Biotechnology and Bioprocess Engineering 2009, 14: 383-390

DOI/10.1007/s12257-009-0082-3





Partial Purification and Characterization of
a Novel Antifungal Compound against
Aspergillus spp. from Synechocystis sp. PCC 6803

Young-sil Yoon and Choul-gyun Lee*
Institute of Industrial and Biotechnology, Department of Biological Engineering, Inha University, Incheon 402-751, Korea


^~ The antifungal compound AK-3 is purified and characterized from intracellular metabolites of the unicellular cyanobacteria,
Synechocystis sp. PCC 6803. AK-3 clearly had antifungal effects upon growth of the fungi Aspergillus spp. From 2 g of dry
cell powder, 4.8 mg of AK-3 was obtained with a yield of 0.24%. Its structure was elucidated by NMR, UV spectra, amino
acid analysis by chemical degradation, and MS measurements, including MALDI-TOF and MS-MS techniques. AK-3 con-
tains a high level of antifungal activity compared to itraconazole and is seemingly a new compound based on its amino acid
composition. Extraction was performed with an acetic acid/water mixture (pH 3.0) while detection at UV 214 nm found AK-3
did not react with ninhydrin. These results suggest that this compound is a cyclic peptide with high hydrophilicity and a phe-
nolic ring. KSBB

hW cyanobacteria, Synechocystis sp., aspergillosis, antifungal, cyclic peptide




INTRODUCTION

Opportunistic fungal infections have increased in frequency
predominantly among the immunocompromised population,
including victims of AIDS, cancer patients receiving aggres-
sive chemotherapy, post-organ transplant patients, and patients
subjected to long hospitalization or broad-spectrum antibiotic
treatment [1]. In particular, fungi belonging to the genus As-
pergillus are some of the most predominant pathogens. Indeed,
invasive aspergillosis is the most common and most insidious
fungal infection worldwide, as it is associated with high mor-
tality rates and virulence [2-5]. Aspergillus spp. is saprophytic,
spore-forming, filamentous fungi ubiquitous throughout the
environment. The most infectious species is A. fumigatus,
accounting for more than 90% of all human infections along
with A. flavus and A. niger [6-8].
Bioactive metabolites are most commonly obtained from
screening of different natural sources. Cyanobacteria are
known to produce a vast array of bioactive compounds, such
as peptides, amides, alkaloids, polyketides, phycobiliproteins,
and potent cyanotoxins, such as mycrocystin, nodularin, and
anatoxin [9-15]. The current need for new antifungal com-
pounds has stimulated research into the toxic properties of

G` ~
Tel: +82-32-860-7518 Fax: +82-32-872-4046
e-mail: leecg@inha.ac.kr
cyanobacterial metabolites. Such cyanobacterial toxins can
be extremely harmful to aquatic predators that feed on
cyanobacteria [12,16]. This indicates that synthesis of highly
active toxins by cyanobacteria is possibly a defense mecha-
nism against attack by eukaryotic organisms such as fungi,
zooplankton, and animals. Although numerous reports on the
bioactive metabolites of cyanobacteria exist, studies that
directly address the antifungal activity of cyanobacterial
extracts are relatively few, including studies concerning in-
hibitory modes. The unicellular cyanobacteria Synechocystis
sp. PCC 6803 is commonly used as a foundational model
system for molecular and biophysical studies on oxygenic
photosynthesis [17,18] since its genome has been completely
sequenced [19]. Research on Synechocystis sp. PCC 6803
has focused mainly on gaining a better understanding of the
metabolic processes of organisms [20], but relatively little
work has been reported on finding its bioactive compounds.
The aim of the present study was therefore to investigate
and purify new antifungal metabolites from Synechocystis sp.
PCC 6803 and characterize the active compound.


MATERIALS AND METHODS

CuItivation of Cyanobacteria

A monoclonal culture strain of the cyanobacteria Synecho-
PUQ

cystis sp. PCC 6803 was obtained from the Pasteur Culture
Collection of Cyanobacteria (PCC, Paris, France). Synecho-
cystis sp. PCC 6803 was cultivated with aeration (filtered air,
0.22 m/min) in a 2 L glass column containing BG-11 me-
dium at 23
o
C, under constant irradiance with cool, white,
fluorescent light at an intensity of 40 E/m
2
. Cells were har-
vested after 20~22 days of incubation by centrifugation at
7,000 rpm. Harvested cells were lyophilized and kept at -20
o
C
until further study.

