Factors affecting utilization of aspartic acid by Leuconostoc Mesenteroides P-60
by Richard S Clark
A THESIS Submitted to the Graduate Faculty in partial fulfillment of the requirements for the degree
of Master of Science in Chemistry at Montana State College
Montana State University
© Copyright by Richard S Clark (1950)
Abstract:
A study of the utilization by Leuconostoc mesenteroides P-60 of the factors affecting this utilization
was made. It was shown that the D-isomer is utilized. An indication of enzymatic adaptation appeared.
A difference in the type and amount of buffer affected the response to D-aspartic acid.... Varying
amounts of alanine had no appreciable affect and D- and L-asparagine would not substitute for D- and
L-aspartic acid. 
FACTORS AFFECTING UTILIZATION OF ASPARTIC ACID 
BI EEUCONOSTOC MESENTEROIDES P-60
Submitted to the Graduate Faculty
in
partial fulfillment of the requirements 
for the degree of 
Master of Science in Chemistry
by
RICHARD S. CLARK
H
A THESIS
at
Montana State College
Approved:
Head Major Department
■5 Graduate Division
' Bozeman, Montana 
May, 1950
''tjWirw
»
I
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TABLE OF CONTENTS
Page
ABSTRACT ............................................................  3
INTRODUCTION ........................................................  U
/  /
HISTORICAL RESUME ................................................... 5
MATERIALS, METHODS AND DATA.........................................  9
EXPERIMENTAL RESULTS ..............................................  3.5
.DISCUSSION..........................................................  29
SUMMARY.............................................................  35
ACKNOWLEDGEMENT ............................................    36
LITERATURE CITED AND CONSULTED......................................  37
3
'ABSTRACT
A study of the utilization by Leuconostoc megenterojdes P-60 of the 
factors affecting this utilization was made. It was shown that the D™ 
isomer is utilized. An indication of enzymatic adaptation appeared. A 
difference in the type and amount of buffer affected the response to D- 
aspartip acid.... Varying amounts of alanine had no appreciable affect and 
D— .and L—asparagine would not substitute for D— and L-aspartic acid.
3
HSTEODDCTION
The D-isomers of amino- acids have long been considered the syn­
thetic or "unnatural" form. ' The possibility of their utilization by- 
microorganisms has been recognized only within the past few years.
The question of whether these forms occur as components of living tissue 
or only as extracellular metabolic products has been debated during the 
past few years and is still being debated.
It now seems apparent that a great many different microorganisms 
can utilize various D-isomers of the amino acids, -The D-isomers of all 
but five or six amino acids have been reported as being utilized partially 
or completely by various microorganisms. The six reported as most gener­
ally utilized are D—alanine, D—valine, D—leucine, h—aspartic acid and D- 
glutamic acid. Of these, D-glutamic acid has been, perhaps, the most 
extensively studied and D-aspartic acid the least.
According to Hydon (19U8) the only lactic acid, producing, organism 
found capable of utilizing D-aspartic acid is Lactobacillus•delhruckii. 
Assays run in the laboratory of the Department of Chemistry Research, 
Montana State College Agricultural Experiment Staiion indicated -the D- 
aspartic acid was substituting, at least partially, for L^aspartic acid 
with Leuconostoc mesenteroides P-60. This organism is of particular in­
terest since it has been used as the principal assay organism for aspartic 
acid. Because of peculiarities which had been noted in its response to 
this amino acid further study seemed advisable.
HISTORICAL EESHIffi
Early investigators. believed that the L-foms of amino acids were 
the only naturally occurring forms of amino acis. The D-forms were 
considered as synthetic compounds and metabolieally .inert. These con­
cepts were based on Fischer and. Abderhalden^ s (1905>) report that only 
the peptide linkages built up from amino acids of the L-series were hydro­
lyzed by enzymes of the pancreas and intestine.
The first experiments with the utilization of D-amino acids were per­
formed by Wohlgemuth (1905) -> He fed racemic tyrosine, leucine, aspartic 
acid and glutamic acid to rabbits. He reported that the corresponding 
D—amino acids were excreted in the urine. Dakin (1909) injected 8 grams 
of optically inactive phenylalanine into a cat and recovered 2 grams of 
inactive phenylalanine from the urine, Dakin (1910) also injected in-, ' 
active tyrosine into a cat. He stated as a result of this experiment,
"the naturally occurring laevo-acid was in every case more readily decom­
posed but the rates of decomposition of the d and I forms' cannot be very 
widely different since when smaller doses of the inactive acid are given, 
no unchanged tyrosine may,be found in the urine." Berg and Potgieter 
(1931) observed that rats fed on a tryptophan deficient diet responded.
