Stable fungal cel6 enzyme variants

Abstract

The disclosure provides variant Ce16a enzymes having increased thermostability, methods of making and using such polypeptides.

What is claimed is: 
     
         1 . A recombinant polypeptide comprising at least 80% identity to SEQ ID NO:4 and having one or more amino acid substitutions at residues selected from the group consisting of N38, S54, V151, V154, M158, C269, Q300, S316, S340, S430, and S437, and wherein the polypeptide has cellulase activity and comprises increased thermostability compared to a wild-type enzyme of SEQ ID NO: 4, 6, or 8. 
     
     
         2 . The recombinant polypeptide of  claim 1 , further comprising a cellulose binding domain (CBD) operably linked to the polypeptide. 
     
     
         3 . The recombinant polypeptide of  claim 2 , wherein the CBD comprises a sequence as set forth in SEQ ID NO:10. 
     
     
         4 . The recombinant polypeptide of  claim 1 , wherein the polypeptide comprises one or more substitutions selected from the group consisting of N38S, S54F, S54M, V151A, V154E, M158L, C269A, C269G, C269L, C269S, Q300L, S316R, S340P, S340W, S430P, S437F, and S437W. 
     
     
         5 . The recombinant polypeptide of  claim 1  comprising SEQ ID NO:4 having substitutions at a positions selected from the group consisting of:
 (a) one or more residue selected from the group consisting of N38, S54, V151, V154, C269, S316, S430 and any combination thereof; 
 (b) M158 and one or more additional residues selected from the group consisting of N38, S54, V151, V154, C269, S316, and S430; 
 (c) Q300 and one or more additional residue selected from the group consisting of N38, S54, V151, V154, C269, S316, and S430; 
 (d) S340 and one or more additional residue selected from the group consisting of N38, S54, V151, V154, C269, S316, and S430; and 
 (e) S437 and one or more additional residue selected from the group consisting of N38, S54, V151, V154, C269, S316, and S430. 
 
     
     
         6 . The recombinant polypeptide of  claim 5 , comprising SEQ ID NO:4 and having having substitutions selected from the group consisting of:
 (a) one or more substitution selected from the group consisting of N38S, S54F, S54M, V151A, V154E, C269A, C269G, C269L, C269S, S316R, and S430P;   (b) M158L and one or more additional substitutions selected from the group consisting of N38S, S54F, S54M, V151A, V154E, C269A, C269G, C269L, C269S, S316R, and S430P;   (c) Q300L and one or more additional substitutions selected from the group consisting of N38S, S54F, S54M, V151A, V154E, C269A, C269G, C269L, C269S, S316R, and S430P;   (d) S340P and one or more additional substitutions selected from the group consisting of N38S, S54F, S54M, V151A, V154E, C269A, C269G, C269L, C269S, S316R, and S430P;   (e) S340W and one or more additional substitutions selected from the group consisting of N38S, S54F, S54M, V151A, V154E, C269A, C269G, C269L, C269S, S316R, and S430P;   (f) S437F and one or more additional substitutions selected from the group consisting of N38S, S54F, S54M, V151A, V154E, C269A, C269G, C269L, C269S, S316R, and S430P;   (g) S437P and one or more additional substitutions selected from the group consisting of N38S, S54F, S54M, V151A, V154E, C269A, C269G, C269L, C269S, S316R, and S430P; and   (h) S437W and one or more additional substitutions selected from the group consisting of N38S, S54F, S54M, V151A, V154E, C269A, C269G, C269L, C269S, S316R, and S430P.   
     
     
         7 . A recombinant polypeptide comprising at least 80% identity to SEQ ID NO:6 and having one or more amino acid substitutions at residues selected from the group consisting of G37, Q53, V155, V158, Q162, L276, I307, K323, P347, T436, and Y443, and wherein the polypeptide has cellulase activity and comprises increased thermostability compared to a wild-type enzyme of SEQ ID NO: 4, 6, or 8. 
     
     
         8 . The recombinant polypeptide of  claim 7 , wherein the substitutions are selected from the group consisting of G37S, Q53F, Q53M, V155A, V158E, Q162L, L276A, L276G, L276S, I307L, K323R, P347W, T436P, Y443F, and Y443W. 
     
     
         9 . The recombinant polypeptide of  claim 7 , wherein the polypeptide comprising SEQ ID NO:6 has up to 50, 25, 10, or 5 conservative amino acid substitutions excluding specific residues G37, Q53, V155, V158, Q162, L276, I307, K323, P347, T436, and/or Y443, wherein at least one or more of these specific residues have substitutions selected form the group consisting of G37S, Q53F, Q53M, V155A, V158E, Q162L, L276A, L276G, L276S, I307L, K323R, P347W, T436P, Y443F, and Y443W. 
     
