A fusion enzyme composed of an Aspergillus flavus-derived flavin adenine dinucleotide glucose dehydrogenase (AfGDH) and an electron transfer domain of Phanerochaete chrysosporium-derived cellobiose dehydrogenase (Pcyb) was previously reported to show the direct electron transfer (DET) ability to an electrode. However, its slow intramolecular electron transfer (IET) rate from the FAD to the heme, limited the sensor signals. In this study, fusion FADGDH (Pcyb-AfGDH) enzymes were strategically redesigned by performing docking simulation, following surface-electrostatic potential estimation in the predicted area. Based on these predictions, we selected the amino acid substitution on Glu324, or on Asn408 to Lys to increase the positive charge at the rim of the interdomain region. Pcyb-AfGDH mutants were recombinantly produced using Pichia pastoris as the host microorganism, and their IET was evaluated. Spectroscopic observations showed that the Glu324Lys (E324K) and Asn408Lys (N408K) Pcyb-AfGDH mutants showed approximately 1.70- and 9.0-fold faster IET than that of wildtype Pcyb-AfGDH, respectively. Electrochemical evaluation revealed that the mutant Pcyb-AfGDH-immobilized electrodes showed higher DET current values than that of the wildtype Pcyb-AfGDH-immobilized electrodes at pH 6.5, which was approximately 9-fold higher in the E324K mutant and 15-fold higher in the N408K mutant, than in the wildtype. Glucose enzyme sensors employing N408K mutant was able to measure glucose concentration under physiological condition using artificial interstitial fluid at pH 7.4, whereas the one with wildtype Pcyb-AfGDH was not. These results indicated that the sensor employed the redesigned mutant Pcyb-AfGDH can be used for future continuous glucose monitoring system based on direct electron transfer principle. (247 words).

由黄曲霉衍生的黄素腺嘌呤二核苷酸葡萄糖脱氢酶（AfGDH）和Phanerochaete chrysosporium-的电子转移域组成的融合酶以前有报道称衍生的纤维二糖脱氢酶（Pcyb）具有向电极的直接电子转移（DET）能力。但是，其从FAD到血红素的分子内电子转移（IET）速度很慢，限制了传感器信号。在这项研究中，融合FADGDH（Pcyb-AfGDH）酶通过对接模拟对策略进行了重新设计，随后对预测区域中的表面静电势进行了估计。基于这些预测，我们选择了Glu324或Asn408上的氨基酸取代Lys，以增加域间区域边缘的正电荷。使用巴斯德毕赤酵母重组产生Pcyb-AfGDH突变体作为宿主微生物，并评估其IET。光谱观察表明，Glu324Lys（E324K）和Asn408Lys（N408K）Pcyb-AfGDH突变体的IET分别比野生型Pcyb-AfGDH快1.70和9.0倍。电化学评估显示，在pH 6.5时，突变型Pcyb-AfGDH固定电极显示的DET电流值高于野生型Pcyb-AfGDH固定电极，在E324K突变体中大约高9倍，而在pH 6.5时高15倍。 N408K突变体，而非野生型。使用N408K突变体的葡萄糖酶传感器能够在生理条件下使用人工组织液在pH 7.4下测量葡萄糖浓度，而具有野生型Pcyb-AfGDH的葡萄糖酶传感器则无法。这些结果表明采用重新设计的突变型Pcyb-AfGDH的传感器可用于基于直接电子转移原理的未来连续葡萄糖监测系统。（247个字）。