Enzymatic production of d-allulose

Abstract

The current disclosure provides a process for enzymatically converting a saccharide into allulose. The invention also relates to a process for preparing allulose where the process involves 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).

What is claimed is: 
     
         1 . A process for preparing allulose, the process comprising:
 converting fructose 6-phosphate (F6P) to allulose 6-phosphate (A6P), catalyzed by an epimerase; and   converting the A6P produced to allulose, catalyzed by a phosphatase.   
     
     
         2 . The process of  claim 1 , further comprising a step of converting glucose 6-phosphate (G6P) to the F6P, wherein the step is catalyzed by phosphoglucoisomerase (PGI). 
     
     
         3 . The process of  claim 2 , further comprising the step of converting glucose 1-phosphate (G1P) to the G6P, wherein the step is catalyzed by phosphoglucomutase (PGM). 
     
     
         4 . The process of  claim 3 , further comprising the step of converting a saccharide to the G1P, wherein the step is catalyzed by at least one enzyme, wherein the saccharide is selected from the group consisting of a starch or derivative thereof, cellulose or a derivative thereof and sucrose. 
     
     
         5 . The process of  claim 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 of  claim 4 , wherein the saccharide is starch or a derivative thereof selected from the group consisting of amylose, amylopectin, soluble starch, amylodextrin, maltodextrin, maltose, and glucose. 
     
     
         7 . The process of  claim 6 , further comprising the step of converting starch to a starch derivative wherein the starch derivative is prepared by enzymatic hydrolysis of starch or by acid hydrolysis of starch. 
     
     
         8 . The process of  claim 6 , wherein 4-glucan transferase (4GT) is added to the process. 
     
     
         9 . The process of  claim 7 , wherein the starch derivative is prepared by enzymatic hydrolysis of starch catalyzed by isoamylase, pullulanase, alpha-amylase, or a combination thereof. 
     
     
         10 . The process of  claim 1 , further comprising:
 the step of converting fructose to the F6P, wherein the step is catalyzed by at least one enzyme; and optionally, the step of converting sucrose to the fructose, wherein the step is catalyzed by at least one enzyme.   
     
     
         11 . The process of  claim 2 , further comprising:
 the step of converting glucose to the G6P, wherein the step is catalyzed by at least one enzyme, and optionally, the step of converting sucrose to the glucose, wherein the step is catalyzed by at least one enzyme.   
     
     
         12 . The process of one of  claim 1 , wherein the epimerase is an allulose 6-phosphate 3-epimerase. 
     
     
         13 . The process of  claim 12 , wherein the allulose 6-phosphate 3-epimerase comprises an amino acid sequence having at 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 with SEQ ID NOs.: 3 or 6, wherein said epimerase catalyzes the conversion of F6P to A6P. 
     
     
         14 . The process of  claim 12 , wherein the allulose 6-phosphate 3-epimerase contains an (α/β) 8 -barrel domain for catalysis, a Ser at the end of the 7th β-strand of the barrel, a Ser at the end of the 8th β-strand of the barrel, a Gly in the active site loop, a His in the 2nd and 3rd β-strands of the barrel, an Asp in the 2nd and 7th β-strand of the barrel, and a His-hydrophobic residue-Asp signature in the 2nd β-strand of the barrel. 
     
     
         15 . The process of  claim 1 , wherein the phosphatase is an allulose 6-phosphate phosphatase. 
     
     
         16 . The process of  claim 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 with SEQ ID NO.: 9, wherein said phosphates catalyzes the conversion of A6P to allulose. 
     
     
         17 . The process of  claim 15 , wherein the allulose 6-phosphate phosphatase is specific for allulose 6-phosphate. 
     
     
         18 . The process of  claim 15 , wherein the allulose 6-phosphate phosphatase contains a Rossmanoid fold domain for catalysis, a Cl capping domain, DxD signature in the 1st β-strand of the Rossmanoid fold, a Thr or Ser at the end of the 2nd β-strand of the Rossmanoid fold, a Lys at the N-terminus of the α-helix C-terminal to the 3rd β-strand of the Rossmanoid fold, and a GDxxxD signature at the end of the 4th β-strand of the Rossmanoid fold. 
     
     
         19 . The process of  claim 1 , wherein the process steps are conducted at a temperature ranging from about 40° C. to about 70° C., at a pH ranging from about 5.0 to about 8.0, and/or for about 8 hours to about 48 hours. 
     
     
         20 . The process of  claim 1 , wherein the process steps are conducted in one bioreactor or in a plurality of bioreactors arranged in series. 
     
     
         21 . The process of  claim 1 , wherein the process steps are conducted ATP-free, NAD(H)-free, at a phosphate concentration from 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 prepared by a process of  claim 1 .