Effect of pH and Ionic Strength on Immobilization

The coupling of enzymes to epoxy-activated carriers is commonly carried out at high ionic strength, because a salt-induced association between the macromolecule and the support surface increases the effective concenhation of nucleophilic groups on the protein close to the epoxide reactive sites, thus favoring the immobilization process [68, 69]. However, the salt concenhation needed for immobilizing an enzyme is highly dependent on the nature of the biocatalyst [69]. 

For the above reasons, the effect of pH and ionic shength on the immobihzation of the fructosyitransferase from A. aculeatus on Sepabeads EC-EP was studied. Although this enzyme constitutes only a minor percentage (0.4% w/w) of the total proteins present in Pectinex Ulha SP-L, the commercial preparation was used directly (without enzyme purification) in order to develop a simple process that could be easily scaleable in the industry. It was considered that, although the other proteins present in Pectinex Ulha SP-L could also be immobilized in Sepabeads EC-EP, they would not interfere in fructooligosaccharide synthesis experiments. 

In order to bind the enzyme to the support using different functional groups, the immobilization was performed at two pH values (5.5 and 9.0), adjusting the pH of commercial Pectinex Ultra SP-L with potassium phosphate or sodium carbonate, respectively. At pH 5.5, the reactive groups of the protein are the carboxylic 

It is worth noting that a low buffer concentration also resulted in higher activity with both supports [56]. The highest activity of the biocatalyst (15.2 U/g) was achieved with Sepabeads EC-EP5, using 0.3 M sodium carbonate (pH 9.0). With Sepabeads EC-EP3, a shghtly acidic pH (5.5) and low buffer concentration (0.3 M) yielded the highest biocatalyst activity (4.2 U/g). The incubation time was also analyzed a further increase from 24 to 72 h did not result in a significant improvement in biocatalyst activity. 

Based on the above results at low ionic strength, the direct immobilization of Pectinex Ultra SP-L, the pH of which is 4.8, on Sepabeads EC-EP was assayed 

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