Stability of Immobilized Enzymes on Polymeric Supports

3.8.1. Stability of Immobilized Multimeric Enzymes Immobilized on Ionic Polymer Coated Supports

Invertase from Saccharomyces cerevisiae was immobilized in 5 mM sodium phosphate, pH 7.0, on DEAE-agarose or PEI supports (see Subheading 3.3.), with an immobilization yield higher than 90% in both cases, after only 5 min of enzyme-support contact. The activity was fully preserved during the immobilization

Enzyme Invertase

Fig. 8. Desorption of invertase from S. cerevisae adsorbed on different anion exchanging supports at growing ionic strength. (A) Activity released from DEAE-agarose; (•) activity released from Sepabeads-PEI by incubation with increasing NaCl concentrations. Enzyme was immobilized at pH 7.0 as described in Subheading 3., with a protein loading of 25 mg/g of support. Desorption was performed at pH 7.0.

Fig. 8. Desorption of invertase from S. cerevisae adsorbed on different anion exchanging supports at growing ionic strength. (A) Activity released from DEAE-agarose; (•) activity released from Sepabeads-PEI by incubation with increasing NaCl concentrations. Enzyme was immobilized at pH 7.0 as described in Subheading 3., with a protein loading of 25 mg/g of support. Desorption was performed at pH 7.0.

protocol in both cases. The salt concentration at which the enzyme began to be desorbed from the support was much higher using PEI supports than a DEAE support (see Fig. 8). Although all the enzyme was desorbed from DEAE-agarose at only 100 mM of NaCl, invertase began to be partially desorbed from the PEI support at more than 200 mM of NaCl (see Subheading 3.4 .).

The invertase from S. cerevisiaeremained fully active at pH levels between 5.5 and 8.5 at 4°C for 24 h. The immobilization could therefore be performed onto PEI supports in this wide range of pH values.

The immobilization rate was very high in all cases and the maximum load of the support was similar (around 30-35 mg of enzyme per gram of support), with activity retentions higher than 95% in all cases.

The stability of the soluble or immobilized (see Fig. 9A) enzyme at acidic pH values was higher than the stability at alkaline pH values. Curiously, the stability of the enzyme immobilized at pH 8.5 was higher than that of the enzyme immobilized at pH 5.5 when both preparations were inactivated at pH 4.5 at either 50°C. 55°C (see Fig. 9B), or 60°C (see Subheading 3.5.).

This result suggests that the different immobilized enzymes could present some differences promoted by the pH value of the immobilization. It has been described that invertase presents different compositions of multimeric structures when the pH value is changed (18-20). Also, it has been reported that PEI-coated supports may permit the stabilization of multimeric enzymes by implicating several sub-units in the immobilization (9). Thus, if the enzyme presented a different aggregation form during the immobilization, this structure seems to be preserved after the immobilization.

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