Note: Descriptions are shown in the official language in which they were submitted.
~fJ~7Z~O
1 BACKGROUND OF TEIE INVENTION
2 The present invention relates to a process for the
3 production o:E L-serine from glycine.
4 L-serine i9 one of amino acicls well known in the
art and is in a great demand as material for medicament.
6 Heretofore, L-serine has been prepared using
7 various methods. For example, it has been obtained by
hydrolysis of proteins.
g As for processes for the production of L-serine
from glycine by fermentation, process using a microorganism
11 belonging to the genus Nocardia is known as described in
12 Japanese Patent Publication No. ~391/76. However, the yield
13 of L-serine is poor.
14 Heretofore, it is reported on page 80 o the summary
`15 of lectures in Congress of Fermentation Teohnology, 3apan 1975,
~:~
16 that the increased yields of L-serine are obtained by culturing
a mutdnt belonging to the species Corynebacterium~glycinophilum
,
18 and having both the ability to convert glycine to L-serine and
1g the lowered ability to decompose L-serine. ~
As other prior methods of producing L-serine by
21 fermentation, processes of culturing a strain belonging to
the genus Arthrobacter, Corynebacterium, Brevibacter1um,~ ~
23 Escherichia, Micrococcus, Pichia, or Candida in a nutrient
24 medium or in a nutrient medium containing glycine are known.
2s ~ However, processes which have a hlgh yield of ~
- L-serine are in demand for utilization in industrial practice.
27 The present inventors have studied about a process
for preparing L-s~rine from glycine. As a results, it has
29 been found that a mutant capable of producing L-serine from
glycine and belonging to the genus Nocardla and having no or
lowered ability to decompose L-serine, when cultured in
., : ' ' ' :
~97Z~L~
1 a nutrient medium containing glycine, produce remarkable
2 amount of L-serine in the culture liquor.
3 Further, it has been found that a mutant capable
4 of produclng L-serine from glycine ancl belonging to the genus
s N~cardia, and having a resistance to aLt least one antagonist
6 selected ~rom those of glycine, serine, methionine,
7 glutamine, histidine, leucine, isoleucine, valine, purine,
8 pyrimidine or folic acid, when cultured in a nutrient medium
g containing glycine, also produce~remarkable amount of L-serine
in the culture liquor.
Further, it has been discovered that more improved
12 yield of L-serine may be attained according to the herein
described processes for the production of L-serine on an
4 industrially feasible level.
16 SUMM~RY OF THE INVENTION
In accordance with the present invention, increased
yields of L-serine are obtained by converting glyc1ne to L-
19~ serine in an aqueous medium containing microbial cells of a
mutant belonging to the genus Nocardia and capable of
21 converting glycine to L-serine.
22 The mutant has no or lowered ability to decompose
23 L-serine and/or a resistance to at least one antagonist
24 selected from those of glycine, serine,
~ .
s methionine, glutamine, histidine, leucine,
26 lsoleucine, valine, purine, pyrimidine or folic acid.
27 The conversion of glycine to L-serine is carried
: ~ .
~ 2B out during the culturing of the mutant in a nutrient medium
; ~ .
`~29 containing glycine ~Process (I)].
The conversion is also carried out by culturing the
31 mutant in a nutrient medium to obtain microbial cells and
- 2 -
~ .
.. . . ..
. : -
~3~72~1D
I presenti.ng the microbial cells in an aqueous medium containing
2 glycine [Process (II)~.
3 Further, in the convexsion of glycine to I,-serine,
~ more increased yields of L-serine are obtained by supplement~
iny an additive such as hydrocarbon , alcohol , ketone ,
6 ether-, ester~, polyalcohol- and the derivative of poly-
7 alcohols to the medium.
s Further, in the culturing of the mutant in a nutrient
g medium containing glycine, more increased yield of L serine is
obtained in the culture liquor by supplementing phosphate to
11 the medium to provide a medium containing phosphate at a high
12 concentration of phosphate ions.
In Process (II), the productivity of L-serine
14 increases by the use of an immobilized cells as the microbial
cells.
DESCRIPTION OF THE INVENTION
As the strain used in the present invention, any
I9 mutant belonging to the genus~ ocardia and capable of converting
0 glycine to L-serine, and having no or lowered ability to
21 decompose L-serine and/or a resistance to at least one metabolic
22 antagonists selected from those of glycine, serine,
23 methionine, glutamine, histidine, leucine, isoleucine, valine,
24 : purine, pyrimidine or folic acid may be used.
Such a strain is obtained by endowing a strain of
26 ~ microorgan1sm belonginy to the genus Nocardia and capable of
27 converting glycine to L-serine with no or lowered ability to
28 deoompose L-serine and/or a resistance to at least one~metabolic
29 antagonist mentioned above.
Further, the strain, is also obtained by endowing a
31 strain of microorganism balonging to the genus Nocardia and
:
24~
1 havi.ng no or lowered ability to decompose L-serine and/or
2 resistance to at least one metabolic antagonist mentioned
3 above with the ability to convert glycine to L-serine.
4 Furthermore, the strain to ~e used in the present
invention may have any other property for contributiny to
6 the L-serine productivity than the properties mentioned
7 above.
8 Examples of metabolic antagonist used for induction
9 of mutants are shown in Table l.
11 :
12 Example of Antagonist
13 Glycine glycine-hydroxamate, amino methylsulfonic acid,
chloxomethyl-glycine, glycidic acid~N-methyl-glycine
Serine serine-hydroxamate, D-serine, homoserine
:
~:16 Methionine ethionine, tri-fluoro-methionine, a-methylmethio-
nine, methionine-hydroxamate, selenomethionine
-- 17
;: Glutamine methionine-sulfone, methionine sulfoximine,
18
azaserine, alanosine, duazomycin
Histidine thiazole-alanine, triazole-alanine, aminomethyl-
triazole, a-methyl-hist.idine, histidine-hydroxamate
:~ 21 Leucine norleucine, tri-fluoroleucine, azaleucine, leucine-
22 hydroxamate
:~ 23 Isoleucine thiaisoleucine, O-methyl-threonine, isoleuc~ine-
~ ~ ~ 24 hydroxamate, D-isoleuc1ne, norleucine
; : : Valine norvaline, valine-hydroxamate, D-valine norleucine
:: 25
Purine 5-mercaptopurine, 8-azaguanine, 2-fluoroadenine
26
~:~ Pyrimidine 5-fluorouracilj 2-thiouracil, 5-fluorocitosine,
7 ~ 6-azauracil
28
Folic acid amethopterin, aminopteri:n, trimethoprim,
29 pyrimethamine
31
- 4 -
: : ` `
Z~L~
I In ordèr to obtain mutan-ts according to the present
2 in~ention, standard procedures for inducing mutation may be
3 followed such as irradiatlon with ultraviolet ligh-t~X-ray,
4 Co 60, etc., or treatment with mutation inducing chemicals
such as N-meth~l-N'-nitro-N-nitrosoguanidine, etc. or the like.
