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Determination of the inherent kinetics of immobilized glucose oxidase using a diffusion cell

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Abstract

The enzyme glucose oxidase (GO) was covalently immobilized onto a poly(vinyl alcohol) hydrogel, cross-linked with glutardialdehyde and a polyazonium salt. To compare the kinetic parameters of immobilized GO with the known kinetic parameters of soluble GO, the diffusion cell method was used.

Between two compartments, containing solutions with different glucose concentrations, a GO-containing hydrogel membrane was placed. Simultaneous diffusion through and enzymatic reaction in the membrane occurred. In this way diffusional effects of the membrane could be eliminated from the effective kinetic parameters to yield the inherent kinetic parameters.

It appeared that the enzymatic reaction is independent of the oxygen concentration at oxygen concentrations ⩾0.22 mol m−3 (Michaelis constant for oxygen < 0.22 mol m−3). Further, the Michaelis constant for glucose does not change dramatically after immobilizing the enzyme. The maximal reaction rate is depending on the enzyme concentration. As the enzyme concentration in the membrane is not exactly known (mainly due to leakage of enzyme out of the membrane during membrane preparation), only an estimation of the turnover number can be made.

The diffusion cell method is easy to carry out. Still, some recommendations can be made on the performance.

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Abbreviations

α g , α 0x :

partition coefficient of glucose and oxygen, respectively

δ :

thickness of the wetted membrane (m)

A m :

surface area of membrane (m−2)

C :

constant (mol2 m−3)

c g , c 0x :

concentration of glucose and oxygen, respectively (mol m−3)

c g,0 c g,δ :

glucose concentration at the filter-paper/membrane interface next to compartment A and B, respectively (mol m−3)

c g, A c g, B :

glucose concentration in compartment A and B, respectively (mol m−3)

c GO :

glucose oxidase concentration (mol m−3)

D eff :

effective diffusion coefficient (m2 s−1)

D m , D sl :

diffusion coefficient in, respectively, the membrane and the solution layer (m2 s−1)

d dl , d df , d sl :

thickness of, respectively, the diffusion layer, the filter-paper and the solution layer (m)

h B :

initial slope of concentration versus time curve of compartment B (mol m−3 s−1)

J :

flux (mol m−2 s−1)

J 0 δ:

flux in the membrane at membrane/filter-paper interface next to compartment A and B, respectively (mol m−2 s−1)

J A , J B :

flux leaving compartment A and entering compartment B, respectively (mol m−2 s−1)

J m :

flux through the membrane (mol m−2 s−1)

k :

total mass transfer coefficient (m s−1)

k 1 , k 2 :

rate constant of a particular reaction step (m3 mol−1 s−1)

k−1, k−2 :

rate constant of a particular reaction step (s−1)

k cat :

(intrinsic) catalytic constant of turnover number (s−1)

k * cat :

inherent catalytic constant, determined by inserting D m (s−1)

k ** cat :

inherent catalytic constant, determined by inserting D eff (s−1)

k m (g) :

(intrinsic) Michaelis constant for glucose (mol m−3)

k m (o) :

(intrinsic) Michaelis constant for oxygen (mol m−3)

k * m (g) :

inherent Michaelis constant for glucose (mol m−3)

k * m (o) :

inherent Michaelis constant for oxygen (mol m−3)

m GO :

number of moles of GO present (mol)

P m :

permeability of glucose in the mebrane (m s−1)

P eff :

effective permeability (m s−1)

V :

volume (m3)

v 0 :

initial reaction velocity (mol m−3 s−1)

V ** max :

inherent maximal reaction velocity, determined by inserting Deff (mol m−3 s−1)

x :

distance (m)

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Author notes

  1. S. A. M. van Stroe-Biezen

    Present address: Unilever Research Laboratory, P.O. Box 114, NL-3130, AC Vlaardingen, The Netherlands

Authors and Affiliations

  1. Laboratory of Instrumental Analysis, Faculty of Chemical Technology, Eindhoven University of Technology, P.O. Box 513, NL-5600, MB Eindhoven, The Netherlands

    S. A. M. van Stroe-Biezen, J. M. H. van der Loo, L. J. J. Janssen & F. M. Everaerts

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  1. S. A. M. van Stroe-Biezen
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  2. J. M. H. van der Loo
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  3. L. J. J. Janssen
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  4. F. M. Everaerts
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van Stroe-Biezen, S.A.M., van der Loo, J.M.H., Janssen, L.J.J. et al. Determination of the inherent kinetics of immobilized glucose oxidase using a diffusion cell. Bioprocess Engineering 15, 87–94 (1996). https://doi.org/10.1007/BF00372982

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