JOURNALOF BIOSCIENCEAND BIOENGWEERING

Vol. 88, No. 2, 153-159. 1999

Purification and Characterization of a Bacteriocin Produced by

Lactococcus lactis subsp. lactis H-559 Isolated from Kimchi
HUN-JO0 LEE,’ YUN-JUNG JOO,’ CHAN-SUN PARK,’ SEUNG-HO KIM,2 IN-KYEONG HWANG,3
JONG-SEOG AHN,‘* AND TAE-ICK MHEEN’
Cellular Response Modifier Research Unit,l Protein Function Research Unit,= Korea Research Institute of
Bioscience and Biotechnology, P. 0. Box 115, Yusong, Taejon 305-600 and Department of
Food Scienceand Nutrition, Seoul National University, Seoul 151-742,’ Korea
Received 18 January 1999/Accepted 20 May 1999

Lactic acid bacteria were isolated from kimchi and screened for bacterlocin production. Strain H-559,
identified as Lactococcus lactis subsp. la&s, exhibited the strongest antibacterial activity among them and was
active against pathogenic bacteria such as Listeria monocytogenes and Staphylococcus aureus as well as many
lactic acid bacteria. The antimicrobial substance produced by L. Zactissubsp. lads H-559 was inactivated by
a-chymotrypsin, and protease type IX and XIV and was confirmed to be a bacteriocin. The bacteriocin activity
was stable from pH 2.0-11.0 and up to 10 min heating at 100°C. The bacteriocin was sequentially purified by
ammonium sulfate precipitation, ion-exchange chromatography, and reversed-phase high-performance liquid
chromatography (HPLC). Its molecular weight was determined to be 3343.7 Da by MALDI-mass spectrometry.
Isoleucine was detected as the first N-terminal amino acid residue but the remaining amino acid sequence could
not be determined by the Edman degradation method. It was different from other bacteriocins in terms of pH
stability, molecular weight, amino acid composition, and the partial amino acid sequencesof peptides obtained
by acid hydrolysis.
[Key words: bacteriocin, Lactococcus lactis subsp. lactis, kimchi]

