J. Microbiol. Biotechnol. (2010), 20(11), 1513–1520
doi: 10.4014/jmb.1002.02006
First published online 11 September 2010
Purification and Physiochemical Characterization of Melanin Pigment
from Klebsiella sp. GSK
Sajjan, Shrishailnath1, Guruprasad Kulkarni1, Veeranagouda Yaligara2, Lee Kyoung2, and T. B. Karegoudar1*
1
Department of Biochemistry, Gulbarga University, Gulbarga - 585 106, Karnataka State, India
Department of Microbiology, Changwon National University, Changwon-si, Kyongnam 641-773, Korea
2
Received: February 4, 2010 / Revised: June 2, 2010 / Accepted: July 21, 2010
A bacterium capable of producing melanin pigment in the
presence of L-tyrosine was isolated from a crop field soil
sample and identified as Klebsiella sp. GSK based on
morphological, biochemical, and 16S rDNA sequencing.
The polymerization of this pigment occurs outside the cell
wall, which has a granular structure as melanin ghosts.
Chemical characterization of the pigment particles showed
then to be acid resistant, alkali soluble, and insoluble in
most of the organic solvents and water. The pigment got
bleached when subjected to the action of oxidants as well
as reductants. This pigment was precipitated with FeCl3,
ammoniacal silver nitrate, and potassium ferricynide. The
pigment showed high absorbance in the UV region and
decreased absorbance when shifted towards the visible
region. The melanin pigment was further charecterized
by FT-IR and EPR spectroscopies. A key enzyme, 4hydroxyphenylacetic acid hydroxylase, that catalyzes the
formation of melanin pigment by hydroxylation of Ltyrosine was detected in this bacterium. Inhibition studies
with specific inhibitors, kojic acid and KCN, proved that
melanin is synthesized by the DOPA-melanin pathway.
Keywords: Klebsiella sp. GSK, L-tyrosine, melanin, pigment,
spectroscopy, 4-hydroxyphenylacetate hydroxylase
Melanins form a diverse group of pigments synthesized in
living organisms in the course of hydroxylation and polymerization of organic compounds. Melanin production is
observed in all large taxa from both the Prokaryota and
Eukaryota [25]. Melanin is nearly a ubiquitous pigment.
Animal melanins may be classified as black eumelanins
and yellow-to-brown pheomelanins, whereas melanins from
plants, fungi, and bacteria are brown-to-black allomelanins
[22]. Melanins are negatively charged, hydrophobic [5],
*Corresponding author
Phone: +91-8472-263289; Fax: +91-8472-245632;
E-mail: [email protected]
and high-molecular-weight compounds. These pigments
are insoluble in both aqueous and organic solvents, and
its is consequently difficult to study their structure by
conventional biochemical and biophysical techniques [23].
The ability to produce melanin is widespread among
microorganisms. From the chemical point of view, the only
common feature of microbial melanins is it being a product
of oxidative polymerization of various phenolic substances.
Melanins form a quite heterogeneous group of biopolymers.
As a consequence, melanogenesis can serve as an example
of evolutionary convergence, besides mimicry, and signaling,
as well as protection against UV and visible light, and extreme
temperatures, and maintaining a proper balance of metal
ions [25]. Melanin pigments are synthesized by organism
representative of all biological kingdoms and have been
implicated in a wide variety of physiological and pathological
processes, including the pathogenesis of some microbial
infections [5, 37]. Production of melanin is one of the most
universal (but at the same time enigmatic) adaptations of
living organisms to the variable conditions of the Earth. The
presence of various kinds of melanins in representatives of
almost every large taxon suggests an evolutionary importance
of melanogenesis [38]. Melanins have great application
potentials in the agriculture, cosmetics, and pharmaceutical
industries. Research has revealed that melanin produced by
Streptomycete showed photoprotection and mosquitocidal
activity of Bacillus thuringiensis subsp. israelensis [19].