FungaI Strains

The following twelve fungal species were obtained from
the Korea Agricultural Culture Collection (KACC, Suwon,
Korea) and used as antifungal test strains: A. niger KACC
6896, A. niger KACC 40280, A. niger KACC 41018, A. ni-
ger KACC 41858, A. flavus KACC 6636, A. flavus KACC
40232, A. flavus KACC 40233, A. fumigatus, KACC 40080,
A. fumigatus, KACC 41016, A. fumigatus KACC 41136, and
A. fumigatus KACC 41143. The fungal strains were grown
on Potato Dextrose Agar (PDA, Difco, Detroit, USA) at
28
o
C for 72 h. Conidia were collected by adding sterile water
and gentle scrubbing with a glass rod. The conidial suspen-
sion was adjusted to the required concentration based on
counting done via a hemocytometer.

f AntifungaI Activities

Antifungal activities against fungal strains were investi-
gated by the paper disk method [21]. Paper disks were
placed on potato dextrose agar seeded with Aspergillus spp.
The test solutions were applied on filter paper disks 8 mm in
diameter. Five milligram per milliliter of concentrated crude
extract solution was dissolved in 5% acetic acid in separate
sterile tubes. A quantity of itraconazole equal to one-fifth the
amount of target compound employed was used as positive
control. The diameter of the growth inhibition zone around
each disk was measured after incubation at 28
o
C for 48 h.
For each extract, three replicate trials were conducted against
each fungus. The minimum fungicidal concentration (MFC)
was defined as the concentration of target compound at
which the growth inhibition zone reached above 1 mm in
diameter. MFCs were also evaluated after incubation for 48 h
at 28
o
C and were adjusted to 10
5
cells/mL.

Extraction and Purification

Cells from 2 L of culture were collected by centrifugation
at 7,000 rpm for 20 min and then lyophilized. Freeze-dried
cells (about 2 g from 2 L of culture) were suspended in 5%
acetic acid (50 mL) for 1 h at 22
o
C, followed by ultrasonic
treatment. Cell debris were filtered out and subjected twice
to the same treatment while the leftover filtrate was evapo-
rated. Impurities in the cell extract were removed using a
C18 solid phase extraction cartridge (Waters Corp., Milford,
MA, USA). Fractionation was performed in three steps with
0, 5, and 10% acetic acid solutions. The eluate was evapo-
rated and passed through an ultrafiltration cartridge (Milli-
pore Corp., Bedford, MA, USA) with molecular weight cut-
off (MWCO) of 5 kDa. Following this, it was loaded on 3
systems: a reverse-phase HPLC equipped with a M930
pump (Younglin Corp., Korea), for further purification a
manually operated loop injection system (IDEX Health &
Science, Oak Harbor, WA, USA) and a 21.4 250 mm C18
semi-preparative column (GE Healthcare, Uppsala, Sweden).
The mobile phase was a mixture of acetonitrile:water (60:40,
v/v) containing 0.05% trifluoroacetic acid and the flow rate
was 2.5 mL/min at room temperature. The target compound
was detected at 214 nm using a M720 UV detector
(Younglin Corp., Korea).

Thin-Iayer Chromatography

The major active fractions obtained throughout purifica-
tion were analyzed by thin-layer chromatography (TLC) on
silica gel plates (10 10 cm, 0.2 mm, Silica gel 60 F
254

plate; Merck, Whitehouse Station, NJ, USA) using butanol-
acetic acid-water (4:1:1) as a solvent. Spots were detected by
a UV transilluminator set at 365 nm or ninhydrin reagent or
6N H
2
SO
4
. The spots were recorded directly on a TLC sheet
by penciled-in circles.