Among a number of antimicrobial compounds pro- MATERIALS AND METHOD

duced by lactic acid bacteria, bacteriocins are peptides
or proteins that show bactericidal activity towards bac-
teria that are closely related to the species producing
them (1). These compounds are of great interest to the
food industry because of their inhibitory activity against
food spoilage and/or pathogenic microorganisms during
food processing and food fermentation. Therefore, bac-
teriocins produced by lactic acid bacteria are potential
candidates for improving the safety of various foods.
The development of new bacteriocins in Korea is promis-
ing because of the relative abundance of traditionally
fermented foods such as kimchi. Kimchi is prepared with
various vegetables and is made palatable and preservable
through proper fermentation using lactic acid bacteria;
Several kinds of kimchi are known in korea.
We have recently isolated a new bacteriocin-producing
strain identified as Lactococcus lacfis subsp. lactis from
kimchi (2). A number of strains of L. lactis have been
reported to produce bacteriocins. Nisin, produced by
some strains of L. lactis subsp. lack, has been the most
extensively studied bacteriocin. Today, nisin is commer-
cially produced and used in processed cheeses and
canned foods (3). Many other lactococcal bacteriocins
have also been reported: lacticin 481 produced by L. lac-
tis subsp. lactis CNRZ 481 (4), lactococcin B produced
by L. lactis subsp. cremoris 9B4 (5), lactococcin G
produced by L. lactis LMG 2081 (6), and diplococcin
produced by L. lactis subsp. cremoris (7).
The purpose of this study was to isolate bacteriocin-
producing lactic acid bacteria from kimchi, and to
characterize and purify the bacteriocin.
* Corresponding author.
153
Bacterial strains and media The bacterial strains
used as indicator microorganisms for the screening of
bacteriocin and the evaluation of antimicrobial activities
are shown in Table 1. The lactic acid bacteria were
propagated in MRS broth (Difco, Detroit, MI, USA) at
30 or 37°C. Bacillus strains were propagated in nutrient
broth (Difco) at 30°C. Streptococcus thermophilus was
grown in tomato-juice medium (tomato juice lOOml/l,
yeast extract 5 g/l, skim milk 100 g/l) at 37°C and Can-
dida albicans was grown in YEPD medium (yeast extract
log/l, peptone 2Og/l, glucose 2Og/l) at 30°C. L. raf-
jinolactis and Listeria monocytogenes were propagated
in blood agar base medium (Difco) at 30°C and BHI
media at 37°C (Difco) respectively. Other indicator
strains were grown in LB medium (Difco). For the isola-
tion of lactic acid bacteria from kimchi, MRS agar (8),
phenylethyl alcohol sucrose agar (9) [trypticase peptone
5 g/l, yeast extract 0.5 g/l, sucrose 20 g/l, MgS04+ 7HZ0
0.244 g/l, (NH&SO4 2 g/l, KH2P04 1 g/l, phenylethyl
alcohol 2.5 g/l, agar 15 g/r], modified Lactobacillus selec-
tion agar (8) (trypticase peptone log/l, yeast extract
5 g/l, glucose 2Og/l, ammonium citrate 2 g/l, sodium
acetate 15 g/l, MgS04.7HzO 0.575 g/l, MnS04 0.12 g/l,
KH2P04 6 g/l, FeS04 0.034 g/l, sorbitan monooleate
1 g/l, agar 15 g/l), and M-Enterococcus agar (9) (trypti-
case peptone 15 g/l, phyton peptone 5 g/l, yeast extract
5 g/l, glucose 2 g/f, KH2P04 4g/l, agar 15 g/l) were
used. The various strains of lactic acid bacteria and
other microorganisms used in this study were obtained
from Korean Collection for Type Cultures (KCTC,
Korea Research Institute of Bioscience and Biotechnol-
ogy, Taejon, Korea). The stock cultures were maintained
at -70°C in MRS broth (Difco) containing 20% (v/v)
