Agric. BioI. Chern.

, 45 (11), 2497~2502, 1981 2497

PolY-L-lysine Produced by Streptomyces.

Part II. Taxonomy and Fermentation Studies

Shoji SHIMA and Heiichi SAKAI

Department of Agricultural Chemistry, College of Agriculture,
University of Osaka Prefecture, Sakai, Osaka 591, Japan
Received April 20, 1981

As a result of screening for a Dragendorff-positive substance of microbial origin, L-lysine

homopolymer (25 ~ 30 residues) with alpha and epsilon linkages was obtained from the culture
filtrate of an actinomycete identified as Streptomyces albulus. Various cultural conditions for the
favorable production of the lysine polymer were studied. The decline of pH during the fermentation
process was an essential condition for the accumulation of lysine polymer. The addition of proline
had a considerable effect.

Various reports concerning the discovery of
a Dragendorff-positive substance of microbial
origin have been published. 1 ,2) However, a
substance of rather high molecular polypep-
tidic nature has not yet been described. We
have obtained homopolymer of lysine consist-
ing of 25 ~ 30 residues connecting with alpha
and epsilon linkage from a culture filtrate of
an actinomycete isolated as a producer of the
Dragendorff-positive substance. 3 ) Although
detailed studies on the structure of the lysine
polymer will be discussed in the following
paper,4) the nature of the product was estab-
lished as being c-polY-L-lysine (c-PL). In this
paper, the taxonomy of the producing or-
ganism, and the cultural conditions for the
accumulation of the lysine polymer will be
described.
Taxonomy of the strain. The taxonomical studies of the
strain No. 346 was carried out mainly by the methods
described by Shirling and Gottlieb. 6 ) The mycelial mor-
phology of the strain was examined with the mycelial
growth on sucrose nitrate agar and inorganic salt-starch
agar media. Utilization of various carbon sources was
tested according to the method of Pridham and Gottlieb. 7 )
The amino acid composition of the cell wall was deter-
mined by the method of Boone and Pine. S ) Type cultures
for comparative studies were obtained from the Institute
of Fermentation, Osaka (IFO) and Research Laboratory
of Kaken Chemical Co. (KCC).
Fermentation. The stock culture of the strain No. 346
was maintained on a modified Bennett's agar at room
temperature. A loopful of the spore from the stock culture
was inoculated into 100 ml of the medium in a 500 ml flask
and cultured at 30°C for 24 ~ 48 hr. The seed culture was
transferred into 100 ml of the medium in a 500 ml shaking
flask and incubated on a reciprocal machine at 30°C for
48hr.