Melanins are heterogeneous polymers of dihydroxy
indole (DHI) and dihydroxy indole carboxylic acid (DHICA)
monomers linked by heterogeneous non-hydrolizable
bonds [9], with only a short-distance ordering [7]. It was
suggested that melanin polymers constitute the building
blocks of melanin granules [39]. The process of granules
formation and their dimension are strongly pH dependent,
where a low pH promotes the aggregate growth and a high
pH induces the break up of the granules to small particles
- oligomers with a lower degree of polymerization. This
process is a consequence of the polyelectrolyte nature of
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Sajjan et al.
melanin, and it is dependent on the ionization state of
melanin groups like carboxylic, phenolic, and aminic
groups as well as on the ionic strength of the environment.
These features make the melanin a very complex absorbing
material [4]. A bacterium capable of producing a high
amount of melanin from L-tyrosine within 3 days of incubation
has been isolated. A key enzyme, 4-hydroxyphenylacetic
acid hydroxylase, involved in the formation of melanin
pigment from L-tyrosine is shown in this bacterium.
Characterization of the physiochemical properties of black
pigment melanin have been carried out. Moreover, inhibitory
studies of melanin synthesis are presented in this paper.
MATERIALS AND METHODS
Chemicals
Synthetic melanin, L-dihydroxyphenylalanine, and L-tyrosine were
procured from Sigma Chemicals Co., St. Louis, USA. Kojic acid
was obtained from HiMedia chemicals, Mumbai, India, and all other
chemicals used were of analytical reagent grade.
Screening of Bacteria Capable of Producing Melanin Pigment
Bacterial strains capable of producing high amounts of melanin
were isolated from various crop field samples. The selected strain
was grown in 250-ml Erlenmeyer flasks containing 100 ml of
minimal medium containing defined components (29.4 mM K HPO ,
10 mM MgSO ·7H O, 5 mM FeSO , 5 mM ZnSO , 5 mM MnSO
50 mM NH NO , and 55 mM glucose) with or without L-tyrosine
(1 g/l) at pH 7.2. Inoculated culture flasks without L-tyrosine as well
as uninoculated flasks containing L-tyrosine served as controls. The
medium was autoclaved at 15 psi (121 C) for 20 min; these flasks
were inoculated with the bacterium and incubated at 37 C on a
rotary shaker at 220 rpm for 72-96 h. Thereafter, the culture was
collected by centrifugation at 8,000 ×g and the melanin present in
the culture spent medium was extracted. For L-tyrosine-dependent
pigment production assay, different concentrations of L-tyrosine
were supplemented to the above media (0 to 2 g/l in 250 mg/l
increments).
The isolated bacterial strain was identified based on morphological,
biochemical, and physiological tests and 16S rDNA sequencing [1].
The sequence was deposited in the National Center for Biotechnology
Information (NCBI) nucleotide sequence database under the accession
number GU066861. The 16S rDNA sequence was compared with
sequences available in public databases, using the BLAST search
program on the NCBI Web site (http://www.ncbi. nlm.nih.gov/) to find
closely related bacterial 16S rDNA gene sequences. Phylogenetic
and molecular evolutionary analyses were conducted using the
MEGA version 4 software [31]. A phylogenetic tree was constructed
by the neighbor-joining method and maximum composite likelihood
model with bootstrap values at 500 replicates. This culture is
deposited in the National Collection of Industrial Microorganisms
(NCIM), Pune, India under the accession number NCIM 5338.
2
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4
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Growth Condition, Pigment Production, and L-Tyrosine Utilization
Glucose was used as carbon source and the growth of the culture
medium was measured as change in optical density (OD) at 660 nm,
optimum pH 7.2, and temperature of 37 C. The formation of melanin
o
pigment from L-tyrosine by this bacterium in the cell-free culture
supernatant was monitored at 400 nm [32]. The absorbance value
was converted by using a standard calibration curve of synthetic
melanin [36]. L-Tyrosine utilization was measured by the method
reported by Arnow [2]. To 1 ml of culture supernatant, 1 ml of
mercuric sulfate reagent and 1 ml of sodium nitrite reagent were
added, and then the absorbance was measured at 546 nm using a
spectrophotometer.
