Polypeptide variants
The present invention relates to polypeptide variants and methods for obtaining variants. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.
1 . A variant of a DNase parent, wherein the variant comprises one or both motifs [D/M/L][S/T]GYSR[D/N] (SEQ ID NO: 25) or ASXNRSKG (SEQ ID NO: 26),
wherein the variant comprises one or more substitution(s) compared to SEQ ID NO: 1, wherein the substitution is one or more substitution(s) are selected from the group consisting of:
T1I, T1V, T1Y, T1M, or T1E,
G4N,
T5F, T5F or T5C,
P6V, P6V or P6G,
S7D, S7T,
K8V,
S9K, S9Q, S9V, S9L, S9F, S9P, or S9R,
A10D, A10M, A10I, A10Q, A10V, A10L, or A10K,
Q12S, Q12V, or Q12E,
Q14M, Q14M or Q14R,
N16S,
A17C, A17V, A17E, or A17T,
T19K, T19L, or T19S, T19I, T19V,
K21E, K21E or K21M,
G24Y,
S25P,
S27N, S27I, S27M, S27D, S27V, S27F, S27A, S27C, S27L, or S27E,
G28L,
Y29W,
S30K, S30D, S30H, or S30T,
D32Q,
I38V, I38V or I38M,
Q40V,
S42C, S42L, S42M, S42F, or S42W,
V49R,
L51I,
K52I, K51I or K52H,
A55S,
D56I, D56L, or D56T,
S57W, S57F, S57H, S57C, S57P, S57V, S57R, or S57T,
Y58A, Y58A or Y58T,
S59C, S59T, S59L, S59Q, S59V, S59K, S59R, S59M, S59I, or S59H,
N61D,
P63A,
T65L, T65I, T65V, T65R, or T65K,
S68V, S68I, S68W, S68Y, S68H, S68C, S68T, or S68L,
V76G, V76L, V76C, V76K, V76H, V76E, V76A, V76Y, V76N, V76M, V76R, V76F, or
T77N, T77Y, T77W, or T77R,
F78I, F78H, F78Y, F78C,
T79G, T79G or T79R,
N80K,
S82L, S82E, S82K, S82R, or S82H,
D83C, D83F, or D83L,
L92T,
A93G,
E94N,
G99S,
S101D, S101D or S101A,
S102M, S102L, S102V, S102A, S102K, S102T, or S102R,
T104P, T104P or T104A,
T105V, T105V or T105I,
K107L, K107C, K107R, K107H, K107S, K107M, K107E, K107A, or K107D,
Q109R, Q109S,
A112S,
S116D, S116R, S116Q, S116H, S116V, S116A, S116E, or S116K,
A125K,
S126I, S126E, S126A, or S126C,
T127C, T127V,
S130E,
G132R,
D135R,
T138Q,
W139R,
R143E, R143E or R143K,
S144Q, S144H, S144L, S144P, S144E, or S144K,
G145V, G145E, or G145D, G145A,
A147Q, A147Q or A147W, A147S,
G149S,
K152H, K152H or K152R,
S156C, S156G, S156K, S156R, S156T, or S156A,
T157S,
Y159F,
K160V,
W161L, W161L or W161Y,
G162Q, G162D, G162M, G162R, G162A, G162S, G162E, G162L, G162K, G162V, or G162H,
S164R, S164R or S164T,
Q166D,
S167M, S167L, S167F, S167W, S167E, S167A, S167Y, S167H, S167C, S1671, S167Q, S167V, or S167T,
S168V, S168E, S168D, or S168L,
K170S, K170L, K170F, or K170R,
T171D, T171E, T171A, or T171C,
A172G, A172S,
L173T, L173A, or L173V,
Q174L,
G175D, G175E, G175N, or G175R,
M176H,
L177I,
N178D, N178E, N178T, N178S, or N178A,
S179E,
S181R, S181E, S181D, S181F, S181H, S181W, S181L, S181M, S181Y, S181Q, S181G, or S181A, and
Y182M, Y182C, Y182K, Y182G, Y182A, Y182S, Y182V, Y182D, Y182Q, Y182F, Y182L, Y182N, Y1821, Y182E, Y182T and or Y182W,
wherein the variant has a sequence identity to the polypeptide shown in SEQ ID NO: 1 of at least 80% and the variant has DNase activity.
2 . The variant of claim 1 , which has an improved stability, measured as half-life improvement factor, HIF, compared to the parent or compared to the DNase having the polypeptide shown in SEQ ID NO: 1.
