Appl Microbiol Biotechnol (1993) 39:250-253
App//ed
An ultraviolet (UV-A) absorbing biopterin glucoside
from the marine planktonic cyanobacterium Oscillatoria sp.
Tadashi Matsunaga 1, J. Grant Burgess 1, Noriko Yamada 1, Kazuo Komatsu 2, Seiichi Yoshida 2, Youji Wachi 2
1 Department of Biotechnology, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184, Japan
2 Shiseido Research Center, Research and Development Headquarters, Kohoku-ku, Yokohama, Japan
Received: 18 September 1992/Accepted: 2 December 1992
Abstract. We have investigated the physiological re-
sponse of marine planktonic cyanobacteria to UV-A
(320-390 nm) irradiation. Here, we report the isolation
of a UV-A absorbing pigment from a UV-A resistant
strain of Oscillatoria. This pigment has been purified,
and its structure determined to be biopterin glucoside
(BG), a compound chemically related to the pteridine
pigments found in butterfly wings. A UV-A sensitive
isolate did not produce significant levels of this chromo-
phore. UV-A radiation was very effective in eliciting
synthesis of BG. In addition, increased UV-A radiation,
increased intracellular levels of BG. These data suggest
that BG may protect the cyanobacterium from adverse
effects of UV-A radiation.
Introduction
The damaging effects of solar ultraviolet UV-B and UV-
C radiation on photosynthesis are well known (Okada et
al. 1976; Sisson 1981). However, the effects of UV-A
(320-390 nm), the major type of UV radiation in sun-
light, are less well understood. In freshwater cyanobac-
teria, UV-A is known to inhibit photosynthesis (Hirosa-
wa and Miyachi 1983) and cause cell death (Shibata et
al. 1991), but the extent to which UV-A inhibits the
growth of marine cyanobacteria has not been reported.
In addition, the mechanisms by which marine cyanobac-
teria protect themselves against UV-A are unknown.
UV-absorbing compounds have been described for
terrestrial and mat-forming cyanobacteria (Scherer et al.
1988; Garcia-Pichel and Castenholz 1991) although the
compounds involved with UV absorption have not had
their structures elucidated. We report here the isolation,
purification and identification of UV-A-absorbing com-
pounds from a marine planktonic cyanobacterium. Such
compounds may have potential industrial application as
sun-screen cosmetics.
Correspondence to: T. Matsunaga
Microbiology
Biotechnology
© Springer-Verlag 1993
Materials and methods
Isolation of marine cyanobacteria. Sea-water samples were col-
lected from coastal areas of Japan and enriched for marine cyano-
bacteria using modified BG-11 medium (Allen 1968) supplemented
with 10 ~tg/1 of vitamin B12 and 2°7oNaCI. Eighty-six strains were
isolated, purified and identified to genus level (Castenholz et al.
1989). The effect of UV-A radiation on these isolates was deter-
mined.
Screening of UV-A-resistant strains of marine eyanobacteria.
Cells were pre-cultured in modified marine BG-11 (pH 7.5) under
cool-white fluorescent light (200 I.tW/cm2), diluted to approxi-
mately 104 cells/ml and 50 ~tl spread evenly on modified BG-11
agar plates. Plates were irradiated with 300 gW/cm z UV-A light
and 200 ~tW/cm -2 cool-white fluorescent light. Control plates
were irradiated with cool-white fluorescent light only. After 14
days, the extent of growth was observed.
Purification of UV-A absorbing compounds. Freeze-dried cells
(20 g) were ground in a mortar and suspended in 100 ml of 80%
aqueous methanol for 1 h. Extracts were centrifuged and the su-
pernatant retained. The pellet of broken cell material was re-
extracted twice with fresh 80% aqueous methanol and all superna-
tants pooled. Solvent was removed by evaporation under reduced
pressure and a yellow powder obtained. The powder was then ex-
tracted with 100 ml deionized water and the concentrated superna-
rant used for Sephadex G-10 (Pharmacia) column chromatogra-
phy with deionized water as the mobile phase. Fractions absorbing
UV-A light were collected, concentrated, and separated by re-
verse-phase HPLC using a CAPCELL PAK C-18 column (Shisei-
do Co. Ltd., Tokyo, Japan, Model) using water: methanol: acetic
acid=94.95:5:0.05, as the mobile phase flow rate; 0.7 ml/min.
The UV-A-absorbing compounds were detected at 345 nm.
Identification of UV-A absorbing compound. UV absorption
spectra were recorded with a Shimadzu UV-2200 spectrophotom-
eter. The nuclear magnetic resonance (NMR) spectra were mea-
sured with a JEOL GX-400. Mass spectra were measured with a
Finnigan-mat MAT 95Q. The mass of the complete molecule was
determined by fast atom bombardment/mass spectrometry (MS).
Results
Screening o f UV-A resistant marine cyanobacteria
Seventy-seven isolates of marine cyanobacteria were
found to be sensitive to UV-A radiation during screen-
 251