6 A simple method to select a strain having no or
7 lowered ability to decomyose L-serine from the colonies
obtained by mutation is as ollows:
A colony is selected from the colonies obtained by
mutation and is cultured in the medium containing L-serine
11 as only one carbon source, only one n1trogen source, or only
2 one other nutrient source.
The strain that does not grow or does grow less
4 than parent strain in the medium mentioned above, has no or
lowered ability to decompose L-serine.
6 Nocardia butanica KY 7985 is an example of the
mutant having a lowered ability to decompose L-serine, which
is obtained~by mutating the L-serine-producing parent strain
9 Nocardia butanica ATCC 2ll97 by method mentioned later.
Further, the strains listed below in Table 2 are
21 examples oE the mutant having a resistance to metabolic
22 antagonists, which are obtained by mutating Nocardia bu-tanica
23 KY 7985 by method mentioned later.
The microbiological properties of the species o~
2s Nocardia butanica are described in Japanese Pa-tent Publication
26 No. 48673/72,
27 The above-noted mutants have been deposited with
28 the Fermentation Research Institute, Agency or Industrial
- 29 Science and Technology, Chiba-ken, Japan.
The strain KY-7985 is deposited under FE~1-P No.
31 3782 and the other strains are deposited under FE~1-P Nos.
,
~ .
5 _
7Z~O
I shown in Table 2.
2 These mutants have also been deposited with the
3 Northern ~egional Research Laboratory, 1815 North ~niversi-ty
4 Street, Peoria, Illinois.
The strain XY 7985 has been accorded accession
6 number NRRL 11189 and the other strains have been accorded
:~ 7 accession numbers`shown in 'rable 2.
- Table 2
o Private FER~l-P NRRL Antagonist
No. No. No.
:
2 KY 7983 3764 11187 trimethopurine (0.35)
13 20;~1P-24 3765 11188 6-mercaptopurlIIe (0.5)
14 21SX-2 3766 11190 serine hydroxama~e (1)
: ~
21GX-1 3767 11191 glycine hydroxamate (1)
IE-36 37 8 glycine hydroxamate (1)
K~ 7988 6 11059 ethionine (5),
~: 18 norleucine (1) ; ~.
l9 IAU-3 glycine hydroxamate (1)
; KY 7989 3769 11192 6-azauracil (0.5)
-~ 36MSF-2 3770 11193 glycine hydroxamate (1)
21 KY 7990 ethionine (5), methyl
sulEone (5)
22
~: 35T~-1 3771 11194 glycine hydroxamate (1)
: 23 KY 7991 ethionine (5), triazole
~: alanine (1)
~: 24
~: 2s Note: Fiyure in the parentheses means concentration (mg/ml)
~: ~ 26 of metabolic antagonist in the medium used for
~: 27 isolation of mutant.
, ~ :
28
~: :
; 29 T~e strain KY 7985, NRRL 11189 is obtained in the
following manner. Microbial cells of the parent strain, i.e.
- Nocardia butanica ATCC 21197, are suspended at a concentration
3l
,
~,
-- 6 --
-
~L~g7;i :4~
I of about 108 cells per ml, in 0.1 M-Tris-malea-te bufEer
2 solution (p~l 6.0). To the suspension i9 added 0.5 mg/ml of
N-me-thyl-N'-ni-tro-N nitrosoguanidine and -the mixture is
4 allowed to stand at room temperature for 30 minutes. Then
the resultant sus2ension is smeared on an agar pla~e of a
~ nutrient medium comprising 0.5 g/dl glucose, 0.5 g/dl yeast
7 extract, 1 g/dl peptone, 1 g/dl meat extract, 0.5 g/dl NaCl
8 and 2 g/dl agar (pH 7.2) and incubated a-t 30C for 2 days
g to form colonies. Cells of the resulting colonies are
smeared on an agar plate of a minimum medium compri ing
Il 0.5 g/dl glucose, 0.2 g/dl (NH4)25O~, 0.15 g/dl KH2PO4,
I2 0.05 g/dl K2HPO~, 0.05 g/dl MgSO4-7H2O, 0.01 g/dl NaCl, 0.001
3 g/dl FeSO4-7H2O, 0.001 g/dl MnSO~-nH2O, 0.001 g/dl CaC12-2H2O,
. .
100 ~g/l biotin, 1 mg/l thiamine hydrochloride, and 2 g/dl
agar (pH 7.2) and also on an agar plate of a medium havlng
l6 ~ the same composition as the minimum medium described above
except using 0.2 g/dl L-serine instead of (NH4)2S04.
8 The strains having an ablllty to produce L-serine
in a good yield are selected from those strains which do not
~ grow on the latter medium, but do grow on the agar plate of
21 the former medium, i.e. minimum medium.
The strain, Nocardia butanica KY 798;, MRRL 11189
22 --
23 iS one of them.
24 The mutants shown in Table 2 are obtalned in the
followlng manner from the strain, Nocardia butanica KY 7985,
NRRL 11189 as the parent strain. '~licrobial cells of the
27 parent strain, i.e. Nocardia butanica KY 7985, NRRL 11189 are
23 suspended at a concentration of about 108 cells per ml, in
O.lM Tris-maleate buffer solution (pH 6.0). To the suspension
is added N-methyl-N'-nitro-N-nitrosoguanidine at a concentra-
31 tion of 0.5 mg/ml and -the suspension is allowed to stand at
:
~ ~ 7 ~
. .
~:
~72~L0
1 room tempera-ture for 30 minutes.
2 Then the suspension is diluted and the diluent is
smearecl on the agar plat:e of the medium havin~ -t~e same
composition as the a~ove-mentioned minimu1ll medium except
further containing the antagonist a-t a concentration shown
6 in Table 2 and incubated at 30C for 2 - lO days.
7 Thus, the mutants shown in Table 2 are selected
~3 from colonies which grow on the medium. The mutants are
g distinguished from the parent strain in possession of a
resistance to the metabolic antagonists.
1~ It may be determined by one or more of the follow-
12 ing methods whether the mutant has a lowered ability to
decompose L-serine compared with that of the parent.
14 A selected strain is cultured in a nutrient medium
containing neither glycine nor L-serine 1n order to confirm
~6 that the strain does not produce L-serine. Then the strain
is cultured in a nutrient medium conta1ning not glycine but
a L-serine in order to determine the ability to decompose
L-serine. After the completion of culturing, if the amount
of L-serine remaining Ln the culture liquor is larger~than
21 that of the parent strain, the strain has no or lowered
22 ability to decompose L-serine.
23 As other methods,~logarithmic phase cells, stational
24 phase cells, extract solution from cells or disruptured cells
is incubated in an aqueous medium containing L-serine for an
26 appropriate period of time. Then, the amount of L-serine in
27 the mixture 1S de-termined and compared with that of ~-serine
23 at the start of incubation.
