 g / L RAC in an ice-water bath for 5 minutes. After centrifugation at 4°C, the supernatant containing β-glucosidase was decanted. RAC combined with cellulase containing endoglucanase and cellobiohydrolase was resuspended in citrate buffer (pH 4.8) for hydrolysis at 50°C, incubated in citrate buffer (pH 4.8) for hydrolysis at 50°C for 12 hours. The RAC hydrolysate was mixed with 5U / mL polyphosphate glucose kinase, 5U / mL cellodextrin phosphorylase, 5U / mL cellobiose phosphorylase, 5U / mL αGP, 5U / mL PGM and 5U / mL PGI in 100mM HEPES buffer (pH 7.2) containing 50mM polyphosphate, 10mM phosphate, 5mM MgCl2 and 0.5mM ZnCl2. The reaction was carried out at 50°C for 72 hours. It was found that the F6P concentration was very high and only a small amount of glucose was now present. F6P was detected using the above-mentioned coupled enzyme assay. Glucose was detected using the hexokinase / G6PDH assay kit as described above.

[0113] Example 8

[0114] To verify the production of psicose by 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 hours. 99% of F6P was converted to psicose by HPLC (Agilent 1100 series) using an Agilent Hi-Plex H column and a refractive index detector. The sample was run at 0.6 mL / min in 5 mM H2SO4.

[0115] Example 9

[0116] In order to verify the production of psicose by maltodextrin, 0.20 mL 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 was incubated at 50 ° C for 24 hours. The reaction was terminated by filtering the enzyme with a Vivaspin 2 concentrator (10,000 MWCO). Agilent 1100 series HPLC with a refractive index detector and an Agilent Hi-Plex H column was used to detect and quantify psicose. The mobile phase was 5 mM H2SO4, which was run at 0.6 mL / minute. Standards of various concentrations of psicose were used to quantify our yield.

[0117] Example 10

[0118] 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 was incubated at 80° C. for 24 hours. This was used to produce 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, which was incubated at 50° C. for 24 hours. The yield of psicose was quantified as in Example 9.

[0119] Embodiment 11

[0120] 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 was incubated at 50°C for 4 hours. This was used to produce 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, which was incubated at 50°C for 24 hours. The yield of psicose was quantified as in Example 9.

[0121] Example 12

[0122] To further increase the psicose yield of maltodextrin, 0.05 U 4-glucanotransferase (4GT) was added to the reaction described in Example 9.

[0123] 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 was incubated at 50° C. for 24 hours. The yield of psicose was quantified as in Example 9.

[0124] Embodiment 13

[0125] To determine the concentration range of phosphate buffered saline (PBS), 0.20 mL of a 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. The short duration ensured that it would not go to completion, and thus the difference in efficiency could be clearly seen. The production of F6P was quantified 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 indicated the production of F6P. For reactions containing 6.25mM, 12.5mM, 25mM, 37.5mM or 50mM phosphate buffered saline pH 7.2, yields of 4.5g / L, 5.1g / L, 5.6g / L, 4.8g / L or 4.9g / L F6P were obtained, respectively (Table 1). These results indicate that a concentration of 25mM PBS pH 7.2 is ideal for these specific reaction conditions. It is noteworthy that even the use of 6.25mM PBS at pH 7.2 results in significant turnover due to phosphate recycling. This indicates that the disclosed phosphate recycling method is able to maintain low phosphate levels even at industrial volumetric productivity (e.g., 200-300g / L maltodextrin).

[0126] Table 1

[0127]

[0128]

[0129] Embodiment 14

[0130] To determine the pH range of the cascade reaction, 0.20 mL of a reaction mixture containing 50 g / L maltodextrin; 50 mM phosphate buffered saline 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. The units were reduced to ensure that they could not be completed and thus the differences in efficiency could be clearly seen. The yield of F6P was quantified as in Example 12. For 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, 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 F6P were obtained, respectively (Table 2). These results indicate that while the system works over a wide pH range, pH 6.8 is ideal for these particular reaction conditions.

[0131] Table 2

[0132] pH of PBS F6P g / L 6.0 4.0 6.2 4.1 6.4 4.2 6.6 4.1 6.8 4.4 7.0 4.1 7.2 3.8 7.3 4.0

[0133] Embodiment 15

[0134] 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 was incubated at 50° C. for 24 hours. The yield of psicose was quantified as in Example 9.

[0135] Example 16

[0136] To further increase the psicose yield of maltodextrin, 0.05 U of maltose phosphorylase was added to the reaction described in Example 9.

[0137] Embodiment 17

[0138] To further increase the psicose yield of maltodextrin, 0.05 U of polyphosphate glucokinase and 75 mM polyphosphate were added to the reaction described in Example 9.

[0139] Embodiment 18

[0140] To produce psicose 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 was incubated at 50° C. for 24 hours. The yield of psicose was quantified as in Example 9.

[0141] Embodiment 19

[0142] To produce psicose 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, 0.05 U A6PE, and 0.05 U A6PP was incubated at 50° C. for 24 hours. The yield of psicose was quantified as in Example 9.

[0143] Embodiment 20

[0144] To produce psicose from sucrose, a reaction mixture containing 10 g / L sucrose, 50 mM phosphate buffered saline pH 7.0, 5 mM MgCl2, 80 μM CoCl2, 0.05 U sucrose phosphorylase, 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 yield of psicose was quantified as in Example 9.

[0145] Embodiment 21

[0146] To further increase the psicose yield of sucrose, 75 mM polyphosphate and 0.05 polyphosphofructokinase were added to the reaction mixture in Example 20. As in Example 9, the yield of psicose was quantified.

Claims

1. A method for preparing psicose, comprising: The conversion of fructose 6-phosphate (F6P) to psicose 6-phosphate (A6P) is catalyzed by epimerase; and The A6P is converted into psicose by phosphatase.

2. The method of claim 1, further comprising the step of converting glucose-6-phosphate (G6P) into the F6P, wherein the step is catalyzed by phosphoglucose isomerase (PGI).

3. The method of claim 2, further comprising the step of converting glucose 1-phosphate (G1P) into said G6P, wherein said step is catalyzed by phosphoglucomutase (PGM).

4. The method of claim 3, further comprising the step of converting a sugar into said G1P, wherein said step is catalyzed by at least one enzyme, wherein said sugar is selected from the group consisting of starch or its derivatives, cellulose or its derivatives, and sucrose.

5. The method of claim 4, wherein the at least one enzyme is selected from the group consisting of α-glucan phosphorylase (αGP), maltose phosphorylase, sucrose phosphorylase, cellodextrin phosphorylase, cellobiose phosphorylase, and cellulose phosphorylase.

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

7. The method of claim 6, further comprising the step of converting starch into starch derivatives, wherein the starch derivatives are prepared by enzymatic hydrolysis of starch or acid hydrolysis of starch.

8. The method of claim 6, wherein 4-glucanotransferase (4GT) is added to the method.

9. The method 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. The method of claim 1, further comprising: a step of converting fructose into said F6P, wherein said step is catalyzed by at least one enzyme; and optionally comprising a step of converting sucrose into said fructose, wherein said step is catalyzed by at least one enzyme.

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