■as well to racemic tryptophan as to L-tryptophan. Whipple and Eobscheit- 
Robbins (1937) showed that D-histidine and D-tyrosine ,are utilized by 
an anemic dog for the regeneration of hemoglobin to the same extent as , 
the L-series. Conrad and Berg (1937) noted that rats fed a histidine .
5
; ; "
6deficient diet supplemented Tfith D-histidine were able to maintain normal 
activity. When the tissues of these rats were examined they were shown 
to contain only 1-histidine, The natural occurrence of a D-isomer of an 
amino acid was first demonstrated by Jacobs and Craig- (1935) who obtained 
a methyl ester of D-proline as a cleaVage product of ergotine. Smith
and Timmis (1937) confirmed the findings of Jacobs and Craig, Ivanovics
/
and Bruckner (1937) isolated D-glutamic acid from Bacillus mesentericus 
vulgatus» Heinsen (1936) reported the isolation of DL-arginine from a 
kidney autolysate. Kotake and Goto (1937) using rat kidney slices found 
an inversion of . D-tryptophan into 1-tryptophan. Hotchkiss and Dubos (195-0) 
reported that nearly half of the amino acids in the acid hydrolysate of 
gramicidin and gramidinic acid occur as the D-isomer. Iipmanns' Hotchkiss 
and Dubos (1951) and Christensen, Edwards and Piersma, (1951) isolated 
D-Ieucihe from gramicidin. Bovarnick (1952) reported a strain of Bacillus 
subtilis that produces a polypeptide composed solely of D-glutamic acid. 
Hanby and Eydoh (1956) showed that the capsular polypeptide of Bacillus 
anthracis contained large amounts of D-glutamic acid. Kogl and Erxeleben 
(1939) claimed to have isolated the D-form of glutamic acid from hydro­
lysates of tumour tissue in amounts of up to' 58 per cent"of the total 
glutamic add. They postulated .that the growth controlling enzymes are- 
deprived of their controlling ability when they have the D-form in their 
structure. The findings and theory of. Kogl and Erxeleben had been postu­
lated in 1907 by Margaret A. Cleaves (1939). Arnow and Opsahl (1939), 
Schroeder (1939), lhite and Vfliite (1939), and others confirmed the findings 
of Kogl and Erxeleben whereas Chibnall, Bees, Tristam, Williams and Boyland
7(1939)5 Graff (1939), Chargaff (1939)5 and others were unable•to find 
' any D-glntamic acid in tumour tissue* Kogl and Erxeleben (1939 A) 
attributed their failure to find D-glutamic acid to differences in 
analytical procedure. Johnson reported finding D-glutamic acid in 
normal rat liver and Chibnall found the compound in a number of plant . 
proteins as well as in normal animal tissue (19I4.O). The utilization 
of D-isomers of amino acids by microorganisms was reported by Webster 
and Bernheim (1936) when they stated that Bacillus pyocyaneus (Pseu­
domonas aeruginsa) utilized both isomers of tyrosine, proline, and 
to a slight extent those of valine and phenylalanine. Dunn, Camien, 
Rockland, Shankman and Goldberg (19Wl) suggested that D-glutamic acid . 
might be utilized by Lactobacillus arabinosus 17-5» Lymam and. Kuiken 
(I9I4.8) found that the ability of Lactobacillus arabinosus to utilize 
the D- forms of the amino acids was greatly increased, by the substi­
tution of pyridoxamine for pyridoxine. They also found that Strep­
tococcus fae calls did not utilize D-methionine in a medium buffered 
with acetate but utilized substantial amounts in a medium buffered • 
with citrate. Camien and Dunn (1989) reported that D— and DL-glutamic 
acids under some conditions are more active than L-glutamic acid in 
promoting growth of Lactobacillus arabinosus 17—5j but that increased 
'amounts of aspartic acid in the basal medium partly or completely sup­
pressed the activity of D-glutamic acid for this organism. Gamien and 
Dunn (1950) showed that Lactobacillus arabinosus 17—5 utilized D-methi- 
opine and DL-methionine equally as well as L-methionine in a synthetic
8medium, containing at least one per cent of pyridoxamine or 10 per cent 
of pyridoxal. At lower concentrations of these vitamins, the utiliza­
tion of D-methionine was reduced or eliminated. Fyridoxine and other 
nutrients in relatively high concentrations were ineffective in pro­
moting utilization of D-methionine.
