Eupergit C For Pga Immobilized

Fig. 4. Blockage of remainder epoxy groups with different compounds reactive with epoxy groups.

4. Assay the enzyme activity of the reference solution using the same time intervals as in step 3. The immobilization process is finished when the activity of the supernatant is zero.

3.3. Desorption of Proteins Noncovalently Immobilized on the Support

1. 2.5 mL Enzymatic suspension were taken and dried by filtration under vacuum filter.

2. The dried support was resuspended in 2.5 mL of distilled water. The suspension was left under mild stirring at 20°C for 30 min.

3. The enzyme activity or the protein concentration of the supernatant was checked.

4. If there are no proteins released, covalent attachment was considered.

3.4. Multi-Interaction Step on Enzyme Immobilization

1. The immobilized preparation was washed 5 times with 3 vol of 100 mMsodium phosphate buffer, pH 10.0, and then resuspended in 3 vol of that solution. Stirring is not necessary in this step.

2. The immobilized protein was left to interact with the support for varying times ranging from 1 d to 1 wk prior to incubation with 3 M glycine, pH 8.5. Activity of the immobilized preparations could be followed over the course of the incubation (e.g., every day) (see Note 8).

3.5. Blocking of Epoxy Groups of the Supports

1. The immobilized preparations were vacuum dried and resuspended in 3 vol 3 M glycine, pH 8.5, under gentle stirring for 8 h at 20°C.

2. Finally, the enzyme preparation was washed with distilled water and stored at 4°C.

3.6. Immobilization of Penicillin G-Acylase on Sepabeads EC-EP1

1. Prepare 90 mL of penicillin G-acylase solution (1 mg/mL) (Antibióticos S.A.) in 1 M sodium phosphate solution, pH 7.0, with 100 mMphenylacetic acid and 20% (v/v) glycerine (see Note 4).

2. Assay the catalytic activity of this solution. Add 10 g of Sepabeads EC-EP1 and assay the enzyme activity of both the suspension and supernatant at different immobilization times.

3. Gently stir the suspension for 24 h at 25°C.

4. After stirring, evaluate the covalent immobilization as described in Subheading 3.3., step 1.

5. Incubate the enzyme suspension in 100 mMsodium phosphate buffer, pH 10, and gently stir the enzyme-support reaction for 5 d. The optimization process should be carried out as described in Fig. 5A,B. The stability was improved when the previously immobilized derivative was incubated at an increasing pH until an optimal pH of 9.0-10.0 was reached, with only a slight decrease in enzyme activity (see Fig. 5A). The stability also improved after incubation for extended periods of time, the optimal being 4-d incubation at pH 9.0 (see Fig. 5B).

6. Filter the suspension and then block the epoxy groups as described in Subheading 3.5., step 1.

7. Wash and filter the suspension with 25 mM phosphate buffer, pH 7.0, and distilled water. Filter to eliminate inter-particle water.

3.7. Immobilization of Penicillin Acylase and Chymotrypsin on Eupergit C

1. Prepare 90 mL of chymotrypsin solution (1 mg/mL) (Sigma-Aldrich) or penicillin G acylase (Antibioticos) in 1 M sodium phosphate solution, pH 8.0.

2. Assay the catalytic activity of this solution. Add 10 g of Eupergit C and assay the enzyme activity of both the suspension and supernatant at different immobilization times.

4. After stirring, evaluate covalent immobilization as described in Subheading 3.3.

5. Incubate the enzyme suspension in 100 mM sodium bicarbonate buffer, pH 10.0, and gently stir the enzyme-support reaction for 5 d (see Note 6).

6. Filter the suspension and then block the epoxy groups as described in Subheading 3.5., step 1.

7. Wash and filter the suspension with 25 mM phosphate buffer, pH 8.0, and distilled water as described in Subheading 3.5., step 2.

8. Filter to dryness.

3.8. Thermal Stabilization of Chymotrypsin- and PGA-Eupergit C

Derivatives (see Table 1)

1. Enzymes were immobilized as in Subheading 3.7.

2. Relative stability refers to the increase in the half life of the enzyme derivative as compared with the half life of the enzyme immobilized under standard conditions (pH 7.0 and 25°C).

4. Notes

1. Sepabeads EC-EP is a commercially available epoxy support (Resindion SRL, Milan, Italy), with different pore sizes: EC-EP1 (small) to EC-EP2 (medium), and finally EC-EP3 (large).

Fig. 5. (A) Effect of the pH of incubation on the stability of penicillin G acylase immobilized on Sepabeads supports. PGA was immobilized in 1 Msodium phosphate at pH 7.0 for 24 h. Afterwards, the pH was increased at the indicated pH value, and the suspension was incubated for 24 h before blocking of the epoxy groups with glycine as described in Subheading 3. (B) Effect of incubation time on the activity/stability of PGA immobilized on Sepabeads EC-EP1. PGA was immobilized at 1 M sodium phosphate at pH 7.0. After 24 h the pH of the immobilization was increased to 9.0 in the presence of phenyl acetic acid and glycerine. Finally, the immobilized preparations were blocked with 3 M glycine at the indicated times. (■) Residual activity; (•) relative stability. Relative stability is given as the ratio between the half-life of the problem preparation divided by the half-life of the preparation just immobilized at pH 7.0 and blocked.

Fig. 5. (A) Effect of the pH of incubation on the stability of penicillin G acylase immobilized on Sepabeads supports. PGA was immobilized in 1 Msodium phosphate at pH 7.0 for 24 h. Afterwards, the pH was increased at the indicated pH value, and the suspension was incubated for 24 h before blocking of the epoxy groups with glycine as described in Subheading 3. (B) Effect of incubation time on the activity/stability of PGA immobilized on Sepabeads EC-EP1. PGA was immobilized at 1 M sodium phosphate at pH 7.0. After 24 h the pH of the immobilization was increased to 9.0 in the presence of phenyl acetic acid and glycerine. Finally, the immobilized preparations were blocked with 3 M glycine at the indicated times. (■) Residual activity; (•) relative stability. Relative stability is given as the ratio between the half-life of the problem preparation divided by the half-life of the preparation just immobilized at pH 7.0 and blocked.

2. Eupergit are oxirane-acrylic commercially available supports from Degussa with different average particle size and pore degrees ranging from C (little) to CM (medium) and finally C 250 L (large).

3. A high concentration of sodium phosphate buffer is required for physical hydrophobic adsorption onto the support.

4. Competitive inhibitor of the enzyme may be required to avoid enzyme inactiva-tion or to preserve the enzyme structure.

Table 1

Effect of Incubation Conditions on the Thermal Stability of Eupergit C-Chymotrypsin and Penicillin G-acylase (PGA)a

Incubation conditions

Table 1

Effect of Incubation Conditions on the Thermal Stability of Eupergit C-Chymotrypsin and Penicillin G-acylase (PGA)a

Incubation conditions

pH

T (°C)

Time (h)

Enzyme

Relative stability

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