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Патент USA US3086926

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3,385,916
United States Patent 0
Patented Apr. 23, 1963
1
2
3,086,916
and Biophysics, 35, 462 [1952], and Journal of Biolog
ical Chemistry, 197, 453, [1952]), but details of the
PROCESS FOR PRODUCING L-GLUTAMIC ACID
FROM RACER/12C GLUTAMIC ACID
research are not apparent.
The present inventors have found that strains belong
Shultuo Kinoshita, Masao Tanaha, and Y0 Kato, Tokyo,
Japan, assignors to Kyowa Hakko Kogyo Co., Ltd,
Tokyo, Japan, a corporation of Japan
No Drawing. Filed Aug. 2, 1961, Ser. No. 128,695
12 Claims. (Cl. 195-29)
ing to the Lactobacillus group, such as Lactobacillus ?er
menti, Lactobacillus arabinosus, Lactobacillus delbm'ickii,
and the like have powerful glutamic acid racemase activ
ity. It has thus been found (a) that enzymatic mate
rials produced from these strains, such as lyaphilized
The present invention relates to a process for producing 10 solid cells, acetone-dried solid cells, cell homogenates
obtained by supersonic vibration, cell suspensions, and
L-glutamic acid from‘ racemic glutamic acid by use of a
speci?c combination of enzymatic materials. More par
the like, speci?cally act for racemization of glutamic acid,
ticularly, it deals with a process for producing L
(b) that there is no change in the total amount of glu
tamic acid after the reaction by their use under whatever
reaction conditions are employed, and (c) that they do
not racemize 2-pyrrolidone-5-carboxylic acid.
The reaction concerned is exempli?ed in Table 1 with
respect to 'actone-dried solid cells of Lactobacillus fer
menti strain using a solution containing D-glutamic acid
glutamic acid from racemic glutamic acid by causing a
dehydration reaction of L-glutamic acid with enzymatic
action of a strain of Pseudomonas cruciviae or its mutant,
and by causing, at the same time, a racemization reaction
of optically active glutamic acid with an enzymatic mate
rial produced from a strain belonging to the Lactobacillus
group, thereby converting the racemic glutamic acid to 20 and L-2-pyrrolidone-5-carboxylic acid.
L-glutamic acid.
-
TABLE 1
An object of the present invention is to provide a
Racemization Ratios of D-Glutamic Acid and
L-Z-Pyrrolidone-S-Carb0xylic Acid
process for producing L-glut-amic acid having a higher
purity by an easy procedure in a short period of timel
with a high yield, excluding ‘such complicated procedure 25
as optical resolution. Other objects will be apparent
from the description which follows.
Production of L-glutamic acid from racemic glutamic
acid has heretofore been conducted by physically or bio
chemically resolving racemic glutamic acid into L- andv
D-glutamic ‘acids or their derivatives, racemizing the
D-glutamic acid remaining after the separation of the
Reaction period (hour)
D-glutamic
acid
'
L-2-pyrroli~
done-51cm
boxylic acid
0
65.0
0
0
82.1
95. 2
0
0
98. 7
0
resolution products, andfurther resolving the racemized
glutamic acid, thus repeating the same procedure. Such
conventional processes, however, have various dit?culties
from technical and economical viewpoints, since such‘re~
pe-ated procedures are extremely complicated and require
I a long period of time; a low yield of L-glutamic acid is
unavoidable.
The present inventors have suggested a process ‘for
producing L-glutamic acid from racemic glutamic acid in
Japanese patent application No. 28,885/ 1959, the gist of
Furthermore, the present inventors have found that
both of glutamic acid-racemase and L-glutamic acid-de
hydrase are powerful and do not affect each other when
they coexist, and, besides, that the actions of both en
zymes help each other through continuous movement of
40 reaction equilibrium when the both reactions take place
at the same time. Thus racemic glutamic acid is rapidly
converted to L-2-pyrrolidone~5-carboxylic acid, which is
‘easily converted to L-glutamic acid by hydrolysis in a
which is in converting racemic glutamic acid into L
high yield. Accordingly, the present invention provides
glutamic acid by a dehydration reaction of L-glut-amic 45 for the ?rst time an extremely advantageous process for
acid with a strain of Pseudomonas cruciviae or its mutant .
commercially producing ~L-g1utamic acid from racemic
: (strain) in the presence of an aromatic aldehyde and a
glutamic acid.