•9
MATERIALS, METHODS AMD DATA
A culture of Leuconostoc mesenterold.es P-60 was obtained from the 
American Type Culture Collection. In order to have a pure strain, it 
was necessary to isolate a single cell of the organism. For this pur­
pose a Chapibers micromanipulator was used. The general procedure fol­
lowed for single cell isolation was that outlined by Hildebrand (1938). 
Two modifications were made on this method. The first was to use a 
different moist chamber than that described by Hildebrand. Hildebrand’s 
moist chamber did not provide sufficient moisture for the dry air of 
this atmosphere. The moist chamber outlined by Richter (19^8) proved 
much more satisfactory. The dilution was made greater" than that de­
scribed by Hildebrand. \ The dilution was such that rarely were two 
organisms contained in a single drop. A drop was placed on the cover 
plate, examined and, if it had only one organism, the cover plate was 
transferred to a hanging drop slide for incubation. If the drop- had 
no organism another drop was deposited and examined for a single or­
ganism. This was repeated until a drop was deposited with • a single 
organism. If more than one organism appeared in a drop the cover plate 
was discarded.
After incubation in the hanging drop slide the organisms were trans­
ferred to a tryptose phosphate medium and maintained, until identification. 
The organisms were identified according to the procedure outlined for 
Leuconostoc mesenteroides in Bergey’ s Manual (19lj.8).
10
After identification the organisms were transferred to an agar 
stab. This stab consisted of basal medium I, enriched according to 
Henderson and Snell (IPljB) by 0.2 per. cent Bacto-tryptone, 0.5 per 
cent Bacto-yeast extract and 2 per cent Bacto-agar. The cultures 
were incubated at. 35>°C for 36 hours when heavy growth was observed.
After incubation the cultures were stored in a"refrigerator uhtil 
used* The inoculum medium was the same as the above, except that agar 
was omitted.
• Three different basal media were. used. Basal medium I (Johnson,
Leon H., unpublished data) is shown in. Table I.
Basal Medium II is the same as that outlined in Table I except 
that 20 grams of sodium acetate was substituted for the 20 grams of 
sodium citrate.
Basal Medium III was the medium described by Horn, Jones and Blum 
(1950). It is shown in Table II. '
' These media were adjusted to a pH of 6.8 as described by Henderson 
and Snell (191(8), and I4. ml was pipetted into each tube with an automatic 
pipette. The volume was doubled by addition of U ml of a solution of 
aspartic acid or water. Forty tubes were placed in a rack and covered 
with a metal cover about two inches deep. The cover was loosely lined 
with gauze to permit it to fit tightly over each tube.. These media 
were steam sterilized under 12- pounds pressure for 10 minutes.
Before inoculation into the assay medium, the organisms were revital­
ized by passing through five transfers. The cultures were incubated 2U■
•11
TABLE I ' 
BASAL IEDIUM I . 
Amino acids irj mg
BL-alpha alanine Soo L-Methionine 100
L-cysteine ' 100 DL-Phenylalanine 100
L-arginine HCL 200 . L-Proline So
L—glutamic Soo , DL-Serine . 100
Glycine 100 DIrThreonine 100
L-Histidine 100 ■ L-Tyrosine 100
DL-Isoleucine 200 DIrTryptophan . 100
L-Leucine .100 LL-Valine 200
Glucose 20 grams
Salts in grams
Sodium citrate 20 KHpPOb ' I
Sodium .acetate I -MgSOli . 0.2
Ammonium chloride 3 FeSOlt 0.01
KpHPOit I
Purines in mg
HaCL 0,01
Adenine 10 Uracil' 10
Guanine 10
Vitamins in mg
1 Xanthine 10
Thiamine o.S Ca pantothenate o.S
Riboflavin 0.5 ■ ■ Niacin 1.0
Hyridoxal 0.3 p-aminobenzoic acid 0.1
Micrograms of the following
Biotin 10 . Folic acid * 10
Eeticulogen 0.2
Quantities are based on 5>00 ml volume
12
hours at 35°C between transfers. After the fifth transfer a centrifuge 
tube containing 3-5 ml of medium was inoculated and incubated for 2U 
hours at 35>°G. Following incubation, the cells were centrifuged and 
washed twice with a normal' physiological saline solution. After the 
final washing the cells were again centrifuged and suspended in 7 ml 
of normal saline solution. Approximately 0.1 ml of this suspension 
was placed in each tube of assay medium.