29 It is understood from the following experiments
that Nocardia butanica KY 7985 has the lowered ability to
decompose L-serine.
.
. ~
~35~7Z~I
EXPERI.~IENT 1
2 The strain oE Nocardia butanlca KY 7985, NRRL 11189
3 is cultured on an agar slant of the medium (pH 7.2) containing
4 0.5 g/dl glucose, 0.5 g/cll yeast extract, 2 g/dl peptone, 0.5
g/dl NaCl, and 2 g/dl agar at 30C foY 2 days.
6 ` One loopful of the resulting seed culture is
7 inoculated into 7 ml of a seed medium (pH 7.2) containing 4
g/dl glucose, 0.15 g/dl KH2P04, 0.05 g/dl K2HP04, 0.05 g/dl
9 MgS04-7H20,0.3 g/dl urea, 50 ~g/dl biotin, 0.5 g/dl yeast
extract and 2 g/dl peptone in a large test tube (20 mm x 190
ll ml). Culturing is carried out with shaking at 30C for 24 hours.
IZ 0.25 ml of the resulting seed culture is inoculated
13 into 5 ml of medium containing L-serine having the following
14 composition in a large test tube:
s glucose 5 g/dl
~ :: :
16 (NH4)2S04 0.2 g/dl
17 urea 0.2 g/dl
KH2P04 ~ 0.15 g/dl
19 K2ETP04 0.05 g/dl
0 MgS04-7~20 0.05 g/dl
1 NaCl 0.01 g/dl
4 7H2 ~0.001 g/dl
3 MnS04 nH20 ~ 0.001 g/dl
biotin 50~g/1
2s ~ yeast extract ~ 0.1 g/dl
L-serine 0.5 g/dl
7 CaC03 3 g/dl
28 (pH 7.2)
29 Culturing is carried ou-t with shaking at 30C for
3 days.
31
_ g _
~ t7~
I The quanti-tative determination of L-serine is
2 carrled out by paper chromato~raphy using Toyo filter paper
3 No. S0 with developing solvent consisting of n~butanol;
l acetone; water and diethyl amine [10 ~ 10 : 5 : 2 by volume].
The reduced amount of L-seri.ne is 1~0 mg/ml. As a
6 control, the same procedures described above the repeated
7 except using Nocardia butanica ATCC 21197 and the reduced
s amount of L-serine is 4.6 mg/ml.
o EXPERIMENT 2
The seed culturing of Nocardia butanica KY 7985,
NRRL 11189 is carried out in the same manner as described in
Experiment 1 and one ml of the resulting seed culture is
14 inoculated into~20 ml of;a fermentation medium having the
.:, : , : : : ~
; 15 following composition in~a 3Q0 mI Erlenmeyer flask:
- ~ 16 glucose 5 g/dl
"
; ; 17 (NH4)2SO~ ~ 0.2 g/dl
glycine 0.5 g/dl
~l9 L~serine ~ ~ 1 g/dl
-~20 urea 0.2 g/dl
.: :
yeast extract 0.5 g/dl
biotin 50 ~g/1
23 ~ ~ ; KH2PO4 ~ 0.15 g/dl
24 ~ ~ K2HPO4 0.05 g/dl
: 25 : MgsO4~7H2o ~ 0.05 g/dl
:
NaC1 0.01 g/dl
FeSO4-7H2o ~ 0.001 g/dl
MnSO4~nH2O 0.001 gjdl
~ ,
~ ~ 29 ~ CacL2~2H2o ~ 0.001 g/dl
`;~ 30 (P~ 7.2)
:~:: : :
31
- I0 -
-
z~
I Culturing is carried out with shaking at 30C for
2 40 hours.
3 S0 ml of the culture bro-th resul-ting from the
4 fermentation is sub~ected to eentrifuqation to recover the
microbial cells: The microblal eells are washed with isotonie-
6 sodium ehloride solution and then are suspended in 0.1 M
7 pyrophosphate buffer solution (pH 9.0) eontaining 5 x 10 5 M
of pyridoxal phosphorie acid, 5 x 10 I M of EDTA and 10
9 of mereapto ethanol.
o The resulting suspension is subjected to disrupt
with ultra sonic disintegrator at 20 K.C. for 30 minutes and
2 then to centrifugation to obtain a supernatant solution.
I3 0.5 ml of the supernatant solution (eoncentration of protein:
14 6.0 mg/ml) and 0.5 ml of 0.1 2~ pyrophosphate buffer solut]on
(pH 9.0) mentioned above containing l.4 g/dl L-serlne are
6 eombined and the mixture 15 allowed to stand~at 37C for 14
hours. : :
18 The reduced amount of L-serine determined by paper
19 ehromatography described ln Experiment 1 is 1.6 mg/ml. As a
0 eontrol, when Nocardla butanica ATCC 21197 is used in the
21 same manner mentioned above, the redueed amount of L-serine
22 is 4.2 mg/ml.
23 In the present invention! when the mutant is
eultured in a nutrient medium eontaining glycine to produce
2s L-serine ln the eulture liquor, the produetivity of 1-serine
26 ean be further enhanced by presenting phosphate at a;eoncen-
27 tration of more than 0.037 M based on the phosphate ion
28 (PO4 ~ to the fermentatlon medium either at the start of the
29 fermentation or during the growth phase of the cells.
An influence on the productivity of L-serine by the
31 presence of a high concentration of phosphate ion in a culture
, ,~
- 11
~o~
I medium is studied by using Nocardia butanica KY 7988 and the
2 result is shown ln the following Experiment 3.
.
4 EXPERIMENT 3
.
Nocardia butanica KY 7988, NRRL 11059 is inoculated
3 into 5 ml of a ~ermentation medium contaIning 5 g/dl glucose,
7 1 g/dl (N~4)2SO4, 2.5 g/dl glycine, 0.05 g/dl K2HPO4 (corIe-
8 spond to 0. 003 r~ of phosphate ion), 0.15 g/dl KH2PO4 (corre-
spond to 0.011 M of phosphate io~), 0.05 g/dl MgSO4~-7H2O,
o 0.001 g/dl FeSO4-7~2O, 0.001 g/dl MnSO4-nH2O, 1 g/dl peptone,
II 3 g/dl CaCO3 and 0 - 2 g/dl Mg3(PO4)2-8H2O (pH 7.2) in a large
?2 test tube. Culturing is carried out with shaking at 28C for
5 days, whereby L-serine is produced in a yield shown in Table 3.
: . : l g
Table 3
~;, 15
Supplemented Total concentration Yield of L-serine
16 Mg3(PO4)2 8H2oof phosphate ion
7 (q~dl W~V) (M); ~mg/ml)
0.014 I.1
~ 0.1 0.019 2.0
.~':
0.2 0.024 2,9
~ 20
;~ 0.3 0.029 ~3.2
~ 21
~ 0 4 0,034 3,5
- ~ 22 ~ ~
0.5 0.039 3.9
23
0.6 0.044 4.5
: 24
; ~ 0.7 0.048 5.0
`~: :25
;~ 0.8 0.053 5.2
~: 26
0.9 0.058 5.5
27
1.0 0.063 6.3
~ 28
1.5 O.o~9 7.3
~ 29
2.0 0.112 7.0
_ -
:
~ 31
: .
