Glutaryl-7aca Acylase Immobilization

Fig. 2. Mechanism of the proteins immobilization on chelate supports. The IDA-Me+ supports are used to immobilize the proteins after they are incubated in the presence of mercaptoethanol to block the remaining epoxy groups and to release the Me2+ from the support.

Fig. 3. Selective adsorption of poly-His-tagged proteins on lowly activated IDA-Co2+ epoxy supports.

Here, we present the preparation and use of this kind of heterofunctional support and its use in the one-step immobilization, purification, and stabilization of several poly-His-tagged proteins.

2. Materials

1. Epoxy Sepabeads® (FP-EP E902 P05) from Resindion SRL (Mitsubishi Chemical, Milan, Italy; see Note 1).

2. Eupergit® 250 from Degussa (40 pmols of epoxy groups per wet gram of support; see Note 1).

3. Iminodiacetic acid disodium salt monohydrate (IDA) was from Fluka (Buchs, Switzerland).

4. Support modification buffer: 0.1 Msodium borate, 2 ^iminodiacetic acid, pH 8.5.

5. Metal containing solution: 0.05 Msodium phosphate buffer, pH 6.0, 1.0 MNaCl, and 5 mg/mL of CoCl2 .

6. Protein immobilization buffer: 0.05, pH 7.0, adjusted with 5 MNaOH.

7. Desorption buffer: 0.1 Mimidazole in 5.0 mM sodium phosphate buffer, pH 7.0.

8. Imidazole was purchased from Merck (Darmstadt, Germany).

9. Incubation buffer: 0.1 Msodium phosphate buffer, adjusted to pH 8.5 or 10.0 with 5 M NaOH. In some instances, some additives could be added to keep enzyme activity, e.g., inhibitors. (See Note 2; not able to react with the epoxy supports.)

10. Blocking solution: 3 Mglycine, pH 8.5.

3. Methods

3.1 Preparation of IDA-Epoxy Support

1. Incubate 10 g of Sepabeads or Eupergit in 18 mL of support modification buffer (see Note 3) under very gentle stirring for 2 h at 25°C.

2. Allow modification of around 5% of the epoxy-groups in the support.

3. Filter and wash the support with an excess of distilled water.

3.2 Preparation of IDA Epoxy-Metal-Chelate Support

1. Resuspended 10 g of IDA epoxy support (see Subheading 3.1.) in the metal containing solution for 2 h.

2. Allow chelation of all IDA groups in the support.

3. Filter and wash the support thoroughly with distilled water.

4. The metal content was analyzed by atomic adsorption spectroscopy.

3.3 Immobilization of Proteins on Epoxy Supports

1. The proteins were dissolved in the immobilization buffer (see Subheading 2.) and a sample was taken as reference (see Note 2).

2. The support was then added to the enzyme solution and submitted to a gently stirring (see Note 3).

3. Samples of supernatant and suspension were periodically taken. Supernatant was achieved by using pipet filter or by centrifugation of the suspension.

4. 2.5 mL Enzymatic suspension were taken and dried under vacuum filter.

5. The wet (but without inter-particle water) support was resuspended in 2.5 mL of desorption buffer. The suspension was left under stirring at 25°C for 30 min.

6. The activity or the protein concentration of the supernatant was checked.

7. If there are no activity releases then covalent attachment was considered.

8. The immobilized preparation was then washed five times with three volumes of incubation buffer and resuspended in three volumes of that buffer. Stirring is not necessary in this step.

9. The immobilized protein was left to interact with the support anywhere from 1 d to 1 wk. Activity of the immobilize preparations could be followed all along the incubation.

10. The immobilized preparations were vacuum dried and resuspended in three volumes of blocking solution under gentle stirring.

11. If the elimination of the metal was desired then the immobilized preparation was incubated with mercaptoethanol or EDTA.

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

3.4. One-Step Purification and Covalent Immobilization of Glutaryl

Acylase

1. 5 g Eupergit 250 IDA-epoxy or Eupergit 250 was suspended in 15 mL of 0.05 M or 1 M sodium phosphate buffer, pH 8.5, respectively, at 25°C. Then, protein extract (glutary acylase [GA]) from Escherichia coli, (1 mg/mL) was added (see Note 4). Periodically, samples were withdrawn and the protein content of the supernatant and GA activity of supernatant and/or suspension was analyzed (17). Other samples of the support were incubated in presence of 0.1 M of imidazole, and both activity of supernatant and suspension were analyzed to study the covalent immobilization. This treatment was enough to desorb the entire enzyme (GA) from the fully modified chelate supports, without any remaining reactive epoxy group. After 24 h at pH 8.5, most of the enzyme was covalently attached to the bifunctional support (see Fig. 4). Interestingly, the immobilization protocol promoted only a slight decrease in enzyme activity (around 25%).

