Serine protease variants and polynucleotides encoding same
AssigneeNovozymes A/S
InventorsDorte M. K. Klitgaard, Roland Alexander Pache, Martin Gudman, Anders G. Sandström
Life of this patent
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Abstract
The present invention relates to serine protease variants, having improved properties compared to the parent protease, in particular variants of a serine protease derived from a strain of Meripilus giganteus belonging to the S53 family. The varinats according to the invention have in particular increased stability, e.g., increased thermo-stability, increased specific activity, and/or increased expression levels, compared to the parent protease. 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.
Claims35
A protease variant, comprising a substitution at one or more positions corresponding to positions 1, 5, 9, 25, 29, 30, 32, 38, 39, 40, 41, 42, 45, 46, 49, 50, 53, 54, 55, 57, 59, 60, 61, 62, 64, 67, 68, 69, 74, 76, 77, 78, 79, 80, 81, 82, 87, 93, 94, 96, 99, 102, 103, 105, 109, 111, 112, 114, 115, 117, 122, 123, 124, 128, 130, 136, 141, 142, 145, 146, 147, 150, 153, 154, 157, 159, 161, 167, 169, 175, 182, 185, 199, 200, 205, 207, 209, 210, 213, 216, 217, 225, 228, 231, 234, 237, 242, 244, 245, 246, 248, 258, 262, 266, 267, 269, 271, 272, 276, 278, 280, 284, 289, 293, 296, 299, 305, 308, 313, 314, 317, 318, 319, 321, 322, 324, 325, 326, 329, 330, 331, 333, 336, 338, 341, 343, 345, 347, 348, 355, 358, 359, 361, 362, 363, and 364 of the polypeptide of SEQ ID NO:
- 3, and the variant has protease activity and wherein the variant has at least 90%, but less than 100% sequence identity to the mature polypeptide of SEQ ID NO:
- 3, and wherein the variant has increased thermo-stability measured as improvement factor, HIF, compared to the protease of SEQ ID NO:
- 3, wherein the at least one substitution is selected from the group consisting of:
- A1T, S5I, S5K, S5L, S5Y, T9H, K25P, S29I, S29P, S29R, S30E, K32W, F38L, I39G, I39L, I39V, I39Y, D40F, D40G, D40H, D40N, D40P, Q41I, Q41V, F42C, F42K, F42S, F42Y, K45L, K45M, K45Y, A46C, A46E, K49P, S50C, S50I, S50L, S50M, S50Q, A53C, A53E, A53V, Q54I, Q54L, Q54M, Q54V, F55H, F55L, K57P, K57S, I59G, I59M, I59P, S60P, S61L, S61P, S62C, S62N, S62P, T64G, T64I, L67V, Q68V, Q68Y, T69C, E74H, E74R, D76L, D76R, Q77L, S78D, P79Y, S80L, E81A, E81H, E81K, E81L, E81P, E81Y, A82F, A82L, A82M, A82P, N87S, T93L, T93S, V94S, L96W, G99C, T102L, T102V, T103I, T103K, T103V, I105Q, D109H, D109K, D109M, D109Y, F111H, F111K, F111S, F111Y, Q112R, G114L, N115A, N115C, N115K, N115P, N115R, N115T, N115Y, E117D, E117G, E117H, E117N, E117Q, E117S, I122K, I122R, I123A, N124L, N124V, G128A, G128E, G128F, G128L, G128S, S130D, S130G, S130L, S130N, S130P, L136K, G141T, Q142A, Q142F, Q142L, Q142N, N145V, T146A, T146I, T146L, T146N, I147S, K150F, K150L, K150R, N153A, N153L, N153M, N153P, N153R, N153Y, Q154A, Q154I, Q154L, Q154M, Q154N, Q154T, Q154V, N157I, N157L, Y159H, Q161E, T167F, I169F, I169S, D175I, D175N, Q182C, Q182T, Q182V, H185P, F199L, F199M, M200S, A205S, Q207H, Q207L, V209F, 5210T, T213L, T213R, A216R, F217I, F217Y, V225Y, I228L, I228T, I228W, Y231I, S234Q, S237F, S237Q, A242I, A242L, A242V, G244S, S245P, T246S, S248Y, F258P, S262P, V266K, V266R, D267A, D267H, D267L, D267N, Q269L, V271R, S272I, S272K, S272L, S272R, S272T, S272V, S272Y, T276L, T276Y, G278P, D280N, D280S, D280T, D280Y, C284A, C284G, F289Y, I293T, V296A, V296I, R299N, K305S, L308V, P313L, F314I, F314L, S317E, S317N, S317W, S318P, A319F, A319Q, K321L, K321Q, K321S, A322D, L324Q, L324S, N325L, D326P, S329C, S329H, S329I, S329L, S329N, S329T, S329V, S329W, G330Q, S331W, S331Y, P333R, S336K, S336N, N338M, N338Q, N338R, P341S, K343A, K343H, K343S, K343T, K343V, G345S, D347I, P348Y, P355I, P355R, A358L, A358P, A358R, A358Y, K359L, K359S, L361M, T362L, T362N, A363G, A363L, and V364I, wherein the positions correspond to amino acid positions in the amino acid sequence set forth in SEQ ID NO:
- 3;
- and wherein the substitution at the one or more positions provides a protease variant having an increase in thermo-stability measured as improvement factor, HIF, of at least 1.4.
