J. Gen. Appl. Microbiol.

, 15, 387-398 (1969)

STUDIES ON THE FERMENTATIVE PRODUCTION
OF L-PROLINE
IV. MECHANISM OF L-PROLINE PRODUCTION BY
BREVIBACTERIUM FBAVUM 2247 NO. 14-51
FUMIHIRO YOSHINAGA
Central Research Laboratories of Ajinomoto Co., Inc.,
Suzuki-cho, Kawasaki, Japan
(Received October 30, 1968)
Addition of L-proline to the production medium for cultivation of strain
No. 14-5, an isoleucine auxotrophic mutant of Brevibacterium flavum 2247,
reduced L-proline accumulation by about 40%. The formation, but not the
activity, of glutamate kinase, the first enzyme required for L-proline bio-
synthesis from L-glutamate, seems to be controlled by L-proline. No dif-
ference was observed in specific activities of this enzyme assayed in vitro
in either strain No. 14-5 or the parent strain.
During the growth of strain No. 14-5 (lacking in threonine dehydratase
activity), the increase of intracellular glycine, methionine, aspartate, and
threonine was considerably greater than in the parent strain. Intracellular
valine, leucine, and lysine also increased. L-Proline produced was reduced
to 42.9% without changing the level of intracellular glutamate by the ad-
dition of L-threonine and L-lysine to the medium before cultivation, but L-
proline production was increased by nearly 20% when both amino acids
were added near before the growth of this strain arrived at the stationary
phase. Glutamate kinase formation was repressed by nearly 20% by the
addition of both amino acids, whereas the activity was not inhibited.
A cell homogenate of the parent strain, which was not able to produce
L-proline in the medium, did produce L-proline from L-glutamate in the
presence of ATP. From these findings, it was concluded that in strain No.
14-5 both the higher level of available ATP resulting from the inhibition
of aspartate and homoserine kinase and the accumulation of intracellular
L-glutamate promoted by biotin-rich condition contributed to the abundant
production of L-proline through stimulation of the glutamate kinase reaction.
In previous papers (1, 2), it was reported that an isoleucine auxotrophic
mutant, Brevibacterium favum 2247 No. 14-5, produced a large amount of
1 This paper was presented at the 16th Symposium on Association of Amino Acid
and Nucleic Acid, October 2, 1967.
387
388 YOSHINAGA
VOL. 15
L-proline directly from sugar and inorganic nitrogen in the medium, and the
conditions favorable for L-proline production by this strain were described.
Evidence was provided for the phosphorylation of L-glutamate (glutamate
kinase reaction) as an intermediate step in the biosynthesis of L-proline from
L-glutamate in this mutant (3, 4). The possibility that L-glutamate was con-
verted into L-proline via L-ornithine after acetylation was excluded by the
finding that all L-ornithine-requiring mutants derived from this isoleucine-
requiring mutant produced L-proline abundantly (4).
The present paper deals with the mechanism of L-proline production by
an isoleucine-requiring mutant and with the elucidation of the relationship
between the mutation site of this strain and its L-proline productivity.
MATERIALS AND METHODS
Microorganism. Brevibacterium flavum 2247 No. 14-5 (ATCC No. 15940)
was usually used throughout this work.
Media and cultivation. Depending on the purpose of studies, various
media, as described in the legends to tables and figures, were employed by
modifying the standard medium for L-proline production (4) which contains,
per liter, 100 g glucose as starch hydrolyzate, 60 g (NH4)2504, 1 g KH2P04,
8 g MgS04.7H20, 0.01 g FeS04.7H20, 0.01 g MnS04.4H20, 150 mg L-isoleucine,
1 ml" Ajieki " (solution of amino acid mixture obtained by the hydrolysis of
soybean protein, total nitrogen 2.4%. Ajinomoto Co., Inc., Tokyo), 1 g D-
tartaric acid, 450 ,gig biotin, 1 mg thiamine hydrochloride, and 50 g CaC03
(sterilized separately). The pH was adjusted to 7.0 with KOH.
The bacterium was cultured as previously described (1).
Preparation of cell free solution. As described in previous papers (3, 4),
cell homogenates prepared by disrupting the cells with alumina followed by
centrifugation at 900 x g to remove alumina were employed exclusively for
determining the formation of L-proline from L-glutamate. Cell-free extracts
obtained by sonic oscillation of these homogenates followed by dialysis for 3
to 5 hr against 0.01 M Tris-H2S04, pH 7.6, were usually used as the source
of crude enzyme.
