# US 7,078,194 B2 · Methods and compositions for synthesis of oligosaccharides using mutant glycosidase enzymes

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- Number: US 7,078,194 B2
- Document ID: US7078194B2
- Status: Granted
- Kind: B2
- Application number: 09/837,711
- Filed: 2001-04-17
- Published: 2003-05-29
- Granted: 2006-07-18
- Earliest claimed: 1995-12-12
- Assignees: The University of British Columbia
- Inventors: Stephen G. Withers; Lloyd MacKenzie; Qingping Wang
- Web page: https://patentgrep.com/patent/US7078194B2

## CPC

- C12P 19/04: Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
- C12N 9/2408: {acting on alpha -1,4-glucosidic bonds}
- C12N 9/2414: {Alpha-amylase (3.2.1.1.)}
- C12N 9/2445: {Beta-glucosidase (3.2.1.21)}
- C12Y 302/01021: Beta-glucosidase (3.2.1.21)
- C07K 2319/02: containing a signal sequence
- 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)
- C12Y: ENZYMES
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

Mutant glycosidase enzymes are formed in which the normal nucleophilic amino acid within the active site has been changed to a nonnucleophilic amino acid. These enzymes cannot hydrolyze disaccharide products, but which can still form them. Using this enzyme, oligosaccharides are synthesized by preparing a mixture of an &agr;glycosyl fluoride and a glycoside acceptor molecule; enzymatically coupling the &agr.glycosyl fluoride to the glycoside acceptor molecule to form a glycosyl glycoside product using the mutant glycosidase enzyme; and recovering the glycosyl glycoside product. Particular enzymes include a mutant form of Agrobacterium &bgr.Glucosidase in which the normal glutamic acid residue at position 358 is replaced with an alanine residue.

## Claims

### Claim 1

A method for synthesizing a glycoside product,
(a) selecting a glycoside product which is a substrate for a wild-type glycosidase enzyme having two catalytically active amino acids with carboxylic acid side chains within the active site;
(b) combining in a reaction mixture
(i) a mutant form of the glycosidase enzyme, said mutant enzyme having one of said catalytically active amino acids of the glycosidase enzyme substituted with a different amino acid of comparable or smaller size, said different amino acid having a non-carboxylic acid side chain, wherein said mutant glycosidase does not catalyze the hydrolysis of the glycoside product and retains the activity to synthesize the glycoside product; and
(ii) a glycosyl donor molecule comprising a sugar linked to a leaving group and a glycoside acceptor molecule, said glycosyl donor molecule having a β configuration and said glycoside acceptor molecule having an α configuration, or vice versa, wherein said glycoside acceptor molecule and said glycosyl donor molecule are selected so as to form a glycosidic bond which is hydrolyzed by the wild-type glycosidase enzyme; and
(c) allowing the mutant glycosidase enzyme to enzymatically couple the glycosyl donor molecule and glycoside acceptor molecule to synthesize the glycoside product.

### Claim 2 (depends on 1)

The method of claim 1, wherein the wild-type glycosidase enzyme is a stereochemistry retaining enzyme in which the carboxylic acid side chain of one of said catalytically active amino acids in the wild-type glycosidase enzyme active site functions as an acid/base catalyst and the carboxylic acid side chain of the other catalytically active amino acid functions as a nucleophile, and wherein the amino acid having the nucleophile carboxylic acid side chain is replaced in the mutant enzyme.

### Claim 3 (depends on 2)

The method of claim 2, wherein the wild-type glycosidase enzyme is a β-glycosidase.

### Claim 4 (depends on 3)

The method of claim 3, wherein the glycosyl donor molecule is α-glycosyl fluoride.

### Claim 5 (depends on 3)

The method of claim 3, wherein the α-glycosyl fluoride is α-glucosyl fluoride.

### Claim 6 (depends on 3)

The method of claim 3, wherein the α-glycosyl fluoride is a α-galactosyl fluoride.

### Claim 7 (depends on 1)

The method of claim 1, wherein the wild-type glycosidase enzyme is a β-glycosidase.

### Claim 8 (depends on 1)

The method of claim 1, wherein the enzyme is a β-glucosidase.

### Claim 9 (depends on 1)

The method of claim 1, wherein the acceptor molecule is an aryl-glycoside.

### Claim 10 (depends on 9)

The method of claim 9, wherein the acceptor molecule is a nitrophenyl-glycoside.

### Claim 11 (depends on 1)

The method of claim 1, wherein the wild-type glycosidase enzyme is a stereochemistry inverting enzyme in which the carboxylic acid side chains of one of said catalytically active amino acids in the active site of the wild-type glycosidase enzyme functions as an acid catalyst and the other carboxylic acid side chain of the other catalytically active amino acid functions as a base catalyst, and wherein the amino acid having the carboxylic acid side chain which functions as a base catalyst is replaced in the mutant enzyme.

### Claim 12 (depends on 1)

The method of claim 1, wherein the wild-type glycosidase enzyme is selected from the group consisting of β-glucosidases, β-galactosidases, β-mannosidases, β-N-acetyl glucosaminidases, β-N acetyl galactosaminidases, β-xylosidases, β-fucosidases, cellulases, xylanases, galactanases, mannanases, hemicellulases, amylases, glucoamylases, α-glucosidases, α-galactosidases, α-mannosidases, α-N-acetyl glucosaminidases, α-N acetyl galactosaminidases, α-xylosidases, α-fucosidases, and neuraminidases/sialidases.

### Claim 13 (depends on 1)

The method of claim 1, wherein the glycoside acceptor molecule is selected from the group consisting of a monosaccharide, an oligosaccharide, and a sugar containing molecule.