IP Library › Granted Patent US 11,492,605
Granted Patent B2
US 11,492,605 · App. 16/605,086 · Granted Nov 8, 2022

Mannanase variants and polynucleotides encoding same

Inventors: Vivek Srivastava (Bangalore, IN); Markus Klinger (Vaerloese, DK); Rakhi Saikia (Bangalore, IN); Vijaya Shankar Nataraj (Bangalore, IN); Sohel Dalal (Bangalore, IN); Jens Erik Nielsen (Bagsvaerd, DK); Rajendra Kulothungan Sainathan (Bangalore, IN)
Assignee: Novozymes A/S
C12N9/2437C11D3/386C12Y302/01078
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Patent No.
US 11,492,605
App. No.
16/605,086
Granted
Nov 8, 2022
Kind
B2
Abstract

The present invention relates to mannanase 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.

Claims (25)

1. An isolated mannanase variant, wherein said variant has mannanase activity and comprises a modification at one position corresponding to a position selected from the positions 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 13, 14, 15, 16, 17, 18, 19, 21, 23, 30, 32, 33, 34, 35, 37, 38, 39, 41, 44, 45, 47, 57, 59, 60, 62, 63, 65, 66, 67, 68, 70, 71, 74, 77, 78, 79, 80, 81, 82, 83, 93, 95, 96, 97, 98, 100, 104, 107, 108, 110, 111, 114, 115, 116, 118, 119, 131, 132, 133, 135, 136, 139, 142, 143, 146, 147, 150, 152, 154, 164, 167, 169, 172, 173, 174, 175, 176, 177, 180, 181, 183, 184, 185, 196, 199, 200, 201, 202, 203, 205, 206, 210, 212, 213, 214, 215, 226, 228, 229, 230, 234, 235, 241, 242, 243, 244, 245, 250, 254, 257, 258, 259, 260, 261, 262, 266, 267, 268, 270, 271, 272, 273, 276, 279, 280, 283, 285, 286, 288, 289, 290, 292, 293, 294, 295, 296, and 298 of the polypeptide of SEQ ID NO: 2, wherein each modification is independently a substitution or a deletion, wherein said variant has at least 80%, but less than 100% sequence identity to the mature polypeptide of SEQ ID NO: 1 or the polypeptide of SEQ ID NO: 2, wherein if the modification is at position 135, the modification is D135H, D135M, D135P, D135Y, D135N, D135G, D135E, D135Q, D135K, D135R, D135V, D135C, D135S, or D135F.

2. The variant of claim 1 , wherein said alteration is a substitution, wherein said substitution of the naturally-occurring amino acid residue at the one position for a different amino acid residue produces a mannanase variant having an Improvement Factor of >1.0 for a measure of stability.

3. The variant of claim 1 , wherein said alteration is a deletion, wherein said deletion of the naturally-occurring amino acid residue at the one position produces a mannanase variant having an Improvement Factor of >1.0 for a measure of stability.

4. The variant of claim 1 , wherein said improved stability is in-detergent stability or thermostability.

5. The variant of claim 1 , wherein said variant comprises a substitution or deletion at one position corresponding to positions 1, 2, 3, 5, 6, 8, 9, 14, 15, 18, 23, 35, 37, 38, 39, 41, 45, 47, 59, 65, 66, 67, 68, 70, 71, 74, 77, 78, 79, 80, 81, 82, 83, 93, 95, 96, 97, 98, 100, 104, 107, 111, 114, 116, 118, 119, 133, 136, 139, 142, 143, 146, 150, 169, 173, 174, 175, 177, 180, 183, 184, 185, 199, 201, 202, 203, 205, 206, 210, 212, 213, 214, 230, 234, 235, 241, 242, 243, 244, 245, 257, 258, 259, 260, 261, 271, 273, 276, 279, 280, 283, 286, 288, 294, and 295 of the polypeptide of SEQ ID NO: 2, and wherein said substitution or deletion of the naturally-occurring amino acid residue at the one position produces a mannanase variant having an Improvement Factor >1.0 for a measure of stability at pH 9.0.

