Note: Descriptions are shown in the official language in which they were submitted.
a
This invention relates to a process for the
microbiological production of L-carnitine.
L-carnitine is an essential substance for human
metabolism, e.g. for the catabolism of fatty acids.
Artificially produced L-carnitine is therefore used in
pharmaceutical preparations as an active ingredient against
corresponding deficiency diseases.
It is known to produce L-carnitine from gamma
butyrobetaine. The gamma-butyrobetaine, in the presence of
sodium-2-oxyglutarate, a reducing agent, an iron ion source
and atmospheric oxygen as a hydroxy group donor is brought
into contact with a hydroxylase enzyme, released from spores
of Neurospora crassa (U. S. Patent No. 4,371,618). This
process has the disadvantage of needing a large number of
cofactors, which have to be fed externally. Thus, in the
reaction, stoichiometric amounts of 2-oxyglutarate are
oxidatively decarboxylated to succinate. There are needed:
Fe+2 as Oz activator, ascorbate to keep the iron ion in the
reduced form and catalase to destroy harmful HZOz arising in
trace amounts.
Lindstedt et al. [Biochemistry, G, 1262-1270,
(1967), "The Formation and Degradation of Carnitine in
Pseudomonas"] isolated a microorganism of the genus
Pseudomonas, which grows with gamma-butyrobetaine as the C and
N source. The first reaction of the catabolic method was the
hydroxylation of the gamma-butyrobetaine to L-carnitine, and
the L-carnitine resulting as intermediate was completely
further catabolized to COZ, HZO and NH3. Also, this
hydroxylase obtained from bacteria, if used for L-carnitine
production, would have the disadvantageous cofactor
requirements described above [Lindstedt et al., Biochemistry,
16, 2181-2188, (1977), "Purification and Properties of- gamma-
Butyrobetaine Hydroxylase from Pseudomonas sp. AK 1"].
It is further known according to U.S. Patent No.
4,708,936 to continuously produce L-carnitine biologically.
It has turned out to be disadvantageous that the strain
stability in the continuous process must be very high (more
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than 1000 hours) . Moreover, relatively low limits are set for
the product concentration in the medium. The product/educt
ratio is also relatively low.
An object of the invention is to avoid the
disadvantages of the known processes and to provide a process
for the production of L-carnitine from crotonobetaine and/or
gamma-butyrobetaine enantioselectively and microbiologically
in high yield.
Accordingly, the invention provides a process for
to the batch production of L-carnitine by microbiological
utilization of erotonobetaine and/or gamma-butyrobetaine. In
the process, gamma-butyrobetaine and/or crotonobetaine as well
as betaine are fed as C and N sources into a culture medium
containing a microorganism of the genus Pseudomonas, genus
Rhizobium or genus Agrobacterium, E. coli or a yeast of the
genus Saccharomyces, and additionall~r a carbon source is fed,
and, after the maximum L-carnitine concentration is reached
(or nearly reached), the L-carnitine is separated.
Suitably microorganisms of the genus Rhizobium are
used, preferably the strain HK 1331b, deposited on February
8, 1985 with the German Collection of Microorganisms (DSM),
Gesellschaft fuer Biotechnologische Forschung mbH [Company for
Biotechnological Research], Griesbachstrasse 8, D-3400
Goettingen, under DSM No., 3225, and the strain HK 13,
deposited on January 23, 1984 in the same collection, under
DSM No. 2903.
Microorganism strains which have been modified by
genetic engineering are also suitable for use in the
invention.
In contrast with the systems known in the prior art,
the microorganisms according to the invention use HZO and not
OZ as the hydroxyl group donor, as has been established by the
inventors' own tests by using ~i2~80 and X802.
The selection and characterization of these
preferred microorganisms are described in European Published
Patent Application No., 0,158,194.
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The process according to the invention for the
production of L-carnitine is suitably performed so that a
first so-called batch phase biomass, capable of production,
is produced. For this purpose, in a known way corresponding
to European Published Patent Application No. 0,158,194, one
of said strains is cultured in a sterilized mineral medium,
preferably containing vitamins [Kulla et al°, Arch.
