Variants of PQQ-dependent glucose dehydrogenase having improved substrate specificity
The invention relates to novel PQQ-dependent soluble glucose dehydrogenases (sPQQGDH) which have an increased substrate specificity compared with the wild type, and also to methods for production and identification thereof.
1. A soluble pyrroloquinoline-quinone-dependent glucose dehydrogenase (sPQQGDH), comprising the amino acid sequence of SEQ ID NO: 22, having, compared with the sPQQGDH wild-type strain LMD 79.41 from Acinetobacter calcoaceticus , an insertion of three to five amino acids between positions Q168 and L169
wherein said insertion is selected from the group consisting of:
(a) an insertion of 3 amino acids, wherein the inserted amino acids have an A or G at position 1, a Y, F, or W at position 2, and a Q, L, V, or I at position 3,
(b) an insertion of 4 amino acids, wherein the inserted amino acids have an A or G at position 1, a G, D, or L at position 2, an R or A at position 3, and an M or V at position 4, and
(c) an insertion of 5 amino acids, wherein the inserted amino acids have an A, M, S, or V at position 1, an E, G, or S at position 2, an H, K, R, S, or T at position 3, an F, H, I, L, N, V or Y at position 4, and an F, H, L, N, Q, T, or Y at position 5.
2. The sPQQGDH as claimed in claim 1 , which arises by mutagenesis from a strain selected from the group consisting of PT16, KOZ62, KOZ65, PTN69, KG106, PTN26, PT15, KGN80, KG140, KGN34, KGN25, and KGN 100.
3. The sPQQGDH as claimed in claim 1 , having an insertion sequence from the group consisting of sequences SEQ ID 1 to SEQ ID 18.
4. A gene which encodes one of the proteins as claimed in claim 1 .
5. A vector, having, in the region of the insertion site, a unique restriction site so that oligonucleotide sequences which encode an sPQQGDH as claimed in claim 1 can be inserted at this site.
6. The vector as claimed in claim 5 having the sequence SEQ ID 27.
7. A method for producing and identifying sPQQGDHs as claimed in claim 1 , said method comprising producing a vector having a unique restriction site in the region of the insertion site in a first step, inserting a multiplicity of different oligonucleotide sequences into the vector in a second step, transforming the recombinant vectors thus obtained into host bacteria and the bacteria are propagated in a third step, screening the sPQQGDHs produced by the bacteria in a fourth step such that both the individual activities of the sPQQGDH on at least two different sugars and the interference of at least one sugar with at least one other one are determined, and selecting those sPQQGDHs which have an improved substrate specificity over the unmodified sPQQGDH of the wild strain in a fifth step.
8. A glucose sensor comprising an sPQQGDH of claim 1 .