Enzymatic production of D-tagatose
The current disclosure provides a process for enzymatically converting a saccharide into tagatose. The invention also relates to a process for preparing tagatose where the process involves converting fructose 6-phosphate (F6P) to tagatose 6-phosphate (T6P), catalyzed by an epimerase, and converting the T6P to tagatose, catalyzed by a phosphatase.
1. An enzymatic process for preparing tagatose from sucrose, the process comprising:
(i) converting sucrose to glucose 1-phosphate (G1P) catalyzed by a sucrose phosphorylase (SP);
(ii) converting G1P to glucose 6-phosphate (G6P) catalyzed by a phosphoglucomutase (PGM);
(iii) converting G6P to fructose 6-phosphate (F6P) catalyzed by a phosphoglucoisomerase (PGI);
(iv) converting fructose 6-phosphate (F6P) to tagatose 6-phosphate (T6P) catalyzed by fructose 6-phosphate epimerase (F6PE); and
(v) converting the T6P produced to tagatose catalyzed by a tagatose 6-phospate phosphatase (T6PP).
2. The process of claim 1 , further comprising converting fructose, generated by the phosphorolytic cleavage of sucrose by SP, to F6P catalyzed by a polyphosphate fructokinase (PPFK).
3. The process of claim 1 , wherein the F6PE is encoded by a polynucleotide comprising a nucleotide sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with SEQ ID NOS. 1, 3, 5, 7, 9, or 10.
4. The process of claim 1 , wherein the F6PE comprises an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with SEQ ID NOS. 2, 4, 6, 8, or 11.
5. The process of claim 1 , wherein the T6PP is encoded by a polynucleotide comprising a nucleotide sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with SEQ ID NOS.: 12, 14, or 16.
6. The process of claim 1 , wherein the T6PP comprises an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with SEQ ID NOS.: 13, 15, or 17.
7. The process of claim 1 , wherein at the process steps are conducted under one or more of the following reaction conditions:
a temperature ranging from about 37° C. to about 85° C.;
a pH ranging from about 5.0 to about 8.0; and
for about 8 hours to about 48 hours.
8. The process of claim 1 , wherein the process steps are conducted in one bioreactor or in a plurality of bioreactors arranged in series.
9. The process of claim 1 , wherein the process steps are conducted under one or more of the following reaction conditions:
ATP-free;
NAD(H)-free;
at a phosphate concentration from about 0 mM to about 150 mM, wherein the phosphate is recycled; and
at least one step of the process involves an energetically favorable chemical reaction.