the capacity to produce antimicrobial peptides ( amps ) is widespread among gram - positive bacteria .
these amps are directed against competitive microorganisms in order to generate a selective advantage for the producer organism .
class i consists of the so - called lantibiotics , which are posttranslationally modified peptides containing ( methyl-)lanthionines , and have a typical size of < 5  kda .
class ii comprises heat - stable , nonmodified peptides of 3758 amino acids ( typical size of < 10  kda ) with the leader peptide , as for class i amps , being removed during maturation .
class iii contains amps that are heat labile and that are generally larger in size ( roughly 30  kda ) . especially heat - stable peptides secreted by lactic acid bacteria have been studied extensively because of their potential use as natural preservatives in the food industry .  
one of the best characterized amps is nisin , which is secreted by lactococcus lactis and is a member of the class i amps ( for comprehensive reviews , see [ 2 , 3 ] ) .
although nisin has been used as a food preservative for more than fifty years , no significant bacterial resistance against nisin has been reported .
nisin , encoded by the nisa gene , is a ( methyl-)lanthionine ring containing peptide that is ribosomally synthesized as a prepeptide consisting of 57 amino acids .
the nisa prepeptide is modified posttranslationally by the dehydratase nisb , which selectively dehydrates serine and threonine residues , and by the cyclase nisc , which catalyzes lanthionine ring formation .
nist finally transports the modified prenisin across the cell membrane , and mature , biologically active nisin is produced upon cleavage of the leader peptide by the extracellular , membrane - anchored protease nisp .
mature nisin harbors three dehydrated amino acids ( one dehydrobutyrine and two dehydroalanines ) , one lanthionine ring , and four methyl - lanthionine rings . in particular
, these intramolecular rings are important for the biological activity of nisin .   it is worth mentioning that nisin induces its own synthesis via interaction with the two - component regulatory system nisrk .
firstly , nisin inhibits cell - wall synthesis by binding to lipid ii , an essential membrane - anchored cell - wall precursor , and secondly , nisin permeabilizes the target membrane .
the binding of nisin to lipid ii induces membrane integration of nisin resulting in the formation of a pore , likely composed of eight nisin and four lipid ii molecules .
this highly specific interaction with lipid ii is reflected by the low nano- to micromolar concentrations of nisin , sufficient to permeabilize the membrane of the target cells .
the bactericidal activity of amps is generally measured by quantifying growth inhibition of an amp - sensitive target organism , grown either on agar plates or in liquid culture . on agar plates , zones of growth inhibition of the indicator organism can be easily visualized , and these so - called halo assays allow determination of the minimum inhibitory concentration ( mic ) of the tested amp . alternatively
, bacterial growth can be monitored in liquid culture in dependence of the amp concentration , by measuring the optical density .
this method allows the simple determination of both the mic and the ic50 , that is , the concentration of amp that inhibits cell growth by 50% .
nisin from l. lactis , like almost all naturally produced amps , can be purified directly from the culture medium [ 10 , 11 ] .
it is a cationic peptide , and therefore , commonly purified using cation exchange chromatography ( ciex ) at acidic ph , using high salt concentration for elution , typically a single - step elution with 1  m  nacl [ 1216 ] . by using nisin purified via such a method , the ic50 and mic values were determined for a variety of bacteria , such as various l. lactis strains , enterococcus faecium , bacillus anthracis , bacillus subtilis , staphylococcus aureus , and streptococcus mutans [ 9 , 17 ] .   whereas e. faecium and s. mutans were equally sensitive to nisin exhibiting an ic50 of 5  m and mic of 12.5  m , l. lactis hp was much more sensitive to nisin , with an ic50 of 14  nm and mic of 32  nm .
