Enzymatic cascade bioreactor
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 system for converting sucrose to, β-D-glucose, comprising:
a reservoir comprising a solution of sucrose;
pumping means in fluid communication with said reservoir;
a first porous packed column comprising invertase, said first porous packed column-in fluid communication with said pumping means;
a second porous packed column comprising glucose isomerase, said second porous packed column in fluid communication with said first porous packed column;
a third porous packed column comprising mutarotase, said third porous packed column in fluid communication with said second porous packed column; and
means for heating said first, second and third porous packed columns.
2. The system of claim 1 , wherein the second porous packed column further comprises an Mg + stabilizer.
3. The system of claim 2 , wherein the Mg + stabilizer comprises MgCl 2 .
4. The system of claim 3 , wherein MgCl 2 is present in an concentration of about 20 mM.
5. The system of claim 1 , wherein:
said first, second and third porous packed columns comprise inlet and outlet ends, wherein said first porous packed column inlet end is in fluid communication with said pumping means, said second porous packed column inlet end is in fluid communication with said first porous packed column outlet end; and said third porous packed column inlet end is in fluid communication with said second porous packed column outlet end.
6. The system of claim 1 , wherein said pumping means is a positive displacement or a dynamic pump.
7. The system of claim 6 , wherein said pumping means is selected from the group consisting of peristaltic pumps, reciprocating piston pumps, rotary gear pumps, helical screw pumps, rotary lobe pumps, gear-within-gear pumps, diaphragm pumps, progressing cavity pumps, and a vane pumps.
8. The system of claim 1 , wherein the means for heating comprises electrically heating each of said first, second, and third porous packed columns.
9. The system of claim 8 , wherein electrically heating said porous packed columns comprises wrapping the porous packed columns with heating tape.
10. The system of claim 1 , wherein the means for heating comprises heating the porous packed columns by contacting said packed columns with a heated jacket.
11. The system of claim 10 , wherein the heated jacket contains a circulating heated liquid.
12. The system of claims 8 or 10 , wherein the porous packed columns are maintained at a temperature between about 43° C. to about 47° C.
13. The system of claim 1 , wherein each of the first, second, and third porous packed columns comprise a glass chromatography column filled with a plurality of silica beads.
14. The system of claim 13 , wherein the silica beads comprise a native surface having a plurality of aminated structures attached thereto.
15. The system of claim 14 , 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.
16. The system of claim 15 , wherein the amine functional group is selected from the list consisting of aminopropyltriethoxysilane, aminopropyltrimethoxysilane, aminopropylmethyldiethoxysilane; aminopropylmethyldimethoxysilane; aminoethylaminopropyltriethoxysilane; aminoethylaminopropyltrimethoxysilane; aminoethylaminopropylmethyldimethoxysilane; diethylenetriaminopropyltriethoxysilane; diethylenetriaminopropyltrimethoxysilane; diethylenetriaminopropylmethyldiethoxysilane; diethylenetriaminopropylmethyldimethoxysilane; cyclohexylaminopropyltrimethoxysilane; haxanedoaminomethyltriethoxysilane; phenylaminomethyltriethoxysilane; phenylaminomethyltrimethoxysilane; diethylaminomethyltriethoxysilane; (dimethylaminomethyl)methyldimethoxysilane; and methylaminopropyltrimethoxysilane.
17. The system of claim 15 , wherein the plurality of amine functional groups comprise an amino alkyl alkyloxy silane.
18. The system of claim 17 , wherein the plurality of amine functional groups comprise 3-aminopropyltriethoxysilane.
19. The system of claim 15 , 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.
20. The system of claim 19 , wherein the amine group cross-linker comprises a mono- or di-aldehyde.
21. The system of claim 20 , wherein the amine group cross-linker is glutaraldehyde.
22. The system of claim 1 , wherein the solution of sucrose comprises a buffer fluid having a pH between about 4.6 and about 5.8.