Microbiol., 135, 1, (1983)] at 20° to 40°C, preferably at
about 30°C, at a suitable pH of 6 to 8, preferably of about
7, for 5 to 80 hours, preferably for 15 to 40 houxs. This
medium suitably contains 0.01 to 10 percent by weight,
preferably O.Ol to 5 percent by weight, of Choline, Glutamate,
acetate, dimethyl glycine and/or betaine as a growth
substrate. It is especially preferred to use betaine together
with glutamate or another carbon source, each in an amount of
0.02 to 5 percent by weight.
In addition, the starting compounds gamma-
butyrobetaine, crotonobetaine or mi~ctures thereof to be
reacted in the batch phase are introduced in amounts 0.01 to
10 percent by weight, preferably 0.1 to 5 percent by weight,
based on reaction medium. ~.Che gamma-butyrobetaine or
crotonobetaine can be present partially as the hydrochloride
salt or as a free inner salt.
Other cultures can be inoculated with the biomass
cultured in the batch phase. These other cultures suitably
have the same composition as tine initial cultures.
In the process according to the invention, the
biomass cultured in the batch phase is the starting point for
L-carnitine production in the so-called "fed-batch" phase.
The culture medium in the ''fed-batch" phase is largely similar
to that of the batch phase.
The "fed-batch" phase is characterized in that the
educt--crotonobetaine and/or gamma-butyrobetaine, betaine as
well as an additional carbon source--is added to the culture
solution.
Compounds usual in the art and known in the
literature, e.g., for Rhizobium strains, can be used as the
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carbon source [The prokaryotes, Chapter 67, the genus
Rhizobium, Springer Verlag, (1981), p. 825]. Suitable carbon
sources are, e.g., sugars such as glucose or fructose, sugar
alcohols such as glycerol or organic acids such as acetic
acid. Preferably glucose or glycerol is used.
The molar ratio of carbon to nitrogen is suitably
selected between greater than 5 mol of C to 1 mol of N and
10,000 mol of C to 1 mol of N, preferably between 10 mol of
C to 1 mol of N and 100 mot of C to 1 mol of N. At very high
1o carbon-nitrogen ratios it can be necessary to add another
nitrogen source besides betaine. As such other nitrogen
source there can be used representatives usual and known in
the art, such as ammonium. Other nutrient media can be added,
such as a sulfur source, a phosphorus source, trace elements,
vitamins or complex nutrients, such as meat extracts, yeast
extracts or corn steep liquor.
The educts--gamma-butyrobetaine or crotonobetaine-
-are suitably added so that a concentration between 0.005 to
5 percent, preferably between 0.02 and 2 percent, is
2o guaranteed in the culture medium.
The rate of addition of the carbon/nitrogen source
depends on the amount of biomass in the bioreactor. A known
method for the determination of the biomass concentration is
the measurement of the dry weight of the culture solution.
Corresponding to this dry weight and in view of the desired
carbon/nitrogen ratio, the carbon source and betaine are added
to the fermenter at a rate of suitably 0.01 to 100, preferably
0.1 to 10 mol, of carbon per kg of dry weight per hour.
The operation is advantageously performed at a
temperature in the range of 20° to 40°C and at a pH between
6 and 8.
After cultivation in the °'fed-batch'° phase of
generally 25 to 250 hours, an L-carnitine concentration in
the culture of more than 6 percent can be achieved.
According to a modification of the process, after
completion of the fermentation, a part of the culture solution
is discharged and the remaining part, brought up to the
5
desired total amount with new culture medium, is used to start
up a fresh "fed-batch" cultivation.
With this so-called "repeated fed-batch' process it
is possible to increase the volumetric productivity of the
fermentation.
Relief of the culture can be achieved if the gamma-
butyrobetaine and/or crotonobetaine to be added is previously
desalted and purified by an ion exchanger is previously
electrodialysis. .
The obtaining of L-carnitine from a culture solution
is known, e.g., from European Published Patent Application
No., 195,944, and can be performed so that, after separation
of the biomass, e.g. by centrifuging, ultrafiltration or '
microfiltration by a laboratory electrodialysis unit, the
solution is freed of charged particles (cations and anions).