nisin shows promising activity towards clinical isolates of the methicillin - resistant staphylococcus aureus ( mrsa ) bacterium , streptococcus pyogenenes , and several of the most severe human pathogens , including the multiresistant streptococcus pneumoniae and vancomycin - resistant e. faecium or e. faecalis , against which new effective antibiotics are most urgently needed [ 1820 ] . in all these ,
studies purified nisin was used and the bactericidal activity of nisin was measured by determining the mic or ic50 . here , we describe a rapid and easy nisin purification protocol , optimized to yield active , monomeric nisin . for this purpose , nisin was either produced from l. lactis strain nz9700 or purchased in the form of a lyophilized powder that contains 2.5% ( w / w ) nisin .
nisin from both sources was purified and tested for bactericidal activity using the nisin - sensitive l. lactis nz9000 strain . during the purification low molecular weight contaminants are removed , which results in purified nisin with high specific activity .
a culture of the l. lactis strain nz9700 was grown overnight in m17 medium containing 0.5% ( w / v ) glucose ( gm17 ) at 30c .
next , 100  ml of the overnight culture was used to inoculate 2  l of gm17 , and cells were grown at 30c . at an od600 of 0.8 ,
the culture was supplemented with nisin ( sigma ) to a final concentration of 1  ng / ml cell culture to ensure nisin production and growth was continued over night .
the supernatant was used and diluted with 1  l of 50  mm lactic acid ph 3 resulting in a 3  l solution with a ph of 5 .
this solution was loaded on a 5  ml hitrap sp hp cation exchange ( ciex ) column ( ge healthcare ) using a flow rate of 4  ml / min . due to the large volume , this step is optimally performed overnight .  
unfortunately , it is not possible to detect nisin at 280  nm , because it does not contain any aromatic amino acids ; therefore , the 215  nm was chosen as wavelength .
the column was subsequently washed with 50  mm lactic acid ph 3 until a stable baseline was reached to remove nonspecifically bound material .
peptides were eluted by increasing the nacl concentration stepwise using a flow rate of 1  ml / min , resulting in elution fractions with 200  mm ( step i ) , 400  mm ( step ii ) , 600  mm ( step iii ) , 800  mm ( step iv ) and 1  m ( step v ) nacl . to remove nacl , protein in the elution fractions
was precipitated with 20% ( v / v ) trichloroacetic acid ( tca ) overnight at 4c .
precipitated protein was washed two times with ice - cold acetone to remove residual tca .
finally , the protein pellet was suspended in 50  mm lactic acid ph 3 .
nisin concentrations were determined by a colorimetric assay ( pierce bca protein assay kit , thermo scientific ) by measuring the absorbance at 584  nm according to the protocol of the manufacturer .
commercial nisin ( sigma ) is available as a lyophilized powder containing 2.5% ( w / w ) nisin .  
1.3  g of nisin powder ( corresponding to 32.5  mg nisin ) was dissolved in 100  ml 50  mm lactic acid ph 3 and filtered through a 0.45  m membrane filter ( pall corporation ) .
the nisin solution was applied to a 5  ml hitrap sp hp cation exchange column ( ge healthcare ) at a flow rate of 2  ml / min , whereas elution was performed at a flow rate of 1  ml / min .
tricine - sds - page was essentially carried out as described in . for analysis , 16  l sample was supplemented with 4  l 5x sds sample buffer ( 0.2  m tris - hcl , ph 6.8 , 10% ( w / v ) sds , 40% ( v / v ) glycerol , 0.02% ( w / v ) bromophenol blue , and 10  mm dtt ) and loaded on a tricine gel consisting of a stacking gel containing 5% acrylamide and a separation gel containing 16% acrylamide . the gel was run at 100  v for 2 hours , and proteins were detected via silver staining . for all purification fractions , 3.2  g of total protein was analyzed .
samples obtained from ciex chromatography were precipitated with tca and washed with acetone as described above .
the protein pellets were then dried for 15  min . at 30c in a vacuum concentrator ( eppendorf concentrator plus ) . dried pellets were stored at 20c until analysis . for mass spectrometric analysis , the samples were dissolved in water with 1% ( v / v ) formic acid .
measurements were performed using a maldi - tof instrument ( voyager - de str , applied biosystems ) with a nitrogen laser (  = 337  nm ) operating in reflector mode with 25  kv acceleration voltage .
