Enzymatic production of hexoses
Disclosed herein are methods of producing hexoses from saccharides by enzymatic processes. The methods utilize fructose 6-phosphate and at least one enzymatic step to convert it to a hexose.
1. A process for preparing mannose from a saccharide, the process comprising:
converting fructose 6-phosphate (F6P) to mannose 6-phosphate (M6P) catalyzed by mannose 6-phosphate isomerase (M6PI) or a phosphoglucose/phosphomannose isomerase (PGPMI); and
converting the M6P to mannose catalyzed by a mannose 6-phosphate phosphatase (M6PP).
2. The process of claim 1 , further comprising a step of converting glucose 6-phosphate (G6P) to the F6P catalyzed by phosphoglucoisomerase (PGI) or PGPMI.
3. The process of claim 2 , further comprising the step of converting glucose 1-phosphate (G1P) to the G6P catalyzed by phosphoglucomutase (PGM).
4. The process of claim 3 , further comprising the step of converting a saccharide to the G1P using catalyzed by at least one enzyme, wherein the saccharide is starch, a starch derivative, or sucrose.
5. The process of claim 4 , wherein the at least one enzyme in the step of converting a saccharide to the G1P is selected from the group consisting of an alpha-glucan phosphorylase (αGP), a sucrose phosphorylase, and mixtures thereof.
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, and mixtures thereof.
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 4 , wherein the starch derivative is prepared by enzymatic hydrolysis of starch catalyzed by an isoamylase, a pullulanase, an alpha-amylase, or a combination thereof.
10. The process of claim 1 , wherein the process steps are conducted at a temperature ranging from about 37° C. to about 95° C., at a pH ranging from about 5.0 to about 9.0, and/or for about 0.5 hours to about 48 hours.
11. The process of claim 1 , wherein the process steps are conducted in a single bioreactor.
12. The process of claim 1 , wherein the process steps are conducted ATP-free, NAD(P)(H)-free, at a phosphate concentration from about 0.1 mM to about 150 mM, the phosphate is recycled, and/or the catalysis by the hexose-specific phosphatase involves an energetically favorable chemical reaction.
13. The process of claim 1 , wherein the M6PI comprises an amino acid sequence having at least 25% sequence identity with any one of SEQ ID Nos: 8-11, and wherein said M6PI catalyzes the conversion of F6P to M6P.
14. The process of claim 13 , wherein the M6PI contains two domains with a core of antiparallel B-strands resembling the cupin fold and a third domain consisting of only a-helixes, and a divalent metal cation.
15. The process of claim 1 , wherein the PGPMI comprises an amino acid sequence having at least 25% sequence identity with any one of SEQ ID Nos: 15-17, and wherein said PGPMI catalyzes the conversion of F6P to M6P.
16. The process of claim 15 , wherein the PGPMI contains two Rossmanoid folds, a GGS motif, a SYSG-X-T-X-ET-hydrophobic motif, an EN signature where the Glu is present for active-site base proton transfer, and an HN signature where the HIS is present for ring opening/closure of the substrate during catalysis.
17. The process of claim 1 , wherein the M6PP comprises an amino acid sequence having at least 30% sequence identity with any one of SEQ ID Nos: 12-14, and wherein the phosphatase catalyzes the conversion of M6P to mannose.
18. The process of claim 17 , wherein the M6PP contains a Rossmanoid fold domain for catalysis, a C1 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.