6. The variant of claim 1 , wherein said variant comprises a substitution or deletion at one position corresponding to positions 1, 2, 3, 8, 9, 10, 13, 16, 17, 18, 19, 21, 35, 37, 38, 41, 44, 45, 47, 59, 60, 62, 67, 70, 71, 74, 77, 78, 80, 93, 95, 97, 100, 107, 110, 111, 115, 118, 132, 133, 135, 136, 139, 142, 143, 147, 150, 152, 154, 164, 167, 169, 174, 176, 177, 180, 181, 183, 184, 185, 200, 202, 203, 205, 206, 210, 212, 213, 215, 226, 229, 235, 243, 244, 254, 257, 260, 267, 270, 273, 276, 279, 283, 285, 286, 289, 290, 292, 293, 294, and 296 of the polypeptide of SEQ ID NO: 2, and wherein said substitution or deletion of the naturally-occurring amino acid residue at the one position produces a mannanase variant having an Improvement Factor >1.0 for a measure of stability at pH 10.8.

7. The variant of claim 1 , wherein said Improvement Factor is at least 1.0 for a measure of stability at pH 9.0 or pH 10.8.

8. The variant of claim 1 , wherein said variant comprises a substitution or deletion at one position corresponding to positions 2, 3, 5, 8, 14, 16, 17, 18, 35, 37, 38, 41, 45, 47, 62, 70, 71, 77, 78, 80, 81, 82, 83, 93, 95, 97, 98, 100, 107, 110, 116, 132, 133, 135, 136, 150, 152, 164, 169, 176, 177, 180, 183, 184, 185, 202, 203, 205, 206, 210, 215, 229, 234, 235, 242, 243, 244, 254, 257, 258, 260, 261, 267, 273, 276, 279, 283, 288, 290, 294, and 295, wherein said substitution or deletion of the naturally-occurring amino acid residue at the one position produces a mannanase variant having an Improvement Factor >1.5 for a measure of stability at pH 9.0 or pH 10.8.

9. The variant of claim 1 , wherein said variant comprises a substitution or deletion at one position corresponding to positions 3, 5, 14, 16, 18, 38, 41, 45, 47, 62, 71, 77, 78, 80, 81, 93, 95, 97, 98, 107, 110, 133, 135, 150, 164, 169, 176, 177, 183, 184, 185, 202, 205, 215, 229, 243, 235, 267, 273, 288, 294, and 295 of the polypeptide of SEQ ID NO: 2, and wherein said substitution or deletion of the naturally-occurring amino acid residue at the one position produces a mannanase variant having an Improvement Factor of >2.0 for a measure of stability at pH 9.0 or pH 10.8.

10. The variant of claim 1 , wherein the different amino acid residue is selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, with the proviso that the different amino acid residue is different from the naturally-occurring amino acid residue.

11. A composition comprising a variant according to claim 1 .

12. The composition of claim 11 , wherein said composition further comprises one or more of a surfactant, a bleaching system, a chelating agents, stabilizing agents, hydrotopes, builders, co-builders, bleach activators, polymers and fabric-huing agents.

13. An isolated polynucleotide encoding the variant of claim 1 .

14. A nucleic acid construct comprising the polynucleotide according to claim 13 .

15. An expression vector comprising the polynucleotide according to claim 13 .

16. An isolated recombinant host cell comprising the polynucleotide according to claim 13 .

17. A method of producing a mannanase variant, comprising:

a. cultivating the host cell according to claim 16 under conditions suitable for expression of said variant; and

b. recovering said variant.

18. A method of dishwashing in an automatic dishwashing machine, comprising the steps of adding said composition of claim 11 in a detergent composition compartment in said automatic dishwashing machine, and releasing said composition during a main-wash cycle.

19. A method of laundering in an automatic laundering machine, comprising the steps of adding said composition of claim 11 in a detergent composition compartment in said automatic laundering machine, and releasing said composition during a main wash cycle.

20. The isolated variant of claim 1 , wherein said variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to the mature polypeptide of SEQ ID NO: 1 or the polypeptide of SEQ ID NO: 2.