2. To determine the degree of purity of the immobilized enzyme, the enzyme derivatives were boiled in the presence of one volume of 2% sodium dodecyl sulfate (SDS). In this way, any molecule not covalently attached to the support was released into the medium. Then, SDS-polyacrylamide gel electrophoresis (PAGE), (18) analysis of the supernatant was performed and the gel was stained with Coomassie blue R-250 and analyzed by densitometry. Figure 5 shows that most of the desorbed enzyme subunits belonged to GA, demonstrating the high selectivity of the Eupergit 250 IDA-epoxy immobilization protocol. However, when using other less specific adsorption protocols (e.g., conventional hydro-phobic Eupergit 250 and an immobilization at high ionic strength), many different proteins can be detected in the supernatant after desorption treatment.

3. The activity of GA was evaluated in the hydrolysis of glutaryl 7-ACA in 0.05 M sodium phosphate buffer, pH 7.5, by titration of the glutaric acid released during the reaction by using 0.025 M NaOH.

3.5. Structural Stabilization of Glutaryl Acylase on Eupergit 250

IDA-Epoxy Supports Via Multipoint Covalent Attachment

1. Immobilized enzymes derivatives obtained in Subheading 3.4. were submitted to different incubation protocols after the adsorption process. Then, to analyze the degree of covalent attachment, they were boiled in the presence of SDS and the supernatants analyzed by SDS-PAGE. After enzyme immobilization at pH 8.5, it was possible to release most of the large P-subunits from the support (see Fig. 6), and there was therefore a lower proportion of small subunit (containing the poly-His-tag), which accounts for the percentage of enzyme physically adsorbed on the support at that time.

Poly His Tagged

Fig. 4. Immobilization course of poly-His-tagged GA on Eupergit 250-IDA-Co support. Immobilizations were performed at pH 8.5 and 25°C in 50 mM sodium phosphate. (A) Enzyme activity of the immobilization suspension; (•) enzyme activity on the supernatant of the immobilization suspension; (■) enzyme activity on the supernatant of the immobilization suspension after addition of 0.1 M imidazole (to release all enzyme molecules adsorbed only through the chelate groups of the support, but not covalently attached via epoxy groups to the support).

Fig. 4. Immobilization course of poly-His-tagged GA on Eupergit 250-IDA-Co support. Immobilizations were performed at pH 8.5 and 25°C in 50 mM sodium phosphate. (A) Enzyme activity of the immobilization suspension; (•) enzyme activity on the supernatant of the immobilization suspension; (■) enzyme activity on the supernatant of the immobilization suspension after addition of 0.1 M imidazole (to release all enzyme molecules adsorbed only through the chelate groups of the support, but not covalently attached via epoxy groups to the support).

Enzyme Immobilization

Fig. 5. Analysis by SDS-PAGE of the proteins (from a crude protein extract containing poly-His-tagged glutarayl acylase) immobilized on differently modified Eupergit 250 supports. Desorption of the proteins from the supports was carried out as previously described, in the case of the immobilized enzymes after the blocking of the epoxides with mercaptoethanol. Lane 1: molecular weight markers; lane 2: crude extracts from E. coli; lane 3: proteins in the supernatant after immobilization of the crude protein extracts on Eupergit 250-IDA-Co2+ at pH 7.0 and 15 mM sodium phosphate; lane 4: proteins released from the previous Eupergit 250-IDA-Co2+ derivatives by boiling it in the presence of SDS; lane 5: proteins in the supernatant after immobilization of crude protein extract on Eupergit 250 at pH 7.0 and 1 M sodium phosphate; lane 6: proteins released from the previous Eupergit 250 derivative by boiling in it the presence of SDS.

Fig. 5. Analysis by SDS-PAGE of the proteins (from a crude protein extract containing poly-His-tagged glutarayl acylase) immobilized on differently modified Eupergit 250 supports. Desorption of the proteins from the supports was carried out as previously described, in the case of the immobilized enzymes after the blocking of the epoxides with mercaptoethanol. Lane 1: molecular weight markers; lane 2: crude extracts from E. coli; lane 3: proteins in the supernatant after immobilization of the crude protein extracts on Eupergit 250-IDA-Co2+ at pH 7.0 and 15 mM sodium phosphate; lane 4: proteins released from the previous Eupergit 250-IDA-Co2+ derivatives by boiling it in the presence of SDS; lane 5: proteins in the supernatant after immobilization of crude protein extract on Eupergit 250 at pH 7.0 and 1 M sodium phosphate; lane 6: proteins released from the previous Eupergit 250 derivative by boiling in it the presence of SDS.

— large subunit

— small subunit

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