Amino acid Composition AnaIysis

Total amino acid composition was determined using an
amino acid analyzer (Biochrom 20; Pharmacia Biotech,
Sweden). Samples were hydrolyzed in 6 N HCl at 110
o
C for
24 h in evacuated sealed tubes. Amino acids were separated
at 55
o
C at a flow rate of 0.3 mL/min by a cation exchange
column (4 150 mm, 5 m particle size; Pickering Lab.,
Mountain View, CA, USA). Detection was performed at 440
and 570 nm following reaction with ninhydrin. Amino acids
were identified after comparing their retention times with
those of standards (Sigma-Aldrich Co., St. Louis, MO, USA).

Mass Spectrometry

MALDI-TOF analysis was performed on a Voyager-DE
STR Biospectrometry Workstation (Applied Biosystems,
Foster City, CA, USA) in linear mode. The MALDI matrix
used for the sample analysis was -cyano-4-hydroxycin-
namic acid (8 mg/mL in 50% v/v acetonitrile, 0.3% v/v
trifluoroacetic acid). Two microliter samples each containing
equal volumes of matrix and sample solution were coated
and air-dried onto a stainless steel target. Positive spectra
were recorded in linear mode using the minimum laser en-
ergy required to produce an observable signal (10:1, sig-
nal:noise). All sample digests were analyzed in at least tripli-
cate using MALDI-TOF. Data from 500 laser shots were
collected using a 337 nm nitrogen laser. Candidate samples
of each target compound were analyzed by an Applied Bio-
systems MS/MS spectrometer (Lagen, Germany) at the Yon-
sei Proteome Research Center (Seoul, Korea). All MS/MS
data were investigated using the MASCOT search engine
(Matrix Science, UK) in conjunction with the NCBI data-
base. Reverse-phase nano-LC-MS/MS was also performed
Biotechnol. Bioprocess Eng. PUR









cK NK Antifungal activity of filtrate with MWCO of 5 kDa against
AspergiIIus niger after 48 h. (C) Control, 100 L of 5% acetic
acid; (5D), 20 g of permeate dissolved in 100 L of 5% ace-
tic acid subjected to ultrafiltration with MWCO of 5 kDa; and
(5U), 20 g retentate dissolved in 100 L of 5% acetic acid
subjected to ultrafiltration with MWCO of 5 kDa.
















cK O. HPLC profiles of the fractionation of the antifungal compound
AK-3. Elution was performed at 2.5 mL/min with a mixture of
acetonitrile:water (60:40, v/v) containing 0.05% TFA.


q~ NK Purification of AK-3
Purification step
Total amount
(mg)
Yield
(%)
Total activity
(U*)
Specific activity
(U*/mg)
Dry cell powder 2,000 100 28,800 32
Set-pack cartridge 900 45 - -
5 kDa cut-off 13.1 0.66 7,860 600
RP-HPLC 4.8 0.24 2,472 515
*One unit refers to 1 mm fungal inhibition diameter.


in this study.


RESULTS

f~ ~ m~ ^hJP

Following purification of the cell extracts by C18 solid
phase extraction (Waters Corp., Milford, MA, USA), the
eluate was dried under a gentle stream of nitrogen at 70
o
C.
The residue was dissolved in 5% acetic acid and passed
through an ultrafiltration cartridge (Millipore Corp., Billerica,

^ _ ` a
























cK PK Thin-layer chromatography of the intracellular extract from
Synechocystis sp. PCC 6803. (A) AK-3 visualized by an UV
transilluminator at 365 nm, (B) visualization with 6N H
2
SO
4

after passing the 5 kDa MWCO, (C) visualization by ninhy-
drin test after the 5 kDa MWCO, and (D) HCl-hydrolysates of
AK-3 for analysis of amino acid composition. Solvent system
used was butanol:acetic acid:water (4:1:1).


MA, USA). Antifungal activity was observed against three
of the Aspergillus strains tested from the 5 kDa MWCO fil-
trate while the remainder of the eluate (= residue) from
Synechocystis sp. PCC 6803 did not have any inhibitory ef-
fect against A. niger (Fig. 1). The growth of A. fumigatus and
A. flavus, however, were inhibited by the residue. Since
these strains were very susceptible to the target antifungal
compound, designated as AK-3, A. niger was selected as the
test strain for evaluating antifungal activity during purifica-
tion. The 5 kDa MWCO filtrate was further purified using
RP-HPLC (Fig. 2) and the active fraction was brought to
homogeneity by SDS-PAGE. The final amount of the AK-3
fraction was 4.8 mg, obtained after lyophilizing the collected
HPLC fractions (Table. 1). Antifungal activity of the sam-
ples throughout purification was evaluated by the paper disk
method and expressed as specific activity (U/mL), where one
unit refers to one millimeter (mm) in inhibition diameter.
In order to visualize AK-3, HPLC fractions obtained
from Synechocystis sp. PCC 6803 extracts after ultrafiltra-
tion (MWCO of 5 kDa) were analyzed by TLC. Fig. 3
shows the TLC plate patterns produced by various visuali-
zation methods. Though the ninhydrin (Fig. 3C) and other
acid reaction (Fig. 3B) was not sufficiently sensitive to
PUS

