Enzymatic production of D-tagatose

• US 10,138,506 B2
• Filed: 09/30/2016
• Issued: 11/27/2018
• Est. Priority Date: 10/02/2015
• Status: Active Grant

• Current Assignee: Bonumose Incorporated
• Original Assignee: Bonumose LLC
• Inventors: Wichelecki, Daniel Joseph
• Primary Examiner(s): Fronda, Christian L
• Application Number:
  US15/743,481
• Publication Number:
  US 20180216146A1
• Time in Patent Office: 788 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 a starch derivative, the process comprising the steps of:

(i) converting a saccharide to glucose 1-phosphate (G1P) using alpha glucan phosphorylase (α

GP), wherein the saccharide is selected from one or more derivatives of starch;

(ii) converting G1P to glucose 6-phosphate (G6P) using a phosphoglucomutase (PGM);

(iii) converting G6P to fructose 6-phosphate (F6P) using a phosphoglucoisomerase (PGI);

(iv) converting fructose 6-phosphate (F6P) to tagatose 6-phosphate (T6P) catalyzed by a fructose 6-phosphate epimerase (F6PE); and

(v) converting the T6P produced to tagatose catalyzed by a tagatose 6-phosphate phosphatase (T6PP)

wherein steps (i)-(v) are conducted in a single reaction vessel.

2. The process of claim 1, further comprising the step of converting starch to a starch derivative wherein the starch derivative is prepared by enzymatic hydrolysis of starch or by acid hydrolysis of starch, wherein the enzymatic hydrolysis of starch is catalyzed by isoamylase, pullulanase, alpha-amylase, or a combination thereof.

3. The process of claim 1, wherein 4-glucan transferase (4GT) is added to the process.

4. 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.

5. 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.

6. 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.

7. 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.

8. The process of claim 1, wherein the process steps are conducted under at least one of the following process conditions:

at a temperature ranging from about 37°

C. to about 85°

C.,

at a pH ranging from about 5.0 to about 8.0, or

for about 8 hours to about 48 hours.

9. The process of claim 1, wherein the process steps are conducted under at least one of the following process conditions:

without adenosine triphosphate (ATP) as a source of phosphate,

without nicotinamide adenosine dinucleotide,

at a phosphate concentration from about 0.1 mM to about 150 mM,

where phosphate is recycled, and

where step (v) involves an energetically favorable chemical reaction.

10. The process of claim 9, wherein phosphate is recycled, wherein phosphate ions produced by T6PP dephosphorylation of T6P are used in the process step of converting a saccharide to G1P.

11. The process of claim 9, wherein the step of converting T6P to tagatose is an energetically favorable, irreversible phosphatase reaction.

12. The process of claim 1, further comprising the step of separating tagatose, wherein the separation is not via chromatography.

13. The process of claim 1, wherein the derivatives of starch are selected from the group consisting of amylose, amylopectin, soluble starch, amylodextrin, maltotriose, maltodextrin, maltose, and glucose.

14. 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, and 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.

15. The process of claim 13, 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, and 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.