Assays were run with various concentrations of D- and L-aspartic 
■ acid with all three media, and with basal medium I varying the amounts 
of glutamic acid and alanine. Varying incubation periods of 12, 2k,
36, U8, 60, 72, and Bk hours were employed.
13
TABLE II 
BASAL MEDIUIifi III
Nutrient Amount Nutrient Amount
Glucose 20gm Biotin .Olmg
Sodium acetate (anhy- Folic acid .002mg
drous) 12gm DL-Alanine . 80mg
Salts A: L-Argipipe hydro- ,
EzEPOlt Igm chloride 96mg
ICH2POU Igm DL-Aspartic acid • ZUOmg
Salts B: L-Cystine UOOmg
MgSOtTH2O UOOmg DL-Glutamic acid H2O 9U0mg
IhSOltliH2O 20mg Glycine UOOmg
NaCL 20mg L-Histidine hydro-
FeSOlJH2O 20mg chloride H2O Ulimg
Adenine IOOmg L-Hydroxyprbline 20mg
Guanine IOOmg DL-Isoleucine UOmg
Uracil IOOmg ■ DL-Leucine . UOOmg.
Thiamine chloride 
Pyridoxamine dihydro-
' 2 .Omg DL-Lysine hydro­
chloride 300mg
chloride .Ipig DL-Methionine 200mg
Calcium pantothenate -Iimg DL-Norleucine UOOmg
Riboflavin, .Umg DL-Phenylalanine IbOmg
Nicotinic acid *8mg L-Proline lUOmg
p-Aminobenzpic acid ■ .Ipng DL-Serine 2U0mg
DL-Tryptophane 'UOOmg DL-Threonine .l80mg
DL-Valine 2U0mg 1-Tyrosine 130mg
Solution brought to 1,000 ml volume, pH 6.8
IU
FIG. I
BURETTE
AIR FOR STIRRING MEDIUM
SUCTION FOR REMOVING 
NEUTRALIZED MEDIUM
ELECTRODESP-H
METER
TITRATION APPARATUS
25
EXPERIMENTAL RESULTS
The response of Lenconostoc mesenteroid.es P-60 in each of the three 
Lasal media to D and L "aspartic acid, varying periods of incubation and 
concentration of aspartic acid, are reported in Tables III to IX. The 
lactic acid produced was titrated with 0.092 I NaOH. All values are cor­
rected to zero blank value. Figures 2 to 18 illustrate the results ex­
pressed in these Tables graphically.
TABLE III 
BASAL MEDIUM I
36 hour culture 60 hour culture
ug of aspartic ml NaOH ml NaOH ml NaOH ml. NaOH
acid per tube D L D L . D L D L .
Uo 0.7 • 1.U 0.6 1.7 1.2 •2.6 1.3 2.3 '
80 1,6 2.8 ■ 1.5 3.0 2.9 U.6 3.1 U.3
160 2 J4 3.7 2,3. 3.6 U.6 6.U u.i 6.1 ■
2U0 - 3.3 5.1 3.3 U.8 5.3 7.U 5.3 7.5
320 3.7 5.2 3.6 U.9 6.7 8.1 6.6 • 8.1
TABLE IV
BASAL MEDIUM I
12 hour 18 hour 36 hour 60 hour 8U hour
ug of aspartic culture culture culture culture culture
acid per tube ml NaOH ml NaOH ml NaOti ml NaOH ml" NaQH
' D L ■ D L D L D L D L
Uo 0..0. 0.7 OJ4 1.0 0.5 1.8 1.1 2.U 2.2 3.5
80 0.0 1.1 O-U 1.8 I.U 3.0 2.9 U-U U-U 5.8
160 0.7 1*3 o.U 2.u 2.3 3.9 U.7 6.3 6.3. 7.U
320 0.8 1.2 1-5 2.6 3.U 5.1 6.5 7.7 7.1 8.5
6U0 0.8 1.1 2.U 2.0 5.0 5.2 6.6 8.1 7.7 8.6
. "';r| MeitI Al,; , ,
:i
; n >
16
TABIE ?
