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4. Monitor the elution spectrophotometrically at 220 nm.

5. Pool the active fractions and concentrate them by evaporation using nitrogen as the carrier gas (TurboVap evaporator).

3.5. Isolation and Purification of Lactocin 705AL

The procedure is similar to that used for purification of Enterocin CRL 10, with the following modifications:

1. The culture medium to grow Lactobacillus is MRS.

2. The final concentration of ammonium sulfate is 44%. Dissolve the resultant precipitate in 100 mL of isopropanol 5%.

3.5.1. Solid-Phase Extraction Columns

1. Equilibrate the column with 2 mL of 5% isopropanol.

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

3. Wash step (see Note 5) 2 mL 10% isopropanol.

4. Elution step: 2 mL 30% isopropanol.

5. Concentrate the active fraction in a TurboVap evaporator at 50°C for 30 min.

6. Additional wash steps to clean and reuse the column. 4 vol of 70% isopropanol.

4 vol of 99% isopropanol.

1. Subject the active extract to RP-HPLC (see Note 7).

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

3. Elute the peptide with a 30 min linear gradient of 5-100% of isopropanol in aqueous 0.1% TFA (v/v)

4. Monitor the elution spectrophotometrically at 220 nm.

3.6. Bacteriocin Activity Assays

The bacteriocin activity of both strains should be determined in all the steps of the purification process using the following assay:

1. Prepare serial twofold dilutions of cell-free supernatant and of all the fractions obtained in the purification steps, in sterile dH2O or sterile culture media (1:2, 1:4, 1:8, 1:«).

2. Prepare Petri dishes with 10 mL of BHI base agar. Let solidify and dry.

3. Mix gently 10 ^L of an overnight culture of the sensitive strain (approx 1.106 CFU/mL) with 7 mL of molten BHI soft agar (45°C) and pour onto the base BHI layer. Slide the plate in circles on the bench top immediately to spread the top agar over the plate. Let solidify and dry.

4. Cut wells 0.5 cm in diameter with sterile hollow punchs in the agar. In each well, seed 30 ^L of undiluted bacteriocin and each dilution of it.

5. Incubate the plates (with the agar side up) overnight at 30°C.

6. Bacteriocin activity is detected by the presence of growth inhibition zones of the indicator strain around the wells.

7. The titer of the bacteriocin is expressed in arbitrary units (AU/mL), as the reciprocal of the highest dilution showing a definite zone of inhibition.

4. Notes

1. TSA (trypticase soy broth) and TSA agar (trypticase soy agar) are also used for maintaining and culturing Listeria sp.

2. This column is recommended for the separation of hydrophobic peptides and proteins, including membrane proteins. Use of an Octyl-Sepharose CL-4B column has also been reported (5,6).

3. A two-step ammonium sulfate (0-25% and 25-70%) precipitation was reported for mundticin purification (4), and 70-75% was also used for Enterocin A (5).

4. The sample could also be applied using a vacuum pump. Apply the sample until the column is saturated. This step could be repeated using a small amount of sample each time.

5. In this step it is very important to know the volume of the column, e.g., for a 5-mL column use 20 mL of the wash solution.

6. The final concentration of NaCl for the elution step may vary between 0.2 and 1 M depending on the bacteriocin that is being purified (7).

7. ISCO HPLC system (2360 Gradient programmer, 2350 HPLC Pump, and V4 Absorbance detector; ISCO, Lincoln, NE).

References

1 Jack, R., Tagg, J., and Ray, B. (1995) Bacteriocins of Gram-positive bacteria. Microbiol. Rev. 59, 171-200.

2. Nes, I. and Holo, H. (2000) Class II antimicrobial peptides from lactic acid bacteria. Biopolymers 55, 50-61.

3. De Vuyst, L. and Vandamme, J. (1994) Lactic acid bacteria and bacteriocins: their practical importance. In: Bacteriocins of Lactic Acid Bacteria. Microbiology, Genetics and Applications (De Vuyst, L. and Vandamme, J., eds.). Blackie , London, pp. 1-13.

4. De Vuyst L. and Vandamme, J. (1994) Antimicrobial potential of lactic acid bacteria. In: Bacteriocins of Lactic Acid Bacteria. Microbiology, Genetics and Applications (De Vuyst, L. and Vandamme, J., eds.). Blackie, London, pp. 91-143.

5 Bennik, M. H. J., Vanloo, B., Brasseur, R., Gorris, L., and Smid, E. (1998) A novel bacte-riocin with YGNGV motif from vegetable-associated Enterococcus mundtii: full characterization and interaction with target organisms. Biochem. Biophys. Acta 1373, 47-58.

6 Aymerich T.; Holo H.; Havarstein L.S.; Hugas M.; Garriga M; Nes I. (1996) Biochemical and genetic characterization of enterocin A from Enterococcus faecium, a new antilisterial bacteriocin in the pediocin family of bacteriocins. Appl. Environ. Microbiol.

62, 1676-1682.

7 Cintas, L. M., Casaus, P., Havarstein, L. S., Hernandez, P., and Nes, I. (1997) Biochemical and genetic characterization of enterocin P, a novel sec-dependent bacteriocin from Enterococcus faecium P13 with a broad antimicrobial spectrum. Appl. Environ. Microbiol.

63, 4321-4330.

8 Uteng, M., Hauge, H. H., Brondz, I., Nissen-Meyer, J., and Fimland, G. (2002) Rapid two-step procedure for large-scale purification of pediocin-like bacteriocins and other cationic antimicrobial peptides from complex culture medium. Appl. Environ. Microbiol. 68, 952-956.

9. Guyonnet, D., Fremaux, C., Cenatiempo, Y., and Berjeaud, J. M. (2000) Method for rapid purification of class Ila bacteriocins and comparison of their activities. Appl. Environ. Microbiol. 66, 1744-1748.

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