the samples were prepared by the standard dried - droplet procedure , by applying 0.5  l of 2,5-dihydroxybenzoic acid ( dhb ) matrix solution ( 10  mg in 1  ml water ) plus 0.5  l of sample solution .
an external calibration with the monomer ion of des - pro - bradykinin , sub p , bombesin , and melittin was used .
the spectrum was obtained by averaging 200 laser shots . to investigate the antimicrobial activity of nisin , the nisin sensitive l. lactis strain nz9000   was grown in a 96-well plate in gm17 medium .
the total volume in each well was 200  l , consisting of 50  l sample and 150  l gm17 containing l. lactis nz9000 cells ( starting od600 = 0.1 ) .
samples were prepared by diluting nisin obtained from various ciex elution fractions in 50  mm lactic acid ph 3 to yield final protein concentrations ranging from 0.15  nm to 300  nm in the wells .
cells were grown at 30c , and the optical density was monitored at 620  nm every 20  min for a period of 8 hours ( 96 plate reader bmg ) . to determine the ic50 values , the optical density was normalized and plotted against the log of the nisin concentration .
data were evaluated according to 
 ( 1)y = od  min  + ( od  max  od  min  ) 1 + 10((log  ( ic50)x)slope ) . 
							
y stands for the normalized optical density value , and x represents the logarithmic concentration of the peptide .
the ic50 value is calculated as the value of the peptide concentration used where the growth inhibition ( or od600 ) is 50% .
the antimicrobial activity of the different nisin preparations was assessed by means of a halo assay .
purified nisin obtained from the different ciex elution fractions were supplemented ( 16  l ) with 4  l of 5x sds sample buffer and tricine - sds - page was carried out as described above .
after electrophoresis the gel was incubated for 30  min in an aqueous solution containing 20% ( v / v ) isopropanol and 10% ( v / v ) acetic acid .
subsequently , the gel was washed two times for 30  min in ddh2o . at this stage ,
finally , the gel was overlaid with gm17-agar ( 0.5%  w / v agar ) containing l. lactis nz9000 cells at an od600 of 0.1 .
after solidification , the overlaid gel was incubated overnight at 30c to allow for bacterial growth .
the bactericidal activity of nisin is readily visualized by the presence of clear zones ( halos ) resulting from growth inhibition .
to determine the antimicrobial activity of commercially available nisin , we used a lyophilized powder , which contains 2.5% ( w / w ) nisin . to further purify nisin , we initially performed sp sepharose cation exchange chromatography using 1  m  nacl to elute the bound nisin .
subsequent sds - page analysis of the eluate revealed a major protein band corresponding to a peptide with a molecular mass of about 3.5  kda ( data not shown ) in line with the calculated mass of 3354  da for mature nisin .
it is of note that these contaminants are not readily visualized by coomassie brilliant blue staining , whereas these impurities are clearly detected by silver staining .
nisin purified via this method showed antimicrobial activity against l. lactis nz9000 exhibiting an ic50 of 30  12  nm .
we first tested elution with a linear gradient ( 50 times the column volume ) ranging from 01  m  nacl .
this approach , however , resulted in   a   broad peak eluting throughout the nacl gradient and further analysis revealed no improvement when compared to the single - step 1  m  nacl elution ( data not shown ) .
in contrast , a substantial improvement was achieved when a five - step nacl step gradient was used to elute nisin from the ciex column ( figure 1(a ) ) .
bound protein eluted at every step as evidenced by the elution profile and subsequent tricine - sds - page analysis ( figure 2(a ) ) .
the step ii elution fraction contained the bulk of nisin as evident by the major protein band with a corresponding molecular mass of 3.5  kda , whereas substantially lower amounts of nisin were detected in elution fractions i , iii , and iv . in the step v fraction
the step i and ii elution fractions contained exclusively nisin , while elution fractions iii 
v contained predominantly higher molecular weight compounds ( ranging from 6  kda70  kda ) . in the latter fractions compounds with molecular weights of 8 
the total protein concentration of the elution fractions was determined to be : 2.1  mg / ml for step i ( 200  mm  nacl ) , 7.5  mg / ml for step ii ( 400  mm  nacl ) , 1.9  mg / ml for step iii ( 600  mm  nacl ) , 0.5  mg / ml for step iv ( 800  mm  nacl ) and 0.4  mg / ml for step v ( 1  m  nacl ) ( table 1 ) . in step ii , 60% of the total purified nisin eluted .
