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

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

    Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

    Resumé

    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.
    OriginalsprogEngelsk
    TidsskriftEnzyme and Microbial Technology
    Vol/bind105
    Sider (fra-til)45-50
    Antal sider6
    ISSN0141-0229
    DOI
    StatusUdgivet - 2017

    Emneord

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

    Citer dette

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    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.",
    keywords = "Pre-steady state kinetics, Burst phase, Transient kinetics, Insoluble substrate, Cellobiohydrolase, Endoglucanase, Pre-steady state kinetics, Burst phase, Transient kinetics, Insoluble substrate, Cellobiohydrolase, Endoglucanase",
    author = "Olsen, {Johan Pelck} and Jeppe Kari and Kim Borch and Peter Westh",
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    A quenched-flow system for measuring heterogeneous enzyme kinetics with sub-second time resolution. / Olsen, Johan Pelck; Kari, Jeppe; Borch, Kim; Westh, Peter.

    I: Enzyme and Microbial Technology, Bind 105, 2017, s. 45-50.

    Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

    TY - JOUR

    T1 - A quenched-flow system for measuring heterogeneous enzyme kinetics with sub-second time resolution

    AU - Olsen, Johan Pelck

    AU - Kari, Jeppe

    AU - Borch, Kim

    AU - Westh, Peter

    PY - 2017

    Y1 - 2017

    N2 - 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.

    AB - 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.

    KW - Pre-steady state kinetics

    KW - Burst phase

    KW - Transient kinetics

    KW - Insoluble substrate

    KW - Cellobiohydrolase

    KW - Endoglucanase

    KW - Pre-steady state kinetics

    KW - Burst phase

    KW - Transient kinetics

    KW - Insoluble substrate

    KW - Cellobiohydrolase

    KW - Endoglucanase

    U2 - 10.1016/j.enzmictec.2017.06.009

    DO - 10.1016/j.enzmictec.2017.06.009

    M3 - Journal article

    VL - 105

    SP - 45

    EP - 50

    JO - Enzyme and Microbial Technology

    JF - Enzyme and Microbial Technology

    SN - 0141-0229

    ER -