RESULTS AND DISCUSSION

MATERIALS AND METHODS
Screening process. Microorganisms newly isolated from
soil samples collected in the Kansai district of Japan, were
cultured on a reciprocal shaker at 30°C for 3 ~ 4 days,
using a medium consisting of 2% glucose, 0.5% polypep-
tone, 0.1 % potassium diphosphate, 0.05% magnesium
sulfate and 0.5% sodium chloride. After cultivation, fil-
trates were subjected to the Dragendorff test.
Dragendorffs reagent was prepared according to the
method of Munier and Macheboeuf. 5 ) The evaluation of
the Dragendorfl\ test were carried out by visual obser-
vation of a spot test on the porcelain spot plate.
Screening
Approximately 2000 actinomycetes were
tested for production of the Dragendorff-
positive substance in their culture filtrate.
Several strains which have similar characteris-
tics of grayish mycelial formation were select-
ed through the screening process. Among
them, the strain No. 346 which was isolated
from a soil sample collected at Hashimoto
City, Wakayama Prefecture, Japan was pro v-
2498 S. SHIMA and H. SAKAI
ed to be the most potent strain for abundant
and stable production of the Dragendorff-
positive substance. Thus, strain No. 346 was
subjected to further investigation.
Taxonomy of the strain No. 346
Morphological characteristics. The vege-
tative mycelium grew well on all media tested
except for sucrose-nitrate agar. Color of the
substrate mycelium was generally pale yellow
but was yellowish brown on tyrosine agar and
glycerin-asparagine agar. The aerial mycelium
was found abundantly on tyrosine agar and
nutrient agar but it showed only poor or no
growth at all on glucose-asparagine agar,
glycerin-asparagine agar and oatmeal agar.
Mature sporulated aerial mycelium was gray
or grayish brown in color. The spore chain
was of a closed spiral formation. Sporangia
were not observed on the tested media, neither
fragmentation nor formation of spores on the
vegetative mycelium was found. The cultural
characteristics on various media are shown in
Table I. Electron microscopy showed that a
mature spore was ellipsoidal (1.2 ~ 1.5 micron)
with spiny surface (Plate 1).
Physiological properties. The strain pro-
duced melanoid pigment only on tyrosine agar
and not on peptone yeast extract iron agar nor
on nutrient agar. Physiological properties and
TABLE r. CULTURAL CHARACTERISTICS
Medium Vegetative mycelium
Sucrose-nitrate agar Poor
(Waksman medium No. I)
Glucose-asparagine agar Moderate, white
(Waksman medium No.5)
Glycerin-asparagine agar Moderate, partly thick
(ISP No.5) brownish wrinkled growth
Tyrosine agar Good, ,yellowish brown
Nutrient agar Moderate, light yellow
(rSp No.7)
Yeast malt ext. agar Good, light yellow
(ISP No.2)
Oatmeal agar Moderate, light yellow
(rSp No.3)
Peptone yeast ext. agar Good, light yellow
(rSp No.6)
the results of the carbon utilization test are
shown in Tables II and III, respectively. The
hydrolyzate of the cell wall contained LL-
diaminopimelic acid.
Comparison with other related species. On
the basis of its morphological features and
presence of LL-diaminopimelic acid in its cell
wall, strain No. 346 seemed to belong to the
genus Streptomyces. Among the species of
Streptomyces described in the 8th edition of
Bergey's manual 9 ) and Shirling's ISP re-
ports/Oj strain No. 346 was identified as the
species related to St. albulus and St. noursei.
Comparative examination of strain No. 346
with type cultures of those related species was
carried out under the same cultural conditions.
The distinguishable characteristics are sum-
marized in Table IV. The characters of the
strain No. 346 are thought to be more similar
to those of St. albulus than St. noursei.
Although strain No. 346 has some slightly
different properties from St. albulus in aerial
mycelium and melanoid formation, these dif-
ferences were not sufficient for us to conclude
that strain No. 346 was a different species.
Thus, strain No. 346 was classified as St.
albulus.
Fermentation
In preliminary fermentation studies to find
OF STRAIN No. 346
Aerial mycelium Soluble pigment
Poor, brown None
None None
None None
Abundant, grayish Brown
brown with white patches
Abundant, white None
Abundant, grayish brown, None
powdery
Poor, brown None
Abundant, brown, powdery None
 Poly-lysine Produced by Streptomyces 2499

PLATE 1. Electron Micrograph of Spores of Strain No. 346.

Spiny surface ( x 10000).

TABLE II. PHYSIOLOGICAL CHARACTERISTICS

OF STRAIN No. 346
reagent for comparison with purified e-PL as
the standard.
Liquefaction of gelatin Positive Medium composition and fermentation proc-
Peptonization of milk Positive ess. Comparative studies for suitable carbon
Coagulation of milk Negative
and nitrogen sources were carried out. The
Hydrolysis of starch Positive
Production of melanoid Positive (on tyrosine agar) results are shown in Tables V and VI, re-
pigment spectively. No accumulation of e-PL was ob-
Temperature for growth served in the medium which lacked additional
carbon sources in spite of the good growth
(Table V). Despite the good growth, sole use
TABLE III. ASSIMILATION PATTERN OF
of organic nitrogen source was not suitable for
CARBON SOURCES BY STRAIN
No. 346 e-PL production (Table VI). Glycerol and
glucose were proved to be the best carbon
L-Arabinose sources, and the combined use of ammonium
o-Xylose
sulfate and yeast extract was preferable for e-
o-Glucose +
o-Fructose + PL production. Consequently, the medium
L-Rhamnose composition for further cultivation was deter-
o-Galactose + mined as follows: 5% glycerol, 1% ammonium
Sucrose
sulfate, 0.05% magnesium sulfate, 0.5% yeast
Raffinose
o-Mannitol + extract, 0.003% ferrous sulfate and 0.004%
i-Inositol + zinc sulfate in M/50 phosphate buffer (pH
Salicin 6.8). The typical fermentation process is
+, assimilation positive; -, assimilation negative.
shown in Fig. 1. After the mycelial growth had
reached its maximum, accumulation of e-PL in
culture broth was observed accompanied by a
suitable cultural conditions for e-PL produc- rapid decrease of mycelial weight. Lowering
tion, various tests were carried out. The pres- the pH value of the medium seemed to be an
ence of e-PL in the culture broth was moni- essential cultural condition for the production
tored by coloring reaction using Dragendorff's of the e-PL. The significant decrease of the
2500 S. SHIMA and H. SAKAI

