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aPGA modified with 1 MED and 10-3 mMEDAC at pH 4.75.

aPGA modified with 1 MED and 10-3 mMEDAC at pH 4.75.

suspension was then gently stirred for 3 h at 25°C, pH 10.0 (see Note 11).

2. When immobilization was performed at pH 10.0, full immobilization of PGA was observed within the first moments, using both nonmodified and aminated PGA. Residual activity was near 100% in both cases.

At pH 9.0, it was not possible to immobilize the nonmodified enzyme. Only the aminated enzyme could be rapidly and fully immobilized on the support (Table 1). This fact could be explained because the pK value of e-NH2 of external lysines is 10.7, whereas the pK value of the artificially introduced primary amino groups is 9.2; therefore, they are even more reactive at pH 9.0 than the superficial lysines at pH 10.0.

3. The activity of both immobilized and soluble PGA was measured as follows: the initial reaction rates were measured using an automatic titrator (DL50 Mettler Toledo) to determine the amount of phenylacetic acid formed. The assays were carried out by adding aliquots of PGA to 10 mL of a 10 mM penicillin G in 0.1 M sodium phosphate/0.5 M NaCl, pH 8.0, at 25°C. The reaction mixture was titrated with 100 |M NaOH, stored at 25°C, and mechanically stirred.

One international unit (U) of PGA activity was defined as the amount of enzyme that hydrolyzes 1 mmol of penicillin G/min at pH 8.0 at 25°C.

3.5.3. Structural Stabilization Via Multipoint Covalent Attachment

1. The aminated PGA immobilized on glyoxyl agarose at pH 10.0, presented a half life around two times higher than the unmodified immobilized PGA (that is 10,000-fold more stable than the one point immobilized PGA) (4) (see Fig. 2).

2. The stability of the derivatives immobilized at pH 9.0 was lower than the ones immobilized at pH 10.0. However, the stability was greatly improved if, after immobilization at pH 9.0, the pH value was increased at pH 10.0 to favor the reaction of the lysines of the protein surface with the aldehyde groups on the support. This derivative was found to be around twice as stable than the aminated PGA directly immobilized at pH 10.0 (see Fig. 3). At pH 10.0, the enzyme is immobilized by the region/s with the highest density of lysines plus primary amino groups introduced by the chemical modification, whereas at pH 9.0, the immobilization proceeds through the area with the highest density of primary amino groups introduced by chemical modification. This different orientation may permit a higher number of enzyme support links to be established and/or the implication in the immobilization of a region critical in the inactivation of the enzyme.

Fig. 2. Thermal inactivation of different PGA glyoxyl derivatives immobilized at pH 10.0. Thermal inactivation was carried out at 66°C, pH 7.0. (A) PGA immobilized onto glyoxyl-agarose; (■) PGA immobilized on glyoxyl-agarose and then modified with 1 M ED and 10-3M EDAC; (•) PGA modified with 1 MEDA in the presence of 10-3 MEDAC and after immobilized onto glyoxyl-agarose.

Fig. 2. Thermal inactivation of different PGA glyoxyl derivatives immobilized at pH 10.0. Thermal inactivation was carried out at 66°C, pH 7.0. (A) PGA immobilized onto glyoxyl-agarose; (■) PGA immobilized on glyoxyl-agarose and then modified with 1 M ED and 10-3M EDAC; (•) PGA modified with 1 MEDA in the presence of 10-3 MEDAC and after immobilized onto glyoxyl-agarose.

Fig. 3. Thermal inactivation courses of PGA derivatives immobilized at different pH values. (♦) Aminated PGA immobilized at pH 9.0 onto glyoxyl-agarose; (•) aminated PGA immobilized at pH 10.0 onto glyoxyl-agarose; (■) aminated PGA immobilized at pH 9.0 onto glyoxyl-agarose and incubated at pH 10.0 for 3 h.

