Академический Документы
Профессиональный Документы
Культура Документы
83-89, 1965]
It has been well known that biotin is a key on its suboptimal level, while only very small
substance playing a very important role in amount of L-glutamic acid was accumulated
required as a growth factor by a number of One of the most important theories on the
bacteria so far as studied which accumulate role of biotin in L-glutamic acid fermentation
a large amount of L-glutamic acid.1•`12) Further which is now generally accepted is as follows:
Main route of glucose catabolism is HMP in ruvic acid to L-glutamic acid, and the role of
biotin-deficient cells, and NADPH produced biotin was not referred to.
by the action of glucose-6-phosphate and 6- The main object of the present experiments
ject was presented by Shiio et al.28) They L-cystine•EHCl, 0.01%; FeSO4•E7H2O, 0.0005%; MnSO4
concluded that glucose catabolism by HMP •E nH2O, 0.0005%, and biotin (pH 7.0). Biotin content
was approximately 10% in the case of Brevi was 0.51ƒÊg/l in biotin-deficient medium (DM) and
Their experiment, however, was carried carried out for 20•`24hrs at 30•Ž on a reciprocal
out only in the presence of arsenite, which shaker (120r.p.m.; 7cm stroke). The grown cells were
Radiorespirometry
Geiger-Muller tube.
method modified by Ishii.30) ƒ¿-Ketoglutaric acid was TABLE II. CO2 EVOLUTION FROM BIOTIN-
estimated by Friedemann and Haugen's method DEFICIENT CULTURE
modified by Shimizu.31)
RESULTS
from Glucose.
acids.
(2) Radiorespirometry using resting cells. in. Then several experiments using resting,
As the above experiments were attended cells were carried out.
with cell growth so the disturbance by other In the first experiment in which cells grown
factors than biotin content would participate on DM were used, glucose entirely disappear
tively.
Additionally, avoiding the disturbance by
the metabolism after pyruvate, the following
FIG. 2. C02 Evolution by Washed Cells Grown FIG. 3. CO2 Evolution by Washed Cells Grown
on Biotin-deficient Medium on Biotin-sufficient Medium
Studies on Amino Acid Fermentation. Part XI 87
formation of ƒ¿-ketoglutaric acid was not ob As shown in Fig. 4, little effect of arsenite
served and the reoxidation of NADPH by re and methylene blue on the CO2 evolution by
ductive amination of ƒ¿-ketoglutaric acid was the cells grown on DM was observed. Evo
eliminated, so the effect of methylene blue lution of CO, from 1-C and 6-C of glucose
which was the hydrogen acceptor from NAD was 25% and zero %o of the initial, respec
PH was also investigated. tively.
The evolution of CO2 from 6-C of glucose In the resting cell experiments in which
by the cells grown on SM was strongly in influence of cell growth was removed, the
hibited by the addition of arsenite and only patterns of CO, evolution were similar to
2% was generated as presented in Fig. 5. those in growing cell experiments. Carbon
The evolution of CO, from 1-C was also de dioxide evolution from 6-C of glucose by the;
creased to 40%. The whole pattern of evolu cells grown on DM was very little but con
tion became similar to that of Fig. 4. Any stant (Fig. 2) as seen in the growing cells,
remarkable effect of methylene blue was not suggesting that complete oxidation of glucose
observed in this case, too. occurs at any time and has no connection
DISCUSSION with cell growth. Carbon dioxide evolution
from 6-C of glucose by the cells grown on SM
Fig. 6 shows the pattern of CO, formation
was, resembling that by growing cells, much
from carbons of various positions of glucose
more higher than that by the cells grown on
molecule through EMP, HMP and TCA cycle.
