# US 2022/0033738 A1 · Polypeptides having nuclease activity

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- Number: US 2022/0033738 A1
- Document ID: US20220033738A1
- Status: Published application
- Kind: A1
- Application number: 17/276,436
- Filed: 2019-10-03
- Published: 2022-02-03
- Earliest claimed: 2018-10-04
- Assignees: Novozymes A/S
- Applicants: Novozymes A/S
- Inventors: Tianqi Sun; Jesper Salomon; Klaus Gori; Morten Gjermansen; Marc Dominique Morant; Mary Ann Stringer
- Web page: https://patentgrep.com/patent/US20220033738A1

## CPC

- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12Y 301/21001: Deoxyribonuclease I (3.1.21.1)
- C11D 3/386: Preparations containing enzymes {, e.g. protease or amylase}
- C11D 3/38636: {containing enzymes other than protease, amylase, lipase, cellulase, oxidase or reductase}
- C11D 2111/12: Soft surfaces, e.g. textile
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES
- C11D: DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL

## Abstract

The present invention relates to polypeptides having nuclease activity, and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and recombinant host cells comprising the polynucleotides as well as methods of producing, recovering and using the polypeptides. The invention further relates to cleaning compositions comprising one or more of the polypeptides having nuclease activity, as well as a cleaning method and use of the polypeptides.

## Claims

### Claim 1

A cleaning composition comprising:
(a) at least 0.001 ppm of a polypeptide have nuclease activity, wherein the polypeptide comprises one or more of the motifs [HQ][FILVY]X[GAQS]DX[HTGSA][QVM]P[LFM]H (SEQ ID NO: 102), G[GA]NX[VILFY]X[VLM] (SEQ ID NO: 103), [SADN]R[GS]H (SEQ ID NO: 104), P[LM]H[VA][GA] (SEQ ID NO: 112), PLH[DN]E (SEQ ID NO: 113), [YV][DN]RGH (SEQ ID NO: 114), YDRGHQ[AV] (SEQ ID NO: 115), [DNA]R[GSC]H[LI] (SEQ ID NO: 116) and/or [RIENLG][YF][RHN]V (SEQ ID NO: 117); wherein the polypeptide belongs to the Pfam family PF02265 (S1-P1_nuclease), PF01223 (Endonuclease_NS) or PF13930 (Endonuclea_NS_2); and
(b) one or more surfactants.

### Claim 2 (depends on 1)

The cleaning composition of claim 1, wherein the polypeptide is an S1-P1 nuclease that belongs to the Pfam family PF02265 (S1-P1_nuclease), and which comprises the motif [HQ][FILVY]X[GAQS]DX[HTGSA][QVM]P[LFM]H (SEQ ID NO: 102) and/or G[GA]NX[VILFY]X[VLM] (SEQ ID NO: 103).

### Claim 3 (depends on 2)

The cleaning composition of claim 2, wherein the polypeptide is of bacterial origin and comprises the motif P[LM]H[VA][GA] (SEQ ID NO: 112).

### Claim 4 (depends on 2)

The cleaning composition of claim 2, wherein the polypeptide is of fungal origin and comprises the motif PLH[DN]E (SEQ ID NO: 113).

### Claim 5 (depends on 1)

The cleaning composition of claim 1, wherein the polypeptide is an EN_NS nuclease that belongs to the Pfam family PF01223 (Endonuclease_NS) or PF13930 (Endonuclea_NS_2), and which comprises the motif [SADN]R[GS]H (SEQ ID NO: 104) and/or [YV][DN]RGH (SEQ ID NO: 114), preferably [YV][DN]RGH (SEQ ID NO: 114).

### Claim 6 (depends on 5)

The cleaning composition of claim 5, wherein the polypeptide is of fungal origin and comprises the motif YDRGHQ[AV] (SEQ ID NO: 115).

### Claim 7 (depends on 5)

The cleaning composition of claim 5, wherein the polypeptide is of bacterial origin and comprises the motif [DNA]R[GSC]H[LI] (SEQ ID NO: 116) and/or [RIENLG][YF][RHN]V (SEQ ID NO: 117).

### Claim 8 (depends on 1)

The cleaning composition of claim 1, wherein said polypeptide is selected from the group consisting of:
(i) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO: 9;
(ii) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO: 15;
(iii) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO: 18;
(iv) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO: 36;
(v) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO: 66;
(vi) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO: 72;
(vii) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO: 81;
(viii) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO: 96; and
(ix) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO: 99.

### Claim 9 (depends on 1)

The cleaning composition of claim 1, wherein the polypeptide is selected from a first group consisting of:
(a) a polypeptide having at least 99% sequence identity to the polypeptide of SEQ ID NO: 3;
(b) a polypeptide having at least 62% sequence identity to the polypeptide of SEQ ID NO: 6;
(c) a polypeptide having at least 97% sequence identity to the polypeptide of SEQ ID NO: 12;
(d) a polypeptide having at least 86% sequence identity to the polypeptide of SEQ ID NO: 21 or 27;
(e) a polypeptide having at least 98% sequence identity to the polypeptide of SEQ ID NO: 30;
(f) a polypeptide having 100% sequence identity to the polypeptide of SEQ ID NO: 33 or 48;
(g) a polypeptide having at least 76% sequence identity to the polypeptide of SEQ ID NO: 39 or 51;
(h) a polypeptide having at least 95% sequence identity to the polypeptide of SEQ ID NO: 42;
(i) a polypeptide having at least 79% sequence identity to the polypeptide of SEQ ID NO: 45 or 24;
(j) a polypeptide having at least 94% sequence identity to the polypeptide of SEQ ID NO: 54;
(k) a polypeptide having at least 72% sequence identity to the polypeptide of SEQ ID NO: 57;
(l) a polypeptide having at least 82% sequence identity to the polypeptide of SEQ ID NO: 60;
(m) a polypeptide having at least 75% sequence identity to the polypeptide of SEQ ID NO: 63; and
(n) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO: 69 or 75;
or is selected from a second group consisting of:
(o) a polypeptide having at least 77% sequence identity to the polypeptide of SEQ ID NO: 78;
(p) a polypeptide having at least 94% sequence identity to the polypeptide of SEQ ID NO: 84;
(q) a polypeptide having at least 99% sequence identity to the polypeptide of SEQ ID NO: 87;
(r) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO: 90; and
(s) a polypeptide having at least 90% sequence identity to the polypeptide of SEQ ID NO: 93;
or is a variant or fragment selected from:
(t) a variant of the polypeptide selected from said first group or said second group wherein the variant has nuclease 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 or 20 positions; and
(u) a fragment of the polypeptide of (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (I), (m), (n), (o), (p), (q), (r) and (s), wherein said fragment has nuclease activity.

