JP2586283B2 - Method for producing L-carnitine by microbiological means - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing L-carnitine by microbiological means.

[0002]

BACKGROUND OF THE INVENTION It is known to produce L-carnitine from γ-butyrobetaine. γ-butyrobetaine is released from Neurospora crassa spores by hydroxylase enzymes in the presence of sodium 2-oxoglutarate, a reducing reagent, a source of iron ions and oxygen in the air as a hydroxyl group donor. (U.S. Pat. No. 4,371,618). This method has the disadvantage of requiring a large number of cofactors. In the reaction, a stoichiometric amount of 2-oxoglutal hydrochloride is oxidatively decarboxylated to succinate. Fe @ 2 + is required as O ₂ activator, ascorbate keeping the iron in reduced state, to destroy harmful H ₂ O ₂ catalase is produced trace amounts.

[0003] Lindstedt et al. [Biochemistry, 6 , 1262-12].
70 (1967)] "Carnitine production and alteration in Pseudomonas" (The Formation and Degrodation of
Carnitin in Pseudomonas) converts γ-butyrobetaine to C
Gattung Pseudomonas microorganisms growing as-and N- sources were isolated. The first reaction in the metabolic process is the hydroxylation of γ-butyrobetaine to L-carnitine, whereby all the L-carnitine produced as an intermediate is CO ₂ , H ₂
It is further decomposed and metabolized to O and NH ₃ .

[0004] In this way, if any hydroxylase obtained from bacteria is added for L-carnitine production, the above-mentioned drawback of the need for cofactors [Lindstedt et al. Biochemist
ry 16 , 2182-1188 (1977)] "Purification and properties of γ-butyrobetaine hydroxylase from Pseudomonas sp. AK1" (Purification and Pro
perties of γ-Butyrobetaine Hydroxylase from Pseu
domonas sp. AK1) can be solved.

[0005]

The object of the present invention, based on this object, is to convert enantioselective L-carnitines, not racemates, into crotonobetaine, butyrobetaine in a simple manner without the disadvantages of the known processes. Another object of the present invention is to find a new type of microorganism which can be produced from a mixture thereof, and to provide a method for producing L-carnitine using the microorganism.

[0006] Unlike the systems known in the state of the art, the microorganisms we have found utilize H ₂ O instead of O ₂ as hydroxyl donor. This could be confirmed in our study using H ₂ ¹⁸ O and ¹⁸ O ₂ .

[0007]

The process for producing L-carnitine by the microbiological means of the present invention belongs to the genus Agrobacterium / Rhizobium and converts L-carnitine from crotonobetaine and / or γ-butyrobetaine. Microorganisms capable of producing and not catabolizing the latter,
It is characterized by culturing in the presence of a growth substrate using crotonobetaine and / or γ-butyrobetaine, and collecting accumulated L-carnitine.

[0008]

As indicated by comparative studies on soil samples from four continents, there are a wide range of microorganisms that degrade and metabolize butyrobetaine and crotonobetaine via L-carnitine. It has also been successfully isolated from activated sludge of sewage purification equipment. All of these strains, when mutated in accordance with the principles of the present invention described below, have the potential as L-carnitine producing strains of interest.

[0009] Such variants are available by the following classification method.

A) mutating a microorganism which grows using betaine, γ-butyrobetaine, crotonobetaine and L-carnitine as a C- and N-source according to a conventional method; b) a culture obtained by inoculation of the mutated microorganism Are stable, do not catabolize L-carnitine, do not grow on L-carnitine, crotonobetaine, and γ-butyrobetaine, but grow on betaine.

Preferably, according to the classification step b), L-
Each microorganism that secretes carnitine and does not grow on L-carnitine, crotonobetaine or γ-butyrobetaine but grows on betaine is selected.

It is advisable to further culture the mutated microorganism in a betaine medium and inoculate the cultured microorganism in the classification step b), preferably on L-carnitine medium. Inoculation of a strain that grows with betaine, gamma-butyrobetaine, crotonobetaine and L-carnitine as C- and N-sources may be performed as follows. That is, a mixed culture is produced from the bacterial mixture by inoculation into a crotonobetaine nutrient solution, from which pure culture of the crotonobetaine-producing microorganism is initiated with the aid of conventional microbial engineering techniques.

