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 galactose from a saccharide, the process comprising:
converting fructose 6-phosphate (F6P) to tagatose 6-phosphate (T6P) catalyzed by fructose 6-phosphate epimerase (F6PE);
converting the T6P to galactose 6-phosphate (Gal6P) catalyzed by galactose 6-phosphate isomerase (Gal6PI); and
converting the Gal6P to galactose catalyzed by galactose 6-phosphate phosphatase (Gal6PP).
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 a phosphoglucoisomerase (PGI) or PGPMI.
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 a 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 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 isoamylase, pullulanase, 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 Gal6PP involves an energetically favorable chemical reaction.
13 . The process of claim 1 , wherein the Gal6PI comprises an amino acid sequence having at least 25% sequence identity with SEQ ID No: 18 or 19, and wherein the Gal6PI catalyzes the conversion of T6P to Gal6P.
14 . The process of claim 13 , wherein the Gal6PI contains a heterodimer consisting of subunits with Rossmann-like αβα sandwich folds.
15 . The process of claim 1 , wherein the Gal6PP comprises an amino acid sequence having at least 25% sequence identity with SEQ ID No: 20, and wherein said Gal6PP catalyzes the conversion of Gal6P to galactose.
16 . The process of claim 15 , wherein the Gal6PP contains a Rossmanoid fold domain for catalysis, a C2 capping domain, D×D 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, and a GDxxxD signature at the end of the 4th β-strand of the Rossmanoid fold.