9
metallic ion, the former havinga radical co-ordinat-able
To produce L-glutamic acid from racemic glutamic
with a metal. Because the racemization of glutamic acid
acid in accordance with the invention, an enzymatic ma
is eliected by an aromatic aldehyde and a metallic ion in 50 terial produced from a strain of glutamic acid-recemase
this process, an amino radical rearrangement reaction
yieldingv bacteria belonging to Lactobacillus and an
tends to take place along with the racemization reaction
enzymatic material prepared from a strain of Pseudomo
according to the condition of the procedure. Part of the
nas cruciviae are added to an aqueous solution containing
glutamic acid is thereby converted to oc-ketoglut-an'c acid,
racemic glutamic acid. The L-glutamic acid in the solu
resulting in a loss of yield of glutamic acid. Besides, the 55 tion is dehydrated by the enzymatic action, and rapidly
activity of glutamic acid dehydrase is inclined to be
converted to L-Z-pyrrolidone-S-carboxylic acid. At the
lowered in a small degree.
same time, the remaining D-glutamic acid is racemized
The present inventors conceived the idea that the use
through the movement of reaction equilibrium, and
of ‘an enzyme would be the best way for speci?cally
changed to racemic glutamic acid. The L-glutamic acid
effecting the racemization, ‘and have searched a racemase
yielding strain.v Consequently, extremely powerful glu
tamic acid racemase-yielding strains have been ‘found
60
newly yielded by the racemization is readily dehydrated
to L-2-pyrrolidone-5¥carboxylic acid. The racemization
reaction is extremely rapid. The dehydration reaction of
L-glutamic acid is also exceedingly rapid, since the pro
and isolated. Thus, an exceedingly superior process is
accomplished, wherein racemic glutamic acid is converted
portional amount of L-glutamic acid against the enzyme
to L-glutamic acid utilizing the enzymatic action of glu 65 is always so small that the enzymatic action is elfective.
Thereby, the mutual action of both reactions of con
tamic acid racemase yielded by such strains.
version of L-glutamic acid to L-2-pyrrolidone-5-carbox
With respect to enzyme racemization of amino acids,
ylic acid and of racemization of D-glutamic acid permits
(See Journal
ofvBiological Chemistry, 190, 403 [1951] and Archives
the whole conversion of racemiciglutamic acid to L-2
of Biochemistry ‘and Biophysics 49, 168, [1954].) As for 70 pyrrolidone-S-carboxylic acid in a short period of time.
L-Z-pyrrolidone-S-carboxylic acid thus obtained readily
glutamic acid, however, there are merely short reports
yields L-glutamic acid by hydrolysis. For production of
of Narrod, Ayengar et al. (see Archives of Biochemistry
there has been some research on alanine.
3,086,916
3
4
L-glutamic acid from racemic glutamic acid in accord
ance with the invention, the preferred concentration of
the racemic glutamic acid in the reaction mixture is
rnatic material produced by cultivation of a glutamic
acid-racemase-yielding bacteria belonging to Lactobacil
lus group and an enzymatic material produced by culti
about 20 to 50 mg./ ml. The pH of the reactive mixture
vation of a strain of Pseudomonas cruciviae (including
its mutant strains) to an aqueous solution containing ra
cemic glutamic acid, thereby causing a reaction to con
,at the beginning is preferably about 7.5. The preferred
temperature of the reaction is from 37 to 40° C. Usually
a reaction period ranging from 4 to 12 hours suffices
to obtainsubstantially quantitative yields.
The separation of the resulting L-2-pyrrolidone-5-car
vert the glutamic acid to L-2-pyrrolidone-carboxylic acid,
and hydrolyzing the resulting L-pyrrolidone-5~carboxylic
acid, and recovering L-glutamic acid from the hydrolysate.
2. A method of producing L-glutarnic acid from race
boxylic acid from the remaining D-glutamic acid may 10
mic glutamic acid which comprises (A) subjecting an
‘be conducted by any of the conventional procedures;
aqueous solution containing racemic glutamic acid (1)
employing .such an ion-exchange resin as may adsorb
to the action of at least one member selected from the
glutamic acid alone and no 2-pyrrolidone-5~carboxylic
group consisting of Lactobacillus fermentz', Lactobacillus
acid is preferred. For instance, when an acidic reaction
mixture having a pH of not more than 3.2 is passed 15 arabinosus and Lacie-bacillus delbriickii and (2) to the
through strongly acidic cation exchange resin, such as
H-cycle sulfonated polystyrene type resin, the remaining
'D-glutamic acid is adsorbed on the said resin while only
v2-pyrrolidone-S-carboxylic acid remains in the effluent.
'Thus, bothD-glutamic acid and 2-pyyrolidone-5-carbox
ylic acid may be separated.
The hydrolysis of the separated L-2-pyrrolidone-5
action of Pseudom-onas cruciviae, thereby converting the
racemic glutamic acid to L-2-pyrrolidone-5-carboxylic
acid, (B) hydrolyzing the resulting L-2-pyrrolidone-5
carboxylic acid, and (C) recovering L-glutamic acid from
the hydrolyzate.
