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US 2003/0199072 A1Published application
Crystal and structure of a thermostable glycosol hydrolase and use thereof, and modified proteins
AssigneeProkaria, Itd.
InventorsSusan J. Crennell, Eva M.N. Karlsson, Gudmundur O. Hreggvidsson, Jakob K. Kristjansson, Arnthor Aevarsson
Life of this patent
Estimating the term…
Abstract
The crystal of a hyperthermostable cellulase from Rhodothermus marinus and the three-dimensional structure of the enzyme are provided. The invention further provides procedures for the identification of structural features that are important for thermostability of the enzyme. Methods based thereon to rationally modify proteins structurally related to R. marinus are disclosed, in particular, methods for increased thermostability are provided. Modified proteins are provided, including modified variants of cellulase from Trichoderma reesei.
Claims34
A crystallizable composition comprising a substantially pure protein having at least 50% amino acids sequence identity with amino acid sequence shown in SEQ ID NO:
- 1.
A crystallized molecule or crystallized molecular complex comprising a protein having at least 50% amino acid sequence identity with the amino acid sequence shown is SEQ ID NO:
- 1.
A crystallized molecule or molecular complex comprising a protein having a crystal structure comprising structural entities that can be independently superimposed on reference structural entities within the structure defined by the structural coordinates set forth in FIGS. 6A-PPP such that the root mean square deviation of Cα atoms being superimposed is less than 0.8 Å, the reference entities comprising (i) residues 18-26, (ii) residues 31-37, (iii) residues 56-64, (iv) residues 84-95, (v) residues 99-112, (vi) residues 122-142, (vii) residues 149-157, (viii) residues 161-173, (ix) residues 196-210, and (x) residues 215-224 of the protein structure defined by said coordinates of FIGS. 6A-PPP.
A method of modifying a clan C glycosyl hydrolase wherein the modification comprises one or more modifications selected from the group consisting of:
- having an Arg, Lys or His residue at one position and an Asp or Glu residue at a second position, wherein the positions are at sequence locations that align with Glu 4 and Arg 47 of SEQ ID NO:
- 1, respectively;
- having an Arg, Lys or His residue at one position and an Asp or Glu residue at a second position, wherein the positions are at sequence locations that align with Arg 8 and Glu 29 of SEQ ID NO:
- 1, respectively;
- having an Arg, Lys or His residue at one position and an Asp or Glu residue at a second position, wherein the positions are at sequence locations that align with Asp 10 and Arg 12 of SEQ ID NO:
- 1, respectvely;
- having an Arg, Lys or His residue at one position and an Asp or Glu residue at a second position, wherein the positions are at sequence locations that align with Asp 10 and Arg 20 of SEQ ID NO:
- 1, respectively;
- having an Arg, Lys or His residue at one position and an Asp or Glu residue at a second position, wherein the positions are at sequence locations that align with Asp 13 and Arg 20 of SEQ ID NO:
- 1, respectively;
- having an Arg, Lys or His residue at one position and an Asp or Glu residue at a second position, wherein the positions are at sequence locations that align with Glu 35 and Arg 216 of SEQ ID NO:
- 1, respectively;
- having an Arg, Lys or His residue at one position and an Asp or Glu residue at a second position, wherein the positions are at sequence locations that align with Arg 47 and Asp 49 of SEQ ID NO:
