# US 10,480,018 B2 · Genus of epimerase enzymes for conversion of fructose to allulose at high temperature and low pH

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- Number: US 10,480,018 B2
- Document ID: US10480018B2
- Status: Granted
- Kind: B2
- Application number: 15/575,636
- Filed: 2016-05-20
- Published: 2018-04-26
- Granted: 2019-11-19
- Earliest claimed: 2015-05-22
- Assignees: Archer Daniels Midland Company
- Applicants: Archer Daniels Midland Company
- Inventors: Padmesh Venkitasubramanian; William Schroeder; Mark Welch; Sridhar Govindarajan
- Web page: https://patentgrep.com/patent/US10480018B2

## CPC

- C12P 19/24: produced by the action of an isomerase, e.g. fructose
- C12N 9/90: Isomerases (5.)
- C12P 19/02: Monosaccharides (2-ketogulonic acid C12P7/60)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Disclosed herein are three geneses of proteins herein established to exhibit a fructose to allulose epimerase activity that are useful for production of allulose from fructose at high temperatures and at low pH in the range of 4.5 to 6.0. Two of the three geneses descend phylogenetically from a common ancestral protein defined herein, and these geneses are distinguished from each other by different parental descendant proteins also defined herein. The proteins with high levels of sequence identity to the parental nodes defining from these two geneses generally exhibit higher levels of specific fructose to glucose epimerase activity than prior known fructose to allulose epimerases and exhibit such activity at low pH. A third genus is not defined by phylogenetic origin except by not descending from the same ancestor as the first two geneses but generally exhibit similar levels of fructose to allulose epimerase activities as prior art epimerases described to be useful for fructose to allulose conversion.

## Claims

### Claim 1

A method of producing allulose comprising:
contacting a solution containing fructose with an enzyme having epimerase activity, the enzyme having a sequence selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 18, SEQ ID NO: 16, and SEQ ID NO: 14 for a time and under conditions suitable to convert at least a portion of the fructose to allulose;
wherein when placed in contact with fructose at a temperature of 50° C. for 2 hours at a pH of 6, the enzyme converts the fructose to the allulose.

### Claim 2 (depends on 1)

The method of claim 1 wherein said enzyme is obtained from a microorganism containing a recombinant nucleic acid vector operably configured with a promoter to express a nucleic acid sequence encoding the enzyme, wherein said promoter is non-native to the nucleic acid encoding said enzyme.

### Claim 3 (depends on 2)

The method of claim 2 wherein said microorganism is selected from the group consisting of Bacillus licheniformis, Saccharomyces cerevisiae, Schizosaccharomyces ombe, Pseudomonas putida, Pichia sp. Aspergillus sp., Trichoderma reesei, Corynebacterium glutamicum, E. coli and B. subtilis.

### Claim 4 (depends on 3)

The method of claim 3 wherein said microorganism is selected from the group consisting of E. coli and B. subtilis.