TABLE IV. COMPARISON OF STRAIN No. 346 WITH RELATED Streptomyces species

St. albulus St. noursei

No. 346
(IFO 13410) (KCC-S-922)

Cultural characteristics
Glucose-asparagine A.M.; none A.M.; none A.M.; thin, white
agar S.P.; none S.P.; none S.P.; none
Tyrosine agar A.M.; abundant, gray A.M.; none A.M.; abundant, gray
S.P.; brown S.P.; none S.P.; brown
Nutrient agar A.M.; white A.M.; light gray A.M.; white
S.P.; none S.P.; none S.P.; none
Yeast malt ext. A.M.; abundunt, A.M.; abundant, A.M.; abundant,
agar grayish-brown grayish-brown grayish-brown
S.P.; none S.P.; none S.P.; none
Oatmeal agar A.M.; poor, brown A.M.; poor, white A.M.; poor, brown
to brown
S.P.; none S.P.; none S.P.; none
Physiological properties
± + +
Liquefaction of gelatin
Hydrolysis of starch + + +
Coagulation of milk +
Utilization of sugar
o-Xylose
L-Arabinone
L-Rhamnose
D-Galactose + +
Sucrose
Raffinose
D-Mannitol + + +
i-Inositol + + +
A.M.; aerial mycelium; S.P., soluble pigment.

TABLE V. COMPARISON OF ADDITIONAL CARBON amount of the c-PL produced in the broth was
SOURCES FOR E-PL PRODUCTION"
not noticeable in prolonged cultivation.
E-PL Other Jactors affecting e-PL production. The
Carbon source b Growth' Final pH ( 11' )
mg Iter effect of cultural temperature on the pro-
duction of the c-PL is shown in Fig. 2.
None +++ 5.5 0 Excellent accumulation was obtained at
Glucose +++ 3.3 300
Glycerin +++ 3.3 300 25 ~ 30°C. The strain could not grow well
Mannitol +++ 3.3 250 under the initial pH 4.0. The maximum accu-
Starch +++ 6.4 5 mulation of the c-PL was attained by fermen-
tation initiated at pH 6.0. When the sole
" Shaking flask culture for 48 hr at 30°C.
Basal medium: Yeast extract 5 g, (NH4)2S04 109, organic nitrogen source was used, the pH
MgS04 · 7H2 0 0.5 g, ZnS04 · 7H20 0.04 g, FeS04 · values of the culture were kept higher than in
7H 20 0.03 g in 1000 ml of MI50 phosphate butTer the case of inorganic nitrogen sources. This
(pH 6.8).
resulted in poor accumulation of c-PL. In the
b Five % of each carbon source was added.
, Estimated by packed cell volume. medium containing calcium carbonate, the pH
value of the culture broth was maintained at 5
to 6 throughout the culture and the pro-
 Poly-lysine Produced by Streptomyces 2501

TABLE VI. COMPARISON OF NITROGEN SOURCES FOR 8-PL PRODUCTIONa

Nitrogen source Concn. (%) Growth b Final pH 8-PL (mg/liter)

(NH4)2 S04 0.5 + 3.3 180

NH4N0 3 0.5 + 3.6 0
NfI4 CI 0.5 + 3.3 90
NaN0 3 0.5 7.1 0
Urea 0.5 7.7 0
Polypeptone 1.0 +++ 5.8 0
Casamino acid 1.0 +++ 4.7 0
Yeast extract 1.0 +++ 5.4 0
Soybean meal 1.0 +++ 5.4 0
Pharmamedia 1.0 +++ 5.8 0
(NH4}zS04 1.0 }
Yeast extract 0.5
+++ 3.2 400

(NH4h S04 1.0 }

Polypeptone 0.5
+++ 4.0 200

a Shaking flask culture for 48 hr at 30°e.