- 1, respectively;
- having an Arg, Lys or His residue at one position and an Asp or Glu residue at a second position, wherein the positions are at sequence locations that align with Asp 51 and Arg 100 of SEQ ID NO:
- 1, respectively;
- having an Arg, Lys or His residue at one position and an Asp or Glu residue at a second position, wherein the positions are at sequence locations that align with His 67 and Glu 203 of SEQ ID NO:
- 1, respectively;
- having an Arg, Lys or His residue at one position and an Asp or Glu residue at a second position, wherein the positions are at sequence locations that align with Arg 79 and Glu 83 of SEQ ID NO:
- 1, respectively;
- having an Arg, Lys or His residue at one position and an Asp or Glu residue at a second position, wherein the positions are at sequence locations that align with Arg 80 and Glu 83 of SEQ ID NO:
- 1, respectively;
- having an Arg, Lys or His residue at one position and an Asp or Glu residue at a second position, wherein the positions are at sequence locations that align with Arg 80 and Glu 196 of SEQ ID NO:
- 1, respectively;
- having an Arg, Lys or His residue at one position and an Asp or Glu residue at a second position, wherein the positions are at sequence locations that align with Asp 86 and Arg 88 of SEQ ID NO:
- 1, respectively;
- having an Arg, Lys or His residue at one position and an Asp or Glu residue at a second position, wherein the positions are at sequence locations that align with Arg 88 and Glu 177 of SEQ ID NO:
- 1, respectively;
- having an Arg, Lys or His residue at one position and an Asp or Glu residue at a second position, wherein the positions are at sequence locations that align with Arg 88 and Asp 179 of SEQ ID NO:
- 1, respectively;
- having an Arg, Lys or His residue at one position and an Asp or Glu residue at a second position, wherein the positions are at sequence locations that align with Arg 100 and Glu 210 of SEQ ID NO:
- 1, respectively;
- having an Arg, Lys or His residue at one position and an Asp or Glu residue at a second position, wherein the positions are at sequence locations that align with Arg 141 and Glu 153 of SEQ ID NO:
- 1, respectively;
- having an Arg, Lys or His residue at one position and an Asp or Glu residue at a second position, wherein the positions are at sequence locations that align with Glu 153 and Arg 167 of SEQ ID NO:
- 1, respectively;
- having an Arg, Lys or His residue at one position and an Asp or Glu residue at a second position, wherein the positions are at sequence locations that align with Asp 179 and Lys 181 of SEQ ID NO:
- 1, respectively;
- having an Arg, Lys or His residue at one position and an Asp or Glu residue at a second position, wherein the positions are at sequence locations that align with Lys 181 and Asp 185 of SEQ ID NO:
- 1, respectively;
- having an Arg, Lys or His residue at one position and an Asp or Glu residue at a second position, wherein the positions are at sequence locations that align with Asp 186 and Arg 190 of SEQ ID NO:
- 1, respectively;
- having an Arg, Lys or His residue at one position and an Asp or Glu residue at a second position, wherein the positions are at sequence locations that align with Arg 194 and Glu 196 of SEQ ID NO:
- 1, respectively;
- having an Arg, Lys or His residue at one position and an Asp or Glu residue at a second position, wherein the positions are at sequence locations that align with Arg 216 and Asp 219 of SEQ ID NO:
- 1.