3 . The variant of claim 1 , wherein the variant has at least 85% but less than 100% sequence identity to the polypeptide shown in SEQ ID NO: 1.
4 . The variant of claim 1 , wherein the total number of alterations compared to SEQ ID NO: 1 is 1-20 alterations.
5 . A detergent composition comprising a variant of claim 1 .
6 . The detergent composition of claim 5 , further comprising one or more detergent components.
7 . The detergent composition of claim 5 , further comprising one or more additional enzymes selected from the group comprising consisting of proteases, amylases, lipases, cutinases, cellulases, endoglucanases, xyloglucanases, pectinases, pectin lyases, xanthanases, peroxidaes, peroxidases, haloperoxygenases, catalases and mannanases, or any mixture thereof.
8 . The detergent composition of claim 6 , in form of a bar, a homogenous tablet, a tablet having two or more layers, a pouch having one or more compartments, a regular or compact powder, a granule, a paste, a gel, or a regular, compact or concentrated liquid.
9 . A method for obtaining a DNase variant, comprising:
a) introducing into a parent DNase an alteration at one or more positions corresponding to positions 1, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 17, 19, 21, 24, 25, 27, 28, 29, 30, 32, 38, 40, 42, 49, 51, 52, 55, 56, 57, 58, 59, 61, 63, 65, 68, 76, 77, 78, 79, 80, 82, 83, 92, 93, 94, 99, 101, 102, 104, 105, 107, 109, 112, 116, 125, 126, 127, 130, 132, 135, 138, 139, 143, 144, 145, 147, 149, 152, 156, 157, 159, 160, 161, 162, 164, 166, 167, 168, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 181 and 182 of SEQ ID NO 1, wherein the variant has DNase activity; and
b) recovering the variant.
10 . The method of claim 9 , wherein the variant comprises 1-20 alterations compared to the polypeptide shown in SEQ ID NO: 1.
11 . The method according to claim 9 , wherein the alteration is a substitution and wherein the substitution is selected from the group consisting of:
T1I, T1L, T1V, T1F, T1Y, or T1M,
T1E,
G4N,
T5F, T5F or T5C,
P6V, P6V or P6G,
S7D, S7D or S7T,
K8V,
S9K, S9Q, S9V, S9L, S9F, S9P, or S9R,
A10D, A10M, A10I, A10Q, A10T, A10V, A10L, or A10K,
Q12S, Q12V, or Q12E,
Q14M, Q14M or Q14R,
N16S,
A17C, A17V, A17E, A17T, or A17S,
T19K, T19N, T19L, T19S, T19I, or T19V,
K21Q, K21E, or K21M,
G24Y,
S25P, S25P or S25T,
S27N, S27I, S27M, S27D, S27T, S27V, S27F, S27A, S27C, S27L, or S27E,
G28L,
Y29W,
S30K, S30D, S30H, or S30T,
D32Q,
I38V, I38V or I38M,
Q40V,
S42G, S42C, S42D, S42L, S42M, S42F, S42N, or S42W,
V49R,
L51I,
K52I, K52Q, or K52H,
A55S,
D56I, D56L, or D56T,
S57W, S57Y, S57F, S57H, S57C, S57P, S57V, S57R, or S57T,
Y58A, Y58A or Y58T,
S59C, S59T, S59L, S59Q, S59V, S59K, S59R, S59M, S59I, or S59H,
N61D,
P63A,
T65L, T65I, T65V, T65R, or T65K,
S68V, S68I, S68W, S68K, S68Y, S68H, S68C, S68T, or S68L,
V76G, V76L, V76C, V76K, V76H, V76E, V76A, V76Y, V76N, V76M, V76R, V761, or V76F,
T77N, T77Y, T77W, or T77R,
F78L, F78I, F78H, F78V, F78Y, or F78C,
T79G, T79G or T79R,
N80K, N80K or N80S,
S82L, S82E, S82K, S82R, or S82H,
D83C, D83F, or D83L,
L92T,
A93G,
E94N,
G99S,
S101D, S101D or S101A,
S102M, S102L, S102V, S102A, S102K, S102T, or S102R,
T104S, T104P, or T104A,
T105V, T105V or T105I,
K107L, K107C, K107R, K107H, K107S, K107M, K107E, K107A, K107Q, or K107D,
Q109K, Q109R, or Q109S,
A112S,
S116D, S116R, S116Q, S116H, S116V, S116A, S116E, or S116K,
A125K,
S1261, S126E, S126A, or S126C,
T127C, T127V, or T127S,
S130E,
G132R,
D135R,
T138Q,
W139R,
R143E, R143E or R143K,
S144Q, S144H, S144A, S144L, S144P, S144E, or S144K,
G145V, G145E, G145D, or G145A,