     
         10 . The recombinant polypeptide of  claim 7  comprising SEQ ID NO:6 having substitutions at a positions selected from the group consisting of:
 (a) a residue selected from the group consisting of G37, Q53, V155, V158, Q162, L276, I307, K323, P347, and T436; and 
 (b) Y443 and one or more additional residue selected from the group consisting of G37, Q53, V155, V158, Q162, L276, I307, K323, P347, and T436. 
 
     
     
         11 . The recombinant polypeptide of  claim 7 , comprising SEQ ID NO:6 and having substitutions selected from the group consisting of:
 (a) a substitution selected from the group consisting of G37S, Q53F, Q53M, V155A, V158E, Q162L, L276A, L276G, L276S, I307L, K323R, P347W, and T436P;   (b) Y443F and one or more additional substitutions selected from the group consisting of G37S, Q53F, Q53M, V155A, V158E, Q162L, L276A, L276G, L276S, I307L, K323R, P347W, T436P; and   (c) Y443P and one or more additional substitutions selected from the group consisting of G37S, Q53F, Q53M, V155A, V158E, Q162L, L276A, L276G, L276S, I307L, K323R, P347W, T436P; and   (d) Y443W and one or more additional substitutions selected from the group consisting of G37S, Q53F, Q53M, V155A, V158E, Q162L, L276A, L276G, L276S, I307L, K323R, P347W, and T436P.   
     
     
         12 . A recombinant polypeptide comprising at least 80% identity to SEQ ID NO:8 and having one or more amino acid substitutions at residues selected from the group consisting of N38, L54, V155, V158, Q162, L276, I307, R323, 5347, T436, and Y443, and wherein the polypeptide has cellulase activity and comprises increased thermostability compared to a wild-type enzyme of SEQ ID NO: 4, 6, or 8. 
     
     
         13 . The recombinant polypeptide of  claim 12  comprising SEQ ID NO:8 having substitutions at a positions selected from the group consisting of:
 (a) a residue selected from the group consisting of N38, L54, V155, V158, Q162, L276, I307, R323, S347, and T436; and 
 (b) Y443 and one or more additional residue selected from the group consisting N38, L54, V155, V158, Q162, L276, I307, R323, S347, and T436. 
 
     
     
         14 . The recombinant polypeptide of  claim 12 , comprising SEQ ID NO:8 and having having substitutions selected from the group consisting of:
 (a) a residue selected from the group consisting of N38S, L54F, L54M, V155A, V158E, Q162L, L276A, L276G, L276S, I307L, S347P, S347W, and T436P;   (b) Y443F and one or more additional substitutions selected from the group consisting N38S, L54F, L54M, V155A, V158E, Q162L, L276A, L276G, L276S, I307L, S347P, S347W, and T436P;   (c) Y443P and one or more additional substitutions selected from the group consisting N38S, L54F, L54M, V155A, V158E, Q162L, L276A, L276G, L276S, I307L, S347P, S347W, and T436P; and   (d) Y443W and one or more additional substitutions selected from the group consisting N38S, L54F, L54M, V155A, V158E, Q162L, L276A, L276G, L276S, I307L, S347P, S347W, and T436P.   
     
     
         15 . The recombinant polypeptide of  claim 12 , wherein the substitutions are selected from the group consisting of N38S, L54F, L54M, V155A, V158E, Q162L, L276A, L276G, L276S, I307L, S347P, S347W, T436P, Y443F, and Y443W. 
     
     
         16 . The recombinant polypeptide of  claim 12 , wherein the polypeptide comprising SEQ ID NO:8 has up to 50, 25, 10, or 5 conservative amino acid substitutions excluding specific residues N38, L54, V155, V158, Q162, L276, I307, R323, S347, T436, and Y443, wherein at least one or more of these specific residues have substitutions selected form the group consisting of N38S, L54F, L54M, V155A, V158E, Q162L, L276A, L276G, L276S, I307L, S347P, S347W, T436P, Y443F, and Y443W. 
     
     
         17 . A polynucleotide encoding a polypeptide of  claim 1 . 
     
     
         18 . A polynucleotide encoding a polypeptide of  claim 7 . 
     
     
         19 . A polynucleotide encoding a polypeptide of  claim 12 .