Basal medium: Glycerin 50 g, MgS04 · 7H2 0 0.5 g, ZnS04 · 7H 2 0 0.04 g, FeS04 · 7H 2 0 0.03 g, yeast extract 0.5 g
in 1000mi of M/50 phosphate buffer (pH 6.8).
b Estimated by packed cell volume.

-=::7
E
OJ
.s5
E
~
E
OJ

:::;
D..
I
U)

05
;,0\ 0--. - 0 - - - 0 - 0 -

,.-.--.~.-
7
5~
3
~0.5
-
OJ
.s 0.3
--'
D..
~ 0.1
•
I~\
•
oJ
/0-0 0- /.20 \
~3 03
15 25 30 35 40
>- Temp ('C)
2
/ FIG. 2. Effect of Temperature on e-PoIY-L-lysine
01
Production.
0 0 20 40 60 130 Cultural conditions were the same as in the legend for Fig.
Time(hr) 1 except temperature.
FIG. 1. Time Course Changes of e-PoIY-L-lysine
Fermentation Process.
0-0, mycelia; e-e, e-poIY-L-lysine (e-PL); 0-0, study on the effects of the addition of amino
pH. acids on the production of the e-PL, various
Medium: Glycerol 5%, (NH4)2S04 1%, yeast extract 0.5% amino acids were solely added to the medium.
MgS04 · 7H 2 0 0.05%, FeS04 · 7H 2 0 0.003%, ZnS04 ·
7H 2 0 0.004%, KH 2 P04 0.136% Na2 HP04 ·12H 2 0
The results are shown in Table VII. Proline,
0.358%, pH 6.8. phenylalanine, diaminopimelic acid, 6-amino-
One % seed culture was added. The organism was culti- caproic acid, arginine and isoleucine seemed
vated at 30°C with shaking. to be effective for e-PL production. While
lysine, the unit amino acid of this polymer, did
duction of e-PL was entirely inhibited (Fig. 3). not show a favorable effect. Among those
To investigate the effect of the aeration, vari- tested, proline was the most effective amino
ous volumes of the culture medium were acid. Murao et al. demonstrated that the
placed into a flask and cultured in the same proline was a stimulatory additive in y-polY-D-
manner as described above. The accumulation glutamate production by Bacillus subtilisY)
of e-PL was in proportion to the increase in Although the mechanism of proline effect on
aeration efficiency. To make a comparative the favorable production of amino acid poly-
2502 S. SHIMA and H. SAKAI

TABLE VII. EFFECT OF AMINO ACIDS

-0-0...
~tJ-D---_
". .
.... D ...... ----··
[7
D.·
5~
ON B-PL P'RODUCTION"

Amino acid b B-PL (g/Iiter)

E 0-0 _ _ 0 _ _ 0_ 3

E05 • _______ 0-
L-Valine 0.63
L-Leucine 1.00
0:0.3 / L-Isoleucine 1.67
0 0 .1 •
L-Proline 2.83
~L-L-LL~W
.. ~.~
.. ~
.. ~L=~~~~
50 100 L-Phenylalanine 1.17
Time(hr) L-Tyrosine 0.42
FIG. 3. Effect of CaC0 3 on B-PolY-L-lysine Production. Yeast extract 0.40
L-Methionine 0.55
0-0, pH; e-e, B-PL in the absence of CaC03 .
L-Arginine 1.22
0-0, pH; . - . , B-PL in the presence of CaC03 .
L-Lysine 0.33
Basal medium was the same as in the legend for Fig. I.
Diamino pimelic acid 1.93
e-Aminocaproic acid 1.67
None 0.27
mer remains uncertain, this phenomena was
thought to be worthy of further investigation. a Shaking flask culture for 48 hr at 30°C.
Basal medium: Glycerin 50 g, (NH4)2S04 109,
yeast extract I g, MgS04 · 7H 20 0.5 g, ZnS04 ' 7H 20
Acknowledgrnerlt. The authors wish to thank Dr. A. 0.04g, FeS04 ·7H20 0.03g in 1000ml OfM/50 phos-
Seino of Kaken Chemical Co. Ltd. for the reference phate buffer (pH 6.8).
culture of actinomycetes. Thanks are also due to Miss E. bOne % of each amino acid was added in the basal
Iguchi of Fujisawa Pharmaceutical Industry Ltd. for her medium.
assistance in the electron photomicrography.

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