The end point of the desalting can be determined
conductometrically. In this case, the salts travel in the
concentrate circuit, while the L-carnitine remains as an inner
salt ("betaine") in the diluate circuit. In this manner,
yields of L-carnitine in the diluate, after desalting, of more
than 95 percent can be achieved.
As an alternative to electrodialysis, the L-
carnitine can also be desalted by means of a strongly acid
cation exchanger in the H~ø~ form [ cf . 3. P. Vandercasteele,
Appl. Environ. Microbiol., 39, 327, (1980)]. In this case the
solution is allowed to flow over an ion-exchange column until
the ion exchanger is exhausted and the L-carnitine breaks
through. The anions go as free acids into the passage. The
rations remain in the ion exchanger. After neutral washing
of the ion exchanger with water, the L-carnitine can be eluted
with aqueous ammonia solution. Thus, yields of L-carnitine
in the ammoniacal eluate of more than 95 percent can be
achieved. The dilute L-carnitine solutions accumulating both
in the electrodialysis and by ion exchanger can be
concentrated either by evaporation or also by reverse osmosis
and the azeotropically dehydrated.
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The resultant L-carnitine can then be converted into
pure which L-carnitine by subsequent recrystallization
suitably from isobutanol/acetone, methanol, ethanol, n-
butanol, or in combination with solvents which dissolve L-
carnitine only slightly, such as acetone, ethyl acetate,
gamma-butyl acetate, isobutyl methyl ketone or acetonitrile,
preferably isobutanol, and additional activated carbon
treatment. According to this process, L-carnitine can be
obtained with a specific rotation of [c~]zsp -30.5° to -31.0°,
c = 1 in HZO, [literature value -30.9°; Strack et al., Hoppe-
Seylers Z.f. physiolog. Chem., 318, (1960), 129] and a content
of more than 99 percent (HPLC).
The following Examples illustrate the invention.
Example 1
0.3 1 of an initial culture of the strain HK 1331b
was cultured in the following nutrient medium at 30°C and pH
7.0 for 24 hours:
Composition of the nutrient medium~
L-glutamate 2 g
betaine 2 g
gamma-butyrobetaine 2 g
buffer solution 100 ml
Mg-Ca-Fe solution 25 ml
trace element solution 1 ml
vitamin solution 1 ml
with water to 1 1
Buffer solution:
NaZS04 1 g
3 0 NaZHP04 ~ 2Hz0 2 5 . 08 g
KHZP04 10 g
NaCl
g
with water to 1 1
Ma-Ca-Fe solution:
MgClz ~ 6H20 16 g
CaCl2~2H20 0.58 g
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FeCl3~6HZ0 0.032 g
with water to 1 1
trace element solution:
ZnSOy7Hz0 100 mg
McCl24HZ0 30 mg
H3B~3 300 mg
CoCl2 6H20 2 00 mg
CuClz 2Hz0 10 mg
NiCl26Hz0 22 mg
NaZMo04 2Hz0 3 0 mg
with water to 1 1
vitamin solution:
pyrodoxal HC1 10 mg
riboflavin 5 mg
nicotinamide 5 mg
thiamine HCl 5 mg
biotin 2 mg
sodium pantothenate 5 mg
p-aminobenzoic acid 5 mg
folic acid 2 mg
vitamin B 12 5 mg
with water to 1 1
'
With the initial culture, 5 1 of nutrient of the same
medium
composition was inoculated in the fermenter cultured at
and
30C and pH 7 for 24 hours. The pH was kept
constant by the
addition of 8 percent phosphoric acid at 7.0
a pH of
(a) Then the "fed-batch" operation was begun. Two
solutions of the following compositions were
continuously
added:
Composition of the carbon-nitrogen feed
betaine 100 g
glucose 135 g
with water to 1 1
carbon/nitrogen ratio 10.3:1
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Composition of the Gamma-butyrobetaine feed
gamma-butyrobetaine 300 g
with water to 1 1
The solution was fed with the carbon and nitrogen sources at
a rate of 4.5 ml/h. This corresponded to a specific feed rate
of 4 mol of C/kg of dry weight/hour at the beginning of the
"fed-batch" phase. The dry weight was determined in the usual
way [e.g., Appl. Microbiol. Biotechnol., 28 (1988), 109f.].