glycerol. Before use, the strains were propagated twice in
154 LEE ET AL.
broth for 18 to 24 h.
Isolation of bacteriocin-producing lactic acid bacteria
Various kinds of kimchi were used as the source of lac-
tic acid bacteria. Samples from each kind of kimchi were
homogenized, IO-fold serially diluted with saline solu-
tion, and plated on MRS agar, phenylethyl alcohol
sucrose agar, modified Lactobacillus selection agar, and
M-Enterococcus agar. Lactic acid bacteria isolated from
kimchi were maintained as frozen stock cultures stored
at -70°C in broth with 20% glycerol. For the detection
of bacteriocin activity, the well-diffusion assay described
by Lyon and Glatz (10) with some modifications were
used. The basal layer of MRS contained 2% agar and
was 5 mm deep. Wells of 7 mm diameter were cut and
the bottoms of the wells were sealed with a few drops
of MRS agar media. After the isolates were grown for
16 h at 3O”C, cells were removed by centrifugation
(10,000 xg, 15 min). Two hundred microliters of super-
natant adjusted to pH 7 with 1 N NaOH were added to
the wells and allowed to diffuse at 4°C. To examine the
proteinaceous nature of the antimicrobial substances,
proteolytic enzyme solutions were spotted adjacent to
the wells. The proteolytic enzymes (all obtained from
Sigma) were a-chymotrypsin (10 mg/ml), trypsin (10
mg/ml), protease type IX (20mg/ml) and protease type
XIV (20mg/ml) and their buffers were used as recom-
mended by the supplier. After the base agar was flipped
into the Petri dish lid, it was covered with 5 ml of soft
agar media inoculated with the indicator strains at the
density of 10’ CFU/ml. After incubation for 20-24 h at
the optimum temperatures for the individual indicators,
the zones of inhibition and partial loss of the indicator
strain were checked.
Bacteriocin-activity assay The antimicrobial activi-
ties of culture supernatants and partially purified bac-
teriocin were determined by the serial dilution method.
The partially purified bacteriocin samples were prepared
as follows. The bacteriocin-producing strain was grown
in MRS broth for 13 h at 30°C. The cell-free super-
natant was brought up to 75% saturation by the addi-
tion of ammonium sulfate. After overnight incubation
at 4°C the precipitate was collected by centrifugation
(1000 x g, 10 min), dialyzed using a Spectra-Por dialysis
membrane (Spectrum medical Industries, CA, USA)
against 10mM phosphate buffer (pH 6.0), and lyophi-
lized. The lyophilized samples were redissolved in 10 mM
phosphate buffer (pH 6.0). The antimicrobial activities
of 2-fold diluted samples (200~1) were examined by the
well-diffusion method as described previously. The indi-
cator strain was Pediococcus acidilactis KCTC 3101. The
activity units (AU) per ml of bacteriocin samples were
calculated from the reciprocal of the highest dilution
that produced a detectable zone of inhibition.
pH, heat, solvents, and enzyme stability of bacterio-
cias The pH stability of bacteriocin was examined
as described by Lyon and Glatz (11). Partially purified
bacteriocin was dissolved in 1OmM phosphate buffer
(pH 6.0) and individually dialyzed for 24 h against 2 1 of
the following buffers: 0.05 M glycine-HCI buffer (pH 2.0,
3.0), 0.05 M acetate buffer (pH 4.0, 5.0), 0.05 M citrate
buffer (pH6.0), 0.05 M Tris-HCl buffer (pH 7.0, 8.0),
0.05 M glycine-NaOH buffer (pH 9.0, lO.O), and 0.05 M
phosphate-NaOH buffer (pH 11.0). Each buffer was
changed three times during dialysis. After dialysis, the
bacteriocin solutions were assayed for activity. The effect
of heat on the activities of the partially purified bacterio-