2.5 a lag period of several days occurred after the UV lamp

A was switched on. However, Synechococcus sp. N K B G
042902 grown under UV-A light and cool-white fluores-
~ 2.0
cent light suffered severe damage and complete cell
death after 15 days.
•~ 1.5
g
~ L~ Purification and identification o f the UV-A absorbing
chromophore
"~ 0.~ After methanol extraction and concentration, pigments
were separated using Sephadex column chromatography
0 and reversed-phase H P L C . One fraction was purified
2.5 with an absorption maxima, in the UV-A range, of
B o 348 nm (pH 4.4, E = 15.8 x 103). Figure 2 shows the ab-
• 2.0
sorption spectra of the UV-A-absorbing compound. The
structure of this compound was determined using a
range of techniques including 1H and 13C-NMR. The
o
•~ 1.5 compound had relative molecular masses (Mr) of 399
and 400. After performing electron ionization/MS and
chemical ionization/MS of isobutan, the Mr was found
to be 399, including an amino group. Seven trimethylsi-
lane-groups combined with one molecule of the UV-A-
~ 0.5 absorbing c o m p o u n d and gave the final Mr of the com-

5 10 15
Time (days)
256nm
Fig. 1A, B. Effect of UV-A light on the growth of A Synechococ-
cus sp. NKBG 042902 (El) and B Oscillatoria sp. NKBG 091600
(©) and grown under cool-white fluorescent light (200 ~W/cm -2).
Cell suspension (200 gl of 1 × 108 cells/ml) was inoculated into
HOCH2
~ 0
(N_N[~NNH2N
H

20 ml modified BG-11 medium in a 50-ml glass petri dish covered

HO/ " ~ ~OH Me 0
with Saran wrap, which is UV-A transparent. After 6 days /
~ HO
OH
growth, Synechococcus sp. NKBG 042902 (~) and Oscillatoria sp.
NKBG 091600 (0) were irradiated from above with 500 gW/cm _2
UV-A light from a UV lamp (Cosmo Bio, Tokyo, Japan, Model;
CSL-30A) and growth was measured for a further 10 days

ing since no growth was observed. The following iso-

lates showed growth in the presence of UV-A radiation:
Nostoc sp. strains N K B G 091911, 096201; Schizothrix
sp. strain NKBG042801; Phormidium sp. strains N K B G
031500, 031504, 031505b, 042404, 060101; and Ana- ' I 362irn
baena sp. strain N K B G 060701. O f these, three isolates
grew well, in particular Oscillatoria sp. N K B G 091600,
whereas the other isolates showed only modest growth.
Oscillatoria sp. N K B G 091600 was employed for further
investigations. A UV-A-sensitive strain, Synechococcus
sp. N K B G 042902 was used to carry out comparative
studies. Cyanobacteria of the genus Synechococcus are
also widespread in the marine environment (Waterbury
et al. 1979), however the effect of UV-A radiation on
marine isolates of this genus has not been investigated. I l
Figure 1 shows the effect o f UV-A irradiation (500 ~tW/ 250 350 450
cm 2) on the growth of Oscillatoria sp. N K B G 091600 W a v e l e n g t h / nm
and Synechococcus sp. N K B G 042902.
Fig. 2. Ultraviolet absorption spectrum o f the UV-A absorbing
The specific growth rate of Osciltatoria sp. N K B G compound from Oscillatoria sp. NKBG 091600 at pH 8.4 showing
091600 in the presence of UV-A irradiation was similar absorbance peaks at 256 and 362 nm. The compound was identif-
to the specific growth rate under normal light, although ied as biopterin glucoside (see insert)