Forms of soluble pyrroloquinoline quinone-dependent glucose dehydrogenase
View Patent ↗The present invention relates to improved variants of soluble pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenases (s-GDH), to genes encoding mutated s-GDH, to mutant proteins of s-GDH with improved substrate specificity for glucose, and to different applications of these s-GDH variants, particularly for determining concentrations of sugar, especially of glucose in a sample.
1. A pyrroloquinoline quinone (PQQ)-dependent soluble glucose dehydrogenase (s-GDH) mutant, comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 24 and wherein said mutant comprises an alanine or a serine substitution for threonine at position 348 of SEQ ID NO: 24 and wherein the mutant has s-GDH activity.
2. The mutant of claim 1 , wherein at least one amino acid residue selected from the group consisting of Gln 76 , Asp 143 , Gln 168 , Leu 169 , and Asn 428 , of SEQ ID NO: 24, is substituted with another amino acid.
3. The mutant of claim 1 , wherein the glutamine at position 76 of SEQ ID NO: 24 is substituted with a different amino acid.
4. The mutant of claim 1 wherein the asparagine at position 428 of SEQ ID NO: 24 is substituted with another amino acid.
5. The mutant of claim 4 , wherein at least one amino acid residue selected from the group consisting of Gln 76 , Thr 127 , and Asp 143 , of SEQ ID NO: 24, is substituted with a different amino acid.
6. The mutant of claim 4 , wherein at least one amino acid residue selected from the group consisting of Glu 245 and Met 341 , of SEQ ID NO: 24, is substituted with a different amino acid.
7. The mutant of claim 3 , wherein the glutamine at position 76, of SEQ ID NO: 24, is substituted with an amino acid selected from the group consisting of alanine, methionine, aspartic acid, proline, serine, glycine, and glutamic acid.
8. The mutant of claim 1 wherein amino acid residues other than glutamine and asparagine are present at positions 76 and 428, respectively, of SEQ ID NO: 24.
9. A PQQ-dependent s-GDH mutant, said mutant comprising an amino acid sequence at least 90% identical to SEQ ID NO: 24 wherein said mutant comprises a serine substitution for threonine at position 348 of SEQ ID NO: 24 and wherein the mutant has s-GDH activity.
10. A PQQ-dependent s-GDH mutant, said mutant comprising an amino acid sequence at least 90% identical to SEQ ID NO: 24 wherein said mutant comprises an alanine substitution for threonine at position 348 of SEQ ID NO: 24 and wherein the mutant has s-GDH activity.
11. The mutant of claim 1 further wherein tyrosine, glycine, and serine residues are substituted at positions 227, 348, and 438, respectively, of SEQ ID NO: 24.