# US 11,896,024 B2 · Method of glycation of a polypeptide

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- Number: US 11,896,024 B2
- Document ID: US11896024B2
- Status: Granted
- Kind: B2
- Application number: 17/687,786
- Filed: 2022-03-07
- Published: 2022-07-28
- Granted: 2024-02-13
- Earliest claimed: 2017-05-15
- Assignees: Novozymes A/S
- Applicants: Novozymes A/S
- Inventors: Jeppe Wegener Tams
- Web page: https://patentgrep.com/patent/US11896024B2

## CPC

- A23C 9/1206: {Lactose hydrolysing enzymes, e.g. lactase, beta-galactosidase}
- C12N 9/2471: {Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase}
- A23C 9/1307: {Milk products or derivatives; Fruit or vegetable juices; Sugars, sugar alcohols, sweeteners; Oligosaccharides; Organic acids or salts thereof or acidifying agents; Flavours, dyes or pigments; Inert or aerosol gases; Carbonation methods}
- A23C 9/1512: {containing isolated milk or whey proteins, caseinates or cheese; Enrichment of milk products with milk proteins in isolated or concentrated form, e.g. ultrafiltration retentate}
- A61K 31/702: Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
- C12P 19/04: Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
- C12P 19/18: produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
- C12Y 302/01023: Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
- C12Y 302/01108: Lactase (3.2.1.108)
- A23C: DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; PREPARATION THEREOF (preservation thereof A23B11/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- 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)}
- C12Y: ENZYMES

## Abstract

The present invention relates to compositions, particularly liquid compositions, comprising enzymes, methods of making the compositions, and uses of the same for making, e.g., dairy products.

## Claims

### Claim 1

A method of modifying by glycation a polypeptide having beta-galactosidase activity, comprising:
contacting the polypeptide with 30-90 wt % of a reducing sugar under conditions sufficient to produce a polypeptide modified by glycation,
wherein the reducing sugar is fructose, galactose, glucose, glyceraldehyde, ribose, xylose, cellobiose, maltose, or lactose, wherein the conditions comprise contacting the polypeptide in a solution at pH 5-8, for a time of 3-100 hours, at a temperature of 50-80° C., and wherein the polypeptide has at least 3% of lysine and arginine residues of the polypeptide modified by glycation.

### Claim 2 (depends on 1)

The method of claim 1, wherein the polypeptide has improved transgalactosylating activity as compared to a polypeptide having beta-galactosidase activity which has not been modified by glycation.

### Claim 3 (depends on 1)

The method of claim 1, wherein the polypeptide has at least 5% of lysine and arginine residues of the polypeptide modified by glycation.

### Claim 4 (depends on 1)

The method of claim 1, wherein the polypeptide is contacted with 40-65 wt. % of a reducing sugar.

### Claim 5 (depends on 1)

The method of claim 1, wherein the polypeptide is from subfamily 5 of glycosyl hydrolase family 2 (GH2_5).

### Claim 6 (depends on 1)

The method of claim 1, wherein the polypeptide is from subfamily 6 of glycosyl hydrolase family 2 (GH2_6).

### Claim 7 (depends on 1)

The method of claim 1, wherein the polypeptide is a Bacillus polypeptide.

### Claim 8 (depends on 1)

The method of claim 1, wherein the polypeptide is a Bacillus circulans polypeptide.

### Claim 9 (depends on 1)

The method of claim 1, wherein the polypeptide is a Bifidobacterium polypeptide.

### Claim 10 (depends on 1)

The method of claim 1, wherein the polypeptide is a Bifidobacterium bifidum polypeptide.

### Claim 11 (depends on 1)

The method of claim 1, wherein the polypeptide is a Kluyvermyces polypeptide.

### Claim 12 (depends on 1)

The method of claim 1, wherein the polypeptide is a Kluyvermyces lactis polypeptide.