21. The isolated variant of claim 1 , wherein said variant comprises one or more of the following substitutions: A1G, A1V, A1I, A1M, A1W, A1S, A1T, A1C, A1Y, A1N, A1D, A1E, A1K, A1R, A1H, A1Q, A1F, N2G, N2R, N2E, N2W, N2V, N2L, N2Q, N2M, N2F, N2A, N2H, N2C, N2Y, 33P, S3V, S3M, S3K, S3W, S3C, S3E, S3Q, S3Y, S3H, S3D, G4D, F5H, F5N, Y6M, Y6H, Y6W, Y6M, Y6F, Y6S, Y6V, Y6K, Y6L, Y6A, Y6R, S8T, S8V, S8E, S8K, S8L, S8A, S8N, S8I, S8F, S8H, S8D, SSW, S8P, S8R, S8Q, S8G, G9E, G9H, G9S, G9K, T10V, T11 R, T1 1 D, T11 E, T11 K, Y13H, Y13F, Y13L, D14S, D14G, D14R, D14W, D14A, D14Q, D14T, D14K, D14N, A15R, N16G, N16I, N16Y, N16V, N16P, N16F, N16K, N16S, G17R, G17N, G17M, G17H, N18R, N18C, N18V, N18K, N18L, N18F, N18T, P19Q, P19N, P19I, P19D, V21T, R23I, A30T, Y32F, Y32W, K33Q, K33P, D34G, D34A, D34E, D34S, D34T, D34I, Q35V, Q35L, Q35K, Q35H, Q35M, Q35C, T37P, T37R, I37A, I37F, T37V, T37Q, T37E, T37Y, T37C, T37H, T37L, T37S, T37N, T381, T38V, A39I, E41V, E41A, E41C, E41R, E41T, E41K, E41Y, E41F, E41S, E41W, E41P, E41N, E41D, E41L, E45K, A44G, N45A, N45S, N45G, N45P, N45Q, N45T, N45R, N45F, G47A, G47S, G47H, G47M, G47N, G47W, G47L, G47R, G47K, G47Y, G47T, G47F, G47Q, G47Q, G47S, G47D, D57N, G59Q, G59S, G59T, G59E, G59W, G59F, Q60R, Q60K, Q60N, Q60C, Q60M, T62D, T62E, T621, T62Q, T62L, T62M, T62V, T62K, T62Y, T62F, T62H, K63R, K63Q, K63L, D65C, D65E, 066H, Q66R, H67S, H67T, H67S, H67Y, H67A, H67D, H67R, H67N, H67Q, H67F, H67G, T68W, T68R, T68D, R70K, R70W, R70M, R70L, R70T, R70Q, R70G, R70N, N71 T, N71S, N71L, N71P, N71Q, N71K, N713, N71 W, N71A, S74H, 374T, S74D, 374L, S74K, S74R, S74W, E77T, E77N, E77G, E77F, E77Y, D78G, D78A, D78Q, D78S, D78E, D78R, D78V, D78F, D78A, D78W, D78L, D78H, D78K, D781, K78Y, K78M, K78N, N79R, N79H, N79C, N79K, H80R, H80S, H80K, H80Y, H80F, H80C, H80W, H80D, L81R, L81N, L81 M, V82S, V821, V82R, A83P, A83S, A83T, Y93W, Y93A, Y93R, Y93L, Y93I, Y93C, Y93V, Y93F, Y93Q, Y93T, Y93M, Y93D, Y93K, Y93S, Y93E, Y93H, S95D, S95E, S95T, S95E, S95C, I96P, I96A, I96F, A97R, A97S, A97D, A97K, A97E, A97N, A97M, S98P, S98D, N100F, N100H, N100Y, N100I, N100L, N100D, N100M, N100C, N100K, R101A, D104S, D104G, D104R, D104Q, D104W, D104A, D104V, D104M, D1041, D104H, I107S, I107A, I107R, I107M, I107K, I107V, E108S, E108Q, E108T, R110T, R110S, R110H, R110G, R110K, R110N, R110V, R110A, R1101, R110Y, R110C, S111P, S111A, S111R, S111W, S111Q, S111G, S111K, S111T, S111G, S111W, S111D, S111H, S111Y, S111V, I114Q, I114M, I114W, I114A, I114E, I114G, I114L, I114R, I114T, 1114V, G115D, G115E, G115F, G115R, K116R, K116H, D118R, D118K, D118A, D118T, D118H, D118L, D118N, D118Q, D118S, T119R, S131T, W132C, W132R, W132L, W132N, W132F, W132P, E133R, E133T, E133M, E133Y, E133A, E133H, E133K, E133V, E133C, E133L, D135H, D135M, D135P, D135Y, D135N, D135G, D135E, D135Q, D135K, D135R, D135V, D135C, A136P, A136G, A136C, A136K, A136P, D139R, D139V, D139W, D139Q, D139G, D139A, D139L, D139P, D139T, D139C, K142R, K142Q, K142M, K142S, K142V, K142I, Q143L, Q143M, Q143A, Q143R, Q143D, Q143I, Q143K, Q143C, Q143E, Q143N, Q143H, Q143T, Q143V, Q143W, P146D, R147T, R147E, N150M, N150H, N150V, N150R, N150W, N150T