DM. (Fig. 3)
It is apparent that no CO, evolution from
For many years differences in metabolic be
glucose is observed when glucose is cataboliz
ed to pyruvate through EMP and that all the havior of biotin-deficient and biotin-sufficient
1-C of glucose, and 2 or 3-C if the recycle of cells were supposed to be caused, at least
HMP is running on, is evolved when glucose partly, by the difference in supply of oxygen.
catabolism proceeds through HMP. Carbon 6 per cell following different cell growth. But
of glucose is evolved only when TCA cycle in the above experiments cell concentrations,
is operative. were nearly the same and cell growth was
From the results presented in this article, it not seen, so the difference in biotin-deficientt
would be possible to assume as follows. and biotin-sufficient cells would be concluded
The weak evolution of CO, from 6-C of to be the difference in cellular activity itself.
glucose in the experiment using DM (Table Arsenite addition experiments revealed some
II) shows that complete degradation of glucose interesting facts. In the presence of arsenite,
occurs continuously while its rate is very low. CO, evolution from 6-C of glucose by the
On the other hand CO, evolution from 1-C cells grown on both DM and SM was almost
of glucose is several-fold higher than that completely inhibited, indicating that most of
from 6-C, which indicates that HMP takes complete degradation of glucose was not per
part in the glucose catabolism in this organi formed by the extensive pentose cycle in both
sm. Considering the constancy of the ratio cases, while the difference in quantity between
of CO, evolution from 1-C and 6-C of glucose CO2 evolved from 1-C and 6-C of glucose did
except that at an early short stage, the path- not show any sharp contrast to that in the
way of glucose catabolism must be constant. absence of arsenite, which suggests that re
In the next experiment using SM (Table actions succeeding pyruvate oxidation do not
III), the similar result was obtained during have any considerable effect on the ratio of
initial 14 hrs, but afterward a large amount EMP to HMP. This is confirmed by the
of CO2 was generated from 6-C of glucose and findings that the addition of methylene blue
the ratio of CO2 from 1-C and 6-C of glucose had no effect on CO2 evolution in the pre
Fell into 1. From these results it would be sence of arsenite. Therefore NADP reduction
possible to consider that biotin promotes the by glucose-6-phosphate and 6-phosphoglucona
extent of complete degradation of glucose via te dehydrogenases in HMP and NADPH
T CA cycle, which is shown in the extent of CO, oxidation by glutamic dehydrogenase could .
evolution from 6-C of glucose, and does not not be considered to be closely connected
a ffect the ratio of EMP and HMP appreciably. with one another in glutamic acid formation.
Studies on Amino Acid Fermentation. Part XI 89
Wang et al.29) proposed the following equa growing culture the participation of HMP is
tion to calculate the pathways of glucose greater at an early stage than at the follow
catabolism. ing one.
Additionally it became apparent from these
glucose catabolized by HMP=x•\y
results that the Entner-Doudoroff Pathway32)
where x is % of 14C evolved as 14CO2 from is not operative in this organism. In this
glucose-l-14C pathway no CO2 evolution is observed during
y is % of 14C evolved as 14CO2 from glucose- pyruvic acid formation while a large amount
6-14C of CO, evolves from 1-C of glucose at the
This equation is available only when the pyruvic acid oxidation, which would disturb
the calculation of EMP and HMP. How-
value of y is not so high, because the CO2
ever, the calculated values of EMP and
evolution from 1-C of glucose in TCA cycle
HMP in the experiments added with arsenite
is not negligible in this case. In the experi
ment using organisms which have a strong (Fig. 4 and 5), where pyruvic acid oxidation
was not proceeded, were the same as those in
activity of TCA cycle such as used here, a
control experiments (Fig. 2 and 3). These re
slightly modified equation should be used
which is as follows. sults show indirectly that the organism used
lacks the activity of the Entner-Doudoroff
When a (%) of 1-C of glucose is converted
to CO2 through HMP and y (%) of 6-C is pathway.
It is of interest that the values in Table IV
generated by TCA cycle, taltal CO2 evolved
are similar to those in several reports using
from 1-C, x, will be expressed as
Escherichia coli,29) Sarcina lutea,33) Bacillus subti
lis,34•`36) and Arthrobacter globiformis.37) From