^ _ `






cK QK Antifungal activity of intracellular extracts of Synechocystis
sp. PCC 6803 against (A) A. fumigates, (B) A. fIavus, and
(C) A. niger. Paper Disks: (a), (b) crude extract 500 g in 100
L DMSO, (c) DMSO 100 L as a negative control, and (p)
itraconazole 100 g in 100 l DMSO as a positive control.


q~ OK Comparison of the inhibitory activities of crude extracts
from Synechocystis sp. PCC 6803 against AspergiIIus spp.
by the paper-disk diffusion method
Fungus Growth inhibition zone (mm)
a

Strain Specimen No. crude extract itraconazole
b

KACC 6896 9 ND
KACC 40280 16 15
KACC 41018 18 ND
^K
KACC 41858 14 ND
KACC 6636 15 15
KACC 40232 14 ND ^K ~
KACC 40233 12 ND
KACC 40080 12 ND
KACC 41016 19 21
KACC 41136 17 ND
^K ~
KACC 41143 16 ND
a
Values are mean (mm) from the experiments in triplicate. The diameter of the
disk (8 mm) is not included.
b
Itraconazole 100 g was dissolved in 100 L of DMSO, while 500 g of crude
extract from p was dissolved in 100 L of DMSO. Please refer
text for detailed explanation.


AK-3, existence of AK-3 could be visualized by a 365 nm
UV lamp (UVItec, Chesterton, UK) (Fig. 3A). Only one
band appeared under UV light, generating an R
f
value of
0.68 (Fig. 3A). The TLC chromatogram clearly confirms the
purity of the fractionated components of AK-3.

^~ ^

In order to confirm that AK-3 was produced by Synecho-
cystis sp. PCC 6803, the intracellular extract of Synechocys-
tis was tested for antifungal activity against several Aspergil-
lus species. The results in Fig. 4 show that even the crude
(not purified) extract of Synechocystis has antifungal activity
equivalent to that of itraconazole, one of the frequently pre-
scribed medications for fungal infections. In Fig. 4, disk p as
a positive control contained 100 g of itraconazole in DMSO,
disk c as a negative control contained just DMSO, and disks a
and b contained 500 g of crude extract Synechocystis.
^ _







cK RK Inhibitory effect of crude extract of Synechocystis sp. PCC
6803 on morphology of A. niger. (A) Normal mycelia of A. ni-
ger (magnification: 100), (B) Abnormal mycelial offshoot of
A. niger after 200 g treatment on a solid medium containing
10
5
spores/mL after 48 h (magnification: 100).


N O







P Q

cK SK Determination of MFC by a modified method using the fun-
gal compound AK-3 against A. niger. A halo 1 mm or larger
outside of the paper disk confirms existence of antifungal
compound. MFC is defined as the lowest concentration with
a halo. (1) 0.01 mg/mL, (2) 0.02 mg/mL, (3) 0.04 mg/mL,
and (4) 0.05 mg/mL.


