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Construction of Mutant Glucose Oxidases with Increased Dye-Mediated Dehydrogenase Activity
Department of Biotechnology, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Nakamachi, Koganei, Tokyo 184-8588, Japan
Ultizyme International Ltd., 1-13-16, Minami, Meguro, Tokyo 152-0013, Japan
* Author to whom correspondence should be addressed.
Received: 20 September 2012; in revised form: 23 October 2012 / Accepted: 26 October 2012 / Published: 2 November 2012
(This article belongs to the Special Issue Flavins
Abstract: Mutagenesis studies on glucose oxidases (GOxs) were conducted to construct GOxs with reduced oxidase activity and increased dehydrogenase activity. We focused on two representative GOxs, of which crystal structures have already been reported—Penicillium amagasakiense GOx (PDB ID; 1gpe) and Aspergillus niger GOx (PDB ID; 1cf3). We constructed oxygen-interacting structural models for GOxs, and predicted the residues responsible for oxidative half reaction with oxygen on the basis of the crystal structure of cholesterol oxidase as well as on the fact that both enzymes are members of the glucose/methanol/choline (GMC) oxidoreductase family. Rational amino acid substitution resulted in the construction of an engineered GOx with drastically decreased oxidase activity and increased dehydrogenase activity, which was higher than that of the wild-type enzyme. As a result, the dehydrogenase/oxidase ratio of the engineered enzyme was more than 11-fold greater than that of the wild-type enzyme. These results indicate that alteration of the dehydrogenase/oxidase activity ratio of GOxs is possible by introducing a mutation into the putative functional residues responsible for oxidative half reaction with oxygen of these enzymes, resulting in a further increased dehydrogenase activity. This is the first study reporting the alteration of GOx electron acceptor preference from oxygen to an artificial electron acceptor.
Keywords: glucose oxidase; glucose dehydrogenase; FAD; oxygen; site directed mutagenesis
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Horaguchi, Y.; Saito, S.; Kojima, K.; Tsugawa, W.; Ferri, S.; Sode, K. Construction of Mutant Glucose Oxidases with Increased Dye-Mediated Dehydrogenase Activity. Int. J. Mol. Sci. 2012, 13, 14149-14157.
Horaguchi Y, Saito S, Kojima K, Tsugawa W, Ferri S, Sode K. Construction of Mutant Glucose Oxidases with Increased Dye-Mediated Dehydrogenase Activity. International Journal of Molecular Sciences. 2012; 13(11):14149-14157.
Horaguchi, Yohei; Saito, Shoko; Kojima, Katsuhiro; Tsugawa, Wakako; Ferri, Stefano; Sode, Koji. 2012. "Construction of Mutant Glucose Oxidases with Increased Dye-Mediated Dehydrogenase Activity." Int. J. Mol. Sci. 13, no. 11: 14149-14157.