Kinetic Parameters

Kineticanalysiswascarriedout by recordingthesolution substrateconcen-tration of the at the column exit. According to the investigation reported previously (Wang et al. 1996), the immobilized enzyme reactivity in the carbon felt was estimated by considering the residence time of the substrate solution in the membrane. The mean residence time, tr, of solute in the porous material can be calculated using the following equation:

Fig. 27. Effect of pH on the enzyme activity of the membrane

Fig. 27. Effect of pH on the enzyme activity of the membrane where L is membrane thickness and J is volume flux of the solution; J is equal to the ratio of flow to membrane surface. The reaction rate is calculated as V = AC/tr, where AC is the concentration decrease of H2O2 in the flowing solution.

Peroxidase Membrane Case

Figure 28A depicts the relationship between the reaction rate and substrate concentration in the case of a POD membrane. The rate follows a classical enzymatic kinetic curve. The first step corresponds to a linear part (weak substrate concentration). For high substrate concentrations, saturation of the reaction rate is observed and the maximal value obtained is due to saturation of active enzyme sites.

Figure 28B shows the corresponding Lineweaver-Burk plot for the immobilized POD membrane. From the obtained slope (KM/VM) and intercept (1/VM) for the linear relationship, both KM and VM are estimated to be 1.45 X 10-4 M and 1.8 X 10-3 M/s, respectively.

Catalase Membrane Case

In order to use the enzyme membrane for H2O2 dismutation, immobilization of catalase has been achieved. A simplified scheme for the mechanism underlying the catalase reaction may be described by the following two equations (Chance 1948; Nicoholls and Schonbaum 1963):

Fig.28. Reaction rate of H2O2 dismutation with POD-polypyrrole membrane (A) and Lineweaver-Burk plot of the POD-polypyrrole membrane (B); residence time, tr = 42 s, membrane thickness 1 cm, flow rate 1 ml/min

where cat(OH)4 is native catalase and cat(OH)3OOH the oxidized enzyme complex.

A kinetic study was carried out both with the peroxidase and the cata-lase membrane in order to understand the effect of immobilization by avidin-biotin technology. In some cases, the treatment of the kinetics re-

Table 6. Kinetic parameters: Michaelis constants (VM and Km) for catalase and peroxidase


k (/s/mg)

Km (mol/l)

Fm (mol/l/s)

Free catalase



3.26x 10-2

Biotinylated catalase

9x 10-4


9.5x 10-3


Immobilized catalase

5x 10-2



3x 10-2

Immobilized peroxidase

1.45x 10-4


suits with a classical Lineweaver-Burk plot gives a negative value for the extrapolated reaction rate. This suggests that the maximum reaction rate of enzymatic reaction is negative, which does not make any sense. For instance, for cases in which zero or negative y-intercepts are obtained, the KM is much larger than the total experimentally accessible substrate concentration and the simplified Michaelis-Menten model does not apply. The same phenomenon has been observed previously with another enzymes such as lipase (Freeman et al. 2000). This is the reason why in Table 6 we preferred to use the catalytic constant (k) to compare the activity of the different enzymes.

The catalytic constant (expressed as per seconds per milligram normalized to a constant amount of enzyme) for the immobilized catalase is defined as the ratio of the slope of the curve reaction rate versus substrate concentration. This value is four times lower than that of the free catalase. This result is in good agreement with those values obtained for catalase immobilized on cellulose (Eremin et al. 1995). However, it is interesting to note that for catalase, biotinylation of the enzyme decreases the rate by a factor of about 10, whilst immobilization enhances the rate as compared to that of the biotinylated enzyme in solution. The KM value drastically increases when catalase is immobilized by avidin-biotin recognition. This value is not a significant physical parameter as it corresponds to an unrealistic concentration of H2O2. At these high concentrations the catalase should be totally deactivated by the substrate (Vasudevan and Weiland 1990). Enzyme immobilization, for instance in polymeric hydrogels (Arica et al. 1999), has been shown to exhibit a large KM value. A strong increase in the value of KM means that the kinetic parameters are affected by enzyme immobilization, presumably due to the steric effects, which significantly decrease the catalytic reaction rate.

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