as the solubility of itraconazole in DMSO was about 1
g/L, the maximum loading amount for a 100 L spot was
therefore 100 g. Likewise, 500 g of crude extract was the
maximum loading amount for the same 100 L spot. Since
AK-3 constituted only a small fraction of the total amount of
crude extract, the amount of active antifungal reagent turned
out to be smaller than that of the positive control (itracona-
zole spot). Table 2 summarizes the in vitro antifungal activ-
ity of cellular extracts from Synechocystis sp. PCC 6803
against typical Aspergillus spp. that cause aspergillosis. It
shows that the crude extract of Synechocystis sp. PCC 6803
hindered the normal development of hyphae by preventing
spore formation from mycelial growth.
The intracellular extracts were quantitatively tested for an-
tifungal activity against the airborne fungi Aspergillus spp.
The MFC was given by the lowest concentration of test
compound in which no fungi grew (spot 2 in Fig. 6). One of
the experimental plates is shown in Fig. 6 as a sample. Sev-
eral disks of varying AK-3 concentration were placed on a
plate of solid medium, containing 10
5
conidia/mL. The size
of the clear zone was then measured after three days of cul
turing. From each plate, the MFC was found for each tested
Aspergillus strain. Table 3 shows that AK-3 can inhibit ger-
mination of Aspergillus spores at a concentration as low as
10 g/m. Specifically, the MFC values of AK-3 against A.
Biotechnol. Bioprocess Eng. PUT

q~ PK MFCs of the antifungal compound AK-3 from Synechocys-
tis sp. PCC 6803 against Aspergillus spp.
Fungal strains MFCsa (mg/mL)
^K KACC 40280 0.02
^K ~ KACC 6636 0.03
^K ~ KACC 41016 0.01
a
MFC denotes minimum fungicidal concentration.


q~ QK Amino acids and their estimated molar ratio of AK-3 from
Synechocystis sp. PCC 6803 by amino acid composition
analysis
Molar of residue per molar of compound
Compound
Ile Glu Phe Ala Gly Cys Val Met Tyr
AK-3 1.16 0.62 0.44 0.45 1.54 0.46 0.49 1.41 0.47


niger, A. flavus, and A. fumigatus were 20, 30, and 10 g/mL,
respectively, when solid media contained 10
5
spores/mL.

^ ^ ` ^~

Ninhydrin reacts with free -NH
2
residues. AK-3 was not
reactive with ninhydrin despite the compound migrating as a
single dot on the silica TLC plate (Fig. 3A). On the contrary,
crude extract and AK-3 HCl-hydrolysates reacted with ninhy-
drin (Fig. 3D). These results suggest that AK-3 may possess
peptide bonds without any free N-terminal. To determine if
AK-3 has peptide bonds, its amino acid composition was ana-
lyzed by an amino acid analyzer after hydrolysis by HCl. AK-
3 was found to be comprised of nine different amino acids: Ile,
Glu, Phe, Ala, Gly, Cys, Val, Met, and Tyr in a molar ratio of
2:1:1:1:3:1:1:3:1, respectively (Table 4). The hypothetical
peptide, Ile
2
GluPheAlaGly
3
CysValMet
3
Tyr, has a mo-
lecular weight of about 1.5 kDa. AK-3 may consist of repeat-
ing units of this peptide, as in (Ile
2
GluPheAlaGly
3

CysValMet
3
Tyr)
n
.

j~ p ~ p

The mass spectra of AK-3 showed three major peaks with
m/z ratios of 679.6, 702.6, and 885.1 (Fig. 7) while that of
the peptides yielded a narrow range of oligomers (5~8 mers
with MH
+
range of 500~1,000 Da). MS/MS spectrum analy-
sis of the 885.1 peak revealed several daughter peaks, as
shown in Fig. 7B. The MASCOT search engine predicted
these smaller peaks as corresponding to one of the sequences
in Table 5. Only the cyclic peptide (Tyr-Gly-Cys-Met-Ile-
Phe-Glu), with sequence coverage of 44%, matched the
amino acid composition analysis shown in Table 4.