In Vitro Melanization Assay
Klebsiella sp. GSK cells were spread on chemically defined
minimal medium containing 1.8% (w/v) bacto agar, pH 7.2, with or
without L-tyrosine, and incubated for 2-3 days at 37 C. Plates were
examined daily to monitor the growth of the bacterium and melanin
pigment production.
o
Pigment Extraction
Aliquots (4 ml) of cells were inoculated into 250-ml conical flasks,
each containing 100 ml of the defined medium with or without Ltyrosine. Cultures were grown until the liquid medium became
darkly pigmented and nearly opaque. The method of pigment
extraction from this Klebsiella strain grown medium was followed
as described by Nicolaus [21]. The 3-days-grown cell suspension
was disrupted by using a Vibracell ultrasonicator (model VC 375;
USA) in an ice bath at a normal power of 70 W for 3-min periods;
each period of disruption was of 30-s cycles followed by a 1-min
off cycle during which the medium and oscillator probe were cooled
in ice. The disrupted broth was acidified with 1 N HCl to pH 2 and
allowed to stand for one week at room temperature. Then this
suspension was boiled for 1 h to prevent the formation of melanoidins
and then centrifuged at 8,000 ×g for 10 min [12]. The formed black
pigment pellet was washed three times with 15 ml of 0.1 N HCl and
then with water. To this pellet, 10 ml of ethanol was added and the
mixture then incubated in a boiling water bath for 10 min and then
kept at room temperature for 1 day. The pellet was washed with
ethanol two times and then dried in air. The extracted pigment
pellets were pooled for use in subsequent analyses.
Chemical Analysis of the Pigment
The chemical analysis of melanin pigment was carried out by the
modified method of Fava et al. [12]. The solubilities of the black
pigment in distilled deionized water, 1 N HCl, 1 N NaOH, ethanol,
acetone, chloroform, benzene, and phenol were checked. Reactions
with oxidizing agents such as 6% sodium hypochlorite (NaOCl) and
30% hydrogen peroxide (H O ) were determined. Reducing agents
such as H S and 5% sodium hydrosulfite (Na S O ) were also tested
for reaction with the black pigment. The pigment was also
precipitated with 1% FeCl , ammoniacal silver nitrite, and potassium
ferricyanide. These tests were carried out in parallel with synthetic
melanin for comparison. Results represent identical outcomes of
qualitative physical and chemical tests for replications.
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UV-Visible Spectroscopic Analysis of the Extracted Melanin
Different concentrations of purified melanin were prepared using the
initial concentrations of 100 mg/l in 0.1 N NaOH, and diluted to 1:1,
1:2, and 1:3. Each alkaline solution was scanned from 180 to
900 nm wavelengths. A 0.1 N NaOH was used as the blank [36].
The spectroscopic property of the melanin pigment obtained from
Klebsiella sp. GSK was compared with synthetic melanin.
PURIFICATION AND CHARACTERIZATION OF MELANIN PIGMENT
FT-IR Spectroscopy Studies
The purified melanin pigment was ground with IR grade KBr (1:10)
and pressed into disks under high pressure using a pellet maker. The
FT-IR spectrum was recorded at 4,000-400 cm- [26] using a
Perkin Elmer FT-IR spectrophotometer (USA).
1
EPR Spectroscopy
One of the unique properties of melanin pigment is the presence of
unpaired electrons in the polymer, which can be detected by EPR
spectroscopy [11]. EPR spectra were obtained with a Bruker EMX
(X-Band) EPR spectrophotometer. The EPR spectral conditions
were frequency, 9.39 GHz; modulation amplitude, 4.0 Gauss; power,
0.211 mW; center field, 3350.0 G; sweep width, 400.0 G; and sweep
time, 83.88 s.
Preparation of Melanin Ghosts
Klebsiella sp. GSK was cultured in chemically defined minimal
medium with L-tyrosine at 37 C in a shaking incubator for 3 days.
Melanin ghosts were isolated from the spent medium by boiling in
HCl and ethanol extraction, as described by Eisenman et al. [10].