The concentrations of gamma-butyrobetaine and L-carnitine were
determined by HPLC. The gamma-butyrobetaine solution was
added so that the gamma-butyrobetaine concentration in the
fermenter was between 0.05 and 0.5 percent by weight. 150
hours after the inoculation of the fermenter, an L-carnitine
concentration of 6.4 percent and a concentration of unreacted
gamma-butyrobetaine of 0.29 percent was reached. This
corresponded to a 95 percent conversion of gamma-
butyrobetaine.
(b) Corresponding to (a) and with the same
composition of the carbon/nitrogen feed and of the gamma-
butyrobetaine feed the "fed-batch" operation was begun. The
solution with the carbon and nitrogen sources was added at a
rate of 4.5 ml/h. This corresponded to a specific feed rate
of 4 mol of C/kg of dry weight/hour at the beginning of the
"fed-batch" phase. The dry weight was determined in the usual
way [e.g. Appl. Microbiol. Biotechnol., 28, (1988), 109f.].
The concentrations of gamma-butyrobetaine and L-carnitine were
determined by HPLC. The gamma-butyrobetaine solution was
added so that the gamma-butyrobetaine concentration in the
fermenter was between 0.05 and 0.15 percent by weight. 155
hours after the inoculation of the fermenter, an L-carnitine
concentration of 6.3 percent and a concentration of unreacted
gamma-butyrobetaine of 0.13 percent was reached. This
corresponded to a 98 percent conversion of gamma-
butyrobetaine.
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Isolation of L-carnitine:
Pure L-carnitine was isolated from the solution
which contained 64 g/1 of L-carnitine, 2.9 g/1 of gamma-
butyrobetaine and inorganic salts, using a procedure
corresponding to that described in European Published Patent
Application No. 195,944. After the stage of purification by
recrystallization, 56.3 g (88 percent) of white L-carnitine,
having an HPLC greater than 99 percent and a specific rotation
of [a]Z~p -30.9°, (c=1, HZO), was obtained.
Example 2
300 ml of a nutrient medium similar to that
described in Example 1, but which instead of the gamma-
butyrobetaine contained 2 g/1 of crotonobetaine, was
inoculated with the strain HK 1331b and cultured at 30°C and
pH 7 for 24 hours. 5 liters of the nutrient medium was
inoculated with the initial culture as in Example 1 and
cultured at 30°C and pH 7.0 for 24 hours. The pH was kept
constant at 7.0 by the addition of 8 percent phosphoric acid.
Then the "fed-batch" operation was begun. As described in
Example 1, the carbon and nitrogen sources as well as a
solution of the following composition were continuously added.
Composition of the crotonobetaine feed'
crotonobetaine 300 g
with water to 1 1
The solution with the carbon and nitrogen sources was added
at a rate of 4.5 ml/h. This corresponded to a specific feed
rate of about 4 mol of C/kg of dry weight/h at the beginning
of the "fed-batch" phase. The samples were treated and
analyzed in the same way as described in Example 1. The
crotonobetaine feed was added so that the crotonobetaine
concentration in the fermenter was between 0.05 and 0.5
percent by weight. 150 hours after inoculation of the
fermenter, an L-carnitine concentration of 6.1 percent and
concentration of unreacted crotonobetaine of 0.17 percent was
achieved. This corresponded to a 95 percent conversion of the
crotonobetaine.
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Isolation of L-carnitine:
Pure L-carnitine was worked up from the solution
which contained 61 g/1 of L-carnitine, 1.7 g/1 of
crotonobetaine and inorganic salts, using a procedure
5 corresponding to that described 9_n European Published Patent
Application No. 195,944. After recrystallization, 52 g (86
percent) of white L-carnitine was obtained with the same
specification as in E~tample 1.
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