Construction of Cellulose Binding Domain Fusion FMN-Dependent NADH-Azoreductase and Glucose 1-Dehydrogenase for the Development of Flow Injection Analysis with Fusion Enzymes Immobilized on Cellulose
Open Access
- 21 May 2019
- journal article
- research article
- Published by The Japanese Society of Applied Glycoscience in Journal of Applied Glycoscience
- Vol. 66 (2), 65-72
- https://doi.org/10.5458/jag.jag.jag-2018_0011
Abstract
The cellulose binding domain (CBD) of cellulosome-integrating protein A from Clostridium thermocellum NBRC 103400 was genetically fused to FMN-dependent NADH-azoreductase (AZR) and glucose 1-dehydrogenase (GDH) from Bacillus subtilis. The fusion enzymes, AZR-CBD and CBD-GDH, were expressed in Escherichia coli Rosetta-gami B (DE3). The enzymes were purified from cell-free extracts, and the specific activity of AZR-CBD was 15.1 U/mg and that of CBD-GDH was 22.6 U/mg. AZR-CBD and CBD-GDH bound strongly to 0.5 % swollen cellulose at approximately 95 % and 98 % of the initial protein amounts, respectively. After immobilization onto the swollen cellulose, AZR-CBD and CBD-GDH retained their catalytic activity. Both enzymes bound weakly to 0.5 % microcrystalline cellulose, but the addition of a high concentration of microcrystalline cellulose (10 %) improved the binding rate of both enzymes. A reactor for flow injection analysis was filled with microcrystalline cellulose-immobilized AZR-CBD and CBD-GDH. This flow injection analysis system was successfully applied for the determination of glucose, and a linear calibration curve was observed in the range of approximately 0.16–2.5 mM glucose, with a correlation coefficient, r, of 0.998.Keywords
This publication has 14 references indexed in Scilit:
- Cellulosome-based, Clostridium-derived multi-functional enzyme complexes for advanced biotechnology tool development: Advances and applicationsBiotechnology Advances, 2013
- Structure of a family 3a carbohydrate-binding module from the cellulosomal scaffoldin CipA ofClostridium thermocellumwith flanking linkers: implications for cellulosome structureActa Crystallographica Section F Structural Biology and Crystallization Communications, 2013
- Enzyme immobilization: an overview on techniques and support materials3 Biotech, 2012
- The Unique Binding Mode of Cellulosomal CBM4 from Clostridium thermocellum Cellobiohydrolase AJournal of Molecular Biology, 2010
- Preparation of novel magnetic cellulose microspheres via cellulose binding domain–streptavidin linkage and use for mRNA isolation from eukaryotic cells and tissuesJournal of Chromatography A, 2009
- Characterization of a NADH:Dichloroindophenol Oxidoreductase fromBacillus subtilisBioscience, Biotechnology, and Biochemistry, 2007
- A Transition from Cellulose Swelling to Cellulose Dissolution by o-Phosphoric Acid: Evidence from Enzymatic Hydrolysis and Supramolecular StructureBiomacromolecules, 2006
- Matrix-Assisted Refolding of Single-Chain Fv– Cellulose Binding Domain Fusion ProteinsProtein Expression and Purification, 1999
- Applications of oxidoreductasesCurrent Opinion in Biotechnology, 1999
- Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4Nature, 1970