Ghosts were fixed on glass slides by gently heating for 1-2 s and
viewed under a Leica A × 80 Ultra zoom microscope (Wetzlar,
Germany) without staining.
o
4-Hydroxyphenylacetic Acid Hydroxylase Assay
Two-days-old grown cells of Klebsiella sp. GSK were subjected to
sonication as described previously. The disrupted cells were
centrifuged for 20 min at 10,000 ×g and the supernatant was used
as the enzyme source. The 4-hydroxyphenylacetic acid dependent
oxidation of NADH was used to monitor the enzyme activity. The
initial rate of oxidation of NADH (∆c =6,220 M- cm- ) was
determined spectrophotometrically by monitoring the decrease in
absorbance at 340 nm. The reaction was performed in a 1-ml quartz
cuvette with a 1-cm light path. The enzyme activity was assayed at
30 C by adding 10 µl of 0.1 M substrate to 0.5 ml of 0.1 M sodium
phosphate buffer, pH 8.0, 0.2 mM NADH, 0.6 µM FAD, and 50 µl
of enzyme. Values were corrected for oxidation of NADH in the
absence of substrate. A unit of activity was defined as the amount of
enzyme that catalyzed the oxidation of 1 µmol of NADH per minute
[33]. The protein concentration of the enzyme solution was
determined by using bovine serum albumin as a standard [20].
1
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1515
Table 1. Morphological and biochemical characteristics of
Klebsiella sp. GSK.
Characteristics
Results
Cell morphology
Small bacilli, nonmotile, pink color
colonies on MacConkey agar, light
yellow colonies on nutrient agar, brown
color colonies on L-tyrosine agar
Gram negative
Aerobic; catalase positive; citrate
utilized; indole negative; gelatin not
liquefied; casein and starch not
hydrolyzed; urease positive; MR
negative; both acid and gas produced
from glucose, lactose, and sucrose;
oxidase positive; H2S gas production
negative, resistant to KCN
6.0 to 8.5
25oC to 40oC
Staining
Physiological
properties
pH range for growth
Growth temperature
The colonies capable of producing black pigment were
picked out and identified as Klebsiella sp. GSK, based on
its morphological and biochemical characteristics (Table 1)
as well as 16S rDNA sequence. This bacterium was resistant
to ampicillin, but sensitive to streptomycin, cefotaxime,
and chloremphenicol. Strain GSK showed 99% homology
with Klebsiella sp. GQ418148.1. The phylogenetic
relationship of this strain is shown in Fig. 1. In vitro
melanization assays were performed to determine whether
Klebsiella sp. GSK produces pigment from L-tyrosine.
Cells were spread onto agar plates with or without Ltyrosine. The bacterial cells turned black colored within
3 days on agar plate containing L-tyrosine (Fig. 2), but
pigmentation was not observed in agar plates lacking Ltyrosine. The liquid medium containing L-tyrosine also
turned black/brown after 2-3 days with the GSK strain.
Cells of Klebsiella sp. GSK darkened within 3 days in
liquid medium supplemented with L-tyrosine. However,
Klebsiella sp. GSK does not produce melanin pigment in
Inhibition Effects of Kojic Acid and KCN on Pigment Production
In order to study the inhibitory effects of kojic acid and KCN on the
pigment production by Klebsiella sp. GSK, cells were cultured in
100 ml of the defined medium with L-tyrosine and then different
concentrations of kojic acid and KCN (0.1 mM to 0.5 mM) were
added to the medium. Control flasks (without inhibitor) were kept
for each test. The inhibition of melanin synthesis was monitored at
regular intervals up to 96 h of incubation. The pigment concentration
was estimated by the spectroscopic method using synthetic melanin
as the standard.
RESULTS AND DISCUSSION
Strain Selection and Pigment Production
A melanin-producing bacterium was isolated from soil
samples placed on L-tyrosine agar plates for several days.
Fig. 1. Phylogenetic tree showing the position of isolate GSK
with reference to related strains.
Strain GSK showed 99% homology with the type strain Klebsiella sp.
GQ418148.1. All 16S rDNA sequences of related strains have been
retrieved from the NCBI database. Genome accession numbers are shown
in parenthesis; 0.001 denotes the genetic distance.
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Sajjan et al.
Fig. 3. Growth curve of GSK ( ■ ), melanin production ( ◆ ), and
L-tyrosine utilization ( ▲ ).
Culture conditions at pH 7.2 and 37oC. Supernatant was taken to determine
the growth, melanin production, and L-tyrosine utilization as described in
Materials and Methods.
Fig. 2. Screening of bacterial strain for pigment production,
where GSK cells were grown with or without L-tyrosine and
incubated for 3 days.
A, C. The dark brown-colored colonies were selected for melanin
production by Klebsiella sp. GSK cells grown with L-tyrosine. B, D.