Assay of enzyme activities. Glutamate kinase activity was assayed by
spectrophotometry, as previously described (3, 4), using the Hitachi Model
139 UV-VIS Spectrophotometer. After incubation at 31 in a water bath,
1.5 volumes of a mixture consisting of 10% FeCl3. 6H2O, 3.3% trichloroacetic
acid, and 0.7 N HCl was added to the reaction mixture and the precipitate
formed was removed by centrifugation. The optical density of the super-
natant at 540 mp was used as a measure of the relative amount of hydro-
xamate formed, after correction for the color value obtained in the absence
of L-glutamate. Aspartate kinase activity was determined according to the
method of BLACK and WRIGHT (5).
Protein was estimated by the method of LOWRY et al. (6), or of
1969 Fermentative Production of L-Proline 389
WARBURG and CHRISTIAN as modified by LAYNE (7).
Extraction of intracellular amino acids. As described by SHIIO et al.
(8), the cells were harvested by centrifugation, washed four times with 0.1 M
phosphate buffer (pH 7.5), and suspended in distilled water. When the cells
were not washed, the cells harvested were suspended directly in distilled
water. Intracellular amino acids were extracted by autoclaving the cell sus-
pension at 120 for 30 min.
Analytical methods. Each analysis was carried out as previously des-
cribed (1). Amino acids were analyzed by the microbioassay method (8, 9).
RESULTS
Relationship between glutamate kinase activity and production of L Proline by
strain No. 14-5
Effect of L-proline on the biosynthetic pathway of L-proline in strain
No. 14-5 was examined, especially with regard to its effect on glutamate
kinase which was described in previous papers (3, 4).
(a) Effect of exogenous L-proline on endogenous L-proline production by
fermentation : As shown in Table 1, L-proline, produced directly from sugar,
in the presence of added L-proline in a concentration of 0.5'3.0%, was
nearly 60% of that produced without exogenous L-Proline. Thus, it appears
that this strain has acquired such a potent proline productivity through
Table 1. Effect of exogenous L-proline on L-proline production by fermentation.
Basal medium : Glucose (as starch hydrolyzate) 100 g, (NH4)2S04 60 g, KH~PO4
1 g, MgSO4.7H2O 5 g, FeSO4.7H2O 0.01 g, MnSO4.4H2O 0.01 g, D-tartaric acid 1 g,
" Ajieki'' 0
.5 ml, L-isoleucine 150 mg, biotin 350 rig, thiamine hydrochloride 500 pg.
CaCO3 50 g, per liter. Fermentation was carried out at 31 for 72 hr.
390 YOSHINAGA VOL. 15
mutation that it is able to produce L-proline in a good yield even in the
presence of substantial concentration of exogenous L-proline. However, no
effect of exogenous L-proline was observed when added at 24 hr, i. e., at the
stationary phase of growth.
(b) Effect of exogenous L-proline on the growth of strain No. 14-5: In
the experiment shown in Table 1, the amount of L-proline produced was
calculated on the assumption that L-proline added would not be dissimi-
lated by this strain. If this strain did dissimilate the added L-proline, it
would be expected that growth would be accelerated by the addition of L-
proline. However, as shown in Table 2, its growth was not promoted by
the addition of L-proline. Moreover, from the fact that the amount of L-
proline produced in each case was about the same, i. e., about 2.0 g/liter,
independent of the amount of L-proline added, it seemed reasonable to cal-
culate the L-proline produced by subtracting the L-proline added from the
total L-proline found in the medium.
(c) Effect of L-proline on the formation of glutamate kinase : The
effect of exogenous L-proline on its biosynthetic pathway, especially in re-
lation to glutamate kinase, was studied in a cell-free system. As shown in
Table 3, glutamate kinase formation appeared to be repressed by nearly 20%
when this strain was cultured in the presence of 0.1% L-proline. This result
seems to be in accord with the results shown in Table 1, namely, that the
amount of L-proline produced was decreased by nearly 40% by the addition
of L-proline to the medium.