### Claim 10 (depends on 1)

The cleaning composition of claim 1, wherein the polypeptide is selected from the group consisting of polypeptides:
(a) comprising or consisting of SEQ ID NO: 3 or the mature polypeptide of SEQ ID NO: 2;
(b) comprising or consisting of SEQ ID NO: 6 or the mature polypeptide of SEQ ID NO: 5;
(c) comprising or consisting of SEQ ID NO: 12 or the mature polypeptide of SEQ ID NO: 11;
(d) comprising or consisting of SEQ ID NO: 21 or the mature polypeptide of SEQ ID NO: 20;
(e) comprising or consisting of SEQ ID NO: 24 or the mature polypeptide of SEQ ID NO: 23;
(f) comprising or consisting of SEQ ID NO: 27 or the mature polypeptide of SEQ ID NO: 26;
(g) comprising or consisting of SEQ ID NO: 30 or the mature polypeptide of SEQ ID NO: 29;
(h) comprising or consisting of SEQ ID NO: 33 or the mature polypeptide of SEQ ID NO: 32;
(i) comprising or consisting of SEQ ID NO: 39 or the mature polypeptide of SEQ ID NO: 38;
(j) comprising or consisting of SEQ ID NO: 42 or the mature polypeptide of SEQ ID NO: 41;
(k) comprising or consisting of SEQ ID NO: 45 or the mature polypeptide of SEQ ID NO: 44;
(l) comprising or consisting of SEQ ID NO: 48 or the mature polypeptide of SEQ ID NO: 47;
(m) comprising or consisting of SEQ ID NO: 51 or the mature polypeptide of SEQ ID NO: 50;
(n) comprising or consisting of SEQ ID NO: 54 or the mature polypeptide of SEQ ID NO: 53;
(o) comprising or consisting of SEQ ID NO: 57 or the mature polypeptide of SEQ ID NO: 56;
(p) comprising or consisting of SEQ ID NO: 60 or the mature polypeptide of SEQ ID NO: 59;
(q) comprising or consisting of SEQ ID NO: 63 or the mature polypeptide of SEQ ID NO: 62;
(r) comprising or consisting of SEQ ID NO: 69 or the mature polypeptide of SEQ ID NO: 68;
(s) comprising or consisting of SEQ ID NO: 75 or the mature polypeptide of SEQ ID NO: 74;
(t) comprising or consisting of SEQ ID NO: 78 or the mature polypeptide of SEQ ID NO: 77;
(u) comprising or consisting of SEQ ID NO: 84 or the mature polypeptide of SEQ ID NO: 83;
(v) comprising or consisting of SEQ ID NO: 87 or the mature polypeptide of SEQ ID NO: 86;
(w) comprising or consisting of SEQ ID NO: 90 or the mature polypeptide of SEQ ID NO: 89;
(x) comprising or consisting of SEQ ID NO: 93 or the mature polypeptide of SEQ ID NO: 92;
(y) comprising or consisting of SEQ ID NO: 96 or the mature polypeptide of SEQ ID NO: 95;
(z) comprising or consisting of SEQ ID NO: 99 or the mature polypeptide of SEQ ID NO: 98;
(aa) comprising or consisting of SEQ ID NO: 108 or the mature polypeptide of SEQ ID NO: 107; and
(bb) comprising or consisting of SEQ ID NO: 111 or the mature polypeptide of SEQ ID NO: 110.

### Claim at position 11

11-13. (canceled)

### Claim 14 (depends on 1)

The cleaning composition of claim 1, wherein the one or more surfactants are selected from anionic surfactants, nonionic surfactants, cationic surfactants, zwitterionic and mixtures thereof.

### Claim 15 (depends on 14)

The cleaning composition of claim 14, wherein the composition comprises at least one anionic surfactant, at least one nonionic surfactant, or both.

### Claim 16 (depends on 1)

A polypeptide having nuclease activity, wherein the polypeptide is as defined in claim 1.

### Claim 17 (depends on 16)

A polynucleotide encoding the polypeptide of claim 16.

### Claim 18 (depends on 17)

A nucleic acid construct or expression vector comprising the polynucleotide of claim 17 operably linked to one or more control sequences that direct production of the polypeptide in an expression host.

### Claim 19 (depends on 17)

A recombinant host cell comprising the polynucleotide of claim 17 operably linked to one or more control sequences that direct production of the polypeptide.

### Claim at position 18

20-21. (canceled)

### Claim 22 (depends on 1)

A method for laundering an item, comprising the steps of:
(a) exposing an item to a wash liquor comprising the cleaning composition of claim 1;
(b) completing at least one wash cycle; and
(c) optionally rinsing the item,
wherein the item is a textile.

### Claim 23

(canceled)