thus , it appears that 400  mm  nacl is sufficient to elute the vast majority of the nisin molecules .
an alternative to purchasing nisin is to produce it in the laboratory , since l. lactis strains that secrete nisin in large amounts are readily available .
we used l. lactis nz9700 grown in gm17 medium to produce nisin ( see section 2 ) .
culture supernatant containing nisin was subjected to ciex chromatography using the same five - step nacl elution gradient as described above . here , a high absorbance at 215  nm occurred at the step i elution ( figure 1(b ) ) , but this did not correspond to nisin or other proteins as evidenced by silver - staining following sds - page analysis ( figure 2(b ) , lane 2 ) .
likely , this high absorbance is due to ingredients from the growth medium , which contains large amounts of peptone , tryptone , and yeast extract .
this was confirmed by a run with only gm17 media ( data not shown ) .
the remainder of the elution profile is similar to that of the lyophilized nisin purification , with two major absorbance peaks observed for the step ii and iii fractions ( figure 1(b ) ) .
analysis of the protein content of the different elution fractions revealed that nisin is exclusively found in the 400  mm  nacl elution fraction ( figure 2(b ) ) .
the total protein concentration of the 400  mm elution fraction was 2.9  mg / ml . however , whereas nisin is most prominent in this fraction , components with a mw 6  kda , 10  kda , and 12  kda are also present .
the 0.61  m elution fractions on the other hand contained compounds with molecular weights ranging from 8  kda70  kda , similar to those observed for the purification of lyophilized nisin ( figures 2(a ) and 2(b ) ) . when compared to the purification of lyophilized nisin , nisin purified from gm17 medium still contained contaminants .
this difference in purity may relate to differences in the loaded material . the lyophilized nisin powder ( also containing denatured milk solids )
was dissolved in 50  mm lactic acid , whereas nisin produced by l. lactis nz9700 was applied to the cationic exchange column as a 1  :  2 mixture of gm17 culture medium and 50  mm lactic acid , respectively .
we considered the possibility that the compounds with molecular weights of 68  kda may represent the unprocessed form of nisin .
immature nisin , that is , nisin still containing the leader sequence , has a molecular weight of 5.9  kda and may arise if inefficient leader cleavage by the protease nisp occurs .
we therefore performed western - blot analysis using a polyclonal antibody raised against the nisin leader sequence .
purified prenisin secreted by a l. lactis strain lacking nisp was used as a positive control for western - blot analysis . for all elution fractions , no signals were observed suggesting that the observed compounds are not derived from prenisin ( data not shown ) . to test the biological activity of the purified nisin , we used l. lactis nz9000 as indicator organism .
l. lactis nz9000 is a derivative of the plasmid - cured l. lactis mg1363 and contains the nisrk genes inserted in the chromosomal pepn locus .
this strain is commonly used as the host for nisin - induced expression system ( nice ) purposes . however , since this strain lacks the nisin immunity genes nisifeg , it is sensitive to nisin .
the antimicrobial activities associated with the different elution fractions obtained as described above were tested in a so - called halo assay . for this , the various fractions containing nisin were analyzed by tricine - sds - page and the tricine gel was overlaid with gm17-agar containing nisin sensitive bacteria ( see section 2 ) .
the biological activity of nisin is visualized by the growth inhibition zones ( halos ) at the position where nisin is present .
the results for the lyophilized and the laboratory produced nisin are shown in figures 3(a ) and 3(b ) , respectively .
