Journal
ANALYTICAL BIOCHEMISTRY
Volume 447, Issue -, Pages 162-168Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2013.11.024
Keywords
Cellulase kinetics; Hypocrea jecorina Cel7A; Cellobiohydrolase; Screen-printed carbon electrodes; Graphene; Nitrophenol
Funding
- Danish Agency for Science, Technology, and Innovation
- Programme Commission on Sustainable Energy and Environment [2104-07-0028]
- Ministry of Education, Culture, Sports, Science, and Technology in Japan [23760746]
- The Scandinavia-Japan Sasakawa Foundation
- Grants-in-Aid for Scientific Research [23760746] Funding Source: KAKEN
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Cellulases hydrolyze cellulose to soluble sugars and this process is utilized in sustainable industries based on lignocellulosic feedstock. Better analytical tools will be necessary to understand basic cellulase mechanisms, and hence deliver rational improvements of the industrial process. In this work we describe a new electrochemical approach to the quantification of the populations of enzyme that are respectively free in the aqueous bulk, adsorbed to the insoluble substrate with an unoccupied active site or threaded with the cellulose strand in the active tunnel. Distinction of these three states appears essential to the identification of the rate-limiting step. The method is based on disposable graphene-modified screen-printed carbon electrodes, and we show how the temporal development in the concentrations of the three enzyme forms can be derived from a combination of the electrochemical data and adsorption measurements. The approach was tested for the cellobiohydrolase Cel7A from Hypocrea jecorina acting on microcrystalline cellulose, and it was found that the threaded enzyme form dominates for this system while adsorbed enzyme with an unoccupied active site constitutes less than 5% of the population. (C) 2013 Elsevier Inc. All rights reserved.
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