title: Modified substrate specificity of pyrroloquinoline quinone glucose dehydrogenase by biased mutation assembling with optimized amino acid substitution.
abstract: A biased mutation-assembling method-that is, a directed evolution strategy to facilitate an optimal accumulation of multiple mutations on the basis of additivity principles, was applied to the directed evolution of water-soluble PQQ glucose dehydrogenase (PQQGDH-B) to reduce its maltose oxidation activity, which can lead to errors in blood glucose determination. Mutations appropriate for the reduction without fatal deterioration of its glucose oxidation activity were developed by an error-prone PCR method coupled with a saturation mutagenesis method. Moreover, two types of incorporation frequency based on their contribution were assigned to the mutations: high (80%) and evens (50%), in constructing a multiple mutant library. The best mutant created showed a marked reduction in maltose oxidation activity, corresponding to 4% of that of the wild-type enzyme, with 35% retention of glucose oxidation activity. In addition, this mutant showed a reduction in galactose oxidation activity corresponding to 5% of that of the wild-type enzyme. In conclusion, we succeeded in developing the PQQGDH-B mutants with improved substrate specificity and validated our method coupled with optimized mutations and their contribution-based incorporation frequencies by applying it to the development.
authors.affiliation: Novartis Pharma K.K., Tsukuba Research Institute, Ohkubo 8, Tsukuba 300-2611, Japan.
authors.forename: Norio
authors.initials: N
authors.lastname: Hamamatsu
authors.forename: Akitoshi
authors.initials: A
authors.lastname: Suzumura
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Nomiya
authors.forename: Masaaki
authors.initials: M
authors.lastname: Sato
authors.forename: Takuyo
authors.initials: T
authors.lastname: Aita
authors.forename: Motowo
authors.initials: M
authors.lastname: Nakajima
authors.forename: Yuzuru
authors.initials: Y
authors.lastname: Husimi
authors.forename: Yasuhiko
authors.initials: Y
authors.lastname: Shibanaka
mesh_terms: D016954:Acinetobacter calcoaceticus / Q000201:enzymology* / Q000235:genetics; D019943:Amino Acid Substitution; D019020:Directed Molecular Evolution; D004789:Enzyme Activation; D004795:Enzyme Stability; D005948:Glucose Dehydrogenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008320:Maltose / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D011994:Recombinant Proteins; D008320:Maltose; D005948:Glucose Dehydrogenases; C033955:glucose dehydrogenase (pyrroloquinoline-quinone)