A protease variant, comprising a substitution at one or more positions corresponding to positions 20, 27, 29, 34, 40, 42, 45, 46, 50, 53, 54, 55, 58, 59, 60, 61, 62, 66, 69, 76, 81, 87, 93, 96, 98, 101, 102, 103, 109, 110, 115, 117, 122, 124, 125, 127, 128, 130, 136, 141, 142, 145, 146, 148, 149, 150, 153, 154, 157, 159, 161, 164, 167, 182, 183, 184, 185, 188, 199, 200, 207, 208, 209, 210, 211, 213, 216, 219, 228, 231, 237, 244, 249, 252, 253, 261, 266, 267, 272, 275, 276, 277, 280, 285, 288, 291, 292, 293, 294, 296, 299, 303, 304, 305, 306, 307, 308, 310, 317, 318, 319, 321, 326, 327, 336, 338, 341, 344, 345, 348, 356, 358, 359, 361, 362, 363, and 364 of the polypeptide of SEQ ID NO:
- 3, and the variant has protease activity and wherein the variant has at least 90%, but less than 100% sequence identity to the mature polypeptide of SEQ ID NO:
- 3, wherein the at least one substitution is selected from the group consisting of:
- G20L, G20R, T27S, S29A, S29C, S29L, S29N, S29P, S29R, A34L, A34N, A34R, D40S, D40Y, F42Y, K45L, K45Y, A46E, A46K, 550A, S50C, S50I, S50L, S50T, A53I, A53K, A53L, A53S, Q54L, F55R, D58M, I59M, I59P, 560C, S60E, S60P, S61D, S61P, S62C, S62N, S66F, S66L, S66M, S66T, T69C, D76A, D76G, D76K, D76L, D76N, D76P, D76V, D76Y, E81S, N87F, T93S, L96F, L96S, L96T, T98L, P101F, P101L, P101M, P101R, T102C, T102I, T103V, D109P, D110W, N115A, N115D, N115G, N115P, E117D, E117F, E117G, E117L, E117N, E117P, E117S, E117W, E117Y, 1122L, N124D, N124I, N124K, N124L, N124M, N124T, N124V, F125K, F125L, F125M, L127C, L127P, G128N, G128R, G128S, G128W, G128Y, S130D, S130F, S130I, S130N, L136C, G141A, G141C, G141F, G141L, G141M, G141Q, G141R, G141V, Q142A, Q142L, Q142M, Q142S, Q142W, N145F, N145G, N145I, N145L, N145P, N145R, N145S, N145V, N145W, T146A, T1460, T146H, T146I, T146M, T146N, T146Q, T146R, T146V, T146W, S148G, A149V, K150F, N153D, N153F, N153G, N153R, N153Y, Q154C, Q154F, Q154G, Q154I, Q154K, Q154L, Q154M, Q154R, Q154T, Q154V, Q154W, Q154Y, N157A, N157E, N157F, N157M, N157Y, Y159F, YI59G, Y159H, Y159S, Q161A, A164L, T167D, T167V, Q182F, S183A, S183F, S183L, S183M, A184D, A184F, A184H, A184I, A184K, A184L, A184Q, A184R, A184S, A184T, A184V, A184W, A184Y, H185A, H185L, H185P, H185S, N188L, N188Q, F199L, M200F, M200L, Q207F, G208H, G208Y, V209F, V209G, V209H, V209L, V209N, V209W, V209Y, S210E, S210L, S210R, S210T, S210Y, P211F, T213F, T213H, T213I, T213L, T213N, T213R, T213S, T213W, T213Y, A216F, A216L, S219M, I228F, I228H, I228L, I228M, I228Q, I228V, I228Y, Y231F, Y231S, S237F, S237I, S237K, S237L, G244L, G244N, G244P, G244R, G249C, N252F, N252K, N252S, R253C, V261A, V261G, V266C, V266R, D267S, S272I, S272K, S272L, S272M, S272Q, S272R, S272T, S272Y, Q275A, Q275F, Q275K, Q275L, Q275R, Q275V, T276F, T276R, T276V, I277L, I277V, D280F, D280H, D280M, D280N, D280S, D280T, A285L, A285S, T288C, T288I, T288V, S291V, V292C, I293M, I293Y, S294T, S294V, V296L, R299P, A303C, A303