A147H, A147R, A147K, A147Q, A147W, A147N, or A147S,
G149S,
K152H, K152H or K152R,
S156C, S156G, S156K, S156R, S156T, or S156A,
T157S,
Y159H, Y159H or Y159F,
K160R, K160R or K160V,
W161L, 161L or W161Y,
G162Q, G162N, G162D, G162M, G162R, G162A, G162S, G162E, G162L, G162K, G162V, or G162H,
S164R, S164H, S164N, or S164T,
Q166D,
S167M, S167L, S167F, S167W, S167E, S167A, S167Y, S167H, S167C, S1671, S167Q, S167V, or S167T,
S168V, S168E, S168D, or S168L,
K170S, K170L, K170F, or K170R,
T171D, T171E, T171N, T171A, T171S, or T171C,
A172G, A172G or A172S,
L173T, L173A, or L173V,
Q174L,
G175D, G175E, G175N, G175R, or G175S,
M176H,
L1771,
N178D, N178E, N178T, N178S, or N178A,
S179E,
S181R, S181E, S181D, S181I, S181F, S181H, S181W, S181L, S181M, S181Y, S181Q, S181V, S181G, or S181A, and
Y182M, Y182C, Y182K, Y182G, Y182A, Y182S, Y182V, Y182D, Y182Q, Y182F, Y182L, Y182N, Y1821, Y182E, Y182T and or Y182W,
compared to the polypeptide shown in SEQ ID NO: 1.
12 . The method according to claim 9 , wherein the DNase parent belongs to the GYS-clade and comprises one or both motifs [D/M/L][S/T]GYSR[D/N] (SEQ ID NO: 25) or ASXNRSKG (SEQ ID NO: 26).
13 . The method of claim 9 , wherein the parent DNase is selected from the group of polypeptides: consisting of:
a) a polypeptide having at least 80% sequence identity to the polypeptide shown in SEQ ID NO: 1,
b) a polypeptide having at least 80% sequence identity to the polypeptide shown in SEQ ID NO: 2,
c) a polypeptide having at least 80% sequence identity to the polypeptide shown in SEQ ID NO: 3,
d) a polypeptide having at least 80% sequence identity to the polypeptide shown in SEQ ID NO: 4,
e) a polypeptide having at least 80% sequence identity to the polypeptide shown in SEQ ID NO: 5,
f) a polypeptide having at least 80% sequence identity to the polypeptide shown in SEQ ID NO: 6,
g) a polypeptide having at least 80% sequence identity to the polypeptide shown in SEQ ID NO: 7,
h) a polypeptide having at least 80% sequence identity to the polypeptide shown in SEQ ID NO: 8,
i) a polypeptide having at least 80% sequence identity to the polypeptide shown in SEQ ID NO: 9,
j) a polypeptide having at least 80% sequence identity to the polypeptide shown in SEQ ID NO: 10,
k) a polypeptide having at least 80% sequence identity to the polypeptide shown in SEQ ID NO: 11,
l) a polypeptide having at least 80% sequence identity to the polypeptide shown in SEQ ID NO: 12,
m) a polypeptide having at least 80% sequence identity to the polypeptide shown in SEQ ID NO: 13,
n) a polypeptide having at least 80% sequence identity to the polypeptide shown in SEQ ID NO: 14,
o) a polypeptide having at least 80% sequence identity to the polypeptide shown in SEQ ID NO: 15,
p) a polypeptide having at least 80% sequence identity to the polypeptide shown in SEQ ID NO: 16,
q) a polypeptide having at least 80% sequence identity to the polypeptide shown in SEQ ID NO: 17,
r) a polypeptide having at least 80% sequence identity to the polypeptide shown in SEQ ID NO: 18,
s) a polypeptide having at least 80% sequence identity to the polypeptide shown in SEQ ID NO: 19,
t) a polypeptide having at least 80% sequence identity to the polypeptide shown in SEQ ID NO: 20,
u) a polypeptide having at least 80% sequence identity to the polypeptide shown in SEQ ID NO: 21,
v) a polypeptide having at least 80% sequence identity to the polypeptide shown in SEQ ID NO: 22,
w) a polypeptide having at least 80% sequence identity to the polypeptide shown in SEQ ID NO: 23, and
x) a polypeptide having at least 80% sequence identity to the polypeptide shown in SEQ ID NO: 24.