tin was determined as described by Lyon and Glatz (IO).
Partially purified bacteriocin samples were boiled for 10,
20, or 30 min in a water bath and autoclaved (121 “C)
for 10min or 20min, and then cooled and assayed for
residual activity. For the examination of the effect of
organic solvents on the bacteriocin activity, partially
purified bacteriocin was mixed with an equal volume
of 100% methanol, ethanol, isopropanol, chloroform,
hexane, acetonitrile, 0.1% (v/v) acetic acid, or 0.2%
(v/v) trifluoroacetic acid (TFA). After incubation at
room temperature for 2 h, the solvents were evaporated
in vacua. Dried samples were then redissolved in IO mM
phosphate buffer (pH 6.0) and the bacteriocin activity
was determined. The following enzymes (all obtained
form Sigma, MO, USA) were used for the determination
of the bacteriocin stability against various classes of
enzymes: catalase (1540 U/mg), glucose oxidase (6000 U/
mg), phospholipase AZ (7 U/mg), lipase (900 U/mg),
peroxidase (78 U/mg), cr-amylase (200 U/mg), ,+amylase
(80 U/mg), pepsin (2100 U/mg), trypsin (17,600 U/mg),
protease type IX (0.7 U/mg), protease type III (0.5 U/
mg), protease type XIV (4.8 U/mg), tr-chymotrypsin
(46 U/mg), subtilisin (15 U/mg), and carboxypeptidase
A (920U/mg). The partially purified bacteriocin was
dissolved in buffers recommended by the supplier
(Sigma, MO, USA) and incubated with each enzyme at a
final concentration of 1 mg/ml for 2 h at 37”C, except
for the samples containing catalase, trypsin, tr-chymo-
trypsin and carboxypeptidase A, which were incubated
at 25°C. Samples containing peroxidase and ;3-amylase
were incubated at 20°C. Separate aliquots with bovine
serum albumin instead of enzymes were included as
controls. After incubation, the samples were boiled for
3 min and the residual activities were determined.
Production and purification of the bacteriocin L.
lactis subsp. la& H-559 was grown in MRS broth for
13 h at 30°C without shaking. After the cells were re-
moved by centrifugation at 8500x g for 20min, ammo-
nium sulfate was slowly added to yield 75% saturation.
The precipitated proteins were collected by centrifuga-
tion at 10,000 xg for 20 min at 4”C, resuspended in
10mM phosphate buffer (pH 6.0), and dialyzed against
the same buffer in Spectra-Por dialysis tubing overnight.
Heat labile proteins were removed by heating the dialy-
sate to 100°C for 5 min and the centrifuging. The partial-
ly purified bacteriocin was applied to a 24.5-ml carbox-
ymethyl sepharose CL-6B column (Pharmacia Biotech.,
Uppsala, Sweden) equilibrated with 10 mM phosphate
buffer (pH 6.0). The column was washed with the same
loading buffer and then the absorbed proteins were elut-
ed by the application of a linear salt gradient (0 to 0.5 M
NaCl, 125 ml) in the same buffer. The tlow rate was
1 ml/min and the fractions (4.5 ml) were monitored for
protein content by measuring the absorbance at 280nm
(12, 13) and then assayed for bacteriocin activity. Frac-
tions containing bacteriocin activity were collected and
freeze-dried.
The lyophilized samples were dissolved in 0.1% (v/v)
trifluoroacetic acid (TFA) at a concentration of 40
mg/ml, and applied to an ODS ultrasphere column (Cr,
colum, 10 x 250 mm, Beckman Instrument Inc., Fuller-
ton, USA) in a HPLC system (Beckman System Gold,
Beckman Instrument Inc.). Elution was carried out with
a 20-min linear gradient from 100% buffer A to 100%
buffer B. Buffer A was 0.1% (v/v) TFA in water and
buffer B was composed of 80% acetonitrile in 0.1”;;
VOL. 88, 1999 PURIFICATION AND CHARACTERIZATION OF BACTERIOCIN 155

