Method for converting sucrose to β-D-glucose
View Patent ↗Disclosed is an apparatus and method for continuously converting sucrose to β-D-glucose. The method comprises a three-stage enzymatic reactor in which an aqueous solution of sucrose is first converted into a solution of fructose and α-D-glucose by passing it through a porous, packed column containing an inert media on which invertase is immobilized. This solution is then sent through a second packed column containing glucose isomerase and finally a third packed column containing mutarotase. Solution temperature and pH are adjusted to maximize glucose output.
1. A method for converting sucrose to β-D-glucose, comprising the steps of:
a) providing a sucrose containing solution having a pH of between about 5.8 and about 4.6;
b) heating the sucrose containing solution to a temperature of about 43° C. to about 47° C.;
c) contacting the sucrose containing solution with invertase, wherein the conditions of pH and temperature are maintained, to provide a fructose and α-D-glucose containing solution;
d) contacting the fructose and α-D-glucose containing solution with glucose isomerase, wherein the conditions of pH and temperature are maintained, to provide a solution containing α-D-glucose and β-D-glucose; and
e) contacting the α-D-glucose and β-D-glucose with mutarotase, wherein the conditions of pH and temperature are maintained, to provide a solution containing a high percentage of β-D-glucose.
2. The method of claim 1 , wherein the step of contacting the fructose and α-D-glucose containing solution with glucose isomerase, further comprises stabilizing the glucose isomerase with a Mg + stabilizer.
3. The method of claim 2 , wherein the Mg + stabilizer comprises MgCl 2 .
4. The method of claim 3 , wherein MgCl 2 is present in a concentration of about 20 mM.
5. The method of claim 1 , wherein said invertase, said glucose isomerase, and said mutarotase are each contained on separate solid substrates having a plurality of aminated structures attached thereto.
6. The method of claim 5 , wherein the separate solid substrates comprise a plurality of silica beads.
7. The method of claim 6 , wherein each of the plurality of aminated structures comprises an amine functional group attached to said native surface of the silica beads, and an amine group cross-linker attached to each of said amine functional groups, wherein said amine group cross-linker provides a site for protein reaction.
8. The method of claim 7 , wherein the amine functional group is selected from the list consisting of aminopropyltriethoxysilane, aminopropyltrimethoxysilane, aminopropylmethyldiethoxysilane; aminopropylmethyldimethoxysilane; aminoethylaminopropyltriethoxysilane; aminoethylaminopropyltrimethoxysilane; aminoethylaminopropylmethyldimethoxysilane; diethylenetriaminopropyltriethoxysilane; diethylenetriaminopropyltrimethoxysilane; diethylenetriaminopropylmethyldiethoxysilane; diethylenetriaminopropylmethyldimethoxysilane; cyclohexylaminopropyltrimethoxysilane; hexanediaminomethyltriethoxysilane; phenylaminomethyltriethoxysilane; phenylaminomethyltrimethoxysilane; diethylaminomethyltriethoxysilane; (dimethylaminomethyl)methyldimethoxysilane; and methylaminopropylltimethoxysilane.
9. The method of claim 7 , wherein the plurality of amine functional groups comprise an amino alkyl alkyloxy silane.
10. The method of claim 9 , wherein the plurality of amine functional groups comprise 3-aminopropyltriethoxysilane.
11. The method of claim 7 , wherein the amine group cross-linker is selected from the group consisting of formaldehyde, glutaraldehyde, mono- and di-aldehyde PEG, and bis(imido esters), bis(succinimidyl esters), diisocyanates and diacid chlorides.
12. The method of claim 11 , wherein the amine group cross-linker comprises a mono- or di-aldehyde.
13. The method of claim 12 , wherein the amine group cross-linker is glutaraldehyde.