Fig. 3. Thermal inactivation courses of PGA derivatives immobilized at different pH values. (♦) Aminated PGA immobilized at pH 9.0 onto glyoxyl-agarose; (•) aminated PGA immobilized at pH 10.0 onto glyoxyl-agarose; (■) aminated PGA immobilized at pH 9.0 onto glyoxyl-agarose and incubated at pH 10.0 for 3 h.

3.6. Improving the Stabilization of Glutaryl Acylase by Multipoint Covalent Attachment on Glyoxyl Supports Via Partial Amination of the Protein Surface

3.6.1. Enzyme Amination 1. 5 mL Soluble glutaryl 7-ACA acylase (GA) from Pseudomonas spp. was added to 45 mL of 1 MED, pH 6.0, containing 10 mM of EDAC (see Note 12). Because of enzyme inactivation the amination process was carried out at pH 6.0 instead of pH 4.7. This yields a modification of the carboxylic groups between 40 and 50%, which means that about 15 amino groups were added to the only 9 Lys presented in the surface of GA. The activity of the aminated enzyme decreased 20%. However, the modification presented a negligible effect on the enzyme stability.

Fig. 4. Immobilization course of nonmodified GA (A) and aminated GA (B) onto glyoxyl-agarose at pH 10.0. ♦, Supernatant; suspension; reference suspension (see Subheading 3.3.). Immobilization was performed in 100 mM sodium carbonate buffer, pH 10.0 at 25°C.

3.6.2. Enzyme Immobilization

1. 10 g Glyoxyl agarose 6 BCL was added to 100 mL of a GA solution (10 mL of aminated or nonmodified GA in 90 mL of 0.1 M potassium bicarbonate buffer [pH 9.0 or 10.0]). The suspension was then gently stirred for 24 h at 25°C, pH 10.0 (see Note 11).

When the immobilization was carried out at pH 10.0, the unmodified enzyme showed a very slow immobilization rate onto glyoxyl agarose support (50% of the immobilization was achieved after 4 h), whereas the modified GA was almost fully immobilized after 1 h (see Fig. 4). The slow immobilization rate of the nonmodified enzyme could be explained by the low amount of lysine residues in the surface of GA, critical when using a support like glyoxyl that requires a multipoint immobilization of the enzyme (24,26). The use of aminated GA, with around threefold more amino groups, permitted a much more rapid immobilization. The immobilization promoted a decrease in GA activity: by 50%, when using the nonmodified enzyme and by 40%, when using the aminated GA.

At pH 9.0, it was not possible to immobilize the nonmodified enzyme. However, the aminated enzyme could be rapidly and fully immobilized on the support (Table 2). This could be explained because the artificially introduced primary amino groups are more reactive at pH 9.0 than the superficial lysines.

2. Enzyme activity was evaluated using an automatic titrator (DL50 Mettler Toledo) to titrate the release of glutaric acid produced by the hydrolysis of 10 mM solution of glutaryl 7-ACA in 0.1 M potassium phosphate buffer, pH 7.0. A 25mM NaOH solution was employed to titrate the reaction mixture.

One unit of GA activity was defined as the amount of enzyme that is necessary to produce 1 |imol of glutaric acid/min in the previously described conditions.

3.6.3.Structural Stabilization Via Multipoint Covalent Attachment

1. The immobilization of the nonmodified GA enzyme on glyoxyl agarose at pH 10.0 improved its stability by 26-fold. Nevertheless, when the aminated enzyme was immobilized onto glyoxyl agarose at the same pH, the stabilization factor was fivefold higher than when prepared with the nonmodified enzyme (see Fig. 5).

Table 2

Immobilization of Soluble Modified GA Onto Glyoxyl-Agarose at pH 9.0 a

Table 2

Immobilization of Soluble Modified GA Onto Glyoxyl-Agarose at pH 9.0 a

Enzyme

Modification

Yield of immobilization after 4 h (%)

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