# US 2020/0399669 A1 · Polypeptides Having Alpha-Galactosidase Activity and Polynucleotides Encoding Same

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- Number: US 2020/0399669 A1
- Document ID: US20200399669A1
- Status: Published application
- Kind: A1
- Application number: 17/014,090
- Filed: 2020-09-08
- Published: 2020-12-24
- Earliest claimed: 2016-05-24
- Assignees: Novozymes A/S
- Applicants: Novozymes A/S
- Inventors: Lone Carstensen; Nikolaj Spodsberg; Morten Gjermansen; Jesper Salomon; Kristian B.R.M. Krogh; Eduardo Antonio Della Pia
- Web page: https://patentgrep.com/patent/US20200399669A1

## CPC

- C12P 19/02: Monosaccharides (2-ketogulonic acid C12P7/60)
- A23K 10/14: Pretreatment of feeding-stuffs with enzymes
- A23K 10/16: Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions (A23K10/12 takes precedence)
- A23K 10/30: from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms (obtained by microbiological or biochemical processes, e.g. using yeasts or enzymes, A23K10/10)
- A23K 20/147: Polymeric derivatives, e.g. peptides or proteins
- A23K 40/10: by agglomeration; by granulation, e.g. making powders
- A23K 50/30: for swines
- A23K 50/75: for poultry
- C12N 9/2465: {acting on alpha-galactose-glycoside bonds, e.g. alpha-galactosidase (3.2.1.22)}
- C12P 19/14: produced by the action of a carbohydrase {(EC 3.2.x)}, e.g. by alpha-amylase {, e.g. by cellulase, hemicellulase}
- C12Y 302/01022: Alpha-galactosidase (3.2.1.22)
- 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)}
- A23K: FODDER
- 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

The present invention relates to methods of releasing galactose from legumes using polypeptides having alpha-galactosidase activity. The invention also relates to polypeptides having alpha-galactosidase activity, polynucleotides encoding the polypeptides, nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing the polypeptides. The invention also relates to compositions comprising the polypeptides of the invention and the use of the polypeptides in animal feed.

## Claims

### Claim 1

A method of releasing galactose from plant based material, comprising treating the plant based material with one or more GH36 polypeptides having alpha-galactosidase activity, wherein the GH36 polypeptide having alpha-galactosidase activity is selected from the group consisting of:
(a) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO: 2;
(b) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO: 5;
(c) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO: 8;
(d) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO: 12;
(e) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO: 14;
(f) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO: 19;
(g) a variant of the polypeptide selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 14 and SEQ ID NO: 19, wherein the variant has alpha-galactosidase activity and comprises one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 positions;
(h) a polypeptide comprising the polypeptide of (a), (b), (c), (d), (e), (f) or (g) and a N-terminal and/or C-terminal His-tag and/or HQ-tag;
(i) a polypeptide comprising the polypeptide of (a), (b), (c), (d), (e), (f) or (g) and a N-terminal and/or C-terminal extension of up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids; and
(j) a fragment of the polypeptide of (a), (b), (c), (d), (e), (f) or (g) having alpha-galactosidase activity and having at least 90% of the length of the mature polypeptide.

### Claim 2 (depends on 1)

The method of claim 1, wherein the GH36 polypeptide is obtained or obtainable from the taxonomic order Bacillales, preferably the family Bacillaceae or obtained or obtainable from the taxonomic order Eurotiales, preferably the family Aspergillaceae.

### Claim 3 (depends on 1, 2)

The method of claim 1 or 2, wherein the GH36 polypeptide comprises or consists of amino acids 1 to 728 of SEQ ID NO: 2, amino acids 1 to 734 of SEQ ID NO: 3, amino acids 1 to 731 of SEQ ID NO: 5, amino acids 1 to 737 of SEQ ID NO: 6, amino acids 1 to 732 of SEQ ID NO: 8, amino acids 1 to 738 of SEQ ID NO: 9, amino acids 1 to 719 of SEQ ID NO: 11 or amino acids 1 to 719 of SEQ ID NO: 12, amino acids 1 to 731 of SEQ ID NO: 14, amino acids 1 to 737 of SEQ ID NO: 15, amino acids 1 to 735 of SEQ ID NO: 18, or amino acids 1 to 735 of SEQ ID NO: 19.

### Claim 4 (depends on 1)

The method of claim 1, wherein the plant based material is soybean, wild soybean, beans, lupin, tepary bean, scarlet runner bean, slimjim bean, lima bean, French bean, Broad bean (fava bean), chickpea, lentil, peanut, Spanish peanut, canola, rapeseed (oilseed rape) or pea or in a processed form such as soybean meal, full fat soy bean meal, soy protein concentrate (SPC), fermented soybean meal (FSBM) or any combination thereof.

### Claim 5 (depends on 1)

The method of claim 1, wherein the GH36 polypeptide releases at least 19 g galactose per kg soybean meal when performed under the reaction conditions 20 mg polypeptide per kg soybean meal in 10% w/v 0.1 M citric acid-phosphate buffer pH 6.5 incubating at 40° C. for 2 hours.

