Covalent Attachment





Fig. 1. Chemical modification for achieving a more stable immobilized preparation.

3. Glycidol (2,3-epoxy propanol) was purchased from Sigma-Aldrich (St. Louis, MO; see Note 2).

4. Support oxidation solution: 0.1 Msodium periodate in water.

5. Protein immobilization buffer: 0.1 Mbicarbonate buffer, pH 10.0; see Note 3).

3. Methods

3.1. Enzyme Amination

1. Add 5 mL of 11 mg/mL soluble enzyme to 45 mL of protein amination solution (see Note 4).

2. Stir gently for 90 min at 25°C and then add 10 mL of a 0.5 M hydroxylamine solution, pH 7.0.

3. Dialyze the enzyme solution five times with 50 volumes of a 25 mM potassium phosphate buffer and store at 4°C.

3.2. Preparation of Glyoxyl-Supports

3.2.1. Activation of Agarose Gel to Glyceryl-Agarose

1. Wash 105 g (150 mL) of commercial agarose 6BCL or 10BCL thoroughly with distilled water (see Note 5).

2. Suspend the agarose in distilled water up to a total volume of 180 mL.

3. Add 50 mL of 1.7 NNaOH solution containing 3.4 g of sodium borohydride to this suspension.

4. Take the vessel to an ice bath; while gently stirring add dropwise 36 mL of glycidol (see Note 6).

5. Gently stir the suspension for 18 h at 25°C.

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

3.2.2. Oxidation of Glyceryl-Agarose to Glyoxyl-Agarose

1. Resuspend 105 g of glyceryl-agarose (see Subheading 3.2.1.) in 1500 mL of distilled water.

2. Add the proper amount of support oxidation solution slowly to this suspension while stirring (see Note 7).

3. Gently stir the suspension for 2 h.

4. Wash the support with an excess of distilled water and filter it to dryness.

3.3. Enzyme Immobilization

1. Incubate 10 g glyoxyl-agarose with 100 mL of chemically aminated or nonmodified enzyme solution prepared in immobilization buffer.

2. Add 1 mL of distilled water to 9 mL of enzyme solution to be used as reference solution (see Note 8).

3. Gently stir the mixtures by end-over-end rotation at 25°C.

4. Aliquots of supernatant and suspension were withdrawn at regular time intervals (see Note 9) to assay enzyme activity.

5. Assay the activity of the reference solution using the same time intervals and aliquot volumes as in step 4.

6. The immobilization process finishes when activity of the supernatant is zero.

7. Then, the immobilized preparation was washed five times with three volumes of immobilization buffer.

3.4. Structural Stabilization by Multipoint Covalent Attachment

1. Resuspend the enzyme derivative (see Subheading 3.3.) in 100 mL of immobilization buffer.

2. Maintain the suspension for several hours (see Note 10). Although stirring is not necessary because the enzyme is already immobilized on the support, stirring is necessary to measure the activity of the suspension.

3. Add 100 mg of solid sodium borohydride and stir the suspension for 30 min at

4. Finally, wash the enzyme derivative with 25 mM sodium phosphate, pH 7.0, while vacuum filtering to eliminate the borohydride.

5. Enzyme derivatives are stored at 4°C.

3.5. Improving the Stabilization of PGA by Multipoint Covalent

Attachment on Glyoxyl Supports Via Partial Amination of the Protein


3.5.1. Enzyme Amination

1. 5 mL Soluble Penicillin G Acylase (PGA) from Escherichia coli was incubated with 45 mL of protein amination solution containing EDAC to a final concentration of 10-3 M. This protocol of amination allows the modification of between 40 and 50% of the external carboxylic groups. This means that the modified enzyme has added to the 41 amines from the Lys, 12-14 new amino groups were generated via the amination. The modification presented negligible effect in the enzyme activity, but decreased the stability of the enzyme (by a factor of eightfold). However, as the full modification of PGA carboxylic groups caused a greater destabilization, the partial modification of the carboxylic groups with ED was chosen to carry out further studies.

3.5.2. Penicillin G Acylase Immobilization

1. 10 g Glyoxyl agarose 10 BCL were added to 100 mL of aminated or nonmodified PGA solution (0.55 mg protein/mL) in 100 mM sodium bicarbonate buffer, pH 9.0 to 10.0, containing 100 mM phenylacetic acid and 20% glycerol (4). The

Table 1

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

Table 1

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



Yield of immobilization after 4 h (%)

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