BASAL MEDIUM I
2U hour 36 hour U8 hour 60 hour
ug of aspartic ' culture culture culture culture
acid per tube ml NaOH ml NaOH ml NaOH ml NaOH
D L • D L D L D L
Uo • 0*6 I. U 0*9 2.1 1.3 2.7 2-U 3.5
160 2.5 U-O 3.8 5-5 5+5 6.6 6.6 7.5
6U0 U-U 5-5 6.2 6.7' 7-5 8.0 9.0 9-9
■ 1280. U-9 5-0 6.U 6.Li ■ 8.3 7,8 ■ 9.7 8.9
TABLE -VI
BASAL MEDIUM I
ORGANISMS PASSED THROUGH 10 TRANSFERS IN MEDIDM' CONTAINING
ONLY THE D-FOHM OF ASPARTIC ACID
60 hour culture
• ug of aspartic ml NaOH ml NaOH'
acid per tube D L
. Uo 1.8 2.7
l6o 6.8 7-3
320 8.2 8-9
6U0 io. U 10.6 ■
960 11.0 ■ 11*1
1280 n.i . 10.3
I
TABLE V I I .
BASAL MEDIUM II
12 hour 2U hour 36 hour 60 hour
ug of aspartic culture culture culture culture
acid per tube ml NaOH ' ml NaOH ml NaOH ml NaOH
■ D L D L D L . D L.
Uo 0.2 0.8 0.5 1.3. 1.1 2.3 2.2 2.6
-160 O.U 2.1 2,1. 3.5 2.8 U-7 U-8 5.7
6U0 O-U 2-2 2.2 ' U-O 3.2 5-8 5-3 7.9
1280 • 0.6 2.2 • 2.3 U-O 3-3 5.8 5.3 7-7
17
TABLE VIII.
BASAL MEDIUM III
36 hour 60 hour
culture culture
ug of aspartic ml HaOH ml NaOH
acid per tube ' D L ' D L
ItO ' . , 'O.li 1.6 O.li 2.U
80 0*9 1.9 1.0 2.9
160 1.5 2.1 ' 1.6 2.9
2L).0 1.8 2.1 1.7 2.9
320 1.9 2.1 2.2 2.9
TABLE' JX 
BASAL MEDIUM I
Glutamic acid varied ---- 60 hour culture
Amount, of glutamic acid per tube
ug of aspartic 50 Ug 100 Ug 500 U& I mg mg ■
acid per tube ml HaOH - ml- HaOH ml MOti ml NaOH ml HaOH
D L D L D L D L D L
• LtO 0.8 1.7 1.5 2.0 1.2 2.0 0.9 2.0 . 1.5 2.0
80 1.2 1.6 2.2 2.8 • 1.8 3.0 2.2 3-It 2.7 3.5
120 0.8 1.2 2.6 3.1 2.5 . it-3 2.8 Lt.3 3.8 it. 7
160 '0.9 0.9 2.8 3.3 : 2.8 lt.9 3.7 5.2 3.9 5-3
TABLE X
 ^ BASAL MEDIUM I
Per cent Response of D-Aspartic Acid in Terms of L-Aspartic Acid' 
at Concentrations of LtQ. to 320 ug per ',Tube
ug of aspartic 
acid per tube
W
80
16.0
21.0
320
36 hour culture 
per cent 
50 
#
1l3
k2
hi
60 hour culture 
' per cent 
60 
70
k$. 
h6 
hh •
18
TABLE II ,
BASAL MEDIUM I
Per cent Response of D-Aspartic Acid in Terms of L-Aspartic Acid 
At Concentrations of ItO to 6ItO ng per Tube
12 hour 18 hour 36 hour 60 hour
ug of aspartic culture culture culture culture
acid per tube per cent per cent . per cent per cent
Uo • 0 UO Uo 50
80 0 20 U6 65
160 25 10. 30 ' 50
320 15 15' 15 U3 '
6U0 7.5
TABLE XII 
BASAL MEDIUM II .
