Construction of Engineered Water-soluble PQQ Glucose Dehydrogenase with Improved Substrate Specificity |
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Authors: | Koji Sode Satoshi Igarashi Akifumi Morimoto Hiromi Yoshida |
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Institution: | Department of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16 Nakamachi, Koganei, Tokyo 184-8588 Japan |
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Abstract: | This was the first study that achieved a narrowing of the substrate specificity of water soluble glucose dehydrogenase harboring pyrroloquinoline quinone as their prosthetic group, PQQGDH-B. We conducted the introduction of amino acid substitutions into the loop 6BC region of the enzyme, which made up the active site cleft without directly interacting with the substrate, and constructed a series of site directed mutants. Among these mutants, Asn452Thr showed the least narrowed substrate specificity while retaining a similar catalytic efficiency, thermal stability and EDTA tolerance as the wild-type enzyme. The relative activities of mutant enzyme with lactose were lower than that of the wild-type enzyme. The altered substrate specificity profile of the mutant enzyme was found to be mainly due to increase in Km value for substrate than glucose. The predicted 3D structures of Asn452Thr and the wild-type enzyme indicated that the most significant impact of the amino acid substitution was observed in the interaction between the 6BC loop region with lactose. |
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Keywords: | Pyrroloquinoline Quinone (PQQ) Glucose Dehydrogenase Substrate Specificity Protein Engineering Glucose Sensor β-Propeller Protein |
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