Without L-tyrosine, the cells were unable to produce pigment. E. The
uninoculated flask shows the autooxidation of L-tyrosine is not likely a
cause.
liquid medium lacking L-tyrosine. Pigment production was
not observed in control flasks, indicating that autooxidation
of chemical constituents in the medium does not likely
cause any pigmentation (Fig. 2).
Increase in the growth of the bacterium increased the
utilization of L-tyrosine and the pigment production, and
maximum growth of the bacterium was observed at 3 days
of incubation along with the maximum amount of melanin
(130 mg/l) production. The maximum utilization of Ltyrosine was observed after 3 days of incubation (Fig. 3).
The melanin production was observed at pH 7.2 and 35oC.
Moreover, L-tyrosine utilization showed that melanin
production was initiated after (24 h) when 50% L-tyrosine
(500 mg/l) was utilized, and 90% L-tyrosine (900 mg/l)
utilization was observed when the culture entered into log
phase (after 48 h), which showed that the maximum Ltyrosine (about 950 mg/l) was utilized before its polymerization
into melanin.
Furthermore, L-tyrosine dependent pigment production
was delayed progressively from 2 g/l to 250 mg/l, and the
concentration of pigment was dependent on the L-tyrosine
concentration, where 2 g/l to 1 g/l L-tyrosine-supplemented
cells produced pigment in 2 to 3 days; below 1 g/l
concentration, the time taken for pigment production was
4-7 days. There was no pigment production in medium
lacking L-tyrosine, even after 30 days (data not shown).
Characteristics of Melanin Produced by Klebsiella sp.
GSK
We have observed that the pigment produced by Klebsiella
sp. GSK was shown to be a true melanin, as revealed by a
number of physical and chemical tests. These tests indicate
that the pigment is likely to be a melanin pigment, as
observed for the melanin isolated from other bacteria such
as Aeromonas media [18, 34] and Escherichia coli [8, 16,
17], and fungi Cryptococcus neoformans [14] and Pleurotus
cystidiosus [29]. The Klebsiella sp. GSK produced a dark
brown pigment in culture medium as a dead-end product.
It is reported that microbes predominantly produce melanin
pigment via tyrosinases, laccases, catecholases, and the
polyketide synthase pathway [6]. However, this strain produced
melanin pigment by the 4-hydroxyphenylacetic acid pathway.
The enzyme 4-hydroxyphenylacetic acid hydroxylase
belongs to a separate family of hydrolases [14]. Besides
its main substrate, 4-hydroxyphenylacetic acid, it also
catalyzes the hydroxylation of other aromatic compounds,
which leads to the formation of dibenzoquinone and other
o-quinone derivatives. These quinone derivatives then
polymerize spontaneously to melanin-like polymers [13].
L-Tyrosine is also a substrate for 4-hydroxyphenylacetic
acid hydroxylase, but unlike tyrosinase this enzyme does
PURIFICATION AND CHARACTERIZATION OF MELANIN PIGMENT
1517
Table 2. Chemical properties of melanin pigment.
Experiments Test
1
2
Result
3
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5
6
7
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9
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11
Water
Solubility in organic solvents
a) Ethanol
b) Chloroform
c) Acetone
d) Benzene
e) Phenol
1 M KOH/NaOH
Color
Precipitation in 3 N HCl
Reaction with oxidizing agent (H2O2)
Reaction with NaOCl
Reaction for polyphenols with FeCl3 test
Reaction with Na2S2O4 and potassium ferricyanide
Reaction with ammoniacal silver nitrate solution
Reduction with H2S gas
12
UV-visible absorption
not contain copper and the presence of copper does not
show any increase in enzyme activity [25]. This strain
showed 4-hydroxyphenylacetic acid hydroxylase activity
only with NADH as cosubstrate. Furthermore, the extracts
prepared from cells grown on L-tyrosine showed 4hydroxyphenylacetic acid hydroxylase activity using
different substrates. The specific activity of this enzyme
for L-tyrosine was greater than 4-hydroxyphenylacetic acid
and 2-hydroxyphenylacetic acid, whereas 3,4-dihydroxy
phenylalanine (L-DOPA) did not show any activity, as
hydroxylation for the 3rd position on L-DOPA was not free
(Table 3). 4-Hydroxyphenylacetic acid hydroxylase catalyzes
L-tyrosine to melanin using the DOPA-melanin pathway.
