Rapid two-step procedure for large-scale purification of pediocin-like bacteriocins and other cationic antimicrobial peptides from complex culture medium

Abstract

A rapid and simple two-step procedure suitable for both small- and large-scale purification of pediocin-like bacteriocins and other cationic peptides has been developed. In the first step, the bacterial culture was applied directly on a cation-exchange column (1-ml cation exchanger per 100-ml cell culture). Bacteria and anionic compounds passed through the column, and cationic bacteriocins were subsequently eluted with 1 M NaCl. In the second step, the bacteriocin fraction was applied on a low-pressure, reverse-phase column and the bacteriocins were detected as major optical density peaks upon elution with propanol. More than 80% of the activity that was initially in the culture supernatant was recovered in both purification steps, and the final bacteriocin preparation was more than 90% pure as judged by analytical reverse-phase chromatography and capillary electrophoresis.

FIG. 1.

Reverse-phase chromatography of the 1 M NaCl fraction obtained in the cation-exchange chromatography step (step 1, Table 1). After addition of propanol and trifluoroacetic acid to final concentrations of 5 and 0.1% (vol/vol), respectively, the 1 M NaCl fraction from step 1 (Table 1) was applied at a flow rate of 5 ml/min on a 3-ml Resource reverse-phase column. Peptides binding to the column were eluted at a flow rate of 1 ml/min with a linear propanol gradient (shown in the figure) in 0.1% trifluoroacetic acid. Only the fractions under the horizontal bar contained bacteriocin activity, which coincided with the absorbance peak under the bar. The first 42 ml represents the flowthrough fraction that contained substances that did not bind to the column. To prevent fouling of the reverse-phase column, samples applied to the column were cleared of gross contaminants by centrifugation at about 4,000 × g for 10 min.

FIG. 2.

The amount of bacteriocin activity in the flowthrough fraction as a function of the amount of bacterial culture (overnight culture of P. acidilactici LMG 2351; produces pediocin PA-1) that has passed through a 3-ml SP Sepharose Fast Flow cation-exchange column. The horizontal, stippled line indicates the activity in the bacterial culture before it is applied to the column.