Variant
Described herein are variants of H. jecorina CBH I, a Cel7 enzyme. The present invention provides novel cellobiohydrolases that have improved thermostability and reversibility.
1. A variant polypeptide, consisting essentially of an amino acid sequence having at least 96.4% sequence identity to the amino acid sequence of SEQ ID NO:2, wherein said polypeptide has CBH 1 cellusase activity and comprises at least one or more amino acid substitutions selected from the group consisting of Q17L, G22D, T41I, N49S, S57N, A68T, A77D, S92T, N103I, A112E, S113(T/N/D), E193V, S196T, M213I, P227(L/T/A), T246(C/A), D249K, Y252(A/Q), T255P, D257E, D259W, S278P, S279N, K286M, L288F, E295K, T296P, S297T, N301(R/K), E325K, T332(K/Y/H), F338Y, S342Y, F352L, T356L, Y371C, T380G, Y381D, V393G, S398T, V403D, S411F, G430F, G440R, T462I, T484S, Q487L, and P491L.
2. A variant polypeptide, consisting essentially of an amino acid sequence having at least 96.4% sequence identity to the amino acid sequence of SEQ ID NO:2, wherein said polypeptide has CBH I cellusase activity and comprises at least one or more amino acid substitutions selected from the group consisting of N49S, A68T, A77D, S92T, S113(N/D), P227(A/L/T), D249K, T255P, D257E, S279N, L288F, E295K, S297T, N301(R/K), T332(K/Y/H), F338Y, T356L, V393G, and G430F.
3. A variant polypeptide, consisting essentially of an amino acid sequence having at least 96.4% sequence identity to the amino acid sequence of SEQ ID NO:2 wherein said polypeptide has CBH I cellusase activity and a a plurality of amino acid mutations selected from the group consisting of:
(a) A112E/T226A;
(b) S196T/S411F;
(c) E295K/S398T;
(d) T246C/Y371C;
(e) V403D/T462I;
(f) T411 and a deletion at T445;
(g) A68T/G440R/P491L;
(h) G22D/S278P/T296P;
(i) T246A/R251A/Y252A;
(j) T380G/Y381D/R394A;
(k) Y252Q/D259W/S342Y;
(l) S113T/T255P/K286M;
(m) P227L/E325K/Q487L;
(n) P227T/T484S/F352L;
(o) Q17L/E193V/M2131/F352L;
(p) S8P/N49S/A68T/S113N;
(q) S8P/N49S/A68T/S113N/P227L;
(r) T41I/A112E/P227L/S278P/T296P;
(s) S8P/N49S/A68T/A112E/T226A;
(t) S8P/N49S/A68T/A112E/P227L;
(u) S8P/T41I/N49S1A68T/A112E/P227L;
(v) G22D/N49S/A68T/P227L/S278P/T296P;
(w) S8P/G22D/T41I/N49S/A68T/N103I/S113N/P227L/S278P/T296P;
(x) G22D/N49S/A68T/N103I/S113N/P227L/S278P/T296P;
(y) G22D/N49S/A68T/N103I/A112E/P227L/S278P/T296P;
(z) G22D/N49S/N64D/A68T/N103I/S113N/S278P/T296P;
(aa) S8P/G22D/T41I/N49S/A68T/N103I/S113N/P227L/D249K/S278P/T296P;
(bb) S8P/G22D/T41I/N49S/A68T/N103I/S113N/P227L/S278P/T296P/N301R;
(cc) S8P/G22D/T41I/N49S/A68T/N103I/S113N/P227L/D249K/S278P/T296P/N301R;
(dd) S8P/G22D/T41I/N49S/A68T/S113N/P227L/D249K/S278P/T296P/N301R;
(ee) S8P/T41I/N49S/S57N/A68T/S113N/P227L/D249K/S278P/T296P/N301R;
(ff) S8P/G22D1T41I/N49S/A68T/S113N/P227L/D249K/S278P/N301R;
(gg) S8P/T41I/N49S/A68T/S92T/S113N/P227L/D249K/V403D/T462I;
(hh) S8P/G22D/T41I/N49S/A68T/S92T/S113N/P227L/D249K/V403D/T462I;
(ii) S8P/T41I/N49S/A68T/S92T/S113N/P227L/D249K/S411F;
(jj) S8P/G22D/T41I/N49S/A68T/S92T/S113N/P227L/D249K/S411F;
(kk) S8P/G22D/T41I/N49S/A68T/S92T/S113N/S196T/P227L/D249K/T255P/S278P/T296P/N301R/E325K/S411F;
(ll) S8P/T41I/N49S/A68T/S92T/S113N/S196T/P227L/D249K/T255P/S278P/T296P/N301R/E325K/V403D/S411F/T462I; or
(mm) S8P/G22D/T41I/N49S/A68T/S92T/S113N/S196T/P227L/D249K/T255P/S278P/T296P/N301R/E325K/V403D/S411F/T462I.
4. A detergent composition comprising a surfactant and a variant of a CBH I cellulase according to any one of claims 1 , 2 and 3 .
5. The detergent according to claim 4 , wherein said detergent is a laundry detergent.
6. The detergent according to claim 4 , wherein said detergent is a dish detergent.
7. A feed additive comprising a variant of a CBH I cellulase according to according to any one of claims 1 , 2 and 3 .
8. The variant according to any one of claims 1 , 2 and 3 which has improved stability, increased processivity, and/or decreased product inhibition as compared to a CBH I protein of SEQ ID NO:2.
9. The variant of claim 8 , wherein said improved stability comprises at least one of: increased pH optimum, increased temperature stability, and/or increased oxidative stability.
10. A fungal cellulase composition enriched in a variant of a CBH I cellulase according to any one of claims 1 , 2 , and 3 .
11. A culture medium comprising a variant of a CBH I cellulase according to any one of claims 1 , 2 , and 3 .
12. The variant of claim 2 , wherein said one or more substitutions includes P227L.
13. The variant of claim 2 , wherein said one or more substitutions includes P227A.
14. The variant of claim 1 , wherein said one or more substitutions includes S342Y.
15. A fungal cellulase composition enriched in a variant polypeptide according to any one of claims 1 , 2 , and 3 .
16. A culture medium comprising a variant polypeptide according to any one of claims 1 , 2 , and 3 .