Bacteriocin Purification

2.2.1. Obtaining the Crude Extract

1. Ammonium sulfate (Sigma).

2. Refrigerated centrifuge.

3. Distilled water.

2.2.2. Solid-Phase Extraction Protocols

1. Octadecyl (C18) Impaq RG1080 columns, pore diameter 100 A, particle size 80 ^m (Sigma), or C8 columns from Supelco (Supelclean LC-8) with 3-mL tubes (or similar; see Note 2).

2. Acetonitrile and isopropanol (HPLC grade; Merck).

3. Turvo-Vap Evaporator (Zymark, Hopkinton, MA).

4. Distilled water.

2.2.3. Ion Exchange Chromatography

1. SP-Sepharose Fast Flow strong cation exchanger (Amersham Pharmacia Biotech).

4. Refrigerated centrifuge.

5. Distilled water.

2.2.4. Reverse-Phase Chromatography 1. Mobile phase.

a. Enterocin CRL10 mobile phase.

Solution A: 0.1% trifluoroacetic acid (TFA) in distilled water. Solution B: 99% acetonitrile/0.1% TFA.

b. Lactocin 705AL mobile phase. Solution A: 5% isopropanol/0.1% TFA. Solution B: 99.9% isopropanol/0.1% TFA.

2. Column for Enterocin CRL 10: ^Bondapack C18, pore diameter 125 A, particle size 10 ^m; 3.9 x 300 mm.

Column for Lactocin 705AL:Vydac 208 TP C8, particle size 10^m, 4.6 mm x 250 mm.

3. Turbo-Vap evaporator.

4. Distilled water.

2.3. Antimicrobial Activity Assays

3. Sterile hollow punch, 0.5-cm diameter.

4. Indicator or sensitive strain.

5. Distilled water.

3. Methods

3.1. Isolation and Purification of Enterocin CRL10

1. Grow the bacteriocin producer strain in 2 L of LAPTg broth (16-h static culture).

2. Remove the cells by centrifugation (12,000g for 15 min) and bring the supernatant to a final concentration of 40% (w/v) saturation of ammonium sulfate. Stir for 12 h at 4°C (see Note 3).

3. Centrifuge the mixture at 17,000g for 20 min at 4°C. Harvest the pellet and the floating material and dissolve them in 100 mL of distilled water. Test the activity of the crude extract using the bacteriocin activity assay (see Subheading 3.6.).

3.2. Reverse-Phase Solid-Phase Extraction Columns

1. Equilibrate the column with distilled water.

2. Apply the sample (crude extract) to the column. (see Note 4).

3. Wash steps (see Note 5): 4 vol of 5% acetonitrile. 4 vol of 10% acetonitrile. 4 vol of 20% acetonitrile.

4. Elution step:

4 vol of 40% acetonitrile.

5. Test the activity of the fractions using the bacteriocin activity assay.

6. Concentrate the active fraction in a rotary evaporator at 40°C for 2 h.

7. Additional wash steps to clean the column: 4 vol of 70% isopropanol.

4 vol of 99% isopropanol.

3.3. Ion Exchange Chromatography

1. Equilibrate the column with 10 mM phosphate buffer, pH 7.0.

2. Apply the sample very slowly.

4 vol of 10 mM of phosphate buffer, pH 7.0, 50 mM NaCl. 4 vol 10 mM phosphate buffer pH 7.0, 100 mM NaCl. 4 vol 10 mM phosphate buffer pH 7.0, 150 mM NaCl.

4. Elution step:

4 vol 10 mM phosphate buffer, pH 7.0, 200 mM NaCl. 4 vol 10 mM phosphate buffer, pH 7.0, 500 mM NaCl.

5. Collect the fractions containing the bacteriocin activity, desalt them with an octadecyl (C18) Impaq column, eluting with 2 vol of 40% acetonitrile. Concentrate the sample in the TurboVap or rotary evaporator.

3.4. Reverse-Phase Chromatography

1. Perform RP-HPLC on the active extract (see Note 7).

2. Equilibrate the column using solution A at a flow rate of 1 mL/min.

3. Elute the antimicrobial peptide (injection volume 2000 ^L) with the following gradient:

Minutes Flow (mL/min) % Solution B
0 0

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