(d) Effect of L-proline on the in vitro activity of glutamate kinase : The
addition of 20 mM L-proline had little or no effect on the activity of glutamate
kinase (Table 4). This finding is also in accord with the results shown in
Table 1, namely, that exogenous L-proline had no effect on endogenous L-
proline production when added at 24 hr. Glutamate kinase activity from the
parent strain was not inhibited either.
(e) Comparison of activity of glutamate kinase in strain No. 14-5 and
Table 2. Effect of L-proline added to the production medium before cultivation
on the growth of strain No. 14-5.
Fermentation was carried out at 31 for 24 hr employing the same medium as
given in Table 1.
1969 Fermentative Production of L-Prohne
391
in parent strain 2247: Because of the increased proline production by the
mutant strain No. 14-5, glutamate kinase activities of both mutant and parent
strains were compared so as to determine whether this activity might be
responsible for the difference in L-proline production. No difference in the
specific activities of glutamate kinase was observed in the two strains (Table 5).
Table 3. Effect of L-proline on the formation of glutamate kinase.
Cells were grown at 31 for 24 hr with or without L-proline (1 mg/ml) in the
standard medium.
The reaction mixture was incubated at 31 for 1.5 hr in a volume of 1.0 ml
containing 100 pmoles of L-glutamate (adjusted to pH 7.6 with KOH), 20 imoles of
ATP, 20 imoles of MgSO4.7H2O, 400 pmoles of NH2OH, 80 ,moles of Tris-H2SO4
(pH 7.6), and 0.5 ml of dialyzed enzyme solutin (0.5 mg as protein).
Table 4. Effect of L-proline on the activity of glutamate kinase.
The reaction mixture was incubated at 31 for 1.5 hr in a volume of 1.0 ml
containing 100 ,moles of L-glutamate (adjusted to pH 7.6 with KOH), 20 pmoles of
ATP, 20 imoles of MgSO4.7H2O, 400 imoles of NH2OH, 80 pmoles of Tris-H2SO4
(pH 7.6), and 0.5 ml of dialyzed enzyme solution (0.50 mg as protein).
Table 5. Activity of glutamate kinase in strain No. 14-5 and parent strain 2247.
The parent strain 2247 was grown at 31 for 24 hr without isoleucine in the
standard medium.
The reaction mixture was incubated at 31 for 1.5 hr in a volume of 1.0 ml
containing 100 imoles of L-glutamate (adjusted to pH 7.6 with KOH), 20 pmoles of
ATP, 20 imoles of MgSO4. 7H2O, 400 j moles of NH2OH, 80 pmoles of Tris-H2SO4
(pH 7.6), and 0.5 ml of dialyzed enzyme solution (0.5 mg as protein).
392 YOSHINAGA VOL. 15
Relationship between the requirement for L-isoleucine and L proline production
Strain No. 14-5 has been found to lack threonine dehydratase activity
(2), and a possible relation of the deficiency of this enzyme in L-glutamic
acid-producing bacteria to the abundant production of L-proline has been
proposed (2). Accordingly, probable changes caused by this deficiency such
as an increase in intracellular L-threonine was examined.
(a) Amino acid composition of the free internal pool in strain No. 14-5
and parent strain 2247 grown in the L-proline production medium : The data
obtained on the intracellular composition of free amino acids in both strain
No. 14-5 and its parent strain are shown in Table 6. Glycine, methionine,
aspartate, and threonine were obviously at a higher concentration in the
mutant form than in the parent strain, especially before washing. In addition,
Table 6. Amino acid composition of the free internal pool of
and parent strain 2247 grown in the proline production
The parent strain 2247 was grown at 31 for 24 hr without
standard medium. A correction was made for contamination of
in cases where the cell paste was analyzed before washing four
phosphate buffer (10).
strain No. 14-5
medium.
isoleucine in the
external medium
times with 0.1 M
1969 Fermentative Production of L-Proline 393
the amino acids, valine, leucine and lysine, also increased, whereas alanine
decreased markedly. If glycine is assumed to be derived from threonine via
the threonine aldolase reaction, it is interesting that all of the intracellular
amino acids (other than proline) which are strikingly increased are those
belonging to the aspartic family.