F, A303H, A303S, A303V, G304H, G304K, G304P, G304R, G304S, G304Y, K3050, K305F, K305H, K305I, K305L, K305M, K305S, K305T, K305Y, S306L, S306M, S306R, P307C, L308P, L308T, F310M, F310P, F310Y, S317A, S317C, S317G, S317H, S317I, S317K, S317L, S317R, S317W, S317Y, S318N, S318R, A319F, A319W, K321R, K321S, D326P, V327C, V327T, S336R, N338H, N338S, N338T, P341I, P341N, P341R, A344K, A344L, A344P, G345K, G345S, P348G, P348L, P348S, N356L, N356Y, A358F, A358L, A358P, K359C, K359L, K359S, K359W, L361E, L361M, L361P, L361R, L361S, L361T, L361V, T362A, T362C, T362H, T362I, T362K, T362L, T362M, T362P, T362Q, T362R, T362V, T362Y, A363E, A363F, A363L, A363M, A363V, V364F, V364I, V364L, V364M, and V364S, and wherein the substitution at the one or more positions provides a protease variant having an increase in specific activity measured as improvement factor, AIF, of at least 1.37.
A protease variant, comprising at least one substitution corresponding to positions 2, 17, 22, 41, 41, 42, 45, 46, 54, 55, 57, 59, 60, 61, 62, 63, 64, 66, 69, 80, 82, 84, 87, 98, 99, 102, 103, 112, 114, 115, 129, 131, 134, 149, 159, 167, 169, 199, 200, 205, 207, 209, 211, 213, 217,242, 250, 266, 267, 269, 270, 271, 272, 274, 278, 279, 280, 284, 288, 291, 292, 294, 296, 301, 307, 314, 329, 331, 333, 336, 362, and 363 of the polypeptide of SEQ ID NO:
- 3, and the variant has protease activity and wherein the variant has at least 90%, but less than 100% sequence identity to the mature polypeptide of SEQ ID NO:
- 3, and wherein the substitution at one or more positions is selected from the group consisting of I2L, A17P, P22V, Q41L, Q41T, Q41V, Q41Y, F42R, K45S, A46L, A46W, Q54R, Q54S, Q54V, Q54Y, F55L, F55N, F55T, F55Y, K57H, K57L, K57V, I59D, I59K, I59R, I59S, I59V, S60I, S60L, S60Q, S60R, S60Y, S61A, S61F, S62H, S62I, S62L, S62R, S62V, T63A, T63E, T63G, T63R, T63V, T64C, T64D, T64M, S66G, T69A, T69F, T69P, T69R, T69Y, S80G, S80L, S80N, S80T, A82H, 184G, N87P, T98C, G99V, T102A, T102H, T103F, T103I, T103V, Q112H, Q112M, Q112R, Q112S, G114A, G114C, G114L, G114P, N115Y, E129L, E129S, E129V, E129Y, N131I, N131S, N131V, Q134A, Q134L, Q134R, A149D, Y159F, Y159L, T167A, T167L, T167S, T167W, 1169S, F199Y, M200L, A205S, Q207N, V209P, P211F, P211S, T213S, F217I, A242E, K250R, V266A, V266F, V266L, V266S, V266T, V266Y, D267F, D267L, D267T, D267V, Q269C, Q269F, Q269I, Q269S, Q269T, Q269V, Q269X, I270A, I270C, I270L, I270S, V271F, V271I, V271K, V271M, V271S, V271Y, S272A, S272N, G274R, G278S, V279I, D280Y, C284A, T288A, T288C, T288G, S291V, V292S, S294G, V296P, V296R, V296T, I301T, P307T, F314L, S329D, S329V, S331A, S331G, S331T, P333V, S336G, S336L, S336T, T362R, and A363T, and wherein the substitution at the one or more positions provides a protease variant having an increase in expression level in Aspergillus oryzae measured as improvement factor, EIF, of at least 1.38.
Dependencies are inferred from the claim text.