Chemical Immobilization of Urease

In order to establish the influence of protein group in avidin-biotin technology on the efficiency of enzyme immobilization, covalent enzyme fixation was carried out with two different routes according to some examples given in literature: using periodate oxidation of cellulose, and using 1,4 phenylenediisothiocyanate as a coupling agent. The first method is based on a direct chemical reaction with cellulose, which brings onto the textile matrix a great number of reactive aldehyde functions. In the second procedure, a coupling reagent intervenes, allowing the grafting of enzymes on the functionalized groups of the textile. The amount of enzyme im-

Table 5. Reaction rate and specific activity for different modified textile measured by spectrophotometry determination of urea concentration (see experimental section)

Table 5. Reaction rate and specific activity for different modified textile measured by spectrophotometry determination of urea concentration (see experimental section)

Periodate

194

52

Tertiary amine

Thiocyanante

40

-

Biotin

131

504

Periodate

156

43

Ammonium

Thiocyanante

38

-

Biotin

140

927

Periodate

189

50

Carboxylic

Thiocyanante

33

Biotin

131

1617

mobilized according these procedures was estimated to lie in the range of 90-95% of the amount of initial enzyme in contact with the textile. The weight of enzyme on a piece of tissue (4 cm2) was about 3.8 mg. The covalent bonding is a highly effective method for irreversible enzyme immobilization; however, experiments devoted to enzyme activity have confirmed that the enzyme is severely deactivated by the chemical treatments involved in the protocols for enzyme covalent bonding onto the textile surface. The large amount of enzyme fixed onto the textile support cannot overcome the effect of deactivation due to these treatments, and as a result, the samples exhibited a poor specific activity as compared to those realized according to the biomimetic route, as depicted in Table 5.

In Table 2, the reaction rate and specific activity values obtained with different immobilization methods are compared. This table shows clearly that avidin-biotin technology provides a good specific activity, in all cases much higher than that obtained with chemical grafting, whichever the chosen functional reactive group brought by the textile. The presence of a protein group such as avidin, close to the enzyme prevents deactivation and as a result preserves the catalytic activity. Moreover, this technology largely extends the stability of the bioactive species over a long period, thanks to the microenvironment created around the immobilized enzyme.

Since urease has been immobilized while maintaining reasonable stability and good efficiency, our immobilization method on the textile support has been generalized to other types of enzymes such as oxidase or decar-boxylase.

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