after overnight incubation at 30c halos were observed for elution step i  iv for the lyophilized nisin , while for the produced nisin , a halo was only observed for elution fraction ii . for both purifications , the highest level of growth inhibition
importantly , the zones of inhibition are located only at the position of the 3.5  kda nisin peptide ( figure 3 ) . thus , purified nisin was biologically active and no growth inhibitory activity is associated with the higher molecular weight compounds . to assess and confirm the presence of nisin in the individual ciex elution fractions ( step ii 
v ) of the lyophilized nisin purification , we applied maldi - tof mass spectrometry .
the 400  mm  nacl elution fraction contained only one peptide with a molecular mass of 3355.09  da ( figure 4 ) , which is in agreement with the calculated mass of 3354.07  da for nisin .
peak integration of the total mass spectrum revealed that the 400  mm elution fraction contains > 98% of nisin , indicating that this fraction is essentially devoid of contaminants .
nisin was also found in the 600 mm and 800 mm elution fractions , whereas nisin was not detected in the 1 m nacl fraction .
these results are in agreement with tricine - sds - page analysis ( figure 2(a ) ) .
however , the 0.61  m  nacl elution fraction contained several peptides with higher molecular masses .
subsequent tandem ms analysis of these peptides yielded in - sequence tags of eight amino acids and six amino acids , which unfortunately could not be assigned to specific proteins .
this was due to the fact that the obtained sequence tags were too short and when blasted gave multiple different protein hits ( data not shown ) .
nevertheless , the obtained sequence tags did not match with the primary sequence of ( pre)nisin .
therefore , it can be excluded that the peptide contaminants with molecular masses of 68  kda are derived from prenisin .
to quantitatively assess the growth inhibitory activity of nisin obtained from the different purification fractions , a liquid culture assay was performed using l. lactis nz9000 as reporter organism .
the optical density of the l. lactis nz9000 cultures after 5 hours of growth was plotted against the total protein concentration of the nisin purification fractions .
results are shown for both , the lyophilized ( figure 5(a ) ) and the produced nisin ( figure 5(b ) ) , respectively .
the resulting growth curves and the calculated ic50 values are shown in figure 5 and table 1 , respectively .   for lyophilized nisin ,
all ciex elution fractions exhibited growth inhibitory activity , however , with very distinct ic50 values ( figure 5(a ) and table 1 ) .
the step ii elution fraction displayed the highest bactericidal activity with an ic50 of 2.6  0.1  nm .
the other elution fractions showed substantially higher ic50 values . whereas the step v elution fraction had only an inhibitory effect at the highest tested concentrations , the step i , iii , and iv elution fractions exhibited an ic50 value of 35.1  0.1  nm , 6.9  0.2  nm , and
thus , the 400  mm  nacl elution fraction contains not only the bulk of nisin , it also contains nisin that displayed the highest specific activity .  
a similar observation can be made for nisin purified from the medium ( figure 5(b ) ) .
however , here only the 400  mm elution fraction shows bactericidal activity , which is in agreement with tricine - sds - page analysis ( figure 2(b ) ) and the halo assay ( figure 3(b ) ) .
the calculated ic50 of 11.2  0.3  nm is , however , 4-fold higher than that of the corresponding fraction obtained from the purification of lyophilized nisin .
we attribute this difference to the contaminants that are still present ( figure 2(b ) , lane ii ) .
nevertheless , for both purifications the highest bactericidal activity is associated with the fraction that contains the highest amount of nisin ( figures 2 - 3 and 5 ) .
taken together the data indicate that nisin obtained from the step ii elution fraction has the highest specific activity .   to determine whether nacl used for elution has an effect on nisin activity , we repeated the experiment and adjusted the concentration of nacl after elution in every fraction to 500  mm either by dilution with buffer without salt or by adding buffer and salt .
precipitated protein from these fractions was subsequently used for growth experiments as described above . in all cases ,
the ic50 values of the  salt experiment  were slightly higher than when measured directly after elution , indicating that more nisin is needed to inhibit cell growth by 50% ( data not shown ) .
this indicates that residual salt does not have a major influence on the activity of nisin .
it can , therefore , be excluded that the differences in ic50 values of the various nisin containing elution fractions are induced by the amounts of nacl used to elute nisin from the column .