- 12 -
,, . . ~ :
- - ; :. ~. . .
- ~ . . ~ .
72~CI
1 As is apparent from Table 3, the yield of L-seri.ne
2 iS i.ncreased with the increasing concentration of phosphate
3 ion ~ma~: 6 times).
The same procedures as mentioned above are repeated
except using 0 - 2 g/dl MgSO4-7H2O instead o~ Mg3(PO4~2-8H2O.
6 As a result, it is found that the increasing of the amount of
7 MgSO~.7H2O does not contribute toward good yield of L-serine.
8 Examples of the phosphate used in the present inven-
tion are NaE2PO4, Na2HPO4, Na3PO~, K~2PO4, R2HPo4~ K3PO~,
NH4 H2PO4, (NH4)2HPO4, (NH4)3PO4, Mg(H2PO4)2, MgHPO4,
11 g3( 4)2~ Ca(H~PO4)2, CaHPO4, ca3(po4)2r Mn(H2PO4~2' MnHPO4
12 Mn3(P04)2/ ZnHP04, Zn3(P04)2, FeHPOj, E'~3(P4)2' Co3(P4)
13 K(NH4)HPO4, Na(NH4)2PO~ and the like, and salt hydrate thereof.
4 As the phosphate, ortho-phosphate is usually used
, ~ :
~ 5 and,. of course, meta-, pyro- and poly-phosph~te may be used.
;,
'~ 16 Further, phosphate containing substances,~ such as
phosphate ore, industrial chemicals, for examplej ion exchange
~: 18 resin adsorbing phosphate ion, activated carbon adsorbing
phosphate ion, etc. may be used.
: 20 These phosphates are used alone or as mixtures of
two or more. ~ : :
~ ~ ~ 2 2 The concentration of phosphates in the medium, which: :
23 promotes the conversion of glycine to L-serine, is usually
: ~ 24 more than 0.037 mole/l, preferably, 0.05 - 0.4 mole/l.
:~: 2s In the present invention, in the conversion of
~ 26 : glycine to L-serine, the produciivity of L-serine can be
: ::
- 27 further enhanced by supplementing to ~he aqueous medium, at
least one additive selected from the group consisting of hydro-
:~ 29 carbon, alcohol, ketone, ether, ester, polyalcohol and deriva-
tives of polyalcohols.
~: 31 of course, when the mutant is cultured in a nutrient
- 13 -
~'97;Z~O
I med.ium containing glyci.ne to produce L-serine ln the culture
2 liquor, the productivity of ~~serine can be enhanced by
3 supplemen-ting the additive to the medialm at any time during
4 culturing the microorganism. In this case, the additive may
be usually supplemented at the time when the microorganism
6 has completed its growth. Further, the additive may be
7 supplemented to the medium containing glycine or after supple-
8 menting the additive to the medium, glycine may be added thereto.
g Examples of additive used in the present invention
o are shown in Table 4.
Table 4
Hydrocarbon: ligroin, petroleum benzine, petroleum spirit,
l3 petroleum ether, cyclohexane, hexane,
4 kerosene, hexadecane
Alcohol: methanol, ethanol, n-propanol, iso-propanol,
n-butanol, iso~butanol, sec-butanol, tert-
6 butanol, n-amylalcohol, cyclohexanol,
17 furfuryl alcohol
18 Ketone: acetone~ methyl ethyl ketone, diethyl ketone
I9 Ether: ethyl ether, isopropyl ether, n-butyl ether,
1,2-propylene oxide, 1,4-dioxane, furan,
furfural, tetrahydrofuran
21
Ester: ethyl formate, methyl acetate, ethyl acetate
tributyl citrate, dioctyl phthalate, ethyl
23 propionate, ethyl benzoate
24 Poly-alcohol: ethylene glycol, propylene glycol,
1,3-butanediol, glycerol
Derivatives of Diethylene glycol, triethylene glycol,
26 poly-alcohols: ethylene glycol monomethyl ether, ethyl
27 celosolve, ethylene glycol monomethyl ether
28 acetate,~ ethylene glycol monobutyl ether,
glycerol triacetate, glycerol ether
31
... .
- 14 -
7~4~
I These adclitives may be usually used at a concentra-
2 tlon of 0 01 - 5 ~ (V/V).
3 As to -the fermentatlon medium employed in the
4 present process for culturing the mutant, any synthetic or
natural medium can be employed, so long as it contains a
6 proper carbon source, a nitrogen source, inorganic materiais,
7 and trace amounts of nutrients necessary for the specific
8 mutant.
9 Any carbon source and nitrogen source can be used
o in the medium, so long as they can be utilized by the micro-
l organism. For example, carbohydrates such as glucose,
12 fructose, sucrose, maltose, mannose, etc.; sugar alcohols
13 such as sorbitol, mannitol; glycerol; starch; starch hydrolyzate
liquor; molasses; etc. may be used. Further, various organic
acids such as pyruvic acid, lactic acid, acetic acid, fumaric
6 acid, gluconic acid, etc. and lower alcohols such as methanol,
l7 ethanol, glycols such as ethylene glycol, hydrocarbons such
l8 as ethane, propane, butane, n-paraffine, kelosene, etc. may
~ -
19 ~ also be used.
0 As a nitrogen source, the following substances~are
21 appropriate: ammonia; various inorganic and organic ammonium
22 salts such as ammonium chloride, ammonium sulfate, ammonium
23 carbonate, ammonium phosphate, ammonium nitrate, ammonium
acetate, etc.; urea and;other nitrogen-containing materials;
2s and nitrogenous organic materials such as peptcne, NZ-amine,
26 meat extract, yeast extract, corn steep liquor, casein hydro-
27 lyzate/ fish meal or its digested product, chrysalis hydrolvzate,
, .
`~ 23 etc.
29 As inorganic materials, monopotassium dihydro~en
phosphate, dipotassium monohydroyen phosphate, magnesium
3I sulfate, sodium chloride, ferrous sulfate, manganese sulfate,
~ ~" ,
- 15 -
.
Z~
I calcium carbonate, etc., may be used.
2 If other nutrients are necessary for the ~rowth
3 of the mutants, they must, of course, be present in the
~ medium. Mowever, it is not necessary that they be separately
s added to the medium so long as they are supplied to the medlum
together with other medium components as desoribed above.
That is, certain natural ingredients may adequately supply
8 the specific growth promoting factors.
g Culturing is carried out under aerobic conditions
such as by shaking or aeration-agitation. Suitable culturing
11 temperature is usually 20 to 40C. It is desirable to keep
the pH of the medium around neutrality throughout culturing
13 in order to obtain a high yield, but these temperature and pH
14 conditions are not essential for the practice of the present;
invention. Culturing is usually carried out for l to 7 days.
