Forms of soluble pyrroloquinoline quinone-dependent glucose dehydrogenase

#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.

#The International Union of Biochemistry and Molecular Biology (IUBMB) has changed the designation of PQQ-dependent glucose dehydrogenase from EC 1.1.99.17 to EC 1.1.5.2. Accordingly, hereafter, the designation of PQQ-dependent glucose dehydrogenase is recited as EC 1.1.5.2.

#Two types of PQQ-dependent glucose dehydrogenase (EC 1.1.5.2) have been characterized: One is membrane-bound (m-GDH), the other is soluble (s-GDH). Both types do not share any significant sequence homology (Cleton-Jansen, A. M., et al., Mol Gen Genet 217 (1989) 430-6; Cleton-Jansen, A. M., et al., Antonie Van Leeuwenhoek 56 (1989) 73-9; Oubrie, A., et al., Proc Natl Acad Sci USA 96 (1999) 11787-91). They are also different regarding both their kinetic as well as their immunological properties (Matsushita, K., et al., Bioscience Biotechnology & Biochemistry 59 (1995) 1548-1555).

#FIG. 1: Nucleotide (DNA) sequence of the Acinetobacter calcoaceticus PQQ-dependent soluble glucose dehydrogenase gene (SEQ ID NO: 23) and the corresponding amino acid sequence (SEQ ID NO: 24).

#FIG. 2: Protein sequences of A. calcoaceticus PQQ-dependent s-GDH (top, SEQ ID NO: 24) and A. baumannii s-GDH (bottom) aligned according to sequence homology.

#FIG. 3: Illustration of pACSGDH vector referred to in Example 1 containing the wild-type or mutated DNA sequences of soluble PQQ-dependent glucose dehydrogenase.

#In a first embodiment the invention relates to a mutant of the soluble form of EC 1.1.5.2 also known as PQQ-dependent soluble glucose dehydrogenase (s-GDH), said mutant characterized in that relative to the corresponding wild-type enzyme and with regard to at least one other selected sugar substrate, it has an at least two-fold improved substrate specificity for glucose.

#The directions given in this invention are easily applied for any known or even yet un-known isolate of s-GDH. These wild-type isolates can be used to assess the relative improvements in specificity for the variants generated therefrom.

#The wild-type enzyme of Acinetobacter calcoaceticus-type strain LMD 79.41 which corresponds to SEQ ID NO: 24 is well-known and well-characterized. In case a different wild-type enzyme under investigation is not well-characterized it is advantageous to use the s-GDH from Acinetobacter calcoaceticus-type strain LMD 79.41 as a reference. In a further preferred embodiment the properties of an improved variant of s-GDH are compared to this wild-type enzyme. The present invention therefore also relates to a mutant of s-GDH said mutant characterized in that relative to the wild-type enzyme of SEQ ID NO: 24 and with regard to at least one other selected sugar substrate, it has an at least two-fold increased substrate specificity for glucose.