Xylanase Variants and Polynucleotides Encoding Same

Abstract

The present invention relates to xylanase variants, polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; compositions comprising the xylanase variants and methods of using the variants.

1 . An isolated polynucleotide encoding a xylanase variant having xylanase activity, wherein said xylanase variant has at least 70% sequence identity to SEQ ID NO: 1 and comprises a substitution at one or more positions corresponding to positions 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 18, 19, 31, 32, 33, 34, 39, 43, 44, 45, 46, 48, 50, 58, 59, 61, 62, 64, 65, 67, 68, 79, 82, 88, 90, 94, 101, 102, 104, 110, 112, 113, 116, 119, 120, 123, 126, 127, 128, 129, 131, 135, 143, 145, 146, 159, 160, 165, 168, 176, 179, 181, 188, 191, 194, 195, 196, 197, 205, 209, 212, 217, 218, 221, 224, 231, 235, 237, 238, 242, 269, 280, 282, 295, 298, 299, 300, 302, 305, 306, 307, 311, 312, 313, 322, 323, 324, 333, 334, 335, 336, 337, 338, 339, 340, 342, 343, 344, 345, 346, 347, 349, 350, 354, 357, 359, 360, 363, 364, 366, 367, 368, 371, 373, 374, 376, 377, 378, 380, 385, 388, 390 and 391 of SEQ ID NO: 1. 
     
     
         2 . The isolated polynucleotide according to  claim 1 , wherein said xylanase variant has improved thermostability compared to a parent xylanase. 
     
     
         3 . The isolated polynucleotide according to  claim 1 , wherein said xylanase variant comprises a substitution 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 substituted amino acid residue is different from the naturally occurring amino acid residue in that position. 
     