After the completion of the culturing, thè resu1tant
7 ~ culture broth is subjected to centrifugation, filtration or
the like to obtain the microbial cel1s. The microbial cells
as they are or the cells disrupted by suitable means such as
ultrasonic disintegrator, etc. are used.
When the culturing of the mutant to obtain L-serine
~: ~
22 in the culture liquor in Process (I)~is carried out in the
23 nutrient medium conta1n~ng glycine,~the same fermentation
medium as tnat used to obtain microbial cells described above
s except containing glycine in the medium may be used, and the
same culturing conditions as those described above may be used.
. : ~
~ 27 In this case, glycine~may be presented in a nutrient
`~ 28 medium at a concentration of O.l - 5 ~ (r~/V) either at the
:~:
;~ ~ 29 start of the fermentation or during the growth phase of the
.,
cells.
31
''' ~ '
- 16 -
~3i7~:4~
1 When the conversion is carried out according to
2 Process (II~, the cells are suspended in -the phosphate buf:Eer
3 solution (pH 7~0) containing 0.5 - 20~ glycine at a concen-
~ tration of 5 - 200 mg/mQ based on dry matter. Then the
conversion is carried out at room temperature for S - 30
6 hours to accumulate L~serine in the aqueous medium.
7 According to the present invention, when immobilized
8 cells prepared by the known method such as entrapment method
9 for example, gel entrapment method, microcapsule entrapment
method, etc., adsorption method, covalent bonding method and
the like axe used as microbial cells, L-serine is produced
advantageously in industrial practice.
In the immobilization of cells by gel entrapment method,
to the suspension of microbial cells are added monomer such
s as acrylic acid amide, N,N'-methylene-bis-acryl1c acid amide,
acrylic acid, metha-acrylic acid amide and the like, polymeri-
zation initiator such as ammonium persuIfate, potassium per-
~:. : : :
sulfate etc., and po1ymerization accelerator such as NrN~N~ ~N~ ~
tetramethylethylene diamine, etc., and suspension polymerization
:
is carried out~at 0 - 40C for about one hour. In the immobili-
zation of microcapsule entrapment method, a substance capable~
22 of forming semipermeable membrane such as ethylcellulose, poly-~
23 styrene etc. is dissolved in an organic solvent unmissible with
24 ~ water and having lower bo1ling point than that of water, such as
benzene, cyclohexane etc.
:
26 Then, to the solution are suspended microbial cells
to form the first water in oil emulsion. Then the emulsion is
28 added to the suspension containing protective colloid substance
2~ such as gelatin, polyvinylalcoholj etc. with stirring to form
- 30 the second emulsion. The organic solvent is removed from the
31 second emulsion whereby microcapsules are ~ormed.
- 17 -
~:
.
~9~;~4al
I The con-tact of glycine with the thus
2 obtained immobilized cells may be carri.ed out in batch
3 system or continuous system such as column method, fluidi~
~ æation method, etc.
s In batch system, the immobilized cells are suspended
at-a concentration of 5 - 15 g/dQ in a glycine solutionO The
7 suspension is subjected to reaction to convert glycine to L-serine
8 with stirring at 20 - 40C, for 5 - 30 hours.
g After the completion of the reation, the resultant
o mixture is subjected to filtration or centrifugation to obtain
11 a filtrate or supernatant containing L-serine.
2 The preferable glycine concentration of the glycine
13 solution is 5 - 50 g/Q based on the total volume of
14 glycine solution and immobilized cells.
The immobilized cells separated from the reaction
mixture may be reused.
In the column method, 5 - 50~g/Q glycine solution is
, ~ ~: : :
passed through the column packed with the immobilized cells to
produce L-serine in the solution.
~;; 20 In the fluidization method, the immobilized cells are
2~ fluidized ln reactor by using alr and phosphate buEer solut1on~
containing 5 - 50 g/Q glycine, 0.5 - 3 g/dQ inorganic compound
such as magnesium sulfate, manganese sulfatet phosphate, etc.
:24 to produce L-serine in the solution. Fluidization reaction is
2s oarried out at 20 - 4~C and at a pH of 5 - 9.
26 Ater~the~completion of conversion, the microbial
27 cells and precipitates are removed from the mixture or culture
liquor by conventional methods. Then L serine is recovered
from the resultant solution by known methods, such as an ion
exchange resin treatment.
3I Practice of certain specific embodiments of the invention is
::
,
- 18 -
7240
:
illustrated by the following representative examples.
2 EXAMPLE
3 Fermentation
Nocardia butanica KY 7985, FERM-P 3782, NRRL 11189
is used. O.ne loopful of the seed culture obtained by culturing
; 6 at 30C for 2 days in an agar plate of the medium containing
7 0.5 g/dl glucose, 0.5 g/dl yeast extract, 2 g/dl peptone, 0.5
8 g/dl NaCl and 2 g/dl agar (pH 7.2) is inoculated into 7 ml o~
9 a seed medium containing 4g/dl glucose, 0.15 g/dl KH2PO~ 0.05
~ 10 g/dl K2HPO4, 0.05 g/dl MgSO4-7H2O, 0.3 g/dl urea, 50 ~g/l biotin
- 11 0.5~g/dl yeast~ extract and 2 g/dl peptone (pH 7.2) in a large
12 test tube (20 mm x 190 mm). Culturinq is carried out with
; 13 shaking at 30C for 24 hours. One ml of the resulting seed culture
~,
14 is inoculated into 20 ml of a fermentation medium containing 3 gfdl
glucose, 1g/dl (NH4)2SO4, 0.2 g/dl yeast extract, lq/dl glycine,
16 0.001 g/dl FeSO4 7H2O,0.001 g/dl MnSO4 nH2O, 0.05~g/dl MgSO4 7H2O,
l7 ~ 0.15 q/dl KH2PO4j~0.~05 q/dl K2HPO~4~and 3~q/dl C~aCO3~(pH~7-0
18 in a;300 ml~Erlenmeyer:flask-;~
9 ~ ~ ~ Culturing ls carried out wlth shakinq at;~30C for
~4;days, whereby L-serine i~s produced in a;yield of 3.5 mg/ml.
1 ~Puriflcatl-on
? ~ After the completion of the culturinq, one liter
23 ~ o~f culture broth resulting from the fermentation is subjected
:
4 to~;centrifugation to remove ~he microbial~cells and~precipi-
;2s~ tates; and~the resultlnq supernatant is passed throuqh~;a
column packed with 400 ml of a strongly acidic cation exchange
7~ ~ resln, Dlaion SK-lA (H+ form, manufactured by Mitsubishl Kasei
a8 ~Kogyo K.K., Japan) to adsorb L-serine thereon.
29 After the resin is washed with 1.5 Q of water, the
:~ :
resin is subjected to elution with 0.5 N aqueous ammonia and
3l then the fractions containing L-serine are collected and
:~
19 -
97~
1 concentrated to 15 ml.
