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Fig. 6. Effect of pH in the multisubunit covalent immobilization of GA on Eupergit 250- IDA-Co supports. Lane 1, crude extract from E. coli; lane 2, supernatant after immobilization on Eupergit 250-IDA-Co supports; lane 3, supernatant after boiling in the presence of SDS of the derivatives incubated during 10 h at pH 8.5; lane 4, supernatant after boiling in the presence of SDS, the previous derivative that was further incubated during 1 h at pH 10.0; lane 5, supernatant after boiling in the presence of SDS, the derivative from lane 3 after incubation during 8 h at pH 10.0; lane 6, supernatant after boiling in the presence of SDS the derivative from lane 3 after incubation during 24 h at pH 10.0. The immobilized preparations were in all cases blocked with mercaptoethanol before the desorptions.

The incubation of the enzyme at pH 10.0 produced a rapid, progressive decrease in the amount of desorbed enzyme subunits. Thus, after 24 h of incubation, there was no detectable release of the two enzyme subunits after desorptions treatment. This result suggests that a multipoint covalent attachment between the enzyme and support was produced; that is, each enzyme molecule was attached by at least one bond per protein subunit to the support (see Fig. 6, lane 6).

2. The structural stabilization of GA was associated with an increase in enzyme stability. Figure 7 shows that the immobilized derivative further incubated at pH 10.0 for 24 h (i.e., containing both subunits covalently attached to the support) was much more stable than the soluble enzyme but also presented multiphase inactivation, suggesting some heterogeneity in the degree of enzyme-support attachment. The average increase in the enzyme's half-life was around 100-fold when compared with the soluble enzyme, although a high percentage of immobilized enzyme presented greatly increased stabilization (>1000-fold more stable than soluble enzyme).

3.6. One-Step Purification and Covalent Immobilization of a Thermophilic Poly-His-Tagged fi-Galactosidase From Thermus sp. Strain T2

1. 1 g Sepabeads FP-EP or Sepabeads FP-EP IDA-epoxy was incubated with 10 mL of crude protein extract containing poly-His-tagged fi-galactosidase dissolved in 50 mM sodium phosphate pH 7.0 (see Note 5). Aliquots were withdrawn at regular time intervals, and the protein content of the supernatant and the activity of supernatant and/or suspension were analyzed (17). The immobilization was

Enzyme Immobilization

Fig. 7. Effect of the immobilization and further enzyme-support incubation of the derivative on the thermal stability of poly-His-tagged GA. Inactivation courses of different GA derivatives were performed at 45°C in 50 mMphosphate buffer, pH 7.5. (•) soluble enzyme; (■) derivative immobilized at pH 8.5; (A) previous derivative further incubated at pH 10.0 during 24 h.

Fig. 7. Effect of the immobilization and further enzyme-support incubation of the derivative on the thermal stability of poly-His-tagged GA. Inactivation courses of different GA derivatives were performed at 45°C in 50 mMphosphate buffer, pH 7.5. (•) soluble enzyme; (■) derivative immobilized at pH 8.5; (A) previous derivative further incubated at pH 10.0 during 24 h.

Fig. 8. Immobilization course of crude extract of poly-His-tagged P-galactosidase from Thermus spp. Strain T2, on IDA-Co-Epoxy-chelate support. (A) enzyme activity of the immobilization suspension; (■) enzyme activity on the supernatant of the immobilization suspension.

stopped when covalent immobilization of the proteins was verified as described in Subheading 3.3., steps 5-7. The use of Sepabeads FP-EP IDA-epoxy allowed the preparation of an immobilized enzyme derivative almost fully loaded with poly-His-tagged protein using as starting material a crude protein extract with only 3 to 5% of enzyme content. Moreover, the residual activity of the enzyme derivative was virtually 100 % (see Fig. 8).

2. To confirm selectivity of immobilization, the enzyme derivatives were boiled in the presence of one volume of 2% SDS, and the supernatants analyzed by SDS-PAGE. The immobilization on the bifunctional support seemed to be much more

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