•
Per cent Response of D-Aspartic Acid in Terms of L-Aspanbic Acid 
At Concentrations of ItO to 160 ug per Tube
ug of aspartic 
acid per tube 
W  
80 
160
36 hour ■ culture 
per cent 
UO 
80
25
60 hour culture 
per cent 
90
75
15
TABLE XIII
BASAL MEDIUM III
Per cent Response of D-Aspartic Acid' in Terms of L-Aspartic Acid 
At ■ Concentrations of ItO to 320 ug per Tube ■
ug of aspartic 
acid per tube 
ItO 
80 
160 
. 2 UO 
320
36 hour culture 
per cent ■ 
18 
20 
23 
25 
25
60 hour culture 
per cent
15 
18 
. IU 
' 10 
11
19
Per cent Response of D-Aspartic Acid in Terms of L-Aspartic Acid 
at Concentrations of UO to 320 ug per Tube 
With Glutamic acid varied
TABLE H V
BASAL MEDIUM I
Amount of glutamic acid per tube
ug of aspartic 50 ug 100 ug 5oo ug I mg it mg
acid per tube per cent per cent per cent per cent per cent
ItO 30 60 ■ 62 ill 70
80 60 ■ iio 58 71
' 120 - 57 53 . 53 75
160 ' 50 50 60 63
8 -
BASAL MEDIUM I
40 TO 320 *1 ASPARTIC ACID
60 HOUR CULTURE / TUBE
ro
o
O
FIG. JTL
BASAL MEDIUM I
4 0  TO 1280 U1 ASPARTIC ACID /  TUBE 
36 HOUR CULTURE
L-ASP
D-ASP
1280
60 HOUR CULTURE
1280OF ASPARTIC/ TUBE
r\D
H
FlG
N
3600 
iO
 
TM
L -  ASPARTIC 
D -  ASPARTIC
BASAL MEDIUM I
ORGANISMS AFTER IO TRANSFERS 
IN D - ASPARTIC ACID.
60  HOUR CULTURE
U e  OF ASPARTIC Z TUBE
M 
L 
OF
 
O 
09
2N
BASAL MEDIUM III
4 0  TO 320 U1 ASPARTIC ACItyz TUBE 
36 HOUR CULTURE
L-ASP
D-ASP
24 0
1 ASPARTICzZ A C ID y z  TUBE
FIG
.
0.
0 
92
BASAL MEDIUM EL
40  TO 320 ASPARTIC ACID /  TUBE
60 HOUR CULTURE
3 -
160.
ASPARTIC A C ID /T U B E
FIG.
12 I
IO -
8,
L- ASP.-------------------------
D-  ASP. ------------------------  c
BASAL MEDIUM Il
4 0  TO 1280 ASPARTIC
3 6  HOUR CULTURE
TUBE
n
o
VT.
® 8 60 HOUR CULTURE
S 6
§
u.
O
Z2
U1 OF ASPARTICyzTUBE
Tl
. H
1280160 I
M 
L 
OF
 
0.
0 
92
 N
 N
» 
OH
12 HOUR
6 -  36  HOUR
6 0  HOUR L-ASR —
D-ASP
TUBEOF ASPATtTIC ACID
BASAL MEDIUM I
4 0  TO 6 4 0  ASPARTIC ACID /  TUBE
f I g. ™xi 
"-XIL 
zxnr 
~x
iy
M
L 
OF
 
0.
09
2N
 N
a
OH
SOn GLUTAMIC ACID 
60 HOUR CULTURE' 
BASAL MEDIUM I
L-ASRTUBE
D -A S P
IOOm1 GLUTAMIC ACiq/TUBE  
60 HOUR CULTURE
BASAL MEDIUM I
4 M.G. GLUTAMIC AClDyZ T U B E  
60  HOUR CULTURE 
BASAL MEDIUM I
T ASPARTIC ACID/ z TUBE
FIO
 
X
S
T
' 
F
ia
T
S
T
 
FIG
. 
Y
B
!]'
MEDIUM I
80 U1 ASPARTIC I  TUBE 
GROWTH LAG WITH D-ASR
i
■n
p
<
<
5
—r-
60
i
4
29
DISCUSSION
In order to explain the results obtained in this work a postu­
lated mechanism for the utilization of the D-form of amino acids will 
be presented.
Horowitz (I9UI4) studying strains of Neurospora expressed the 0- 
pinion that cells normally synthesize DL-amino acids and the D-amino 
oxidase is present to oxidatively deaminate the D-forms. The a-lceto 
analogue is then resynthesized to the L-form. He also stated that 
this D-amino acid oxidase enabled the organism to utilize D-amino 
acids from unnatural sources.