2 0.1 M citrate buffer sol.ution having a pH of 3.41
3 is prepared by dissolving 21.01 g of citric acid in 200 ml
4 of 1 N caustic soda, adding 110 ml of 1 N hydrochloric acid
and 5 ml of thioglycol and making the resultant solution up
to 1 Q with water.
7 After the pH of the concentrate is adjusted to 3.41
8 with citric acid, the resulting solution is passed through a
g column (3.2 cm x 85 cm) packed with Diaion SK-lA (Na form)
equilibrated with the citrate buffer solution to adsorb
11 L-serine thereon and elution is carried out with the citrate
12 buffer solution having a pH of 3.41 mentloned above.
13 The column is kept at a temperature of 37.5C during the;elution.
l4 The fractions containing L-serine are collected and
passed through a column packed with 385 ml of Diaion SK-lA
16 (H form).
17 ~ The resin 15: washed with water and subjeeted~to~
18 ~ elution with 0.5 N aqueou`s ammonia. Then, the fractions~con-
19 talning L-serine are eolleeted~and coneentrated to 100 ml under
redueed pressure. The;resulting eoneentrate lS deeolorlzed
1 with activated carbon~and concentrated to 20 ml. The resulting
: ~ :
~: 22 solution is cooled to 5C and 70 % (V/V) ethanol cooled to 5C
23 is slowly added thereto to crystallize L-serine. The resulting
, ~
crude crystals are recrystallized from alcohol, whereby l.9 g
of L-serine crystals is~obtained.
As a control, the same fermentation procedures as
27 : ~ ~ mentioned above are repeated exeept~using Nocardia butanica
ATCC 21197, whereby L-serine is produced in a yield of 2.0
mg/ml
31
- 20 -
~ ~ :
l~g~
1 EXAMPLE 2
-
2 The same Eermentation procedures as in Example 1
3 are repeated except using medium containing 8 g/dl glucose
and 2.5 g~dl glycine instead of 3 g/dl glucose and 1 g/dl
glycine as a fermentation medium and adding 1 ~ (V/V) prolyl-
eneglycol 50 hours after the inoculation and then culturing
7 is carried out for 73 hours, whereby L-serine is produced in
a yie~d of 9.5 mg/ml.
9 ':
o EXAMPLE 3
l~ In this example, L-serine producing mutant, Nocardia
12 butanica IE-36, KY 7988, NRRL 11059 and Nocardia butanica KY
13 7985, NRRL 11189 are used.
14 The mutant is inoculated into 7 ml of a seed medium
S (pH 7.2) containing 4 g/dl glucose, 0.3 g/dl urea, O.I5 g/dl
16 KH2PO4, 0.05 g/dl K2HPO4, 0.05 g/dl MgSO4-7H2O, 0.5 g/dl yeast
17 extract, 2 g/dl peptone and 50 ~g/1 biotin in a~50 ml~ of large
18 test tube (20 mm x 190 mm) and culturing is carried~out at 30C
19 for 24 hours. Two ml of the resu1ting seed culture~is inocu-
lated into 20 ml of a fermentation medium containing 5 g/dl
1 glucose, 0.5 g/dl NH4C1, 2 g/dl glycine, 0.15 g/dl KH2PO4,
22 0.05 g/dl K2HPO~, 0.05~g/dl MgSO4-7H2O, 0.01 g/dl NaCl, 1 mg/dl
EeSO4-7H2O, l mg/dl MnSO4-nH2O, 1 g/dl peptone,~2 g/dl
Mg3(PO4)2~8H2O, (pH 8.2) in a 300 ml Erlenmeyer flask.
Culturing is carried out with shaking at 30C for 5
days, whereby E-serine is produced in a~yield of 8~6 mg/ml
:
27 and 6.5 mg/ml respectively. The amount of the remaining glycine
~ 28 is 1.0 mg!ml and 6.0 mg/ml respectively.
;: 29 As a control, the same procedures descr:ibed above are
repeated except using Nocardia butanica ATCC 21197, whereby
~; 3I L-serine is produced in a yield of 4.8 mg/ml.
- 21 -
.
;24(;~
I After completion of the culturing of Nocardia
2 butanica NRRL 11059~similar purifying procedures as descxibed
3 in Example 1 are repeated, whereby 5.1 g of L-serine crystals
~ is obtained.
6 EXAMPI,E 4
7 In this example, L~serine producing mutants shown
8 in Table 5 are used.
9 As a control, Nocardia butanica ATCC 21197 is also
o used.
11 0.25 ml of seed culture obtained by culturing micro-
organisms shown in Table 5 in the same manner as described in
Example 3 is inoculated into 5 ml of a fermentation medium
(pH 6.1) containing 5 g/dl glucose, 1 g/dl (NH4)2SO4, 2~gjdl
glycine, 0.15 g/dl KH2PO4, 0.05 g/dl K2HPO4, 0.05 5/dl M~SO4-
7H2O, 0.01 g/dl NaCl, 1 mg/dl FeSO4-7H2O, 1 mg/dl MnSO4.nH2O,
1 g/dl peptone and 3 g/dl CaCO3 in a~large~test tube.
Culturing is carried~out with shaking at~30C for
9 4 days, whereby L-serine is produced in a yield sho~n in Table
Table 5
22 Microorganism ~ Yield of L-serine~ (mg/ml)
23 Nocardia KY 7983, NRRL 11187 4.1
butanica
s ~ " KY 7984, NRRL 11188 4.1
KY 7986, NRRL 11190 2.9
KY 7987,;NRRL 11191 2.8
s " XY 7985, NRR~ 11189 2.0
" ATCC 21197 0.5
_ _
....}~
: ~ 31
:
- 22 -
~!397240
I - RXAMPLR S
2 In this example, microorganisms shown in Table 6
3 are used. The same procedures as described in Exa~.nple 3 are
4 repeated except 0.Z5 ml o the seed culture obtained by
cul-turing in the same manner as described in Example 3 is
6 inoculated into 5 ml o~ the same ferme.ntation medium as
7 described in Example 3, whereby L-serine is produced in a
8 yield shown in Table 6.
Table 6
10 . ~
Microorganism Yield of L-serine (mg/ml)
Nocardia KY 7989, NRRL 11192 7.0
l2butanica
13 " KY 7990, NRRL 11193 9.5
lt " KY 7991, NRRL 11194 ~ 9.0
IS " KY 7985, NRRL 11189 6.0
16 ATCC 21197 4.5
:~ ~ 17
18
EX~'IPLE 6 ~ -
Nocardia butanica IE-36, KY 7988, FERM-P No. 3768,
NRRL 11059 is used. One loopful of the seed culture obtained
22 by culturing at 30C for 2 days in a yeast bouillon slant~
; containing 0.5 g/dl yeast extract, 1 g/dl peptones, 1 g/dl
meat extract, 0.5 g/dl NaCl and 2 g/dl agar (pH 7.2) lS
inoculated into 7 ml of a seed medium containing 4 g/dl
glucose, 0.15 g/dl KHzPO~, Q~0$ g/dl KzHPO~, 0~05 g/dl MgSO4-
27 7Hzo, 0.5 g/dl yeast extract and 2 g/dl peptones (pH 7.Z) in
~: 28 a 50 ml - large test tube (20 mm x 190 mm). Culturlng is
carried out~at 30C for~24 hours.
