# US 11,304,425 B2 · Glycosylated beta-galactosidase compositions having improved transgalactosylating activity

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- Number: US 11,304,425 B2
- Document ID: US11304425B2
- Status: Granted
- Kind: B2
- Application number: 16/609,276
- Filed: 2018-05-15
- Published: 2020-07-23
- Granted: 2022-04-19
- Earliest claimed: 2017-05-15
- Assignees: Novozymes A/S
- Applicants: Novozymes A/S
- Inventors: Jeppe Wegener Tams
- Web page: https://patentgrep.com/patent/US11304425B2

## 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 polypeptides having beta-galactosidase activity, methods of making said compositions, and uses of the compositions for making e.g. dairy products. The polypeptides having beta-galactosidase activity are modified by glycation of lysine and/or arginine residues by incubating the enzyme in the presence of reducing sugars, optionally combined with a heat treatment. Thereby, transgalactosylating activity is increased.

## Claims

### Claim 1

A formulation comprising:
(a) 30-90 wt. % of a reducing sugar, wherein the reducing sugar is fructose, galactose, glucose, glyceraldehyde, ribose, xylose, cellobiose, maltose, or lactose, and
(b) a polypeptide of glycoside hydrolase family 2 and enzyme class EC 3.2.1.23 or 3.2.1.108 comprising at least one lysine and/or arginine residue which is glycated by the reducing sugar, wherein the polypeptide has beta-galactosidase activity and transgalactosylating activity.

### Claim 2 (depends on 1)

The formulation of claim 1, wherein the reducing sugar is fructose, galactose, glucose, glyceraldehyde, ribose, or xylose.

### Claim 3 (depends on 2)

The formulation of claim 2, wherein the reducing sugar is galactose.

### Claim 4 (depends on 2)

The formulation of claim 2, wherein the reducing sugar is fructose.

### Claim 5 (depends on 2)

The formulation of claim 2, wherein the reducing sugar is glucose.

### Claim 6 (depends on 1)

The formulation of claim 1, wherein the reducing sugar is cellobiose or maltose.

### Claim 7 (depends on 1)

The formulation of claim 1, wherein the reducing sugar is lactose.

### Claim 8 (depends on 1)

The formulation of claim 1, wherein at least 1% of the lysine and arginine residues of the polypeptide are glycated by the reducing sugar.

### Claim 9 (depends on 1)

The formulation of claim 1, wherein at least 3% of the lysine and arginine residues of the polypeptide are glycated by the reducing sugar.

### Claim 10 (depends on 1)

The formulation of claim 1, wherein at least 5% of the lysine and arginine residues of the polypeptide are glycated by the reducing sugar.

### Claim 11 (depends on 1)

The formulation of claim 1, wherein at least 10% of the lysine and arginine residues of the polypeptide are glycated by the reducing sugar.

### Claim 12 (depends on 1)

The formulation of claim 1, wherein at least 20% of the lysine and arginine residues of the polypeptide are glycated by the reducing sugar.

### Claim 13 (depends on 1)

The formulation of claim 1, which comprises 40-90 wt. % of the reducing sugar.

### Claim 14 (depends on 1)

The formulation of claim 1, which comprises 40-65 wt. % of the reducing sugar.

### Claim 15 (depends on 1)

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

### Claim 16 (depends on 1)

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

### Claim 17 (depends on 1)

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

### Claim 18 (depends on 17)

The formulation of claim 17, wherein the polypeptide is a Bacillus circulans polypeptide.

### Claim 19 (depends on 1)

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

### Claim 20 (depends on 19)

The formulation of claim 19, wherein the polypeptide is a Bifidobacterium bifidum polypeptide.

### Claim 21 (depends on 1)

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

### Claim 22 (depends on 21)

The formulation of claim 21, wherein the polypeptide is a Kluyvermyces lactis polypeptide.

### Claim 23 (depends on 1)

The formulation of claim 1, wherein the polypeptide has at least 500 amino acid residues.

### Claim 24 (depends on 1)

The formulation of claim 1, wherein the polypeptide has at least 750 amino acid residues.

### Claim 25 (depends on 1)

The formulation of claim 1, wherein the polypeptide has at least 1000 amino acid residues.

### Claim 26 (depends on 1)

The formulation of claim 1, wherein the polypeptide has at least 1250 amino acid residues.

### Claim 27 (depends on 1)

The formulation of claim 1, which has an activity of 200-20,000 LAU(C)/g.

### Claim 28 (depends on 1)

The formulation of claim 1, which is a liquid formulation and has an activity of 200-15,000 LAU(C)/g.

### Claim 29 (depends on 1)

The formulation of claim 1, which is a solid formulation and has an activity of 1,000-20,000 LAU(C)/g.

### Claim 30 (depends on 1)

The formulation of claim 1, which is substantially free of glycerol.

### Claim 31 (depends on 1)

The formulation of claim 1, which further comprises sodium chloride or potassium chloride in an amount of 0.01-5 wt. %.

### Claim 32 (depends on 1)

A method for producing galacto-oligosaccharides (GOS) comprising contacting the formulation of claim 1 with lactose at conditions to produce the galacto-oligosaccharides, wherein the conditions are a pH of 5-8, a temperature of 50-80° C., and a time of 3-100 hours.

### Claim 33 (depends on 32)

The method of claim 32, wherein the reducing sugar is fructose, glucose, galactose, or lactose.

### Claim 34 (depends on 32)

The method of claim 32, wherein the conditions are a pH of 6-7, a temperature of 50-70° C., and a time of 15-80 hours.

### Claim 35 (depends on 1)

The formulation of claim 1, wherein the polypeptide has a ratio of transgalactosylating activity to beta-galactosidase activity of at least 1:1.