The DOPA-melanin pathway in this strain was further
confirmed by the use of kojic acid, a specific inhibitor of
the DOPA-melanin pathway. The biosynthesis of melanin
from L-tyrosine via 4-hydroxyphenylacetic acid hydroxylase
is an unusual pathway of melanogenesis [13, 14].
Table 3. Activity of 4-hydroxyphenylacetic acid hydroxylase
from Klebsiella sp. GSK grown on L-tyrosine-supplemented
mineral salt medium.
Substrate
L-Tyrosine
4-Hydroxyphenylacetic acid
2-Hydroxyphenylacetic acid
L-DOPA
IU
Specific activity
87.4
30.8
28.7
ND
17.1
5.9
4.7
ND
ND: Not detected.
The enzymatic activity values are expressed as the average of four
determinations.
Insoluble
Insoluble
Insoluble
Insoluble
Insoluble
Soluble
Soluble
Blackish brown
Precipitated readily
Decolorized (black to colorless)
Decolorized
Brown flocculent precipitate
Decolorized and turned brown with addition of potassium ferricyanide
Formed a grey-colored silver precipitate lining on the sides of the test tube
Reduced
Linear relationship between log absorbance and wavelength
Between 400 and 600 nm
Our present strain GSK appears to be the best melanin
producer when compared with the previous reports on
Klebsiella spp. or any other natural bacteria. The supplementation
of 50 mg/l of L-DOPA along with L-tyrosine enhanced the
production of melanin up to 540 mg/l within 84 h of
incubation, which was 4 times greater than that of Ltyrosine alone. Wenlin et al. [36] reported that the melanin
synthesis from Frankia strain Cel5 was about 180 mg/l
with both 1.4 mM L-tyrosine and 10-8 M L-DOPA after 14
days of incubation, whereas only 20 mg/l of melanin
pigment production was observed in the medium lacking
L-DOPA. A fungus Cryptococcus neoformans produces
pigment, on induction by Klebsiella aerogenes. Whereas,
Klebsiella aerogenes or Cryptococcus neoformans were
unable to produce pigment individually [30].
The chemical analysis of the melanin pigment is
summarized in Table 2. The pigment was black colored,
and insoluble in water, acid, ethanol, benzene, chloroform,
and acetone. The pigment was soluble in alkaline solution
and in phenol. The dissolved pigment was decolorized
by oxidising and reducting reagents such as NaOCl, H2O2,
H2S, and Na2S2O4. The pigment tested positive for polyphenols
with FeCl3, producing a flocculent brown precipitate, and
reduced ammonical silver nitrate [3, 36].
Spectroscopic Analysis of the Pigment
The UV-visible wavelength scan (180 to 900 nm) of the
pigment is shown in Fig. 4A. The absorption was highest
in the UV region at 200-300 nm, but decreased towards
the visible region, which is the characteristic property of
melanin. This phenomenon is due to the presence of the
very complex conjugated structure in melanin. This property
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Sajjan et al.
Fig. 5. FT-IR spectrum of the melanin pigment from Klebsiella
sp. GSK.
Fig. 4. UV-visible spectroscopic properties of melanin pigment.
One g/l melanin was diluted to 1:1, 1:2, and 1:3 dilutions, showing the
differential effect of melanin concentration on absorbance at varying
wavelengths. A. UV-Visible absorption spectra of melanin pigment. B.
Log absorbance of melanin pigment in different concentrations.
of melanin is confirmed by comparing with the previous
descriptions of melanin pigment and measurements of
synthetic melanin [25, 27, 28, 34]. An increase in wavelength
decreases the absorbance of melanin pigment progressively.
Hence, the slopes of linear plots are often used to identify
melanin pigments (Fig. 4A). There was a linear relationship
between log absorbance and wavelength from 400 to
600 nm, which is one of the most important criteria for the
characterization of melanin, Schaeffer [28] showed that the
log of optical density of a melanin solution, when plotted
against wavelength, produces a linear curve with negative
slopes. Such characteristic straight lines with negative
slopes have been obtained for melanin produced by some
fungi [26, 29, 36]. The pigment produced by Klebsiella sp.
