Enzymatic transphosphorylation of sugar substrates
The present invention relates to methods of producing sugar phosphates by enzymatic transphosphorylation, wherein the phosphoryl transfer from phosphate donor substrates to sugar substrates is catalyzed by an enzyme having alpha-glucose-1-phosphatase activity. Furthermore, the present invention relates to a biocatalyst having sucrose phosphorylase as well as alpha-glucose-1-phosphatase activity and its use. The production of an enzyme having alpha-glucose-1-phosphatase activity and its use in the transphosphorylation of sugar substrates is also presented herein.
1. A method of producing one or more sugar-1-phosphates by enzymatic transphosphorylation comprising:
(i) providing one or more sugar substrates selected from the group consisting of aldoses and ketoses,
(ii) providing one or more phosphate donor substrates selected from the group consisting of phosphorylated carbohydrates,
(iii) providing an alpha-glucose-1-phosphatase or an enzyme having alpha-glucose-1-phosphatase activity,
(iv) incubating the one or more sugar substrates provided in step (i) and the one or more phosphate donor substrates provided in step (ii) with the alpha-glucose-1-phosphatase or, respectively, the enzyme having alpha-glucose-1-phosphatase activity provided in step (iii) under conditions which allow transphosphorylation to obtain one or more sugar-1-phosphates,
(v) optionally, purifying the one or more sugar-1-phosphates obtained in step (iv).
2. The method according to claim 1 , wherein the one or more sugar substrate is selected from the group consisting of glucose, fructose, galactose, fucose, mannose, sorbose, xylose, arabinose, and rhamnose.
3. The method according to claim 1 , wherein the one or more phosphate donor substrate is selected from the group consisting of alpha-glucose-1-phosphate, glucose-6-phosphate, fructose-6-phosphate and fructose-1-phosphate.
4. The method according to claim 1 , wherein the phosphate donor substrate or one of the phosphate donor substrates provided in step (ii) is alpha-glucose-1-phosphate.
5. The method according to claim 4 wherein step (ii) comprises an enzymatic conversion of sucrose to D-fructose and alpha-D-glucose-1-phosphate by a sucrose phosphorylase or an enzyme having sucrose phosphorylase activity in the presence of phosphate or one or more sources of phosphate.
6. The method according to claim 5 , wherein step (ii) and step (iv) and, optionally, step (iii) are carried out simultaneously.
7. The method according to claim 5 , wherein the method is performed in the presence of a biocatalyst, having sucrose phosphorylase activity and alpha-glucose-1-phosphatase activity and/or comprising a sucrose phosphorylase and an alpha-glucose-1-phosphatase.
8. The method according to claim 1 , wherein step (iii) comprises the expression of an alpha-glucose-1-phosphatase or an enzyme having alpha-glucose-1-phosphatase activity by a host cell.
9. The method according to claim 8 , wherein the alpha-glucose-1-phosphatase or the enzyme having alpha-glucose-1-phosphatase activity, respectively, is expressed as a recombinant fusion protein comprising a tag suitable for isolation of the alpha-glucose-1-phosphatase or the enzyme having alpha-glucose-1-phosphatase activity.