their uniqueness of especially the lantibiotic group of amps , resides in the posttranslational modifications , such as dehydration of amino acids and intramolecular thioether bridges .
one of the best - characterized amp is nisin , a compound used for more than 40 years in up to 80 countries as an effective agent to combat food - borne pathogens .
commonly , nisin and other cationic amps are purified using a single 1  m  nacl elution step , from a ciex column at an acidic ph [ 1214 , 16 , 26 ] .   the activity of amps is strictly dependent on the target organism .
for example , the ic50 value of nisin ranges from 14  nm for l. lactis hp to 5  m for vancomycin - resistant e. faecium , with respective mics of 32  nm and 12.5  m . in all these studies
, it is noticeable that there is a large variation in the sensitivity to nisin between isogenic strains of gram - positive bacteria , whereas some bacteria are inherently resistant to nisin .
the most prominent example is the enzymatic destruction or modification of the antibiotic , thereby rendering it ineffective .
a second important mechanism of resistance is shielding of the target such that the antibiotic can not get access to it  for example , by cell - surface alterations ( capsules s - layers ) or by active extrusion by efflux pumps .
moreover , the resistance of the amp producer organism towards its secreted amp ( autoimmunity ) is typically based on atp - binding cassette ( abc ) transporters which expel the amp from the membrane .   in l.
lactis nz9700 , cells autoimmunity is mediated by the scavenger protein nisi and the abc transporter nisfeg .
l. lactis cells lacking the nisifeg defense system ( e.g. , l. lactis nz9000 ) are sensitive to nisin and can be used as indicator organism to measure the biological activity of nisin .
the nisi and nisfeg genes are part of the nis operon and are expressed in concert with the genes involved in nisin production and secretion .
recently , in nisin - non - producing l. lactis , nisin resistance was shown to be conferred by a specific nisin resistance gene ( nsr ) , which encodes a 35  kda nisin resistance protein ( nsr ) .
nsr proteolytically removes the last six amino acids of nisin , thereby reducing its bactericidal activity by a factor of 100 .  
the level of intrinsic resistance and the employed mechanisms of antibiotic resistance may differ greatly among microorganisms .
it is , therefore , difficult , if not impossible , to directly compare ic50 values for a given amp when comparing strains .
moreover , the purity of the amp preparation is of great importance to determine accurately the bactericidal activity of the amp .   in this study , we optimized the purification of the amp nisin and determined its ic50 values against the nisin - sensitive l. lactis strain nz9000
. nisin typically purified via a 1  m  nacl one - step elution yields high levels of active nisin ( see above and ) but contains a substantial amount of contaminants .
we show that these contaminants , which are mainly proteinaceous in nature , are largely removed by using a five - step nacl elution .  
notably , 400  mm nacl ( step ii ) was sufficient to elute the bulk of the nisin molecules , while the majority of contaminants remain bound to the column . in this manner
when tested against the nisin - sensitive l. lactis nz9000 , this highly active nisin exhibited an ic50 of 2.6  0.1  nm , which is a 10-fold improvement as compared to the nisin obtained via the one - step elution .
the potent bactericidal activity of nisin against l. lactis nz9000 lacking nisi and nisfeg suggests that these autoimmunity proteins are of great importance for l. lactis cells that produce nisin ( e.g. , l. lactis nz9700 ) .
amps get more and more into the focus of biochemical , biophysical , and medical studies due to their antimicrobial activity against a wide variety of bacteria . here , we report an easy and rapid protocol for the purification of highly active nisin , purified either directly from the culture medium or from a commercially available lyophilized powder .
a pure , homogenous , and biologically active preparation will ensure reliable determination of the efficacy of amps towards their microbial target(s ) . due to the similar chemical and biophysical properties of lantibiotics , our manner of purification may also apply to amps other than nisin .
 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 ic50 of ~3  nm , which is a tenfold improvement as compared to nisin obtained via the one - step elution procedure . 
1. Introduction
2. Material and Methods
3. Results
4. Discussion