Info

Fig. 7. Effect of the presence of lactose on the inactivation of Sepabeads-boronic-epoxy-ß-galactosidase derivative during the incubation with aldehyde-dextran. The assay was carried out in Novo buffer, pH 6.5) at 25°C. Values were reproduced in two separate experiments. All other conditions are detailed in Subheadings 2. and 3. (•) Sepabeads-boronic-epoxy-ß-galactosidase derivative cross-linked with aldehyde-dextran incubated in the presence of 1 M lactose. (■) Sepabeads-boronic-epoxy-ß-galactosidase derivative cross-linked with aldehyde-dextran.

Enzyme Immobilization

Fig. 8. Functional inactivation of optimal cross-linked derivative. Thermal stability course of Sepabeads-boronic-epoxy-P-galactosidase derivative at 80°C. (•) cross-linked with aldehyde-dextran Sepabeads-boronic-epoxy-P-galactosidase; (■) Sepabeads-bo-ronic-epoxy-P-galactosidase derivative cross-linked with aldehyde-dextran and protected with 1 M lactose; and (♦) soluble P-galactosidase.

Fig. 8. Functional inactivation of optimal cross-linked derivative. Thermal stability course of Sepabeads-boronic-epoxy-P-galactosidase derivative at 80°C. (•) cross-linked with aldehyde-dextran Sepabeads-boronic-epoxy-P-galactosidase; (■) Sepabeads-bo-ronic-epoxy-P-galactosidase derivative cross-linked with aldehyde-dextran and protected with 1 M lactose; and (♦) soluble P-galactosidase.

3.7. Stabilization of the Quaternary Structure of a-Amino Acid Ester Hydrolase From Acetobacter turbidans Via Immobilization and Postimmobilization Techniques

3.7.1. Functional Stability

The stability of this tetrameric enzyme in soluble form is quite low and decreased with decreasing enzyme concentration (see Fig. 9), suggesting that some dissociative process may be the key step of the enzyme inactivation. The immobilization of the enzyme onto supports with low-activation degree has a negligible effect on its stability, even when using longer immobilization times (e.g., 24 h). When using these low-activated supports, the dependence of the immobilized enzyme stability on the protein concentration was quite similar to that of the soluble counterpart (see Fig. 9).

On the contrary, when using highly activated supports, the thermal stability of the enzyme was significantly improved. Such stabilization increased when the enzyme-support reaction time increased, reaching its maximum after 24 h of contacting the enzyme with the support. In this derivative, the dependence of the enzyme stability on the protein concentration becomes smaller than in the soluble enzyme, but it is still quite significant.

The modification of such immobilized and partially stabilized derivative with aldehyde-dextran promoted a further stabilization of the enzyme (see Fig. 9). Moreover, the stability of the enzyme derivative did not depend on the concentration of protein. This derivative was much more stable than both the soluble enzyme and the enzyme that was immobilized onto low-activated supports (see Fig. 9). These results suggest that dissociation of the enzyme subunits may be responsible for the low stability of the soluble enzyme.

On the other hand, Fig. 10 shows that the stability of the soluble enzyme was extremely dependent on the phosphate concentration. The enzyme stability was

Enzyme Immobilization

Fig. 9. Thermal inactivation of different preparations of a-amino acid ester hydrolase from A. turbidans. Inactivation was carried out in 25 mM phosphate at 25°C, pH 6.5, using purified enzyme. Solid lines, solid symbols: concentration of protein was 1 |g/mL, Dashed lines, empty symbols: concentration of protein was 10 |g/mL. (A) Soluble enzyme and immobilized in low-activated glutaraldehyde supports. (■) Enzyme immobilized on highly activated glutaraldehdye supports. (♦) Previous derivative further modified with polyaldehyde-dextran.

Fig. 9. Thermal inactivation of different preparations of a-amino acid ester hydrolase from A. turbidans. Inactivation was carried out in 25 mM phosphate at 25°C, pH 6.5, using purified enzyme. Solid lines, solid symbols: concentration of protein was 1 |g/mL, Dashed lines, empty symbols: concentration of protein was 10 |g/mL. (A) Soluble enzyme and immobilized in low-activated glutaraldehyde supports. (■) Enzyme immobilized on highly activated glutaraldehdye supports. (♦) Previous derivative further modified with polyaldehyde-dextran.

very low in the absence of phosphate, and the effect of the protein concentration was even more relevant than in 100 mM phosphate. However, the immobilized and chemically cross-linked derivative remained fully active even in the absence of phosphate ions for very long incubation times (see Fig. 10). These results suggest that the phosphate ions may be somehow related to the maintenance of the quaternary structure of the enzyme.

The immobilization and modification of the enzyme promoted only a slight decrease in its activity (~15-20%) while increasing the operational stability of the diluted enzyme by several orders of magnitude.

3.7.2. Structural Stabilization

Figure 11 shows the SDS-PAGE gel obtained after boiling the different derivatives in the presence of SDS, offering in all cases similar amounts of protein. Lane 2 shows the purified enzyme with a main band corresponding to a MW of around 70 KD (corresponding to the MW of each monomer of this enzyme). After being subjected to the same treatment, the derivative prepared in low-activated glutaral-dehyde support still produced that band, with an intensity of about 70% of that observed for the purified enzyme. This result indicated that only one of the four protein subunits has been covalently attached to the support and may explain why the stability of this derivative was almost identical to that of the soluble enzyme (the possibilities of subunit dissociation would be almost identical).

When analyzing the derivative prepared using highly activated agarose-glutaral-dehyde, after 24 h of enzyme support multi-interaction, some protein could still be released from the support by the desorption treatment, but the relative intensity of the band was now between 30 and 40% (regarding the pure enzyme), suggesting

Enzyme Immobilization

Fig. 10. Effect of phosphate concentration on the stability of a-amino acid ester hydrolase from A. turbidans. Inactivations were performed at 25°C, pH 6.5, with a fixed concentration of protein (5 |g/mL). (♦) Stabilized enzyme derivative at 0 or 100 mM of sodium phosphate. (A) Soluble enzyme in 100 mM phosphate, (A) Soluble enzyme in 100 mM NaCl or water.

Fig. 10. Effect of phosphate concentration on the stability of a-amino acid ester hydrolase from A. turbidans. Inactivations were performed at 25°C, pH 6.5, with a fixed concentration of protein (5 |g/mL). (♦) Stabilized enzyme derivative at 0 or 100 mM of sodium phosphate. (A) Soluble enzyme in 100 mM phosphate, (A) Soluble enzyme in 100 mM NaCl or water.

94,000 67,000

43,000

30,000

20,100

14,400

Was this article helpful?

0 0
Detoxify the Body

Detoxify the Body

Need to Detoxify? Discover The Secrets to Detox Your Body The Quick & Easy Way at Home! Too much partying got you feeling bad about yourself? Or perhaps you want to lose weight and have tried everything under the sun?

Get My Free Ebook


Post a comment