A graphene screen-printed carbon electrode for real-time measurements of unoccupied active sites in a cellulase

Nicolaj Cruys-Bagger, Hirosuke Tatsumi, Kim Borch, Peter Westh

    Research output: Contribution to journalJournal articlepeer-review

    Abstract

    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 idetification 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 measuments. 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.
    Original languageEnglish
    JournalAnalytical Biochemistry
    Volume447
    Pages (from-to)162-168
    Number of pages6
    ISSN0003-2697
    DOIs
    Publication statusPublished - Feb 2014

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