Fungi-derived flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenases (FADGDHs) are the most popular and advanced enzymes for SMBG sensors because of their high substrate specificity toward glucose and oxygen insensitivity. However, this type of FADGDH hardly shows direct electron transfer (DET) ability. In this study, we developed a new DET-type FADGDH by harboring Cytochrome b₅₆₂ (cyt b₅₆₂) derived from Escherichia coli as the electron transfer domain. The structural genes encoding fusion enzymes composed of cyt b₅₆₂ at either the N- or C-terminus of fungal FADGDH, (cyt b₅₆₂-GDH or GDH-cyt b₅₆₂), were constructed, recombinantly expressed, and characteristics of the fusion proteins were investigated. Both constructed fusion enzymes were successfully expressed in E. coli, as the soluble and GDH active proteins, showing cyt b₅₆₂ specific redox properties. Thusconstructed fusion proteins showed internal electron transfer between FAD in FADGDH and fused cyt b₅₆₂. Consequently, both cyt b₅₆₂-GDH and GDH-cyt b₅₆₂ showed DET abilities toward electrode. Interestingly, cyt b₅₆₂-GDH showed much rapid internal electron transfer and higher DET ability than GDH-cyt b₅₆₂. Thus, we demonstrated the construction and production of a new DET-type FADGDH using E.coli as the host cells, which is advantageous for future industrial application and further engineering.

真菌来源的黄素腺嘌呤二核苷酸（FAD）依赖性葡萄糖脱氢酶（FADGDHs）是SMBG传感器最流行和最先进的酶，因为它们对葡萄糖和氧气不敏感的底物特异性很高。但是，这种类型的FADGDH几乎不显示直接电子转移（DET）的能力。在这项研究中，我们通过携带源自大肠杆菌的细胞色素b ₅₆₂（cyt b ₅₆₂）作为电子转移域，开发了一种新的DET型FADGDH 。编码由真菌FADGDH的N或C端的cyt b ₅₆₂组成的融合酶的结构基因（cyt b ₅₆₂ -GDH或GDH-cyt b ₅₆₂），构建，重组表达和研究融合蛋白的特性。两种构建的融合酶均在大肠杆菌中成功表达为可溶性和GDH活性蛋白，显示cyt b _562的特定氧化还原特性。如此构建的融合蛋白在FADGDH的FAD和融合的cyt b ₅₆₂之间表现出内部电子转移。因此，cyt  b ₅₆₂ -GDH和GDH-cyt  b ₅₆₂均显示出对电极的DET能力。有趣的是，与GDH-cyt b ₅₆₂相比，cyt b ₅₆₂ -GDH具有更快的内部电子传递和更高的DET能力。。因此，我们证明了使用大肠杆菌作为宿主细胞的新型DET型FADGDH的构建和生产，这对于将来的工业应用和进一步的工程设计是有利的。