Such a mutation in a culture that grows using betaine, γ-butyrobetaine, crotonobetaine and L-carnitine as C- and N-sources can be carried out according to known methods. [J. H. Miller, "Experiments in Molecular Genetics",
Cold Spring Harbor Laboratory (Cold)
Spring Harbor Laboratory), 1972] Suitable methods for producing stable mutants are a frame shift method, a deletion method and a transposon-insertion method. Microorganisms so mutated are further cultured on betaine media and transferred to L-carnitine media and subjected to the classification step b), where they are known to have a known "cross-classification" (Counter-Selektionierenden Agenzi).
en) [P. Edited by Gerhardt et al., "Manual of Methods for General Bacteriol"
ogy), American Society for Microbiology (Am. Soc. for Microbiology), 1981]
, Each microorganism is classified, and stable, without catabolizing L-carnitine, L-carnitine, crotonobetaine, γ-carnitine
One that does not grow on butyrobetaine but grows on betaine is obtained.

A preferred microorganism which grows on betaine, γ-butyrobetaine, crotonobetaine and L-carnitine as C- and N-sources is HK4 (DSM No. 293).
8) The strain and its progeny and variants.

These strains were established on March 3, 1984 in Deutschen Sammlung von Mikroorganismen, DS.
M), Gesellschaftfuer Biofechonllgisc
he Forschung) mbH, Göttingen-4300-Griesebachstrasse 8, Germany
Deposited under the DSM2938 number (international deposit, the same applies hereinafter).

Academic description of HK4 (DSM No. 2938) strain Cell morphology Bacillus, partly deformed Length 1-2 μm Width 0.5-0.8 μm Motility + Flagellar periphery Gram reaction-spore-poly-β- Hydroxybutyrate formation-Oxidase + catalase + growth Anaerobic-Macconkey agar + 37 ° C + SS agar-41 ° C-Cetrimide agar-pH 5.6-Pigmentation Non-diffusible-Diffusive-Fluorescent-Acid formation (OF test ) aerobic glucose - anaerobic - aerobic fructose - ASS glucose + xylose + trehalose + ethanol - gas formation from glucose - ONPG + arginine dihydrolase - lysine decarboxylase - phenylalanine deaminase - ornithine decarboxylase - H ₂ S-Forges Roscauer-indole-nitrite to nitrite + denitrification + levanization-lecithinase-urease + growth starch-gelatin-casein-tyrosine-Tween 80-DNA + esculin + substrate conversion acetate-citrate-malonate-glycine-norleucine - xylose + fructose + glucose + autotrophic growth presence of H ₂ - 3-keto lactose - there in suitable microorganisms classified mutated from developmental betaine + L-carnitine + .gamma.-butyrobetaine + crotonobetaine + the microorganism HK13 is stable, does not catabolize L-carnitine, but secretes it, and does not grow on L-carnitine, crotonobetaine and γ-butyrobetaine but grows on betaine.

On January 23, 1984, the above-mentioned strain was
Deutsche Sangmulng von Microorganismen (DSM), Geselshaft Huer Biotechniquesche Forsung mbH, Göttingen-4300-Griesebachstrasse 8, Germany; Deposited under the number DSM2903.

Academic description of HK13 (DSM No. 2903) strain Cell morphology Bacillus, partly deformed Length 1-2 μm Width 0.5-0.8 μm Motility + Flagella periphery Gram reaction-spore-poly-β- Hydroxybutyrate formation-Oxidase + catalase + growth Anaerobic-Macconkey agar + 37 ° C + SS agar-41 ° C-Cetrimide agar-pH 5.6-Pigmentation Non-diffusible-Diffusive-Fluorescent-Acid formation (OF test ) aerobic glucose - anaerobic - aerobic fructose - ASS glucose + xylose + trehalose + ethanol - gas formation from glucose - ONPG + arginine dihydrolase - lysine decarboxylase - phenylalanine deaminase - ornithine decarboxylase - H ₂ S-Forges Proscour-Indole-nitrite to nitrite + denitrification + levanization-lecithinase-urease + growth starch-gelatin-casein-tyrosine-Tween 80-DNA + esculin + substrate conversion acetate-citrate-malonate-glycine-norleucine - xylose + fructose + glucose + autotrophic growth presence of H ₂ - 3-keto lactose - growth betaine + L-carnitine - .gamma.-butyrobetaine - crotonobetaine - L-glutamate + crotonobetaine ± L-glutamate + butyrobetaine ± L-glutamate + L-carnitine ± progeny of the microorganism HK13, which is stable and does not catabolize L-carnitine but secretes it, and L-carnitine, crotonobetaine and γ-butyro Does not grow on betaine, but does not produce betaine, L-glutamate and crotonobetaine, L-glutamate and butyrobetaine, L
-Growing on glutamate and L-carnitine is the HK1331b strain. It was isolated as a well-growing mutant colony by spontaneous mutation on the surface of nutrient medium containing L-glutamic acid and γ-butyrobetaine solidified on agar.

