# US 2008/0187970 A1 · Beta-Fructofuranosidase Variants

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- Number: US 2008/0187970 A1
- Document ID: US20080187970A1
- Status: Published application
- Kind: A1
- Application number: 10/591,751
- Filed: 2005-03-04
- Published: 2008-08-07
- Earliest claimed: 2004-03-04
- Assignees: Meiji Seika Kaisha, Ltd.
- Inventors: Hirofumi Nakamura; Akitaka Nakane; Hidetoshi Kubota
- Web page: https://patentgrep.com/patent/US20080187970A1

## CPC

- C12P 19/18: produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
- C12N 9/2408: {acting on alpha -1,4-glucosidic bonds}
- C12Y 302/01026: Beta-fructofuranosidase (3.2.1.26), i.e. invertase
- 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

## Abstract

An object of the present invention is to provide a β-fructofuranosidase variant whose reaction specificity is improved to be suitable for the production of fructooligosaccharides. According to the present invention, there is provided a β-fructofuranosidase variant consisting of a mutated amino acid sequence of SEQ ID NO: 2 or a mutated homologue thereof, which has mutations at specific amino acid residues.

## Claims

### Claim 1

A β-fructofuranosidase variant consisting of the following amino acid sequence:
(a) a mutated amino acid sequence of SEQ ID NO: 2, which has at least one mutation in amino acid residues at positions 62, 122, 128, 165, 221, 395, and 550, or
(b) a mutated homologue of the amino acid sequence of SEQ ID NO: 2, which has at least one mutation in amino acid residues corresponding to the amino acid residues at positions 62, 122, 128, 165, 221, 395, and 550 of SEQ ID NO: 2.

### Claim 2 (depends on 1)

The variant according to claim 1, wherein the mutation is a substitution.

### Claim 3 (depends on 2)

The variant according to claim 2, wherein the substitution is
a substitution of the amino acid residue at position 62 with an acidic amino acid selected from the group consisting of aspartic acid and glutamic acid;
a substitution of the amino acid residue at position 122 with an amino acid selected from the group consisting of methionine, isoleucine, leucine, and valine;
a substitution of the amino acid residue at position 128 with an amino acid selected from the group consisting of asparagine and glutamine;
a substitution of the amino acid residue at position 165 with an aromatic amino acid selected from the group consisting of tryptophan, phenylalanine, and tyrosine;
a substitution of the amino acid residue at position 221 with an aromatic amino acid selected from the group consisting of tryptophan, phenylalanine, and tyrosine;
a substitution of the amino acid residue at position 395 with an amino acid selected from the group consisting of leucine, methionine, isoleucine, and valine; or
a substitution of the amino acid residue at position 550 with a hydroxy amino acid selected from the group consisting of serine and threonine.

### Claim 4 (depends on 1, 2)

The variant according to claim 1, 2, or 3, which further has a mutation in at least one amino acid residue at positions 170, 300, 313, and 386 of the amino acid sequence represented by SEQ ID NO: 2 and its homologue.

### Claim 5 (depends on 4)

The variant according to claim 4, wherein the mutation is a substitution.

### Claim 6 (depends on 5)

The variant according to claim 5, wherein the substitution is
a substitution of the amino acid residue at position 170 with an aromatic amino acid selected from the group consisting of tryptophan, phenylalanine, and tyrosine;
a substitution of the amino acid residue at position 300 with an amino acid selected from the group consisting of tryptophan, phenylalanine, tyrosine, and valine;
a substitution of the amino acid residue at position 313 with a basic amino acid selected from the group consisting of lysine, arginine, and histidine; or
a substitution of the amino acid residue at position 386 with a basic amino acid selected from the group consisting of lysine, arginine, and histidine.

### Claim 7 (depends on 4)

The variant according to claim 4, which has mutations in the amino acid residues at positions 165, 300 and 313.

### Claim 8 (depends on 7)

The variant according to claim 7, wherein the mutations are substitutions.

### Claim 9 (depends on 8)

The variant according to claim 8, wherein the substitutions are
a substitution of the amino acid residue at position 165 with an aromatic amino acid selected from the group consisting of tryptophan, phenylalanine, and tyrosine;
a substitution of the amino acid residue at position 300 with an amino acid selected from the group consisting of tryptophan, phenylalanine, tyrosine and valine; and
a substitution of the amino acid residue at position 313 with a basic amino acid selected from the group consisting of lysine, arginine, and histidine.

### Claim 10

A β-fructofuranosidase variant consisting of the following amino acid sequence:
(c) a mutated amino acid sequence of SEQ ID NO: 2, which has at least one mutation in amino acid residues at positions 40, 379, and 381, or
(d) a mutated homologue of the amino acid sequence of SEQ ID NO: 2, which has at least one mutation in amino acid residues corresponding to the amino acid residues at positions 40, 379, and 381 of SEQ ID NO: 2.

### Claim 11 (depends on 10)

The variant according to claim 10, wherein the mutation is a substitution.

### Claim 12 (depends on 11)

The variant according to claim 11, wherein the substitution is
a substitution of the amino acid residue at position 40 with an acidic amino acid selected from the group consisting of aspartic acid and glutamic acid;
a substitution of the amino acid residue at position 379 with cysteine; or
a substitution of the amino acid residue at position 381 with an amino acid selected from the group consisting of methionine, isoleucine, leucine, and valine.

### Claim 13 (depends on 1, 10)

A polynucleotide encoding the β-fructofuranosidase variant of claim 1 or 10.

### Claim 14 (depends on 13)

A recombinant vector comprising the polynucleotide of claim 13.

### Claim 15 (depends on 14)

A transformant comprising the recombinant vector of claim 14.

### Claim 16 (depends on 1, 10, 15)

A method for producing a fructooligosaccharide, which comprises the step of bringing the transformant of claim 15 or the β-fructofuranosidase variant of claim 1 or 10 into contact with sucrose.