Immobilization of Some Industrial Enzymes on Polymeric Supports

3.7.1. Immobilization of P-Galactosidase From Aspergillus oryzae on

Polyethyleneimine Support

1. P-Galactosidase from A. oryzae preparation (see Subheading 3.3.). was incubated on PEI-Sepabeads (see Subheading 3.1.).

Figure 4 shows the immobilization course of P-galactosidase from A. oryzae onto the optimized PEI support. The immobilization proceeded very rapidly (almost all enzymes were immobilized in less than 1 h), whereas the activity of the immobilized enzyme was kept completely unaltered after the immobilization.

Figure 5 shows that P-galactosidase from A. oryzae remained fully adsorbed on the PEI support even when it was incubated in 150 mM NaCl, whereas most enzymes (around 90%) were released to the medium using commercial DEAE-support at only 90 mM NaCl. In fact, 20% of the P-galactosidase from A. oryzae remaining adsorbed on PEI support even at 1 MNaCl. This immobilized preparation retained more than 80% of the immobilized activity after incubation for one month in 25 mM sodium phosphate, pH 7.0, at 37°C. Therefore, this derivative seemed to have quite good properties if it was to be used under very different conditions. The desorption process was carried out as described in Subheading 3.4.

3.7.2. Immobilization of Chymotrypsin From Bovine Pancreas on Sulfate-

Dextran Support

Chymotrypsin from pancreas was offered (see Subheading 3.3.) onto the sul-

fate-dextran (see Subheading 3.2.) and CMC supports at pH 7.0. The chymot-

Enzyme Immobilization

Fig. 4. Immobilization of P-galactosidase from A. oryzae on optimized PEI support. Immobilization experiment was carried out at pH 7.0 at 4°C in 5 mM sodium phosphate buffer: (•) activity of the suspension; (■) activity of the supernatant. The experiment was carried out as described in Subheading 3.

Fig. 4. Immobilization of P-galactosidase from A. oryzae on optimized PEI support. Immobilization experiment was carried out at pH 7.0 at 4°C in 5 mM sodium phosphate buffer: (•) activity of the suspension; (■) activity of the supernatant. The experiment was carried out as described in Subheading 3.

Fig. 5. Desorption of P-galactosidase from A. oryzae adsorbed on different anion exchanger supports at growing ionic strenghth. (■) Percentage of P-galactosidase activity released from optimized PEI support. (♦) Percentage of activity released from DEAE-agarose. The experiments were carried out as described in Subheading 3.

Fig. 5. Desorption of P-galactosidase from A. oryzae adsorbed on different anion exchanger supports at growing ionic strenghth. (■) Percentage of P-galactosidase activity released from optimized PEI support. (♦) Percentage of activity released from DEAE-agarose. The experiments were carried out as described in Subheading 3.

rypsin preparation (see Subheading 3.3.) was incubated with sulfate-dextran support in immobilization buffer (5 mM phosphate buffer, pH 7.0). The enzyme was rapidly immobilized onto both supports, keeping its activity completely unaltered (see Fig. 6).

Only 35% of adsorbed chymotrypsin on sulfate-dextran was released when it was incubated with 300 mMNaCl at pH 7.0, whereas the chymotrypsin adsorbed on CMC was fully released under these conditions (see Fig. 7).

In all cases, these facts seemed to suggest that this immobilization system was very mild for the enzyme structure, most likely because of the possibility of the polymer becoming adapted to the enzyme instead of forcing the enzyme to be adapted to the support (9-12,17).

Bovine Pancreas

Fig. 6. Immobilization course of chymotrypsin from bovine pancreas on optimized Sul-fate-dextran support and carboxymethyl support. Immobilization experiment was carried out at pH 7.0 at 4°C in 5 mM sodium phosphate buffer. The percentage of adsorbed proteins were measured at pH 7.0 at 25°C as described in Subheading 3. (♦) Activity of all suspensions; (A) activity of the supernatants during immobilization of chymotrypsin on CMC; (•) activity of supernatants during the immobilization of chymotrypsin on optimized sulfate-dextran support.

Fig. 6. Immobilization course of chymotrypsin from bovine pancreas on optimized Sul-fate-dextran support and carboxymethyl support. Immobilization experiment was carried out at pH 7.0 at 4°C in 5 mM sodium phosphate buffer. The percentage of adsorbed proteins were measured at pH 7.0 at 25°C as described in Subheading 3. (♦) Activity of all suspensions; (A) activity of the supernatants during immobilization of chymotrypsin on CMC; (•) activity of supernatants during the immobilization of chymotrypsin on optimized sulfate-dextran support.

Bovine Pancreas

Fig. 7. Desorption of chymotrypsin from bovine pancreas adsorbed on different cation exchanger at growing ionic strength. Immobilization was performed at pH 7.0 in 5 mM sodium phosphate buffer as described in Subheading 3. The enzyme was released from supports by incubation on growing concentration of NaCl. (♦) Activity of all suspensions; (A) activity released from CMC; (■) activity released from optimized sulfate-dextran composites. The experiments were carried out as described in Subheading 3.

Fig. 7. Desorption of chymotrypsin from bovine pancreas adsorbed on different cation exchanger at growing ionic strength. Immobilization was performed at pH 7.0 in 5 mM sodium phosphate buffer as described in Subheading 3. The enzyme was released from supports by incubation on growing concentration of NaCl. (♦) Activity of all suspensions; (A) activity released from CMC; (■) activity released from optimized sulfate-dextran composites. The experiments were carried out as described in Subheading 3.

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