A quenched-flow system for measuring heterogeneous enzyme kinetics with sub-second time resolution

Johan Pelck Olsen, Jeppe Kari, Kim Borch, Peter Westh

    Research output: Contribution to journalJournal articleResearchpeer-review

    Abstract

    Even though many enzyme processes occur at the interface of an insoluble substrate, these reactions are generally much less studied than homogenous enzyme reactions in the aqueous bulk. Interfacial (or heterogeneous) enzyme reactions involve several reaction steps, and the established experimental approach to elucidate multi-step reactions is transient (or pre steady-state) kinetics. A key requirement for pre steady-state measurements is good time resolution, and while this has been amply achieved in different commercial instruments, they are generally not applicable to precipitating suspensions of insoluble substrate. Perhaps for this reason, transient kinetics has rarely been reported for heterogeneous enzyme reactions. Here, we describe a quenched-flow system using peristaltic pumps and stirred substrate suspensions with a dead time below 100 ms. The general performance was verified by alkali catalyzed hydrolysis of 2,4-dinitrophenyl acetate (DNPA), and the applicability to heterogeneous reactions was documented by two cellulases (Cel7A and Cel7B) acting on suspensions of microcrystalline cellulose (Avicel) at different loads up to 15 g/l. The results showed distinctive differences between the two enzymes. In particular, we found that endo-lytic Cel7B combined very quickly with the substrate and reached the maximal activity within the dead-time of the instrument. Conversely, exo-lytic Cel7A showed a much slower initiation with maximal activity after 5–8 s and a 10-fold lower turnover. We suggest that the instrument may provide an important tool in attempts to elucidate the mechanism of cellulases and other enzymes’ action on insoluble substrate.
    Original languageEnglish
    JournalEnzyme and Microbial Technology
    Volume105
    Pages (from-to)45-50
    Number of pages6
    ISSN0141-0229
    DOIs
    Publication statusPublished - 2017

    Keywords

    • Pre-steady state kinetics
    • Burst phase
    • Transient kinetics
    • Insoluble substrate
    • Cellobiohydrolase
    • Endoglucanase

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