[nife]-hydrogenases having an improved resistance to...

Details [nife]-hydrogenases having an improved resistance to... [nife]-hydrogenases having an improved resistance to...

C - Chemistry, Metallurgy

12

N

C12N 9/02 (2006.01) C12N 15/00 (2006.01) C12N 15/53 (2006.01) C12N 1/15 (2006.01) C12N 1/21 (2006.01) C12N 5/10 (2006.01)

Patent

CA 2695491

The invention relates to [NiFe] -hydrogenases having an improved resistance to dioxygen, said [NiFe]-hydrogenases may be obtained by: - providing an initial polynucleotide comprising a sequence encoding a large subunit of a [NiFe] -hydro genase, said large subunit comprising the following peptide motifs: .cndot. L1 : RGXE, wherein X = L, I, F, V or M .cndot. L2: [R/K]X1C[G/R]X2C, wherein Xi is any amino acid residue, X2 = L, V, I or M; L1 and L2 being separated by 16 any amino acid residues; .cndot. L3: X1X2X3X4X5X6X7X8X9X10X11X12[D/S/E], wherein X1 = D, S, N or E, X2 = H, D, S, N or L, X5 = H, S, A, Q or W, X6 = F, T, Y or G, X9 = L, F, M or Y, the other X n being any amino acid residue; .cndot. L4: D[P/I/S]CX1X2CX3X4[H/R], wherein X2 = A.S.V.G or T, X1, X3 and X4 are any amino acid residue .cndot. and optionally comprising a motif LO: R[I/V/A]EG[H/D/A]. - modifying said initial polynucleotide in order to substitute at least one of the residues X2 of motif L2 andZor X4 of motif L3 andZor X9 of motif L3 of said large subunit by a methionine.

L'invention concerne des hydrogénases [NiFe] présentant une meilleure résistance au dioxygène, lesdites hydrogénases [NiFe] peuvent être obtenues : - en fournissant un polynucléotide initial contenant une séquence codant une grosse sous-unité d'hydrogénase [NiFe], ladite grosse sous-unité contenant les motifs peptidiques suivant : L1 : RGXE, où X = L, I, F, V ou M L2 : [R/K]X1C[G/R]X2C, où Xi est n'importe quel résidu d'acide aminé, X2 = L, V, I ou M ; L1 et L2 étant séparés par 16 résidus d'acide aminé quelconque ; L3 : X1X2X3X4X5X6X7X8X9X10X11X12[D/S/E], où X1 = D, S, N ou E, X2 = H, D, S, N ou L, X5 = H, S, A, Q ou W, X6 = F, T, Y ou G, X9 = L, F, M ou Y, les autres Xn étant n'importe quel résidu d'acide aminé ; L4 : D[P/I/S]CX1X2CX3X4[H/R], où X2 = A.S.V.G ou T, X1, X3 et X4 sont n'importe quel résidu d'acide aminé et contenant facultativement un motif LO : R[I/V/A]EG[H/D/A]. - et en modifiant ledit polynucléotide initial pour substituer au moins un résidu choisi parmi le résidu X2 du motif L2 et/ou le résidu X4 du motif L3 et/ou le résidu X9 du motif L3 de ladite grosse sous-unité par une méthionine.

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