[NiFe]-hydrogenases having an improved resistance to dioxygen, process for obtaining them and their applications

A [NiFe]-hydrogenase encoded by a polynucleotide obtained by a process comprising:

• providing an initial polynucleotide comprising a sequence encoding a large subunit of a wild-type O2 sensitive [NiFe]-hydrogenase having a hydrogenase activity, said large subunit comprising the following peptide motifs:
• L1:
• RGXE, wherein X=I, F, V or M
• L2:
• [R/K]X1C[G/R]X2C, wherein X1 is any amino acid residue, X2=L, V, I or M;
• L1 and L2 being separated by 16 any amino acid residues;
• 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 Xn being any amino acid residue;
• 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
• and optionally comprising a motif L0:
• R[I/V/A]EG[H/D/A];
• modifying said initial polynucleotide in order to substitute the residues X2 of motif L2 of said large subunit by a methionine, wherein said initial polynucleotide is an operon further comprising the sequence(s) encoding the other subunit(s) of said [NiFe]-hydrogenase;
• wherein said [NiFe]-hydrogenase has an equivalent hydrogenase activity and an improved dioxygen tolerance when compared with the O2, sensitive [NiFe]-hydrogenase from which it is derived.