Surface charge engineering of PQQ glucose dehydrogenase for downstream processing.
The ion-exchange chromatography behavior of recombinant glucose dehydrogenase harboring pyrroloquinoline quinone (PQQGDH) was modified to greatly simplify its purification. The surface charge of PQQGDH was engineered by either fusing a three-arginine tail to the C-terminus of PQQGDH (PQQGDH+Arg3) or by substituting three residues exposed on the surface of the enzyme to Arg by site-directed mutagenesis (3RPQQGDH). During cation exchange chromatography, both surface charge-engineered enzymes eluted at much higher salt concentrations than the wild-type enzyme. After the chromatography purification step, both PQQGDH+Arg3 and 3RPQQGDH appeared as single bands on SDS-PAGE, while extra bands appeared with the wild-type protein sample. Although all tested kinetic parameters of both engineered enzymes are similar to those of wild type, both modifications resulted in enzymes with increased thermal stability. Our achievements have resulted in the greater production of an improved quality PQQGDH by a simplified process.
Koh
Hideharu
H
Department of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan.
Igarashi
Satoshi
S
Sode
Koji
K
D002852:Chromatography, Ion Exchange / Q000379:methods*; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D024861:Genetic Enhancement / Q000379:methods; D005948:Glucose Dehydrogenases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D055672:Static Electricity; D013379:Substrate Specificity; D013499:Surface Properties
D011993:Recombinant Fusion Proteins; D005948:Glucose Dehydrogenases; C033955:glucose dehydrogenase (pyrroloquinoline-quinone)