Hydon (19U8) states that it seems the synthesis and utilization 
of D-amino acids involve a-keto or a-imino acids as intermediates. He 
generalizes the scheme thus: Where (a) and (b) are alternate routes.
Smaller Molecules
P
L-
HgN —  C--H
R
D-
COgH
CO2H
This is presented as one possibility in the utilization of the D- 
aspartic acid. If this is correct it must be assumed that the aspartic
30
acid is used in the synthesis of the protein material of the cell. This- 
does not seem to be an unreasonable assumption since other energy sources 
should be available and it is unlikely that Leuconostoe mesenteroides 
P-60 should require aspartic acid as a specific source of energy.
Until this work no evidence had been offered to show that D-aspartic 
acid was utilized by Leuconostoc mesenteroides P-60, Bydon, (I9J48). That 
it is utilized is shown by Tables III to IX. At high concentrations,(i.e. 
1160 ug per tube) the response to D-aspartic acid exceeds that of' L-as- 
parfic acid. (See. Figures V and VI).. It will be noted that this is 
caused by a decrease in the response of L-aspartic acid and a continued 
increase in the response of D—aspartic acid at these higher concentrations♦
It is interesting that in practically all cases, the percent response 
to D- in terms of L-aspartic acid is greater at lower concentrations of 
the aspartic acid. This may be a function of pH, because as the' concen­
tration of aspartic acid increases the acid production increases. It 
would also be anticipated that at the greater periods of time, where acid 
production is greater there would be less D- utilized in terms of L-as­
partic. acid-. This is not true, however, but when the lag in -utilization 
of tfte D-form is considered the results may not be out of keeping with 
what might be expected.
The response obtained with the D-form at 60 hours and a concentration 
of ii.0 ug of aspartic acid per tube are shown in Table XV.
Medium II exhibits a higher utilization of D-aspartic acid than either 
Medium I or III* Ih the case of .Medium III low acid production with both, -
31
TABLE XV
Comparative Response-at Concentration of 
hO ug of Aspartic Acid Per Tube
60 hour culture
Basal Medium ml NaOH Percent of D- in "
D L Terms of L-aspartic acid
I 1.3 2.3 • ' 60
II 2.2 2.6 90
III 0,1* 2.1* 13
D- and L-aspartic acid was noted. This may be attributed to insufficient
buffering. The pH fell very rapidly in this medium and perhaps inactivated 
the enzyme system before much acid could be produced. An explanation for 
the difference between Medium I and II is not as apparent. A difference 
in response to the D-form of methionine by Streptococcus faecalis between 
media buffered with citrate and acetate was reported by Lyman and ICuiken
(191*8). They found no utilization of the D-form in a medium buffered 
with acetate, but a considerable ■ utilization in a medium buffered with 
citrate. The variation in utilization appears to be due to the difference 
in buffers which is usually not considered as having any influence except 
on pH. Additional investigations would be helpful here. The difference 
in i esponse between Medium-1 and Medium II may also be due to unlike rates 
of change of pH. Cohen and Lichstein (19U3) report that in certain trans­
amination reactions there is an optimal pH and temperature.
If it is -assumed that pH has an important effect on the utilization 
of D—aspartic acid it would appear that at the earliest times the greatest
32
utilization of D—aspartic acid in terms of L—aspartic acid should occur. ■ 
This is not so. An examination of Tables IV and U  and Figures XI, XII, 
and XVIII shows that there is practically no utilization of the D-isomer 
during the first few hours of incubation. This slowness in utilization 
of the D-form has been noted previously. Eydon (I9I4.8) mentions that ■ 
D-amino acids are utilized quite slowly with reference to L-amino acids, 
and that the organisms must be growing actively. He reported that Konakova 
and Micolle found that D-alanine will substitute for L-alanine with both 
Salmonella typhimurium and Proteus X~19, but that growth is slower and 
preceded by a considerable lag. He stated that they were able to train 
the organisms to utilize D-alanine as readily as the- D-isomer. An attempt 
was made to train Leuconostoc mesenteroides P-60 to D-aspartic acid by 
passing the■organism through ten transfers in a medium containing only the 
D-isomer of aspartic acid. Although this may not have been sufficient to 
train, the organisms it should have been sufficient to eliminate all but 
the variant'causing utilization of the D-form, if there were such a. variant. 
