Urease Immobilization by Molecular Recognition

The first step for enzyme entrapment is the synthesis of a biotinylated ure-ase. The amount of enzyme immobilized is calculated by kinetic measurements. Assuming that the enzyme keeps its activity after immobilization, the weight of immobilized enzyme is given by:

Wt = Wi - Wim - Wr, where Wt, Wi, Wim, and Wr are the weight of enzyme attached to the textile, the weight of enzyme in the initial solution, the weight of enzyme in the solution after textile immersion, and the weight of enzyme in the rinsed solution, respectively.

Textile activity is obtained by spectrophotometric measurement of the urea consumed by the enzyme reaction. The kinetic parameters corresponding to the activity of the immobilized enzyme are calculated from the absorbance curve versus time (Fig. 13).

As far as the specific activity is concerned, this parameter is determined by the ratio of enzyme textile activity to the weight of enzyme. With an initial weight of enzyme in solution of 0.41 mg, absorbance measurements gave a final weight of enzyme on the textile of 0.081, 0.151, and 0.26 mg for carboxylic acid, quaternary ammonium, and tertiary amine textile, respectively. The corresponding specific activities are 1617,927, and 504 U/mg for carboxylic acid, ammonium, and tertiary amine textiles, respectively.

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Fig. 13. Urea concentration versus time at the exit of the column; flow rate = 1 ml/min. N Tert Tertiary amine, Carbox carboxylic acid, N Quat quaternary ammonium

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Fig. 14. Stability curve obtained from the free urease and immobilized urease on three different textile supports: carboxylic acid (carbox, pink squares), quaternary ammonium (Quater,yellow triangles), and tertiary amine (tert, green crosses)

Figure 14 represents the variations with time of the specific urease activity of textiles bearing different reactive groups in comparison with that of the corresponding enzyme in solution. The activity of free enzyme in solution decreases almost linearly in the first days of storage. Conversely, the textile activity remains more stable after 24 h (or a few days) and was even shown to be almost constant over a period of 45 days in the case of carboxylic acid textile. The biomimetic immobilization process allows the maintenance of good stability for the enzyme by protecting the it from deactivation. The enzyme immobilized on the textile kept 57% of its initial activity in solution. This result demonstrates the efficiency of this immobilization method as compared to the covalent grafting method (Yakup Arica 2000). An increase in enzyme activity after a few days of storage has already been observed in the case of glucose oxidase immobilized in a polymer matrix (Coche-Guerente et al. 1995).

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