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Expression, characterization and mutagenesis of an FAD-dependent glucose dehydrogenase from Aspergillus terreus

Paper ID Volume ID Publish Year Pages File Format Full-Text
16967 42627 2015 7 PDF Available
Title
Expression, characterization and mutagenesis of an FAD-dependent glucose dehydrogenase from Aspergillus terreus
Abstract

•FAD-GDH from A. terreus was successfully expressed in soluble form in E. coli.•Chaperone coexpression and osmotic stress induced by simple carbon sources both proved effective for improving the yield of soluble FAD-GDH.•Enzyme variants with improved substrate specificity were generated using site-directed mutagenesis.

An FAD-dependent glucose dehydrogenase (FAD-GDH) from Aspergillus terreus NIH2624 was expressed in Escherichia coli with a yield of 228 ± 16 U/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 ± 563 U/L after optimization. FAD-GDH was purified in two steps with the specific activity of 604 U/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 52 min at 42 °C. Km and Vmax were calculated as 86.7 ± 5.3 mM and 928 ± 35 U/mg, and the molecular weight was approximately 65.6 kDa 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.

Keywords
FAD-GDH; Chaperones DnaK/DnaJ/GrpE; Osmotic stress response; Site-directed mutagenesis; Substrate specificity
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Expression, characterization and mutagenesis of an FAD-dependent glucose dehydrogenase from Aspergillus terreus
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 68, January 2015, Pages 43–49
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
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Full-text PDF Download
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