title: Expression, characterization and mutagenesis of an FAD-dependent glucose dehydrogenase from Aspergillus terreus.
abstract: 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.
authors.affiliation: 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.
authors.forename: Yufeng
authors.initials: Y
authors.lastname: Yang
authors.affiliation: Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, PR China.
authors.forename: Lei
authors.initials: L
authors.lastname: Huang
authors.affiliation: School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, PR China. Electronic address: jufwang@scut.edu.cn.
authors.forename: Jufang
authors.initials: J
authors.lastname: Wang
authors.affiliation: Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, PR China. Electronic address: znxu@zju.edu.cn.
authors.forename: Zhinan
authors.initials: Z
authors.lastname: Xu
mesh_terms: D019943:Amino Acid Substitution; D001230:Aspergillus / Q000201:enzymology* / Q000235:genetics; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D005947:Glucose / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D009005:Monosaccharides / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D017354:Point Mutation; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D005656:Fungal Proteins; D009005:Monosaccharides; D011993:Recombinant Fusion Proteins; D005182:Flavin-Adenine Dinucleotide; D005947:Glucose
keywords: Chaperones DnaK/DnaJ/GrpE; FAD-GDH; Osmotic stress response; Site-directed mutagenesis; Substrate specificity