### Claim 6

An isolated polypeptide having alpha-galactosidase activity, selected from the group consisting of:
(a) a polypeptide having at least 80%, e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 2;
(b) a polypeptide having at least 82%, e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 5;
(c) a polypeptide having at least 82%, e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 8;
(d) a polypeptide having at least 84%, e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 12;
(e) a polypeptide having at least 80%, e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 14;
(f) a polypeptide having at least 80%, e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 19;
(g) a polypeptide encoded by a polynucleotide having at least 80%, e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1;
(h) a polypeptide encoded by a polynucleotide having at least 82%, e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 4;
(i) a polypeptide encoded by a polynucleotide having at least 82%, e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 7;
(j) a polypeptide encoded by a polynucleotide having at least 84%, e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 12;
(k) a polypeptide encoded by a polynucleotide having at least 80%, e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 14;
(l) a polypeptide encoded by a polynucleotide having at least 80%, e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 19;
(m) a variant of SEQ ID NO: 2 wherein the variant has alpha-galactosidase activity and comprises one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 positions;
(n) a variant of SEQ ID NO: 5 wherein the variant has alpha-galactosidase activity and comprises one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 positions;
(o) a variant of SEQ ID NO: 8 wherein the variant has alpha-galactosidase activity and comprises one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 positions;
(p) a variant of SEQ ID NO: 12 wherein the variant has alpha-galactosidase activity and comprises one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 positions;
(q) a variant of SEQ ID NO: 14 wherein the variant has alpha-galactosidase activity and comprises one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 positions;
(r) a variant of SEQ ID NO: 19 wherein the variant has alpha-galactosidase activity and comprises one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 positions;
(s) a polypeptide comprising the polypeptide of (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (l), (m), (n), (o), (p), (q) or (r) and a N-terminal and/or C-terminal His-tag and/or HQ-tag;
(t) a polypeptide comprising the polypeptide of (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (l), (m), (n), (o), (p), (q) or (r) and a N-terminal and/or C-terminal extension of up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids; and
(u) a fragment of the polypeptide of (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (l), (m), (n), (o), (p), (q) or (r) alpha-galactosidase activity and having at least 90% of the length of the mature polypeptide.

### Claim 7 (depends on 6)

The polypeptide of claim 6, wherein the polypeptide comprises or consists of amino acids 1 to 728 of SEQ ID NO: 2, amino acids 1 to 734 of SEQ ID NO: 3, amino acids 1 to 731 of SEQ ID NO: 5, amino acids 1 to 737 of SEQ ID NO: 6, amino acids 1 to 732 of SEQ ID NO: 8, amino acids 1 to 738 of SEQ ID NO: 9, amino acids 1 to 719 of SEQ ID NO: 11 or amino acids 1 to 719 of SEQ ID NO: 12, amino acids 1 to 731 of SEQ ID NO: 14, amino acids 1 to 737 of SEQ ID NO: 15, amino acids 1 to 735 of SEQ ID NO: 18, or amino acids 1 to 735 of SEQ ID NO: 19.

### Claim 8 (depends on 6)

A composition comprising one or more polypeptides of claim 6.

### Claim 9 (depends on 8)

The composition of claim 8, further comprising one or more components selected from the group consisting of:
one or more formulating agents;
one or more additional enzymes; and
one or more microbes.

### Claim 10 (depends on 6)

A granule comprising one or more polypeptides of claim 6.

### Claim 11 (depends on 6)

An animal feed additive comprising
(a) one or more polypeptides of claim 6; and
(b) one or more components selected from the group consisting of:
one or more vitamins;
one or more minerals;
one or more amino acids;
one or more prebiotics;
one or more phytogenics;
one or more organic acids; and
one or more other feed ingredients.

### Claim 12 (depends on 6)

A liquid formulation comprising the polypeptide of claim 6.

### Claim 13 (depends on 6)

An animal feed comprising plant based material and one or more polypeptides of claim 6.

### Claim 14 (depends on 6)

A method of improving one or more performance parameters of an animal comprising administering to one or more animals one or more polypeptides of claim 6.

### Claim 15 (depends on 6)

A method for improving the nutritional value of an animal feed, comprising adding to the feed one or more polypeptides of claim 6.

### Claim 16 (depends on 6)

A method of preparing an animal feed, comprising mixing one or more polypeptides of claim 6.

### Claim 17

(canceled)

### Claim 18 (depends on 6)

A polynucleotide encoding the polypeptide of claim 6.

### Claim 19 (depends on 18)

A nucleic acid construct or expression vector comprising the polynucleotide of claim 18 operably linked to one or more control sequences that direct the production of the polypeptide in an expression host.

### Claim 20 (depends on 18)

A recombinant host cell comprising the polynucleotide of claim 18 operably linked to one or more control sequences that direct the production of the polypeptide.

### Claim 21 (depends on 20)

A method of producing a polypeptide having alpha-galactosidase activity, comprising:
(a) cultivating the recombinant host cell of claim 20 under conditions conducive for production of the polypeptide; and
(b) recovering the polypeptide.