Training did not occur and that no culture of a variant developed is evi­
denced from a comparison of Figures V and VI.
' The slowness of response to- D-aspartic acid indicates that perhaps 
an enzymatic adaptation is'taking place. SpiegeMan (Cold Spring Harbor 
Symposia on Quantitative Biology, XI) defines an enzymatic adaptation 
thus: ("A population of cells placed in contact with some substrate ac­
quires, after the lapse of some time, the enzymes necessary to metabolize 
the added substrate".) The production of an enzyme for this assimilation
33
could be a function of both time and the concentration of the D-fora in- 
the substrate. If this is true then the organism meets both of these 
requirements and the definition of Spiegelman. It appears to the author 
that this may be an example of enzymatic adaptation.
The effect of varying amounts of glutamic acid upon the utilization 
of aspartic acid was -studied. The results are shown in Figures XV, XVI3 ' 
and XVIII3 and tabulated in Tables IX and XIV. Green3 Leloir and Nicito 
(19l).3) reported an enzyme which they called aspartic-glutamic acid trans­
aminase. They offered the following reaction. Glutamic acid +  Oxalacetic 
acid— > ar-ketoglutaric acid-f-Aspartic acid. This reaction was also re­
ported by Cohen and Lichstein (I9li5)<>
No evidence of transamination was found in this report. An - unex­
pected decrease in the response to L-aspartic acid was noted at IiO ug 
of aspartic acid per tube with increasing concentrations of glutamic acid.
A surlier decrease was noted at 80 and 120 ug of D-aspartic acid per tube, 
apd tjie increase anticipated at 160 ug was not observed. The concentration 
of h mg of glutamic acid3 Tihich is used in this laboratory for assay, pro­
vided the most constant results. It is interesting to note (see Table IX) 
that at the lowest levels of glutamic acid the utilization of L-aspartic 
acid decreased as the amounts of this isomer increased. This phenomenon 
1 was not apparent with the D-isomer which would seem to indicate that per­
haps different mechanisms were being affected.
Alanine was also varied, but it showed no significant changes in 
response of the organisms at various concentrations.
3h
D- and L-asparagine were substituted for D- and L-aspartic acid in 
■Basal Medium I. No growth was evident with either isomer of asparagine.
Although this work accomplished its major purpose by establishing 
that Leuconostoc mesenteroides P-60 utilizes the D-form of'aspartic acid 
it left many questions unanswered. No clear explanation as to the mech­
anism of the utilization of L-aspartic acid was provided. The author1 s 
belief is that it is utilized by being oxidatively deaminated and then 
resynthesized. It is further believed that this oxidation is caused by . 
an enzyme which is elaborated only under special conditions. Part of 
those conditions must be the presence of D-aspartic do id. A further ■ 
study adding small amounts of L- to D-aspartic acid using DL-aspartic 
and the a-keto analogue would be interesting.
"If titrations were made at close intervals and the pH controlled 
by varying the amounts of buffers used until the optimum was obtained, 
the extent of the lag, or period of adaptation could be much more sharply
defined,,
35
SUMMflBr
A study of utilization of D-aspartic acid by Leuconostoc iaesen- 
teroides P-60 was made. The following conclusions were reached:
I* Lepconostoc mesenteroides P-60 utilizes the D-isomer of 
aspartic acid.
2« The utilization of the D-isomer is affected by the kind and 
quantity of the buffers present in the medium. The affect 
of buffers is in part, at least, due to differences in the 
rate of change of pH.
3» It is believed that the utilization is made possible by.the 
organism adaptively elaborating enzymes perhaps capable of 
oxidative deamination and resynthesis.
U. Glutamic acid was varied TdLth different concentrations of 
both D- and L-aspartic acid, but no final conclusions were 
drawn.
5« Varying the amounts of alanine has no appreciable affect on 
the utilization of D- or L-aspartic acid by this organism.
6. D- or L-asparagine will not substitute for D- or L-aspartic
acid.
36
ACKWOWLEDGEMEHT
The author gratefully acknowledges the invaluable counsel of Dr. 
Ieon H. Johnson, whose help and guidance made this investigation possi­
ble; and furthermore,' to Mr. W. G. Walter for his advice in the iso­
lation of single cells., ,, ■
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