GSK also gave a straight line with a negative slope
indicating that it was melanin (Fig. 4B). When the melanin
was subjected to gradual dilutions, the absorbance decreased
unevenly from the UV region to near the red region. These
results are in accordance with the earlier reports [29, 36].
FT-IR spectroscopy was chosen for further characterization
of the pigment, since it is regarded as the most informative,
well-resolved, and non-destructive method, providing
information on functional groups and detailed structural
analysis of melanin [24]. The IR spectrum of the melanin
pigment showed a broad absorption at 3,329.08 cm-1, which
revealed the presence of the -OH group. The broadening
of the band might be due to the hydrogen bonding of the
-OH group with the -NH group. The peak occurred at
2,921.14 cm-1, which indicates as -CH. Absorption at
1,627.73 cm-1 was attributed to aromatic ring C=C stretching
(Fig. 5). These characteristic properties of the IR spectrum
of this pigment were similar to earlier reports [15, 29]. The
spectroscopic properties of the melanin pigment from
Klebsiella culture correlated with those of melanin produced
by various microorganisms as reported previously [10, 12,
36] as well as with the properties of the synthetic melanin.
Apart from UV-visible and FT-IR spectral study, EPR
spectroscopic studies were conducted to see the
paramagnetic properties and free radicals present in the
melanin pigment. The major defining features of all the
melanins is the presence of a stable organic free radicals,
which results in the characteristic electron paramagnetic
resonance behavior. This property of melanin was
exploited by studying EPR spectrum [11]. In the present
study, the EPR spectrum of the melanin pigment indicated
the presence of free radicals (Fig. 6). These results
demonstrate the usefulness of spectroscopy in studying
diverse aspects of melanin and melanization in microorganisms.
Fig. 6. EPR spectral analysis of the melanin pigment from
Klebsiella sp. GSK.
PURIFICATION AND CHARACTERIZATION OF MELANIN PIGMENT
Fig. 7. Microscopy of melanin ghosts isolated from Klebsiella
sp. GSK.
Cells were grown in the presence of L-tyrosine and subjected to chemical
degradation as described in Materials and Methods. The resulting particles
were imaged by light microscopy without staining.
Melanin Ghosts
Klebsiella sp. GSK strain was grown in the presence of Ltyrosine and melanin ghosts were recovered by acid
treatments and removal of unwanted cellular materials.
The resulting particles were visualized under microscope
(Fig. 7). The isolated melanin ghosts were similar to those
in previous reports [10, 30].
Inhibition by Kojic Acid and KCN
Kojic acid and KCN are the inhibitors of melanin synthesis
and were evaluated by in vitro melanization assay. Kojic
acid is an inhibitor of the DOPA-melanin synthesis pathway
[34], and KCN also inhibits melanin synthesis [36], which
confirmed that this organism catalyzes L-tyrosine via the
DOPA-melanin synthesis pathway. Melanin production
was effectively inhibited (about 90%) by kojic acid at
0.2 mM and KCN at 0.3 mM concentrations. Below these
concentrations of inhibitors, there was a slight increase in
pigment production (Fig. 8).
In conclusion, melanin pigment from Klebsiella sp.
GSK is very similar to that of a typical melanin, which
constitutes a diverse group of aromatic polymers with
many different potential applications in cosmetic and
pharmaceutical industries. Bacterial synthesis of melanin
pigment is an alternative option for commercial-scale
production. To our knowledge, this is the first report so far
on Klebsiella sp. alone. This strain produced a high
amount of melanin pigment (130 mg/l) as compared with
other reported strains, and this culture produced melanin in
3 days without any inducers added to the growth medium.
Furthermore, the melanin production was enhanced to
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Fig. 8. Melanin production and inhibition in broth media by
different concentrations of KCN and kojic acid.
540 mg/l when the medium was supplemented with 50 mg/l
of L-DOPA along with L-tyrosine.
Acknowledgments
The authors wish to thank the Department of Biotechnology
(DBT), Ministry of Science and Technology, New Delhi,
India for financial support, and the University Grant
Commission (UGC) for supporting the Department through
the UGC-SAP programme. We are thankful to Prof. S. V.
Bhat, Department of Physics, Indian Institute of Science
(IISc), Bangalore for his help in the EPR analysis.
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