43 more elements
An isolated clan C glycosyl hydrolase that comprises one or more substituted residues selected from the group consisting of:
- having an Arg, Lys or His residue at one position and an Asp or Glu residue at a second position, wherein the positions are at sequence locations that align with Glu 4 and Arg 47 of SEQ ID NO:
- 1, respectively;
- having an Arg, Lys or His residue at one position and an Asp or Glu residue at a second position, wherein the positions are at sequence locations that align with Arg 8 and Glu 29 of SEQ ID NO:
- 1, respectively;
- having an Arg, Lys or His residue at one position and an Asp or Glu residue at a second position, wherein the positions are at sequence locations that align with Asp 10 and Arg 12 of SEQ ID NO:
- 1, respectively;
- having an Arg, Lys or His residue at one position and an Asp or Glu residue at a second position, wherein the positions are at sequence locations that align with Asp 10 and Arg 20 of SEQ ID NO:
- 1, respectively;
- having an Arg, Lys or His residue at one position and an Asp or Glu residue at a second position, wherein the positions are at sequence locations that, align with Asp 13 and Arg 20 of SEQ ID NO 1, respectively;
- having an Arg, Lys or His residue at one position and an Asp or Glu residue at a second position, wherein the positions are at sequence locations that align with Glu 35 and Arg 216 of SEQ ID NO:
- 1, respectively;
- having an Arg, Lys or His residue at one position and an Asp or Glu residue at a second position, wherein the positions are at sequence locations that align with Arg 47 and Asp 49 of SEQ ID NO:
- 1, respectively;
- having an Arg, Lys or His residue at one position and an Asp or Glu residue at a second position, wherein the positions are at sequence locations that align with Asp 51 and Arg 100 of SEQ ID NO:
- 1, respectively;
- having an Arg, Lys or His residue at one position and an Asp or Glu residue at a second position, wherein the positions are at sequence locations that align with His 67 and Glu 203 of SEQ ID NO:
- 1, respectively;
- having an Arg, Lys or His residue at one position and an Asp or Glu residue at a second position, wherein the positions are at sequence locations that align with Arg 79 and Glu 83 of SEQ ID NO:
- 1, respectively;
- having an Arg, Lys or His residue at one position and an Asp or Glu residue at a second position, wherein the positions are at sequence locations that align with Arg 80 and Glu 83 of SEQ ID NO:
- 1, respectively;
- having an Arg, Lys or His residue at one position and an Asp or Glu residue at a second position, wherein the positions are at sequence locations that align with Arg 80 and Glu 196 of SEQ ID NO:
- 1, respectively;
- having an Arg, Lys or His residue at one position and an Asp or Glu residue at a second position, wherein the positions are at sequence locations that align with Asp 86 and Arg 88 of SEQ ID NO:
- 1, respectively;
- having an Arg, Lys or His residue at one position and an Asp or Glu residue at a second position, wherein the positions are at sequence locations that align with Arg 88 and Glu 177 of SEQ ID NO:
- 1, respectively;
- having an Arg, Lys or His residue at one position and an Asp or Glu residue at a second position, wherein the positions are at sequence locations that align with Arg 88 and Asp 179 of SEQ ID NO:
- 1, respectively;
- having an Arg, Lys or His residue at one position and an Asp or Glu residue at a second position, wherein the positions are at sequence locations that align with Arg 100 and Glu 210 of SEQ ID NO:
- 1, respectively;
- having an Arg, Lys or His residue at one position and an Asp or Glu residue at a second position, wherein the positions are at sequence locations that align with Arg 141 and Glu 153 of SEQ ID NO:
- 1, respectively;
- having an Arg, Lys or His residue at one position and an Asp or Glu residue at a second position, wherein the positions are at sequence locations that align with Glu 153 and Arg 167 of SEQ ID NO:
- 1, respectively;
- having an Arg, Lys or His residue at one position and an Asp or Glu residue at a second position, wherein the positions are at sequence locations that align with Asp 179 and Lys 181 of SEQ ID NO:
- 1, respectively;
- having an Arg, Lys or His residue at one position and an Asp or Glu residue at a second position, wherein the positions are at sequence locations that align with Lys 181 and Asp 185 of SEQ ID NO:
- 1, respectively;
- having an Arg, Lys or His residue at one position and an Asp or Glu residue at a second position, wherein the positions are at sequence locations that align with Asp 186 and Arg 190 of SEQ ID NO:
- 1, respectively;
- having an Arg, Lys or His residue at one position and an Asp or Glu residue at a second position, wherein the positions are at sequence locations that align with Arg 194 and Glu 196 of SEQ ID NO:
- 1, respectively;
- having an Arg, Lys or His residue at one position and an Asp or Glu residue at a second position, wherein the positions are at sequence locations that align with Arg 216 and Asp 219 of SEQ ID NO:
- 1;
42 more elements
A crystallized molecule or molecular complex comprising a family 12 glycosyl hydrolase obtainable from Rhodotermus marinus.
Dependencies are inferred from the claim text.