3. .A method of producing L-glutamic acid from race
mic glutamic acid which comprises (A) bringing (1) an
aqueous solution containing racemic glutamic acid into
carboxylic acid may be carried out using aqueous acid
or alkali solution in conventional procedure, the detailed
contact with (2) an aqueous suspension of a microor
explanation of which is unnecessary since it is apparent 25 ganism selected from the group consisting of Lactobacil
to those skilled in the art.
lus fermenti, Lactobacillus arabinosus and Lactobac‘illus
The present invention will more fully be explained with
delbrilckii and with (3) Pseudomonas cruciviae, whereby
respect to the ‘following examples, which are provided
the racemic glutamic acid is converted to L-2-pyrrolidone
by way of illustration, and not by way of limitation.
S-carboxylic acid, (B) hydrolyzing the resulting L-2~pyr
30 rolidone-S-carboxylic acid, and (C) recovering the L
EXAMPLE 1
:glutamic acid from the hydrolyzate.
To a solution containing 100 .g. of racemic glutamic
4. A method of producing L-glutamic acid from race
acid and adjusted‘to pH 7.5, there are added 2 g. of dried
mic glutamic acid which comprises (A) bringing (1) an
solid cells of Lactobacillus fermenti and 2 g. of dried solid
aqueous solution containing racemic glutamic acid into
cells of Pseudomonas cruciviae. The glutamic acid in 35 contact with (2) cells of a microorganism selected from
the solution is converted almost completely .to L2
the group consisting of Lactobacillus fermenti, Lacto~
pyrrolidone-‘S-carboxylic acid after being allowed to react
bacillus arabinosus and Lactobacillus delbrt‘lckii and with
at the temperature of 40° C. for 4 hours. The pH is
(3) Pseudomonas cruciviae, whereby the racemic glu
adjusted to 2.0 after the termination of the reaction. The
- solution is passed through a column ?lled with 100 ,g. 40
of strongly acidic ion exchange resin having previously
been regenerated with 10% hydrochloric acid and then
Washed, thereby unreacted D-glutamic acid being re
moved by adsorption. The effluent is concentrated and,
at :the same time, hydrolyzed, followed by ?ltration. The
pH of the ?ltrate is adjusted to 3.2, and the crystals
isolated are separated therefrom, and are dried to yield
97 1g. of L-glutamic acid. {Further 1.5 .g. of L-glutamic
' vacid are recovered by concentration of the mother liquor.
EXAMPLE 2
To a solution containing .82 g. of racemic glutamic
. acid, there is added 1 g. of acetone-dried solid cells of
Lactobacillus arabinosus and 300 mg. of a crude enzy
matic material produced from Pseudomonas cruciviae at
. the pH of 7.5. The glutamic acid in the solution is sub
stantially completely converted to L-2-pyrrolidone-5
.tamic acid is converted to L-2-pyrrolidone-S-carboxylic
acid, (B) hydrolyzing the resulting L-2-pyrrolidone-5
carboxylic acid, and (C) recovering L-glutamic acid
from the hydrolyzate.
5. A method of producing L-glutamic acid from race
.mic glutamic acid which comprises (A) bringing (1) an
aqueoussolution containing racemic glutamic acid into
contact with (2) cell homogenate of a microorganism
selected from the group consisting of Lactobacillus fer
;menti, Lactobacillus arabinosus and Lactobacillus d‘el
bra'lckii and with (3) Pseudomonas cruciviae, whereby
the racemic glutamic acid is converted to L-2-pyrroli
done-S-carboxylic acid, (B) hydrolyzing the resulting L
2~pyrrolidone-5-carboxylic acid, and (C) recovering L
glutamic acid from the hydrolyzate.
6. A method of producing L-glutamic acid from race
mic glutamic acid which comprises (A) bringing (1) an
aqueous solution containing racemic glutamic acid into
contact with (2) enzyme of a microorganism selected
carboxylic acid after being allowed to react at the tem
from the group consisting of Lactobacillus fermenti,
’ perature of 37° C. for 6 hours. The reaction liquor is
processed as in Example 1, and the crystals isolated at 60 Lactobacillus arabinosus and Lactobacillus delbriickii
and with (3) Pseudomonas cruciviae, whereby the race
the pH of 3.2 are separated and dried, to yield 81.1 g.
mic'glutamic acid is converted to L-2-pyrrolidone-5-car
of Lrglutamic acid.
EXAMPLE 3
boxylic acid, (B) hydrolyzing the resulting L-Z-pyr
rolidone-S-carboxylic acid, and '(C) recovering L-glu
To a solution containing 28 g. of racemic glutamic 65 tamic acid from the hydrolyzate.
