Yang Yufeng, Huang Lei, Wang Jufang, Xu Zhinan
School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, PR China; Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, PR China; Zunyi Medical College (Zhuhai Campus), Zhuhai, 519041, PR China.
Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, PR China.
Enzyme Microb Technol. 2015 Jan;68:43-9. doi: 10.1016/j.enzmictec.2014.10.002. Epub 2014 Oct 23.
An FAD-dependent glucose dehydrogenase (FAD-GDH) from Aspergillus terreus NIH2624 was expressed in Escherichia coli with a yield of 228±16U/L of culture. Co-expression with chaperones DnaK/DnaJ/GrpE and osmotic stress induced by simple carbon sources enhanced productivity significantly, improving the yield to 23883±563U/L after optimization. FAD-GDH was purified in two steps with the specific activity of 604U/mg. Using d-glucose as substrate, the optimal pH and temperature for FAD-GDH were determined to be 7.5 and 50°C, respectively. Activity was stable across the pH range 3.5-9.0, and the half-life was 52min at 42°C. Km and Vmax were calculated as 86.7±5.3mM and 928±35U/mg, and the molecular weight was approximately 65.6kDa based on size exclusion chromatography, indicating a monomeric structure. The 3D structure of FAD-GDH was simulated by homology modelling using the structure of A. niger glucose oxidase (GOD) as template. From the model, His551, His508, Asn506 and Arg504 were identified as key residues, and their importance was verified by site-directed mutagenesis. Furthermore, three additional mutants (Arg84Ala, Tyr340Phe and Tyr406Phe) were generated and all exhibited a higher degree of substrate specificity than the native enzyme. These results extend our understanding of the structure and function of FAD-GDH, and could assist potential commercial applications.
来自土曲霉NIH2624的一种依赖黄素腺嘌呤二核苷酸（FAD）的葡萄糖脱氢酶（FAD-GDH）在大肠杆菌中表达，培养物产量为228±16U/L。与伴侣蛋白DnaK/DnaJ/GrpE共表达以及由简单碳源诱导的渗透胁迫显著提高了生产力，优化后产量提高到23883±563U/L。FAD-GDH通过两步法纯化，比活性为604U/mg。以d-葡萄糖为底物，FAD-GDH的最佳pH和温度分别确定为7.5和50°C。在pH值3.5 - 9.0范围内活性稳定，在42°C下半衰期为52分钟。根据尺寸排阻色谱法计算，Km和Vmax分别为86.7±5.3mM和928±35U/mg，基于此分子量约为65.6kDa，表明其为单体结构。以黑曲霉葡萄糖氧化酶（GOD）的结构为模板，通过同源建模模拟了FAD-GDH的三维结构。从该模型中，His551、His508、Asn506和Arg504被确定为关键残基，并通过定点诱变验证了它们的重要性。此外，还产生了另外三个突变体（Arg84Ala、Tyr340Phe和Tyr406Phe），它们都表现出比天然酶更高程度的底物特异性。这些结果扩展了我们对FAD-GDH结构和功能的理解，并有助于潜在的商业应用。