(b) Effect of L-threonine and L-lysine added to the medium :
The striking increase of L-threonine suggested the possibility that the
accumulation of this amino acid might be related to the increase in proline
production by strain No. 14-5. Therefore, the effects of L-threonine and L-
lysine, which have been reported (11) to be concerted feedback inhibitors of
aspartate kinase in the parent strain 2247, were examined in detail. It was
found that L-proline produced was reduced to 42.9%, by the addition of L-
threonine (0.6 g/liter) and L-lysine (3.0 g/liter as its hydrochloride) to the
medium. As shown in Table 7, no change was observed, under the same
conditions, in the level of intracellular glutamate, a precursor of proline,
which was not discharged as extracellular glutamate depending on the biotin-
rich condition (8) just for the production of L-proline.
(c) Effect of L-threonine and L-lysine added to the medium during cell
growth on L-proline production : The accumulation of L-proline was increased
by nearly 20% when threonine and lysine were added to the production
medium before the growth of these cells arrived at the stationary phase
(Table 8). This result implies that the level of intracellular threonine plays
an important role in the abundant production of L-proline, and that an in-
crease in proline productivity in strain No. 14-5 could be related to an in-
crease of intracellular threonine. In turn, this increase of threonine with
the growth of this microorganism depends on the deficiency of threonine
dehydratase activity.
(d) Effect of L-threonine and L-lysine on the formation of glutamate
kinase : In the dialyzed cell-free extracts from the cells grown under the
conditions described in Table 7, the specific activity of glutamate kinase was
repressed by 22.1% (Table 9). This decrease of enzyme activity might be
sufficient to explain the decrease in L-proline accumulation caused by the
Table 7. Effect of L-threonine, L-lysine, and L-methionine added to the
medium on L-proline production by fermentation.
Fermentation was carried out at 31 for 72 hr employing the standard medium.
394 YOSHINAGA VOL. 15
addition of both amino acids as shown in Table
(e) Effect of L-threonine and L-lysine on
kinase and aspartate kinase : As demonstrated
7.
the activities
by the data in
of glutamate
Table 10, the
Table 8. Effect of L-threonine and L-lysine added to the medium during
cell growth on L-proline production by fermentation.
Fermentation was carried out at 31 for 72 hr employing the standard medium.
Table 9. Effect of L-threonine and L-lysine on the formation of glutamate kinase.
Cells were grown at 31 for 24 hr with or without L-threonine (0.6 g/liter) and
L-lysine HCl (3.0 g/liter) in the standard medium.
The reaction mixture was incubated at 31 for 1.5 hr in a volume of 2.0 ml
containing 200 ~imoles of L-glutamate (adjusted to pH 7.6 with KOH), 40 tmoles of
ATP, 40 pmoles of MgSO4.7H2O, 800 ,moles of NH2OH, 80 pmoles of Tris-H2SO4
(pH 7.6), and 1.0 ml of dialyzed enzyme solution (1.5 mg as protein).
Table 10. Effect of L-threonine and L-lysine on the activities of
glutamate kinase and aspartate kinase.
The reaction mixture was incubated at 31 for 1.5 hr in a volume of 2.0 ml
containing 200 ,moles of L-glutamate or 400 pmoles of L-aspartate (adjusted to pH
7.6 with KOH), 40 ,moles of ATP, 40 ,moles of MgSO4.7H2O, 800 ,moles of NH2OH,
80 ,moles of Tris-H2SO4 (pH 7.6), and 1.0 ml of dialyzed enzyme solution (1.50 mg
as protein).
1969 Fermentativ e Production of L-Proline 395
addition of both amino acids had no effect on glutamate kinase activity,
whereas the inhibition of aspartate kinase in this mutant as well as in the
parent strain was confirmed.
(f) Effect of ATP on proline formation by cell homogenate of parent
strain 2247: Referring to Fig. 1, the findings reported above lead to the
following proposals for the mechanism of L-proline production in strain No.
14-5. In this strain, an increase of intracellular threonine (whose level was
very low in parent strain 2247 (8)) caused by the deficiency in threonine
dehydratase would bring about an inhibition of aspartate kinase. This in-
hibition would be further increased by lysine, and would result in the spar-
ing of ATP. In addition, the inhibition of homoserine kinase by threonine,
which was observed in the parent strain (11), might be sufficient to spare
ATP as well. The higher level of available ATP resulting from the in-
hibitions of both aspartate and homoserine kinase might act to more effectively
promote the glutamate kinase reaction which requires ATP as a cofactor.