On February 8, 1985, the above-mentioned strains were found in Deutschen-Sanmurung-Hur Microorganisms (DSM), Geselshaft-Hur Biotechniquesche Forsung-mbH, Göttingen-4300-Germany. No. 8 in Griesebachstrasse 8 Deposited under the DSM 3225 number.

Academic description of HK1331b (DSM No. 3225) strain Cell morphology Bacillus, partly deformed Length 1-2 μm Width 0.5-0.8 μm Motility + Flagella periphery Gram reaction-spore-poly-β- Hydroxybutyrate formation-Oxidase + Catalase + Proliferation Anaerobic-Macconkey agar + 37 ° C + SS agar-41 ° C-Cetrimide agar-pH 5.6-Pigmentation Non-diffusible-Diffusive-Fluorescent-Acid-forming Good from glucose vapor manner - anaerobic - aerobic fructose - ASS glucose + xylose + trehalose + ethanol - gas formation from glucose - ONPG + arginine dihydrolase - lysine decarboxylase - phenylalanine deaminase - ornithine decarboxylase - H ₂ S - Fogesu & Pros Wore-indole-nitrite to nitrite + denitrification + levan formation-lecithinase-urease + growth starch-gelatin-casein-tyrosine-Tween 80-DNA + esculin + substrate conversion acetate-citrate-malonate-glycine-norleucine - xylose + fructose + glucose + autotrophic growth presence of H ₂ - 3-keto lactose - growth betaine + L-carnitine - .gamma.-butyrobetaine - crotonobetaine - L-glutamate and crotonobetaine + L-glutamate and butyrobetaine + L-glutamate and L-carnitine + In taxonomic identification, all three microorganisms exhibit characteristics that should belong to the genus Agrobacterium and at the same time, Then also it showed to be characteristic. Therefore, they were described as being "Agrobacterium / Rhizobium" microorganisms. The method for producing L-carnitine is preferably carried out as follows. That is, a microorganism, preferably a microorganism of the number HK13,
Sterile preculture, preferably vitamin-containing mineral medium [Kulla et al., Archif microbiorosy
(Arch. Microbiol.), 135, 1 (1983)]
The culture is carried out at a suitable pH of 6 to 8, preferably at pH 7, at 0 to 40 ° C, preferably 30 ° C, for 20 to 50 hours, advantageously 30 to 40 hours. This preculture typically contains 0.1 to 10% by weight, especially 0.5 to 5% by weight, of choline, glutamate, acetate, dimethylglycine or betaine as growth substrate. Particularly preferred are those containing 0.5 to 5% by weight of betaine.

Furthermore, as is customary in microbiological technology, in addition to the starting compound added to this preculture, γ-butyrobetaine, crotonobetaine or a mixture thereof is added to the reaction medium in an amount of 0.1%. It is added in an amount of from 10 to 10% by weight, preferably from 0.5 to 5% by weight. γ-butyrobetaine or crotonobetaine may be a hydrochloride, a free internal salt, or a derivative thereof. The preculture produced by the method described above can be inoculated for another culture. It is preferable to use the same composition as the pre-culture for this another culture.

The amount of crotonobetaine, γ-butyrobetaine or a mixture thereof to be added is 0.1 to 10% by weight, preferably 0.5 to 5% by weight.

The growth substrates, choline, glutamate, acetate, dimethylglycine and betaine can be advantageously used in the concentrate used in the preculture. It is advisable to match the culture conditions of the subsequent culture with those of the preculture.

Therefore, the temperature is preferably 20 to 40 ° C.,
Especially at 30 ° C., the pH value is in the range from 6 to 8, especially at pH
Hold at 7.

The production of L-carnitine carried out in this way reaches a halt after 20 to 30 hours. The concentration of L-carnitine corresponds to the range of the amount of γ-butyrobetaine or crotonobetaine added. The cells can be centrifuged or filtered and available for new cultures as inoculum. L-carnitine can be synthesized by a known method [J. P. Vande Castile
casteele), Applied environ mental Microbiology (Appl. Environ. Microbiol.) 39, 3
27 (1980)], can be recovered from the supernatant by means of cation exchange chromatography and purified by recrystallization.

The production of L-carnitine can also be carried out by a continuous process, in which the cells are placed in a chemical device, advantageously at a dilution rate of 0.04 to 0.2 / h, preferably 0.1 to 0.2 / h.
At a rate of 06 to 0.08 / h, the cells can be grown under the same conditions as in the batch culture.