One ml of the resultlng seed culture is lnoculated
into 20 ml of a fermentation medium having the following
: '
- 23 -
' :
~tt3724(1~
1 composition in a 250 ml Erlenmeyer flask:
2 glucose 5 g~dl
3 (NH4)2So4 1 ~/dl
4 glycine 2.5 g/dl
I~H2PO~ 0.15 g/dl (0.011 M as phosphate ion)
K2HPo4 0.05 g/dl (0.003 M as phosphate ion)
Mg3(po4)2 8H2o 3 g/dl (0.147 M as phosphate ion)
8 Mgso4.7H2o 0.05 g/dl:
9 FeSO4.7H2O 0.001 g/dl-
o MnSO4 nH2~ 0.001 g/dl
peptone- 1 g/dl :
l2 CaCO3 3 g/dl
l3 (pH ~ 7.0)
Culturing is carri d out with shaking at 30C for 5
days, whereby L-serine is~produced~in a yield of 8O4 mg/ml.
As a control, the same procedures described:above
are repeat~d except using the~same fermentation medium
3 excluded Mg3(PO4)28H2O which contains O.OI4 M of the phos-
phate ion, whereby L-serine is produced in a yield of 2.5
mg/ml. After the completion of:the culturing, 1 Q of culture
2l broth~is subjected to the similar purlfying procedures as
described in Example 1 and 4.0 g of L-serine crystals is
tained- :
EXAMPLE 7
In this example, Nocardia butanica KY 7988 NRRL
27 11059 is used. The same seed culture procedures as described
28 ~ ln Example 6 are repeated. ~0.25 ml of the resulting seed
culture is inoculated into 5 ml:o fermentation mediums
having the following compositions (r~edium I and II) in a
.
31 50 ml - large test tubes.
.
24 -
- : . , - .
~97~4~
I Medium I
-
2 glucose 5 g/dl
3 (N~I4)2So~} 0.4 g/dl
.I KH2P04 0.15 g~dl
K2HP0~ 0.05 g/dl
MgS04 7H2 0.05 g/dl
FeS04-7~20 0.001 g/dl
8 MnS4 nH2 0.001 g/dl
9 glycine 2 g/dl
o peptone 1 g/dl
CaC03 3 g/dl
7~0
3This medium contains 0.014 M of phosphate ion~
Medium II
I5The same composition as in~Medium I except further
16containing phosphate shown in Table 6. ~ :
Culturing is carried out in the same manner as in
Example 6, whereby L-serine lS produced in a yield shot~n in
Table 7.
22
23
24
2s
26
27
28
29
::
; 31
- 25 -
7240
Table 7
Supplemented Total concentra- .
3 Phosphate tion oE p~losphate ~leld of L-serlne
% (w/v~ ion (M) (mg/ml)
. . . ~ _ ~
Mg3(PO4)2~8H2o (2) 0.112 7.0
MgHPO4-3H2O (~ 0.129 7.5
6 Ca3(P4)2 (2, 0.143 2.5
Mg2(P2o7) 3H2o (2) 0.159 1.8
8 Zn3(PO4)2 4H2O (2) 0.102 2.0
9 K3PO4-nH2O (2) 0.089* 6.0
~ Mg2SO4-7H2O (3)
Il (none) 0~,014 1.0
12
* K3PO4-nH2O is calculated as tri-hydrate.
14
EXAMPLE 8 ~:
1~ :
In this example, Nocardia butanica KY 7990 NRRL
I6
: : 11193 is inoculated into 30 ml of a seed medium containing
17
4 g/dl glucose, 0.1S~g/dl KH2PO4, 0.05 g/dl K2HPO4, 0O05 g/dl
: 1 ~
;~ MgSO4-7H2O, 0.5 g/dl yeast extract,~and 2 gjdl peptone (pH 7O2)
~ 19
: in a 300 ml Erlenmeyer flask. Culturing is carried out with
shaking at 30C for 24 hours. One ml of:the resulting seed
21
culture is inoculated into 30 ml of a fermentation medium
2~ .
containing 8 g/dl glucose, 1 g/dl (NH4)2SO4, 1 g/dl pe~tone,
: 23
24 3 g/dl Mg3(PO4)2-8H2O, 0.15 g/dl KH2PO4, 0.05 g/dI K2HPO4,
0.05 g/dl MgSO4-7H2O, 0.001 g/dl FeSO~-7H2O, and 0.001 g/dl
2s
MnSO4-4H2O (pH 7.2~ in fi~teen 30Q ml of Erlenmeyer
flask provided with buffle~
: 27
Culturing is carried out with shaking at 30C for
24 hours.
29
Then, 2 ml of 2 g/dl glycine solution is added -to
~ the medium and culturing is carried out for 16 hours.
: 31
- 26 -
.
7~9LO
1 The resulting culture bro-th is combined ancl the mixture lS
2 subjected to centrifugation to obtain microbial cells. The
3 resulting cells are suspended to 0.2 M phosphate buffer
solution containing 2.5 g/dl glycine to obtain 60 mg/ml
s suspension based on dry cells. Then, the suspension is
6 poured into 300 ml Erlenmeyer flask and the conversion
7 is carried out with shaking at 30C for 20 hours, whereby
8 L-serine is produced in a yield of 12 mg/ml.
o EXAMPLE 9
In this example, Nocardia butanica KY 7988, NRRL
2 11059 is used.
The same seed culture procedures as described in
Example 6 are repeated.
Two ml of the resulting seed culture is inoculated
into 20 ml of a fermentation medium containing 5 g/dl glucose,
1 g/dl (NH4)2SO4, 2.5 g/dl glycine, 0.15 g/dl KH2PO~, 0.05
,
g/dl K2HPO4, 0.05 g/dl MgSO4-7H2O, 0.001 g/dl FeSO4-7H2O,
~` 0.001 g/dl MnSO~-nH2O, 1 g/dl peptone, 3 g/dl Mg3(PO~)2-8H2O,
zO ~pH 7.0) in a 300 ml Erlenmeyer flask. Culturing is carried
21 out with shaking at 30C for 48 hours. The concentration of
22 glucose is less than ~ g/dl after culturing.
23 At thls time, it is understood from the following
that the growth of microorganism has completed.