(v/v) TFA. The flow rate was maintained at 2ml/min TABLE I. Antimicrobial activity spectrum of the partially purified
and the eluate was monitored at 220nm. Active frac- bacteriocin through ammonium sulfate precipitation from the
tions were collected, freeze-dried and redissolved in buf- culture supernatant of L. lactis subsp. lactis H-559
fer A. The bacteriocin samples were rechromatographed Antimicrobial
on the same column. Indicator strain activity”
SDS-PAGE The active fractions eluated from the Lactobacillus acidophilus MT-21
final HPLC were subjected to urea-SDS-PAGE. Elec- Lactobacillus brevis subsp. brevis KCTC 3498
trophoresis was performed according to the Swank and Lactobacillusplantarum KCTC 3099 i
Munkres method (14) using 15% polyacrylamide gels Lactobacillus plantarum MT-13 i-
containing urea at a constant voltage of 90 V for 5 h. Lactobacillus delbrueckii-lactis ATCC 4191 T+-
The gels were fixed in 40% (v/v) methanol-lo% glacial Lactobacillus viridescens KCTC 3504 +-
acetic acid for 30 min and silver-stained (Bio-Rad Lab- Lactobacillus hilgaridi KCTC 3500 -4
Lactobacillus parabuchneri KCTC 3503 -1
oratories Inc., Hercules, USA).
Lacfobaciltus animalis KCTC 3501 -t
UV-absorption spectrum and mass spectrometry Lactobacillus vaginalis KCTC 3515 -
The UV-absorption spectrum of the bacteriocin was ex- Lactobacillus delbrueckii-lactis KCTC 1058 --
amined from 210 nm to 700 nm using a spectrophotome- Lactobacillus confusus KCTC 3499 .-
ter (Beckman Du-64, Beckman Instruments). The molecu- Lactobacillus paracasei subsp. paracasei KCTC 3510 -
lar mass was determined by MALDI-mass spectrometry Leuconostoc paramesenteroides KCTC 3531
for consistency (Kompact Maldi II, Kratos Analytical, Leuconostoc carnosum KCTC 3525 5-
England). The matrix used was a-cyno-4-hydroxycinamic Leuconostoc lactis KCTC 3528
acid and other experimental conditions were as follows: Leuconostoc mesenteroides KCTC 3 100 i-
positive mode, room temperature, and lo-kV accelerat- Leuconostoc mesenteroides subsp. mesenteroides +
KCTC 3505
ing voltage. -
Leuconostoc mesenteroides subsp. cremiris KCTC 3529
Amino acid composition and sequence analysis Af- Leuconostoc mesenteroides subsp. dextranicum
ter the purified bacteriocin was hydrolyzed in concentrat- KCTC 3530
ed HCl under vacuum at 110°C for 24 h, the amino acid Leuconostoc citreum KCTC 3526 .-
composition of the bacteriocin was analyzed by the Leuconostoc amelibiosum KCTC 3524 -
Pica-Tag method (15) using a HPLC system (Waters, Leuconostocpseudomesenteroides KCTC 3532 -
Milipore Co., MA, USA). For partial acid hydrolysis, Pediococcus acidilactici KCTC 3 101 -
Pediococcus dextrinicus KCTC 3506 -
the purified bacteriocin was dissolved in 30% TFA and
hydrolyzed at 110°C for 3 h. After evaporation of the Pediococcus pentosaceus KCTC 3507 i
Lactococcus rafinolactis KCTC 3509 -
solvent, the dried sample was redissolved in 0.1% TFA
Listeria monocytogenes KCTC 3444 t
and the partially hydrolyzed peptides were separated Staphylococcus aureus KCTC 1916 i-
using a HPLC as described previously. The amino acid Staphylococcus aureus KCTC 1928 +
sequence analysis of intact bacteriocin and three hydro- Escherichia coli KCTC 1924 -
lyzed peptides was carried out by Edman degradation Salmonella typhimorium KCTC 1926 -
using a gas phase protein sequencer (Model 431A, Applied Streptococcus thermophilus KCTC 2185 -
Biosystem Inc., Foster, USA). Bacillus subtilis KCTC 1023 -
Bacillus cereus KCTC 1013 t
Candida albicans KCTC 1940 -
RESULTS AND DISCUSSION
a Antimicrobial activity was determined by the well-diffusion assay
Isolation of bacteriocin-producing strain Approxi- as described in Materials and Methods.
mately about 4000 lactic acid bacteria were isolated from - , No inhibition zone; + , radius of inhibition zone < 3 mm; t-,
many kinds of kimchi using various isolation media. radius of inhibition zone 3 to 6mm; +-t, radius of inhibition zone
These isolates were tested for antimicrobial activity >6mm.
against 25 indicator strains. Only 10 strains showed inhi-
bition zones when cell-free supernatants were analyzed tive to the bacteriocin, but another, KCTC 3507, was
by the well-diffusion assay. The antagonistic activities of weakly inhibited by the bacteriocin. Nine species of Lac-
these 10 strains were sensitive to proteolytic enzymes, tobacillus and 7 species of Leuconostoc tested were sensi-
indicating that they were due to bacteriocins. tive. Among the pathogenic and food-spoiling bacteria
The antimicrobial activity spectrum of strain H-559 tested, Listeria monocytogenes, Staphylococcus aureus
was broader than that of any of the other isolates (data and Bacillus cereus were inhibited, but no inhibitory
not shown), and this strain was chosen for further activity was observed against Candida albicans and
characterization. This strain was identified as L. la&is Bacillus subtilis. The bacteriocin was not active against
subsp. lactis from phylogenetic analysis based on the gram-negative bacteria, such as Escherichia coli and
16s rDNA sequence and by comparison of morphologi- Salmonella typhimorium. From the standpoint of its
cal, cultural and biochemical characteristics with Ber- inhibitory spectrum, the bacteriocin appeared to take
gey’s Manual of Systematic Bacteriology (16) and Ber- an intermediate position between the lantibiotic nisin,
gey’s Manual of Determinative Bacteriology (17). There- which inhibits most gram-positive bacteria (18) and several
fore it was named L. la&is subsp. lack H-559 (2). bacteriocins from Lactobacillus such as lactacin B (19),
Antimicrobial spectrum The antimicrobial activity helveticin (20), and caseicin 80 (21), whose activity spectra
of partially purified bacteriocin was tested against vari- are narrow and include strains belonging to the same
ous gram-negative and gram-positive bacteria, including genus.
other lactic acid bacteria and several pathogenic strains. pH, heat, solvents, and enzyme stability of bacterio-
The results are shown in Table 1. Two strains of tins The partially purified bacteriocin was stable in
Pediococcus tested, KCTC 3101 and 3506, were insensi- wide pH range from 2 to 11 (data not shown). The in-
156 LEE ET AL.