, N150S, N150Q, N150L, N150K, N150G, N150D, N150A, N150E, G152M, G152R, G152D, G152H, N154G, N154C, N154R, N154M, W164G, W164N, W164Q, W164F, W164L, W164M, W164S, F167Y, Q169A, Q169D, Q169T, Q169R, Q169K, H172R, H172W, H172F, H172R, D173E, D173C, Y174R, Y174W, Y174F, Y174L, Y174K, Y174M, G175A, R176A, R176Q, R176E, R176C, R176D, R176V, R176L, R176P, R176K, R176Y, R176S, R176M, R176G, R176H, R1761, E177G, E177S, E177Y, E177Q, E177N, E177H, E177T, E177V, E177M, E177C, E177W, N180A, N180V, N180R, N180A, N180C, N180G, N180K, N180E, N180M, N180Y, N180S, N180T, A181R, A181T, A181V, A1811, A181E, A181K, A181H, P183M, P183T, P183G, P183E, P183H, P183F, P183L, P183A, P183V, P183W, P183D, P183S, Q184T, Q184A, Q184R, Q184S, Q184W, Q184E, Q184K, Q184G, Q184H, Q184C, Q184N, Q184E, Q184F, R185D, R185A, R185V, R185G, R185L, R1851, R185S, R185T, R185C, R185E, R185Y, R185K, Y196W, Y196F, G199A, N200T, N200S, A201′, A201 E, S202H, S202T, S202V, S202C, S202N, S202E, S202D, S202W, S202K, S202M, S202R, S202Y, S202I, S202F, S202Q, Q203M, Q203L, Q203V, Q203T, Q203A, R205K, R205I, R205V, T206R, T206C, T206L, T206I, T206P, T206D, T206M, R210N, R210C, R210K, R210M, R210G, R210L, L212F, L212I, N213I, N213V, N213C, N213D, N213E, N213K, Q214E, D215G, D215W, D215C, D215F, D215N, D215L, R226K, T2285, T228M, N229D, N229C, N229H, N229W, N229Y, G230′, E234F, E234H, E234Y, E234W, A235R, A235K, A235T, A235C, A235E, S241 C, E242Q, E242H, E242Y, Q243E, Q243K, Q243C, Q243A, Q243Y, Q243H, Q243R, Q243A, Q243T, Q243A, Q243L, R244K, R244V, G245K, A250G, K254Y, K254T, K254H, K254F, K254W, G257W, G257E, G257A, G257F, G2575, G257Y, G257Q, G257D, G257L, G257M, G257H, P258Q, P258V, P258M, P258W, P258F, E259F, E259C, W260L, W260V, W260F, W260T, W260Y, E261 C, E261 D, E261A, Y262F, S266A, N267C, N267Y, N267Q, N267F, N267E, N267M, N267V, N267K, N267D, N167W, D268N, A270D, A270E, A270P, A270Y, A270M, A270C, A270Q, G271 I, N272M, N272K, N272E, N273E, N273H, N273Y, N273K, N273C, N273Q, N273F, N273D, N273S, N273W, N273A, N273V, N273M, A276N, A276T, A276C, A276E, A276P, A276D, A276W, A276Q, N279D, N279E, N279Q, N279D, N279H, N279Q, T280L, T280C, N283H, N283W, N283Y, N283Q, N283F, P285D, P285S, P285R, P285K, Y286C, Y286N, Y286W, Y286F, Y286K, L288I, R289D, R289Q, R289L, R289G, R289C, R289E, E290D, E290A, E290Q, S292A, R293T, R293E, R293H, R293S, L294R, L294V, L294P, L294H, L294K, L294I, L294T, S295V, S295P, S295C, S295F, S295I, S295L, S295K, S295Q, S295A, S295M, S295N, S295R, T296S, and F298Y.

22. The composition according to claim 11 , wherein said composition further comprises an additional enzyme.

23. The composition according to claim 22 , wherein the additional enzyme is a protease.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Oct 14, 2019
From: SRIVASTAVA, VIVEK; KLINGER, MARKUS; SAIKIA, RAKHI; NATARAJ, VIJAYA SHANKAR; DALAL, SOHEL; NIELSEN, JENS ERIK; SAINATHAN, RAJENDRA KULOTHUNGAN
To: NOVOZYMES A/S
Reel/Frame 050704/0835 →
Priority Claims (2)
IN 201741016140 · May 8, 2017 · national
EP 17180182 · Jul 7, 2017 · regional
Continuity (1)
Related Publication 20210130801A1 · May 6, 2021
Cited By (1)
US 12,655,368