US 7,732,179 B2 · Mutants of pyrroloquinoline quinone dependent soluble glucose dehydrogenase

Abstract

A mutant of PQQ-dependent soluble glucose dehydrogenase (s-GDH; EC 1.1.5.2) is provided with improved specificity for glucose as compared to maltose, having a substitution of threonine at position 348 by either glycine, alamine or serine, wherein said mutant additionally comprises, at least one mutation for improving the stability of the mutant and one or more mutation(s) for improving the affinity of the mutant to glucose, and/or one or more mutation(s) for further improving the specificity of the mutant for glucose as compared to maltose, and wherein position 348 correspond to the amino acid positions known from the A. calcoaceticus s-GDH wild-type sequence. Also disclosed are genes encoding such mutant s-GDH, and different applications of these s-GDH mutants, particularly for determining the concentration of glucose in a sample.

Claims15

Dependent claims

An isolated mutant of PQQ-dependent soluble glucose dehydrogenase (s-GDH) that relative to the A. calcoaceticus s-GDH wild-type polypeptide of SEQ ID NO:

2 has improved specificity for glucose as compared to maltose, said mutant comprising an s-GDH polypeptide, said s-GDH polypeptide consisting of an amino acid sequence having at least 90% sequence identity with the A. calcoaceticus s-GDH wild-type sequence of SEQ ID NO:
2, wherein said mutant differs from SEQ ID NO:
2 by a substitution at position 348 of either glycine, alanine or serine;
and
a) at least one stabilizing mutation selected from the group consisting of D87R;
N122K;
S124K;
S146G;
V298L and L386F;
b) at least one glucose affinity mutation selected from the group consisting of L110H L110Y;
N229A, N229G, N229S, Q246H, Q246M, Q246N, Y333A;
G339T, M341V, V349A, V349G and V436P, and optionally;
c) one or more glucose specificity mutation(s) selected from the group consisting of Q145P, D163G, D163N, A164F, L169F, Y171G, I208L, I208V, T224I, E245D, G276S, A294D, A294E;
V300A, V300S, V300N, V300Y, V300I, T307G, T323V, A354Y, A354E, A354L, R378I, R378M, R378A, R378D, N428P and insertion 429 P;
and wherein the mutant has s-GDH activity and the designated positions correspond to the amino acid positions known from the A. calcoaceticus s-GDH wild-type sequence of SEQ ID NO:
2.

13 more elements

Dependencies are inferred from the claim text.

Mutants of pyrroloquinoline quinone dependent soluble glucose dehydrogenase

Life of this patent

Mutants of pyrroloquinoline quinone dependent soluble glucose dehydrogenase

Life of this patent

Abstract

Claims15