7. A method of producing L-glutamic acid from race
acid, there is added a crude enzymatic material obtained
mic glutamic acid which comprises (A) bringing (1) an
by supersonic vibration of 2 g. of solid cells of Lacto
aqueous solution containing racemic glutamic acid into
bacillus delbmickii and ‘500 mg. of dried solid cells of
contact with (2) enzyme of a microorganism selected
, Pseudomionas cruciviae. After a reaction at a tempera~
. ture of 35° C. for 12 hours, and a treatment of the re 70 from the group consisting of Lactobacillus fermenti, Lac
tobacillus arabinosus and Lactobacillus delbrt'tckii and
sulting reaction liquor as in Example '1, 26.6 g. of L
with (3) enzyme of Pseudomionws curciviae at a tem
glutamic acid are yielded.
perature of from 37° to 40° C., whereby the racemic
What we claim is:
glutamic acid is converted to L-2-pyrrolidone-5-car
.1. A process for producing L-glutamic acid from ra
cemic glutamic acid, which comprises adding an enzy 75 boxylic acid, (B) hydrolyzing the resulting L-2-pyr
3,086,916
5
6
rolidone-S-carboxylic acid and (C) recovering L-glutamic
consisting of Pseudomonas cruciviae and enzyme there
of, whereby the racemic glutamic acid is converted to L
acid from the hydrolyzate.
2-pyrrolidone-5-carboxylic acid, (B) hydrolyzing the re
8. A method of producing L-glutamic acid from race
sulting L-2-pyrrolidone-S-carboxylic acid, and (C) re
mic glutamic acid which comprises (A) bringing (1) an
aqueous solution containing racemic glutamic acid into 5 covering L-glutamic acid from the hydrolyzate.
11. A method of producing L-glutamic acid from race
contact with (2) enzyme of a microorganism selected
mic glutarnic acid which comprises (A) subjecting an
aqueous solution containing racemic glutamic acid ( 1)
from the group consisting of Lactobacillus fermen'ti, Lac
tobacillus arabinosus and Lactobacillus delbmickii and
with (3) enzyme of Pseudo-moms curciviae at a pH of
to the action of a member selected from the group con
about 7.5, whereby the racemic glutamic acid is con 10 sisting of Lactobacillus arabinosus and enzyme thereof
and (2) to the action of a member selected from the
group consisting of Pseudomonas cruciviae and enzyme
verted to L-2-pyrrolidone-S-carboxylic acid, (B) hy
drolyzing the resulting L- 2 -pyrrolidone-S-carboxylic
acid, and (C) recovering L-glutamic acid from the hy
thereof, whereby the racemic glutamic acid is converted
to L-Z-pyrrolidone-S-carboxylic acid, (B) hydrolyzing the
9. A method of producing L-glutamic acid from race 15 resulting L-Z-pyrrolidone-S-carboxylic acid, and (C) re
covering L-glutamic acid from the hydrolyzate.
mic glutamic acid which comprises (A) forming a reac
drolyzate.
12. A method of producing L-glutamic acid from ra
tion mixture of (1) an aqueous solution containing ra
cemic glutamic acid, (2) an aqueous suspension of a
cemic glutamic acid which comprises (A) subjecting an
aqueous solution containing racemic glutamic acid (1)
microorganism selected from the group consisting of
Lactobacillius fermenlz', Lactobacillus arabinosus and
Lactobacillus delbrt'ickii and ( 3) Pseudomonas ci‘uciviae,
the concentration of racemic glutamic acid in the reac
tion mixture being from 20 to 50 milligrams per milli
liter, whereby said racemic glutamic' acid is converted to
L-2-pyrrolidone-5-carboxylic acid‘, (B) hydrolyzing the
to the action of a member selected from the group con
sisting of Lactobacillus delbm‘ickii ‘and enzyme thereof
and (2) to the action of a member selected from the
group consisting of Pseudomonas crucivz'ae and enzyme
thereof, whereby the racemic glutamic acid is converted
25
resulting L-Z-pyrrolidone-S-carboxylic acid, and (C) re
covering L-glutamic acid from the hydrolyzate.
10. A method of producing L-glutamic acid from ra
cemic glutamic acid which comprises (A) subjecting an
aqueous solution containing racemic glutamic ‘acid (1) to 30
the action of a member selected from the group consist
ing of Lactobacillus fermem‘i and enzyme thereof and
(2) to the action of a member selected from the group
the resulting L-2-pyrr0lidone-5-carboXylic acid, (B) hy
drolyzing the resulting L-Z-pyrrolidone-S-carboxylic acid,
and (C) recovering L-glutamic acid from the hydrolyzate.
References Cited in the ?le of this patent
UNITED STATES PATENTS
3,003,921
3,003,922
3,003,923
Kinoshita et al. ______ __ Oct. 10, 1961
Kinoshita et a1. ______ __ Oct. 10, 1961
Kinoshita et a1. ______ __ Oct. 10, 1961
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