     
         4 . The isolated polynucleotide according to  claim 1 , wherein said xylanase variant comprises one or more of the following substitutions: A2D, A2Q, A2G, A2W, A2P, A2L, A2Y, S3W, S3F, S3H, S3L, S3G, S3M, S3T, S3P, D4L, D4C, D4Y, D4Q, D4A, D4K, D4P, V5H, V5G, V5P, T6K, T6Q, T6L, T6N, T6R, T6D, T6F, T6H, V7F, V7S, V7C, V7A, V7W, N8Q, N8R, N8D, N8F, N8W, N8Y, N8S, N8M, N8L, N8V, N8T, N8I, N8A, V9H, V9R, V9M, S10A, S10H, S10L, S10I, S10D, S10K, S10V, A11P, A11I, A11N, A11G, A11Y, A11C, A11S, A11F, A11M, A11D, A11L, A11H, A11W, A11E, A11V, A11R, A11Q, E12G, E12R, E12K, E12T, E12V, E12W, E12C, K13Q, K13H, K13N, K13L, K13S, Q14K, Q14Y, Q14A, Q14V, Q14W, Q14F, Q14S, Q14P, Q14G, Q14R, Q14M, Q14H, Q14C, Q14D, V15F, V15L, V15N, V15K, V15H, G18H, G18Y, G18W, G18F, G18C, G18S, G18A, F19H, F19Y, L31Q, L31H, L31P, L31G, L31S, L31R, L31N, L31I, L31V, T32N, T32D, A33Q, A33H, A33M, A33Y, A33K, A33E, A33R, A33C, A33N, A34F, A34N, A34C, A34L, A34P, A34S, A34Q, A39S, G43W, G43A, G43N, Q44D, Q44N, Q44R, Q44Y, Q44K, N45D, N45W, N45S, N45F, N45I, N45E, N45H, N45Q, N45G, N45P, N45T, N45Y, Q46D, Q46H, Q46C, Q46S, Q46T, Q46N, Q46K, G48L, G48V, G48I, G48Q, G48C, S50H, S50F, S50R, S50K, S50I, S50L, S50Q, S50Y, S50N, S50V, S50M, S50P, S50A, S50C, S50G, S50D, S50E, S50T, E58P, E58K, E58R, N59C, N59V, N59D, N59K, N59L, N59S, N61W, N61L, N61D, N61E, N61H, N61M, N61Y, N61I, N61T, N61F, N61Q, N62A, N62V, N62L, N62C, N62T, N62Y, N62F, N62M, N62W, N62Q, Y64F, Y64N, K65I, K65L, V67A, V67S, V67F, V67G, V67K, E68W, E68V, E68I, E68A, E68H, E68N, I79K, I79R, I79V, I79A, A82G, P88M, P88F, P88T, P88W, P88Q, D90R, T94H, T101L, T101R, S102R, K104R, K104H, K110E, K110M, A112G, A112T, A112V, A113E, A113Q, A113L, A113D, A113S, Q116E, Q116A, Q116G, N119G, N119S, N119V, N119L, N119C, N119R, D120R, D120A, D120S, D120Y, D120L, D120W, D120T, D120G, T123E, T123Y, T123C, T123H, T123Q, T123K, T123V, T123G, T123R, T123I, K126R, K126A, K126Y, K126L, K126F, K126W, K126E, K126H, K126Q, N127P, N127W, N128H, N128K, G129M, G129Q, G129A, G129F, G129W, N131K, N131R, I135T, Y143H, Y143R, Y143N, H145Y, H145C, H145K, H145A, H145W, E146R, E146H, E146Y, E146F, E146W, E146A, E146K, E146G, E146S, M159C, R160I, S165Y, S165M, S165I, A168I, A168R, F176H, F176R, F176Y, F176K, F176W, L179D, L179N, L179C, L179A, L179R, L179K, L179S, L179Q, L179W, N181C, N181A, N181T, N188M, N188E, N188L, Q191I, Q191K, Q191V, Q191R, Q191L, Q191M, A194H, A194R, A194K, N195H, M196L, M196I, D197A, D197S, D197Q, D197G, D197P, D197T, D197N, G205C, G205Q, S209N, S209C, S209W, P212A, P212K, P212Q, P212R, P212E, P212S, P212N, K217Q, K217M, Q218H, Q218I, A221R, A221K, A221H, A221Q, A221C, A221N, D224Q, Y231T, Y231V, Y231S, Y231A, Y231C, S235A, S235G, T237P, T237R, T237K, N238G, R242I, Y269Q, Y269M, Y269I, Y269F, Y269L, D280S, D280N, D280R, T282M, T282H, T282R, T282C, T282V, T282L, T282E, T282F, K295W, K295T, K295I, K295V, R298Q, R298A, R298E, R298M, R298T, R298N, R298C, R298I, R298D, R298L, R298G, R298W, R298S, R298P, R298Y, P299W, P299A, P299K, P299F, P299Y, P299Q, P299N, P299H, P299E, P299D, P299R, P299M, P299C, P299G, P299S, G300A, G300R, V302I, V302S, V302R, V302T, V302A, V302Q, V302G, V302C, V302K, V302W, V302P, V302D, V302F, D305W, D305F, D305I, D305M, A306I, A306T, T307I, T307N, T307R, T307Q, T307K, T307F, T307M, T307D, T307V, T307W, T307C, T307S, T307H, T307E, T307Y, N311R, N311M, N311I, N311C, N311V, A312I, A312R, A312M, A312F, N313D, N313R, N313I, N313L, N313C, N313G, N313F, D322F, D322G, D322L, N323A, N323C, N323Q, N323L, N323G, N323R, N323E, N323S, N323Y, N323P, K324S, K324P, S333I, S333R, S333T, N334I, N334A, N334L, T335I, T335A, G336C, G336A, G336E, V337A, V337Q, V337D, V337M, V337E, V337G, N338H, Q339I, Q339T, Q339A, N340S, N340A, N340C, V342A, V342L, V342I, V342R, V342D, L343C, L343Q, L343I, L343A, L343P, L343S, L343D, L343Y, L343F, L343K, L343H, L343E, L343N, Q344I, Q344R, Q344S, N345C, N345A, N345H, N345W, N345Q, N345R, N345I, N345V, N345P, G346H, S347R, S347H, S347I, S347A, S347G, S347K, S347Y, S347W, S347T, S347L, S347F, S349R, S349C, S349A, S349V, S349I, S349F, S349Y, S349T, S349M, S349D, N350K, N350A, W354L, S357V, S357Q, S359H, S359Q, S359F, S359R, S359I, S359G, S359Y, S359A, S359P, S359N, S359W, S359E, S360R, S360G, Q363V, Q363R, Q363A, Q363G, Q363H, Q363L, Q363N, Q363F, P364Q, P364H, P364W, P364I, P364L, T366S, T366N, T366W, T366Q, T366C, T366V, T366A, T366S, T366L, T366K, T366R, T366G, T366I, T366Q, N367Y, N367P, N367L, N367A, N367F, N367Q, N367W, N367D, N367E, L368D, L368H, S371F, S371W, S371V, S371E, S371R, S371H, S371Q, S371D, S371I, N373D, N373I, N373E, N373W, N373Y, N373A, N373H, N373Q, H374E, H374F, H374D, H374T, H374S, H374I, H374L, H374W, W376A, W376Q, W376D, W376M, W376N, W376P, W376H, W376Y, W376L, W376E, W376G, W376F, W376R, A377I, A377V, A377M, H378A, H378T, H378I, H378R, H378C, H378M, H378Q, H378N, H378G, H378L, H378V, H378S, H378Y, H378K, H378D, H378P, H378E, H378F, P380M, P380D, P380E, P380C, P380V, P380I, P380L, P380F, P380G, P380K, P380H, T385F, T385M, T385R, V388E, V388D, V388Y, V388K, V388H, V388L, V388Q, N390R, N390M, N390P, R391C, R391M, R391G, R391P, R391H and R391V. 
     
     
         5 . The isolated polynucleotide according to  claim 1 , wherein said xylanase variant has at least 70% sequence identity to SEQ ID NO: 1 and comprises substitutions at Q14, G18, S50, H145, E146, F176, L179, D197, R298 and P299. 
     