TABLE 2. Effect of heat treatment on the bacteriocin activity TABLE 3. Effect of treatment with various enzymes on the
bacteriocin activity
Heat treatment Residual activity
(AU/ml) Residual activity
Enzyme (AU/ml)
100°C 10 min 1280
20 min 640 Catalase 1280
30 min 640 Glucose oxidase 1280
121°C 1Omin 640 Phospholipase Az 1280
20 min 320 Lipase 640
Control 1280 Peroxidase 1280
tr-Amylase 1280
;?-Amylase 1280
hibitory activity was unaffected by heating at moderate Pepsin 1280
temperatures and at 100°C for IOmin. However the in- Trypsisn 1280
hibitory activity was reduced during subsequent heating, Protease type 111 0
Protease type 1X 0
but still not completely inactivated even at 121°C for Protease type XIV 0
20min (Table 2). The heat stability may be due to the tr-Chymotrypsin 0
formation of small globular structures and the occur- Subtilisin 1280
rence of strongly hydrophobic regions, stable cross-link- Carboxypeptidase 1280
ages, and a high glycine content (22). This heat stability Control 1280
would be a very useful characteristic if the bacteriocin
was to be used as a food preservative, because many
food-processing procedures involve a heating step. There- sulfate precipitation, ion-exchange chromatography and
fore, the bacteriocin produced by L. lactis subsp. lactis reversed-phase HPLC. The purification steps and the
H-559 demonstrates potential as a food preservative in recovery values of the bacteriocin are given in Table
pasteurized products or canned foods. Furthermore, the 4. When ion-exchange chromatography using CM-
pH stability, in particular at neutral and basic pHs, con- Sepharose CL-6B was carried out, the shoulder portion
stitutes an advantage over nisin. The maximal solubility of the protein peak eluted at about 0.25 to 0.3 M NaCl
and stability exhibited by nisin at pH 2 decrease sig- contained 74% of the bacteriocin activity originally
nificantly as the pH increases, and this is a considerable applied to the column (Fig. 1). The elution profile of
disadvantage for its use as an additive in nonacidic HPLC is shown in Fig. 2. The bacteriocin activity eluted
foods (23). with 70% (v/v) acetonitrile was represented as a major
To select the appropriate acid and organic solvents for peak. The final specific activity of the purified bacterio-
the purification of the bacteriocin, the effects of various tin was increased approximately 3795-fold compared to
organic solvents and acids on the bacteriocin activity that in the culture supernatant and the recovery was
were examined. Incubation of the partially purified bac- 11%. When the purity of the fractions containing bac-
teriocin with various solvents and acids for 2 h did not teriocin activity was assessed by SDS-PAGE, a single
affect the activity (data not shown). The bacteriocin was protein band with an apparent molecular weight of 2500
insensitive to treatment with chloroform and hexane, was detected (Fig. 3).
suggest that the bacteriocin do not contain a lipid. UV-absorption spectrum The UV-absorption spec-
The antibacterial activity was not inactivated in the trum of the purified bacteriocin was examined. The puri-
presence of catalase, which excluded inhibition by hydro- fied bacteriocin showed maximum absorbance at 220 nm,
gen peroxide (Table 3). Treatment with protease types characteristic of peptide bonds. Minimal absorbance was
III, IX, and XIV, and cw-chymotrypsin caused the loss of observed at 280 nm suggesting a low content of aromatic
bacteriocin activity. When the partially purified bacterio- amino acids.
tin was treated with lipase, the activity was reduced, but Molecular mass MALDI-mass spectrometry con-
it did not exhibit a time- and concentration-dependent firmed the purity of the sample and determined the
kinetics (data not shown). These results confirm the pro- molecular mass of the bacteriocin to be 3343.7 Da (Fig.
teinaceous nature of the antimicrobial substance and 4). In SDS-PAGE, the bacteriocin migrated, with an ap-
suggest that neither lipid nor carbohydrate moieties are parent molecular weight of about 2500Da (Fig. 3). This
essential for the bacteriocin acitivity. However lipid moie- discrepancy could be attributed to the nonlinear migra-
ties may stabilize the bacteriocin activity. tion of small peptides on SDS-PAGE gels, and probably
Purification of the bacteriocin The bacteriocin results from the hydrophobicity of the bacteriocin (4).
produced by L. lactis subsp. lactis H-559 was purified Similarly, the hydrophobic bacteriocins, lacticin 481 and
from the supernatant fraction of cultures by ammonium lactococcin A, migrated to positions of 1.7 kDa and