~ ~ 2s That is, 0.5 ml of 6N-hydrochloride is added to
-~ 26 0.5 ml of culture liquor to solve the insoluble material
27 other than microbial cells in the culture liquor. Then, the
28 optical density of the suspension obtained by addin~ 24 ml
29 of water to the resultant mixture, is measured at a wave
length of 660 m~ with spectrophotometer (101 type, produced
; 3I by Hitachi, Ltd.) to provide 0.27. This value is the same
- 27 -
. ' - ~: , -- : '
.
~L~39~Z~
with t.hat measured 2 hours before in the same manner
as described abobe.
3 Then, the additives listed in Table 8 are supple-
4 men-ted at a concentra-tion shown in Table 8 and cul-turing is
s continued for 72 hours. As a result, L-serine is produced in
6 a yield sho~n in Table 8.
7 ~fter the completion of the culturing, 1 Q of
8 culture liquor obtained by culturing using acetone as the
g additive is subjected to purification.
o The similar purifying procedures as described in
Il Example 1 are repeated and 6.2 g of L-serine crystals is
2 obtained.
3 Table 8
; Concen-tration Yield of L-serine
Addltlve ~% v/v ) (mg/ml)
6 llgroln 3 13.0
cycIohexane 3 lQ.4
methanol l 10.8
ethanol l 9.0
acetone 0.5 ~11.9
21 methylethylketone l 9.5
22 ethyl acetate O.S 10.5
23 methyl ace-tate 1 lO.O '
2~ tributyl citrate 0.3 lO.0
2s ~ l,4-dioxane 2 9.5
26 tetrahydrofuran ; l 9.8
~27 propylene glycol 0.5 lO.O
28 1,3-butanediol 0.5 9.5
29 ethyl celosolve 0.13 9.0
ethylene glycol mono- O 13 9.O
methyl ether acetate
31 no additive - 8.2
- 28 ~
:
~7~4(:1
1 Example 10
2 In this example, the same seed culture procedures
3 as described in Example 8 are repeatecl. 100 mQ of the seed
culture is inoculated lnto 3 Q of the fermentation medium
having the same composition as described in Example 8 except
6 further containing 0.01 g/dQ of CaCQ2~2H2O in a 5 Q jar
7 fermenter. Culturing is carried out with aeration and
8 agitation at 30C for 48 hours. The culture liquor is
subjected to centrifugation to obtain microbial cells and
o the cells are washed twice with 0.1M phosphate buffer solution
ll (pH 7.0) and preserved in phosphate buffer solution
2 in a frozen state. 5 g of ethylcellulose (50 - 100 cps)
13 is dissolved in a mixed solvent consisting of 72.5 g of
14 benzene and 25.5 g of n-hexane containing Span-20 (sorbitan
monolauliric acid, trade mark, Kanto Kagaku Co.~ Ltd.) as a
6 dispersing agent.
On the other hand, 5 g of frozen microbial cells
8 (by dry weight) is suspended ln 50~mQ of 0.lM phosphate
9 buffer solution (pH 7.0) containing l00 mg o~ Mg(H2PO4)2 and
100 mg of Mg3(PO4~2. The resultant suspension is added to
21 the ethylcellulose solution and the mixture is stirred
.
22 vigorously to form a first emulsion. Then, the emu1sion is
; 23 added to 300 mQ of 1% (V/W) polyethyleneglycol (average
; 24 molecular weight: 6j000) solution having a pH of 8 - 9 and
cooled with ice (10 - 15C) t and the mixture is stirred to
26~ form a second emulsion. To the emulsion is continuously added
the n~hexane cooled with ice water (5 - 10C) at a rate of
28 10 - 15 mQ/min to remove benzene and to crystallize ethy1-
29 cellulose.
After an hour, the immobilized cells entrapped in
~ 3l ethylcellulose having a particle diameter of 5 - 100 ~ are obtained.:::
.
~ - 29 -
: ~ ... . . . .
37Z~
1 The activity of the immobilized cells is about
2 50 - 60% of that of the cells before :immobilization and the
3 amount of the entrapped cells is 300 - 350 mg cells/mQ micro-
4 capsule. lO0 mQ of the immobilized cells is fluidized in a lQ-
reactor (90 mm x lS0 mm~)by using air and O.lM phosphate
6 bufar solution (pH 6.1). The phosphate buffer
7 solution contains 0.2 g/mQ of glycinel 5 g/mQ
8 of Mg(H2PO4)2 and 5 g/mQ of Mg3(PO4)2r and is fed
g to the reactor at a retention time of lS - 20 hours. As a
result, L-serine is produced continuously in the overflow
11 solution at a concentration of lO - 13 mg/mQ for 50 hours.
12
Example ll
In this example, 500 mQ of cyclohexane as dispersing
medium and 4 g of Span-20 as the dispersing agent are mixed and
6 stirred at lO - 15C under ice cooling and nitrogen gas
17 atmosphere. Then, to the mixture are added l9.5 g of acryl-
18 amide and 0.5 g of N,N' bis-acrylamide dissolved or suspended
19 in lO0 mQ of O.lM phosphate buffer solution (pH 6.1) and lO g
of frozen cells (by dry matter) prepared as in the same manner
21 as in Example lO. To the resultant mixture are added
22 lO mQ of 2.5% (W/V) N,N,N',N'-tetramethylethylenediamine a~ the
23 polymerization stabilizer, 125 mQ of 2.5% (W/V) o~ ammonium
persulphate as the polymerization initiator. Polymerization
reaction is carried out for one hour to form immobilized
26 cells entrapped in gel having a diameter of 300 - 400 ~.
27 The activity of the immobilized cells is about 30 -
28 40% of that of the cells before immobilization and the amount
29 of cells entrapped in gel is 60 - 70 mg cell/mQ gell.
Then, lO0 mQ of gel and 800 mQ of 25 mg/mQ glycine
31 solution is poured into a l Q reactor and ~he reaction is carried
.
~ - 30 -
:: .
72~
1 out at 30C for 25 hours in batch system to produce L-serine
2 in the reaction mixture at a concentration of 12.5 mg/mQ.
3 The gell is recovered from the reaction mixture and the above
,l reclction procedure is r~peated twice u5ing the recovered yell.
The results are shown in Table 9.
6 Table 9
_ . __ .
: Number Concentration of Concentration of Conversion 8 of use glycine (mg/mQ) L-serine (mg/mQ) ratio (%)
g _ _ _ _
. 1 25 12.5 50.0
2 25 11.8 47.2
3 25 10.5 42.0
13
14 One Q of the first reaction mixture is subjected to filtration
to remove the immobilized cells and the pH of the supernatant :
filtrate is adjusted to Z.0 with hydrochloride. Then, the
filtrate is passed t~rough the column packed with strongly
: la acidic cation exchange resin Diaion SX-lB (trademark), and :
elution is carried out~with 2N-aqueous ammonia. The eluate
is concentrated in vacuo to obtain 8.5 g of L-serine. ~ :
. 21
22
~ 23
; 2~
:~ 25
~ 26
~ 27
~ 28 ~
~ 29
' ~
3~ : :
31
~ ,
. . ,~
.
- - .
