Enzymatic production of D-tagatose

• US 11,034,988 B2
• Filed: 01/07/2020
• Issued: 06/15/2021
• Est. Priority Date: 10/02/2015
• Status: Active Grant

• Current Assignee: Bonumose LLC
• Original Assignee: Bonumose Incorporated
• Inventors: Wichelecki, Daniel Joseph
• Primary Examiner(s): Fronda, Christian L
• Application Number:
  US16/735,961
• Publication Number:
  US 20200199637A1
• Time in Patent Office: 525 Days
• Field of Search: None
• CPC Class Codes: C12N 9/90 Isomerases (5.)
• CPC Class Codes: C12P 19/02 Monosaccharides 2-ketogulonic acid C12P7/60
• CPC Class Codes: C12Y 207/01144 Tagatose-6-phosphate kinase (2.7.1.144)

Abstract

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.

Claims

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.