TABLE 4. Purification of the bacteriocin produced by L. lactis subsp. luctis H-559

Total bacteriocin activity Specific activity Purification fold Yield
Sample Total Azsob
WJ)a (SS)
Culture supernatant 40000 9280 4.3 1 100
Ammonium sulfate precipitation and dialysis 8832 1669.8 5.3 1.2 22
Cation-exchange chromatography 6560 41.7 137.5 31.9 16.4
Reversed-phase HPLC 4455 0.3 16319 3795 11.1---
a Total bacteriocin activity is the activity unit multiplied by the volume in mililiters.
b Total Asso is the Asso multiplied by the volume in mililiters.
c Specific activity is AU divided by the Azso.
VOL. 88, 1999 PURIFICATION AND CHARACTERIZATION OF BACTERIOCIN 1.57

.5 - 2.0

.4 - 4-66.2 kDa
1.5

.3-d
zc x
8 g 1.0

= .2-f -16.9
i
.5 - 8.1
.1 - 6.2

- 2.5

FIG. 1. Chromatogram of the bacteriocin produced by L. lucks

subsp. luctis H-559 using CM-Sepharose CL-6B column chro- FIG. 3. Silver-stained urea-SDS-PAGE gel of the bacteriocin
matography. Lines are: -, absorbance at 280nm; ..., bacteriocin purified by reversed-phase HPLC. Lanes: 1, BSA; 2, low-molecular-
activity (Au/ml); --m,concentration of NaCl (M). weight standards; 3, purified bacteriocin; 4, BSA.
3.4 kDa, respectively, in SDS-PAGE, whereas the calcu-
lated molecular sizes deduced from their amino acid com- interactions of these peptides with lipids. It appeared
positions were 2.7 kDa and 5.8 kDa, respectively (4, 12). that the cruder the bacteriocin, the higher the apparent
Amino acid composition and sequence analysis molecular weight, suggesting that it associated with itself
The amino acid composition of the bacteriocin was deter- or other macromolecules. Ultrafiltration of the culture
mined (Table 5) and does not coincide with any other supernatant suggested that the apparent molecular
known bacteriocin compositions. No unidentified com- weight of the bacteriocin was above 50,000 Da. However,
pound was observed in the acid hydrolysate. The mass spectrometric analysis of a pure bacteriocin prepa-
amount of tryptophan, which is acid-labile, could not be ration revealed a molecular weight of 3343.07 Da. The
determined by the Pica-Tag method (15). If the bacterio- hydrophobic nature of a number of bacteriocins includ-
tin contained tryptophan residues, they were undetected. ing leucocin A-UAL 187 (26), lactacin F (27), mesenteri-
Calculation of the probable number of amino acid tin Y105 (28), lacticin 481 (4) and curvaticin FS47 (25),
residues in the bacteriocin molecule with a molecular and the apparent hydrophobicity of the bacteriocin
weight of 3343.7 Da reveals that it most likely contains produced by L. lack subsp. lactis H-559 also allow their
34 amino acids. Neutral (Gly, Asx, Ser, Thr) residues purification by reversed-phase HPLC.
and hydrophobic residues (Ala, Pro, Val, Ile, Leu, Met, Amino acid sequence Isoleucine was detected as
Phe) constitute a significant portion of the bacteriocin, the N-terminus of the bacteriocin by Edman degrada-
i.e., 29% and 53%, respectively. Many bacteriocins of tion. After the first N-terminal amino acid, the remain-
lactic acid bacteria have been shown to have high ing sequence could not be determined. Therefore, after