14 . The method of claim 9 , wherein the parent DNase is obtained from the Bacillus genus.
15 . The method according to claim 9 , wherein the DNase variant has at least 80% sequence identity to the polypeptide shown in SEQ ID NO: 1.
16 . A method, comprising,
exposing a textile or hard surface to the detergent composition of claim 5 , completing at least one wash cycle, and
optionally rinsing the textile or hard surface.
17. The variant of claim 1 , which comprises one or more substitution(s) selected from the group consisting of:
T1I, T1V, T1Y, or T1E,
S25P,
S27N, S27M, S27D, S27V, S27F, S27A, S27C, S27L, or S27E,
S42C, S42L, S42M, S42F, or S42W,
D56I, D56L, or D56T,
S57W, S57F, S57H, S57C, S57P, S57V, S57R, or S57T,
S59C, S59T, S59L, S59Q, S59K, S59R, S59M, S59I, or S59H,
T65L, T65I, T65V, T65R, or T65K,
V76G, V76C, V76K, V76H, V76E, V76A, V76Y, V76N, V76M, V76R, or V76F,
T77N, T77Y, T77W, or T77R,
Q109S,
S116D, S116R, S116Q, S116H, S116V, S116A, S116E, or S116K,
T127C,
S144Q, S144H, S144L, S144P, S144E, or S144K,
G145V, G145E, or G145D,
A147Q or A147W,
S167M, S167L, S167F, S167W, S167E, S167Y, S167H, S167C, S167I, S167Q, S167V, or S167T,
G175D, G175E, G175N, or G175R, and
S181R, S181E, S181D, S181F, S181H, S181W, S181L, S181M, S181Y, S181Q, S181G, or S181A.
18. The variant of claim 1 , which further comprises one or more additional substitution(s) compared to SEQ ID NO: 1, wherein the one or more additional substitution(s) are selected from the group consisting of:
S7T,
T19I or T19V,
S27I,
S59V,
V76L,
F78I,
K107R,
Q109R,
T127V,
G145A,
A147S,
G162D, and
A172S.
19. The variant of claim 17 , wherein the variant has at least 85% but less than 100% sequence identity to the polypeptide shown in SEQ ID NO: 1.
20. The variant of claim 1 , wherein the variant has at least 90% but less than 100% sequence identity to the polypeptide shown in SEQ ID NO: 1.
21. The variant of claim 1 , wherein the variant has at least six of the substitutions.
22. The variant of claim 1 , wherein the variant has at least seven of the substitutions.
23. The variant of claim 1 , wherein the variant has at least eight of the substitutions.
24. A variant of a DNase parent, wherein the variant comprises one or both motifs [D/M/L][S/T]GYSR[D/N] (SEQ ID NO: 25) or ASXNRSKG (SEQ ID NO: 26),
wherein the variant comprises one or more substitution(s) compared to SEQ ID NO: 1, wherein the one or more substitution(s) are selected from the group consisting of:
S7T,
T19I or T19V,
S27I,
S59V,
V76L,
F78I,
K107R,
Q109R,
T127V,
A147S,
G162D, and
A172S,
wherein the variant has a sequence identity to the polypeptide shown in SEQ ID NO: 1 of at least 85% and the variant has DNase activity.
25. The variant of claim 24 which has an improved stability, measured as half-life improvement factor, HIF, compared to the parent or compared to the DNase having the polypeptide shown in SEQ ID NO: 1.
26. The variant of claim 24 , wherein the variant has at least 90% but less than 100% sequence identity to the polypeptide shown in SEQ ID NO: 1.
27. The variant of claim 24 , wherein the total number of alterations compared to SEQ ID NO: 1 is 1-20 alterations.
28. A detergent composition comprising a variant of claim 24 .
29. The detergent composition of claim 28 , further comprising one or more detergent components.
30. The detergent composition of claim 28 , further comprising one or more additional enzymes selected from the group consisting of proteases, amylases, lipases, cutinases, cellulases, endoglucanases, xyloglucanases, pectinases, pectin lyases, xanthanases, peroxidases, haloperoxygenases, catalases and mannanases, or any mixture thereof.
31. The detergent composition of claim 28 , in form of a bar, a homogeneous tablet, a tablet having two or more layers, a pouch having one or more compartments, a regular or compact powder, a granule, a paste, a gel, or a regular, compact or concentrated liquid.