DISCUSSION

The present study confirmed the presence of one or more
antifungal reagents within the blue green alga Synechocystis
sp. PCC 6803, along with the structures of these antifungal
agents. Specifically, the antifungal compound purified from
Synechocystis sp. PCC 6803 was designated AK-3. This
compound showed inhibitory activity against several fungal
strains: A. niger, A. flavus, and A. fumigatus.
Assessment of numerous solvent systems such as metha-
nol led to use of an acetic acid/water mixture as extractant
[22]. The TLC chromatogram confirmed the identity of the
fractionated components as the target compound, AK-3. The
migration pattern of AK-3 in the butanol/acetic acid/water
(4:1:1) solvent system revealed a high level of hydrophicity.
This indicates that the fungicidal compound extracted from
Synechocystis sp. PCC 6803 is a hydrophilic toxin similar to
microcystin from other cyanobacteria [23-25]. A reliable and
reproducible method for the purification of AK-3 was devel-
oped in this study. The method starts with ultrafiltration to
separate small molecules such as AK-3 from higher MW
impurities, followed by RP-HPLC for separation of the ac-
tive fraction. Finally, TLC is performed to identify the active
compound.
The MW of AK-3 is obviously lower than 5 kDa since the
filtrate, which had a MWCO of 5 kDa, contained the active
fraction. On the contrary, filtrate having a MWCO of 1 kDa
lost antifungal activity (data not shown), suggesting that the
MW of AK-3 is between 1 and 5 kDa, or that the antifungal
compound consisted of several subunits or monomers [26].
More detailed chemical analyses must be performed to
elucidate the complete structure of AK-3 from Synechocystis
sp. PCC 6803. According to the obtained UV-spectra, the
antifungal compound showed maximum absorption at 214
nm (data not shown), suggesting AK-3 contains an aromatic
ring compound. These results are in agreement with a report
by Gromov et al. [26], who found that the cyanobacterium
LU-2 from Nostoc sp. has a maximum UV absorbance at
210 nm, producing a cyclic peptide with a phenolic ring. The
structure of the active antifungal compound was therefore
theorized to contain an aromatic ring that affects fungal
growth [27,28].
The mass spectra of purified AK-3 showed reproducible
(MH)
+
ion peaks with m/z ratios of 679, 702, and 885. The
mass spectra of AK-3 consistently yielded a narrow range of
oligomers (5~8 mers with MH
+
range of 500~1,000 Da).
Edman degradation sequencing was performed on AK-3, but
no degradation was observed (data not shown). This result
suggests that no free amino groups were present in AK-3,
confirmed by the fact that AK-3 did not react with ninhydrin.
Though more detailed studies should be conducted to deter-
mine the structure of AK-3, there is a probability that AK-3
is a cyclic peptide, such as a cyanotoxin. Partial sequencing
could be performed by MS/MS and an amino acid composi-
tion analysis. Based on these observations, there may be a
general sequence of cyclic (Tyr-Gly-Cys-Met-Ile-Phe-Glu),
including some aromatic amino acids such as Phe and Tyr.
For cyclic peptides, a mixture of various linear peptide ions
of identical mass could arise due to ring opening in many
places [29]. The structure of a cyclic peptide, anabaeno-
peptin B(1), isolated from the cultured cyanobacterium Os-
cillatoria agardhii (NIES-204) was determined by extensive
PUU

^
















_
















cK TK MS of the antifungal agent AK-3. (A) High-resolution MALDI-TOF MS spectrum of AK-3, (B) the MS/MS spectra of molecular ion
at m/z 885.


q~ RK Results of Q-TOF mass spectra and database searching for peptide identification
Predicted sequence Theoretical mass (Da) linear form/cyclic form Sequence coverage
*v`djLHH 857/839 46.6%
vd`jfbc 860/842 44.0%
vdN^jSAc 893/875 6.6%
*The amino acids written in bold letters are in accordance with those of Table 4.


2D NMR spectroscopy and chemical degradation [30]. The
chemical structure of nostofungicidine, a cyanobacterial cy-
clic peptide from Nostoc commune with allelopathic activity,
was elucidated by chemical degradation as well as by de-
tailed NMR and mass spectroscopic analyses [31]. However,
this study failed to elucidate the structure of AK-3 even after
analysis by 1H-NMR and COSY, partly because of smaller
quantities and/or impurities.
This study describes the distinct antifungal activity of
Synechocystis sp. PCC 6803, confirming that the active
compound may contain cyclic peptide. Though the structure
of this antifungal compound could not be determined, it is
believed to resemble antifungal cyclic peptides from other
cyanobacteria. The antifungal activity of AK-3 was found to
be very high since the crude extract was even more effective
than itraconazole.


^ This work was supported by the 21C
Frontier Microbial Genomics and Applications Center Pro-
gram, Ministry of Education, Science and Technology, Re-
public of Korea.
Biotechnol. Bioprocess Eng. PUV

Received April 9, 2009; accepted April 13, 2009


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