Easy and rapid purification of highly active nisin

Abstract

Nisin is an antimicrobial peptide produced and secreted by several L. lactis strains and is specifically active against Gram-positive bacteria. In previous studies, nisin was purified via cation exchange chromatography at low pH employing a single-step elution using 1 M NaCl. Here, we describe an optimized purification protocol using a five-step NaCl elution to remove contaminants. The obtained nisin is devoid of impurities and shows high bactericidal activity against the nisin-sensitive L. lactis strain NZ9000. Purified nisin exhibits an IC(50) of ~3 nM, which is a tenfold improvement as compared to nisin obtained via the one-step elution procedure.

Figure 1

Purification of nisin via cation exchange chromatography. The elution profiles of the purification of commercial nisin (a) and nisin produced by L. lactis NZ9700 (b) are shown. In both cases, nisin is eluted from the column using a five-step gradient with 200 mM (Step I), 400 mM (Step II), 600 mM (Step III), 800 mM (Step IV) and 1 M NaCl (Step V). The different elution steps and corresponding NaCl concentrations are indicated by the dashed line and the right y-axis, respectively. Protein was detected by measuring the absorbance at 215 nm.

Figure 2

Tricine-SDS-PAGE analysis of the cIEX purification of nisin. Purification of commercial nisin (a) and nisin secreted by the L. lactis NZ9700 strain (b). M, marker proteins; I, elution with 200 mM NaCl; II, elution with 400 mM NaCl; III, elution with 600 mM NaCl; IV, elution with 800 mM NaCl; V, elution with 1 M NaCl. Protein was visualized by silver staining. The three lowest marker proteins are indicated with molecular weights (kDa).

Figure 3

Bactericidal activity of the various nisin purification fractions. Equal amounts of protein of the different elution fractions (Step I–V) from the purification of commercial nisin (a) and from nisin secreted by the L. lactis NZ9700 strain (b) were run on a tricine-SDS-PA gel and overlaid with nisin-sensitive L. lactis NZ9000 cells (see Section 2). The position of marker proteins with known molecular weight (kDa) are indicated on the left. The growth inhibition zones are visible as dark areas. Lanes I–V represent the five different elution fractions of the cation exchange chromatography. For both purifications, maximum growth inhibition is observed for the Step II elution fraction (400 mM NaCl). Notably, the growth inhibition zone is only visible at a position of ∼3.5 kDa.

Figure 4

MALDI-TOF mass spectrometry analysis of purified nisin. Mass spectrum of the Step II elution fraction (400 mM NaCl) from the lyophilized nisin purification (for corresponding tricine-SDS-PAGE analysis, see Figure 2(a), lane II).

Figure 5

IC₅₀ determination of the nisin purification fractions. Growth inhibition experiments were performed with nisin obtained from the different elution fractions of the purifications of commercial nisin (a) and of nisin secreted by the L. lactis NZ9700 strain (b). The log of the used nisin concentration of each elution fraction is plotted against the normalized optical density of L. lactis NZ9000 after five hours of growth. Shown are the inhibition curves for the NaCl elution fractions of 200 mM (o), 400 mM (∆), 600 mM (∇), 800 mM (◊), and 1 M (*). Data was fitted and evaluated according to (1).