Finally, intracellular L-glutamate, the accumulation of which was found to
be promoted by biotin-rich condition (8), would serve as a substrate for the
production of L-proline in the medium. That ATP might indeed be limiting
for L-proline production in strain No. 14-5 seems to be indicated by the fact
that a cell homogenate of the parent strain, which was not able to produce
L-proline in the medium, did produce L-proline from L-glutamate in the pre-
sence of ATP as shown in Table 11.
Fig.
bacterium
1. Relationship between
flavuin.
the glutamate and aspartate families in Bievi-
396 YOSHINAGA
VOL. 15
DISCUSSION
It has been suggested by STRECKER (12), BAICH and PIERSON (13), and by
TRISTRAM and THURSTON (14) from experiments with intact cells of E, coli that
one of the early steps (probably the first step) in the biosynthetic pathway of L-
proline from L-glutamate is subject to end-product inhibition, thus controlling
L-proline biosynthesis. However in Brevibacterium flavum, glutamate kinase,
the first enzyme of L-proline biosynthesis, was not inhibited by L-proline.
If the metabolic pathway for the reduction of L-glutamate to L-proline, which
was observed in strain No. 14-5 (4), is not the same as the pathway for
oxidation of L-proline to L-glutamate, as suggested by S TRECKER (15), bio-
synthesis of L-proline is presumably controlled in this microorganism by some
mechanism other than the feedback inhibition of glutamate kinase by L-pro-
line. If the concentration of biotin in the medium is low in such a case,
intracellular L-glutamate reduced from L-proline will be excreted as extra-
cellular L-glutamate, but, at a higher concentration of biotin just for the
production of L-proline, it is impossible that intracellular L-glutamate is dis-
charged as extracellular L-glutamate itself or L-glutamine (the latter is known
to be produced very easily at high NH4+ concentration (16)). In other words,
this fermentative production of L-proline might be regarded as a kind of
detoxication of intracellular L-glutamate by converting less permeable L-
glutamate into more permeable L-proline (4). BAICH and PIERSON (13) and
TRISTRAM and THURSTON (14) proposed also that overall control of L-proline
biosynthesis, besides involving end-product inhibition, also involved enzyme
repression. In our experiments, glutamate kinase formation in Brevibacterium
flavum was repressed by nearly 20% when 0.1% L-proline was added to the
culture medium.
Further interesting data were obtained by comparing the intracellular
free amino acid concentration in strain No. 14-5 with that of the parent
Table 11. Effect of ATP on the proline formation by the cell
homogenate of parent strain 2247.
Cells were grown at 31 for 24 hr without isoleucine in the standard medium.
The reaction mixture was incubated at 31 for 5 hr in a volume of 3.0 ml con-
taining 100 pmoles of L-glutamate (adjusted to pH 7.6 with KOH), 60 pmoles of
MgSO4 7H2O, 300 pmoles of Tris-H2SO4 (pH 7.6), and 1.0 ml of cell homogenate
(37.0 mg as protein).
1969 Fermentative Production of L-Proline 397
strain. In addition to the expected increase of intracellular threonine result-
ing from threonine dehydratase deficiency, the amount of aspartate and
methionine also increased markedly. The increase of aspartate is probably
due to inhibition of aspartate kinase by threonine and lysine, while the in-
crease of methionine appears to be due to inhibition of homoserine kinase
by threonine (11). This proposed role for the relation of intracellular thre-
onine to the abundant production of L-proline appears to be supported by the
characteristic decrease of L-proline production resulting from such decrease
of intracellular threonine, as is obtained by increasing the concentration of
L-isoleucine, reported to be an inhibitor of homoserine dehydrogenase activity
(11), or by adding L-methionine to the medium which is known to repress
homoserine kinase (17). Thus, it is concluded that ATP plays the most im-
portant role in the fermentative production of L-proline in strain No. 14-5,
although the effect of cofactors ether than ATP also has to be taken into
account. The proposed mechanism might be called fermentation of metabolic
control, reflecting the complexity of metabolic control in microorganisms.
In connection with this mechanism, it is interesting that an increase of
L-proline biosynthesis by ATP in plants also has been reported recently. In
tobacco leaves formation of L-proline from L-glutamate was promoted by
light irradiation (18), and it was reported that this phenomenon was chiefly
due to ATP synthesis caused by noncyclic photophosphorylation. No effect
of ATP or ADP was seen in young leaves with potent biosynthesis of L-
proline but L-proline formation in old leaves with weak biosynthesis of L-
proline was accelerated by the addition of either cofactor (19).