     
         6 . The isolated polynucleotide according to  claim 1 , wherein said xylanase variant has at least 70% sequence identity to SEQ ID NO: 1 and comprises one or more of the following substitutions: Q14K, Q14Y, Q14A, Q14V, Q14W, Q14F, Q14S, Q14P, Q14G, Q14R, Q14M, Q14H, Q14C, Q14D, G18H, G18Y, G18W, G18F, G18C, G18S, G18A, S50H, S50F, S50R, S50K, S50I, S50L, S50Q, S50Y, S50N, S50V, S50M, S50P, S50A, S50C, S50G, S50D, S50E, S50T, H145Y, H145C, H145K, H145A, H145W, E146R, E146H, E146Y, E146F, E146W, E146A, E146K, E146G, E146S, F176H, F176R, F176Y, F176K, F176W, L179D, L179N, L179C, L179A, L179R, L179K, L179S, L179Q, L179W, D197A, D197S, D197Q, D197G, D197P, D197T, D197N, R298Q, R298A, R298E, R298M, R298T, R298N, R298C, R298I, R298D, R298L, R298G, R298W, R298S, R298P, R298Y, P299W, P299A, P299K, P299F, P299Y, P299Q, P299N, P299H, P299E, P299D, P299R, P299M, P299C, P299G and P299S. 
     
     
         7 . The isolated polynucleotide according to  claim 1 , wherein said xylanase variant has at least 70% sequence identity to SEQ ID NO: 1 and comprises substitutions S50H, H145Y, F176H, L179D and M196L. 
     
     
         8 . The isolated polynucleotide according to  claim 1 , wherein said xylanase variant has at least 70% sequence identity to SEQ ID NO: 1 and comprises substitutions A2D, A26E, S50H, S102R, H145Y, F176H, L179D, M196L, D197A and Y269F. 
     
     
         9 . The isolated polynucleotide according to  claim 1  wherein said xylanase variant has at least 70% sequence identity to SEQ ID NO: 1 and comprises substitutions A2D, D4L, A26E, S50H, S102R, H145Y, S165Y, N167I, F176H, L179D, M196L, D197A, Y269F, P299W and V302I. 
     
     
         10 . The isolated polynucleotide according to  claim 1  wherein said xylanase variant has at least 70% sequence identity to SEQ ID NO: 1 and comprises substitutions A2D, D4L, Q14K, A26E, S50H, N61W, T94H, S102R, H145Y, S165Y, N167I, F176H, L179D, M196L, D197A, D224Q, Y269F, P299W, V302I and S333I. 
     
     
         11 . A nucleic acid construct or expression vector comprising the isolated polynucleotide of  claim 1  operably linked to one or more control sequences that direct the production of the isolated polypeptide in an expression host. 
     
     
         12 . A recombinant host cell comprising the isolated polynucleotide of  claim 1  operably linked to one or more control sequences that direct the production of the isolated polypeptide. 
     
     
         13 . A xylanase variant having at least 70% sequence identity to SEQ ID NO: 1 and comprising substitutions at Q14, G18, S50, H145, E146, F176, L179, D197, R298 and P299. 
     
     
         14 . A xylanase variant having at least 70% sequence identity to SEQ ID NO: 1 and comprising one or more of the following substitutions: Q14K, Q14Y, Q14A, Q14V, Q14W, Q14F, Q14S, Q14P, Q14G, Q14R, Q14M, Q14H, Q14C, Q14D, G18H, G18Y, G18W, G18F, G18C, G18S, G18A, S50H, S50F, S50R, S50K, S50I, S50L, S50Q, S50Y, S50N, S50V, S50M, S50P, S50A, S50C, S50G, S50D, S50E, S50T, H145Y, H145C, H145K, H145A, H145W, E146R, E146H, E146Y, E146F, E146W, E146A, E146K, E146G, E146S, F176H, F176R, F176Y, F176K, F176W, L179D, L179N, L179C, L179A, L179R, L179K, L179S, L179Q, L179W, D197A, D197S, D197Q, D197G, D197P, D197T, D197N, R298Q, R298A, R298E, R298M, R298T, R298N, R298C, R298I, R298D, R298L, R298G, R298W, R298S, R298P, R298Y, P299W, P299A, P299K, P299F, P299Y, P299Q, P299N, P299H, P299E, P299D, P299R, P299M, P299C, P299G and P299S. 
     
     
         15 . The xylanase of  claim 14 , comprising substitutions S50H, H145Y, F176H, L179D and M196L. 
     
     
         16 . The xylanase variant of  claim 14 , comprising substitutions A2D, A26E, S50H, S102R, H145Y, F176H, L179D, M196L, D197A and Y269F. 
     
     
         17 . The xylanase variant of  claim 14 , comprising as substitutions A2D, D4L, A26E, S50H, S102R, H145Y, S165Y, N167I, F176H, L179D, M196L, D197A, Y269F, P299W and V302I. 
     
     
         18 . The xylanase variant of  claim 14 , comprising substitutions A2D, D4L, Q14K, A26E, S50H, N61W, T94H, S102R, H145Y, S165Y, N167I, F176H, L179D, M196L, D197A, D224Q, Y269F, P299W, V302I and S333I. 
     
     
         19 . A liquid composition comprising the xylanase variant of  claim 13 . 
     
     
         20 . An animal feed comprising the xylanase variant of  claim 13 .