hydrophobicity. For example, about 50% of amino acids the bacteriocin was partially hydrolyzed, three peptides
are hydrophobic in lactococcin A (12), lactocin S (24), were purified from the hydrolysate and then amino acid
and curvaticin FS 47 (25) as well as lantibiotics such as sequencing was performed. Alanine was identified as the
nisin and lacticin 481 (4). The hydrophobic properties of N-terminal amino acid of one of the peptides but the
these bacteriocins most likely contribute to interprotein sequence after that was obstructed. The N-terminals of the
interactions that form multimeric complexes and to the
TABLE 5. Amino acid composition of the bacteriocin produced by
I I r 1500 L. lactis subsp. lactis H-559
Amino acid % Amount Residues per molecule
Asx (Asn + Asp) 8.40 2.86 (3)
Glx (Gln + Glu) 4.08 1.39 (1)
Ser 5.95 2.02 (2)
GUY 12.65 4.30 (4)
His 3.97 1.35 (1)
Arg 1.91 0.65 (1)
Thr 2.23 0.76 (1)
Ala 8.91 3.03 (3)
Pro 7.26 2.47 (3)
Val 5.35 1.82 (2)
Ile 10.55 3.59(4)
Time (min) Leu 10.49 3.57 (4)
FIG. 2. Cls reversed-phase HPLC chromatogram of the bac- LYS 11.68 3.97 (4)
teriocin produced by L. lacks subsp. luctis H-559. Elution was carried Met 3.79 1.29 (1)
Phe 2.02 0.69 (1)
out with a linear gradient of 0 to 80% acetonitrile in 0.1% TFA.
Lines are: p, absorbance at 220 nm; ‘. , concentration of acetonitrile Total number of amino acids 34
(%); ----, bacteriocin activity (AU/ml).
158 LEE ET AL.
100% = 123 mV [sum= 3704 mV] S s l-30 Smooth Av 30
3: 7
4wo
Mass/Charge
FIG. 4. Mass spectrum of the purified bacteriocin.
other two peptides were identified as methionine. Valine
and lysine were detected as the second residues. However
further amino acid sequencing was impossible by the
Edman degradation method. An explanation for the
obstruction could be that a disulfide bond was established
between the amino acid in the obstructing position and
another one located further down in the sequence (29).
However this explanation is not valid, since cysteine was
not detected by amino acid composition analysis. Lan-
thionine and P-methyllantionine introduce blank cycles
during sequencing by Edman degradation, while dide-
hydroalanine and didehydrobutyrine produce obstruction
of the reaction (30). The CP and p-unsaturated amino
acids (dehydroalanine and dehydrobutyrine) could not
be identified in acid hydrolysates of the bacteriocin,
since they are degraded during acid hydrolysis. The detec-
tion of unusual amino acids requires special methods
such as the o-phthaldialdehyde derivatization method.
Nisin is very similar to the bacteriocin studied here in
terms of molecular weight, the characteristics of the pro-
ducing strain, and the first amino acid at the N-terminus.
However nisin does not contain the sequence of -Met-
Val- and these two bacteriocins are different in amino
acid composition and pH stabiIity.
ACKNOWLEDGMENT
This work was supported by grants from the Korea Ministry of
Science and Technology. We thank Dr. Jong-Shin Yoo, Zee-Won
Lee, and Soo-Mi Kweon (Korea Basic Research Institute) for skill-
ful assistance with mass spectrometry, determination of amino acid
composition, and amino acid sequencing analysis.
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