Recently, fermentative production of L-proline by histidine auxotrophic
mutants of Brevibacterium flavum (20) and Brevibacterium sp. No. 7996 (21)
has also been reported. L-Proline productivity of Brevibacterium sp. No. 7996
YS-48 was said to be far better than its parent (22), but its mechanism
remains unsolved. However, the leading role of ATP in L-proline production
indicated for an isoleucine auxotrophic mutant might perhaps be applicable
also to these histidine auxotrophic mutants in relation to the biosynthetic
pathway of histidine.
The author is grateful to Dr. H. J. Strecker, Professor of Biochemistry, Albert
Einstein College of Medicine, Yeshiva University, for his valuable suggestions. The
author also wishes to thank Drs. T. Yoshida, T. Tsunoda, N. Katsuya and S. Okumura
of this laboratories for their interest and encouragement during the course of the
present work. Much appreciation is expressed to Mr. Y. Takeda and Mrs. M. Sato for
their technical assistance.
REFERENCES
1) F. YOSHINAGA, S. KONISHI, S. OKUMURA and N. KATSUYA, J. Gen. Appl. Microbiol.,
12, 219 (1966).
2) F. YOSHINAGA, Y. YOSHIHARA, S. OKUMURA and N. KATSUYA, J. Gen. Appl. Micro-
biol., 13, 25 (1967).
398
3)
4)
5)
6)
7)
8)
9)
10)
11)
12)
13)
14)
15)
16)
17)
18)
19)
20)
21)
22)
YOSHINAGA VOL. 15
F. YOSHINAGA, Y. TAKEDA and S. OKUMURA, Biochem. Biophys. Res. Commun., 27,
143 (1967).
F. YOSHINAGA, Nippon Nogeikagaku Kaishi, 42, 703 (1968), in Japanese.
S. BLACK and N.G. WRIGHT, J. Biol. Chem., 213, 39 (1955).
OH. LOWRY, N.J. ROSEBROUGH, AL. FARR and R.J. RANDALL, J. Biol. Chem., 193,
235 (1951).
E. LAYNE, In Methods in Enzymology, 3, ed. by S.P. COLOWICK and NO. KAPLAN,
Academic Press Inc., New York (1957), p. 447.
I. SHIIO, K. NARUI, N. YAHABA and M. TAKAHASHI, J. Biochem. (Tokyo), 51, 109
(1962).
T. TSUNODA, In Chemistry of Proteins, 1, ed. by S. Akabori and S. Mizushima,
Kyoritsu Shuppan Co., Ltd. Tokyo (1954), p. 282.
I. SHIIO, S. OTSUKA and M. TAKAHASHI, J. Biochem. (Tokyo), 51, 56 (1962).
R. MIYAJIMA, S. OTSUKA and I. SHIIO, J Biochem. (Tokyo), 63, 139 (1968).
H.J. STRECKER, J. Biol. Chem., 225, 825 (1957).
A. BAICH and D.J. PIERSON, Biochim. Biophys. Acta, 104, 397 (1965).
H. TRISTRAM and C.F. THURSTON, Nature, 212, 74 (1966).
H.J. STRECKER, J. Biol. Chem., 235, 3218 (1960).
K. OSHIMA, K. TANAKA and S. KINOSHITA, Amino Acids, 7, 73 (1963), in Japanese.
R. MIYAJIMA, S. OTSUKA and I. SHIIO, Proc. Ann. Meet. Agr. Chem. Soc. Japan, p.
272 (1968), abstract in Japanese.
S. MIZUSAKI, M. NoGucHI and E. TAMAKI, Arch. Biochem. Biophys., 105, 599 (1964).
M. NOGUCHI, A. KOIWAI, M. YOKOYAMA and E. TAMAKI, Plant & Cell Physiol., 9,
35 (1968).
France Patent 1,427,534 [cited from Chem. Abstr., 65, 16030 (1966)].
S. YAMATODANI, M. SUZUKI and Y. NAKAO, Amino Acid and Nucleic Acid, 16, 126
(1967), in Japanese.
M. SUZUKI, Y. NAKAO and S. YAMATODANI, Amino Acid and Nucleic Acid, 16, 134
(1967), in Japanese.