[0027]

【Example】

Example 1 Isolation of Crotonobetaine-Growing Microorganisms Microorganisms were extracted from soil using a neutral phosphate buffer solution under agitation, and finally, large components were separated by filter paper. The bacterial mixture obtained as described above was inoculated into the solution until it became slightly turbid.
After 9 days, turbidity had increased to a 90-fold cell concentration population. Crotonobetaine is completely lost from this solution,
Ammonia was detected as a by-product of the production.

In this case, pure cultures of crotonobetaine-growing microorganisms were carried out from the cultures with the aid of conventional microbiological means (solidified agar-nutrition medium). The culture was further processed and selected and named HK4.

This strain also grows on γ-butyrobetaine, L-carnitine and betaine.

Example 2 Isolation of Stable Carnitine-Dehydrogenase Negative Mutants Such mutants do not degrade and metabolize L-carnitine produced from γ-butyrobetaine or crotonobetaine any more. In the ideal case they are rather secreted.

A culture of HK4 was incubated with 5 μg / ml of “Acridine Mutagen ICR 191” in a succinate medium according to guidelines [J. H. Miller, "Molecular Biology Experiments", Cold Spring Harbor Laboratory,
(1972)], and the cells were then promoted in a "vegetable broth" according to the standard to promote the appearance of the mutation, and subsequently performed in betaine medium. The aged culture was inoculated into L-carnitine medium.

After several hours, the culture showed logarithmic growth. At this point, penicillin G (15 mg / ml) and D-cyclozerin (0.5 mg / ml) were added [Ornston et al., Biochim. Biophys. Res. Comm.
un. 36, 179 (1969)]. This "counter-sorted" reagent killed only the growing bacteria. Can no longer grow on L-carnitine,
Mutants of interest to us remained and increased relatively.

After 30 hours, the number of living cells is 100
Retreated by a factor of one. The antibiotic was washed off and the culture was performed in betaine medium. After growth, the corresponding culture dilutions on solidified nutrient agar were dispensed. The cells so individualized were expanded into colonies and tested individually. The mutant HK13 was selected. It is stable and no longer contains carnitine dehydrogenase, and therefore did not grow on L-carnitine, gamma-butyrobetaine or crotonobetaine but did grow on betaine.
Upon growth on betaine, dimethylglycine, choline, glutamate or acetate, this strain converted crotonobetaine or γ-butyrobetaine to L-carnitine and secreted it.

Example 3 5 l of the preculture of HK13 was
Vitamin-containing mineral medium [Kura et al., Arch. Microbio
l, 135 , 1 (1983)] and 1% by weight of betaine
And 0.5% by weight of crotonobetaine chloride at 30 ° C. and pH 7.0,
Cultured for a time. This was inoculated into 15 l of the same composition and, as in the preculture (30 ° C., pH 7.0, pO ₂
= Ppm) for 24 hours. When production ceased, cells were centrifuged and used as inoculum for a new batch. The concentration of L-carnitine in the supernatant (19.8 l) was determined by enzymatic analysis.

This supernatant contains 4.26 mg of L per ml.
-Carnitine was included. This was calculated to correspond to a 95.0% yield, based on the amount of crotonobetaine added.

No intermediates or other contaminants were detected in the NMR spectrum.

As described [J. P. Vande Castile, Appl. Environ. Microbiol. 39 , 327
(1980)], L-carnitine can be recovered from the supernatant by means of cation exchange chromatography,
Purified by recrystallization.

Example 4 5 l of a preculture of HK13 was
Cultured in a vitamin-containing mineral medium (according to Example 1) containing 1% choline and 0.6% γ-butyrobetain at pH 7.0, temperature 30 ° C. for 32 hours. In this preculture, 15 l of medium of the same composition was inoculated and cultured under the same conditions as in feasibility 1.

After about 30 hours, the production was stopped, and the cells were separated by microfiltration (Amicon hollow fiber cartridge). This cell population could be used for the next L-carnitine production. The concentration of L-carnitine in the filtrate (19.6 l) was determined enzymatically. The filtrate contained 5.3 mg L-carnitine per ml. This was calculated to correspond to an analytical yield of 97.6% based on the amount of γ-butyrobetaine chloride added.

No intermediates or other different organic by-products were detected in the filtrate by expanded NMR analysis. From this solution, L-carnitine could be isolated by known techniques, for example azeotropic distillation (German Patent No. 2300492).

Example 5 A fermenter equipped with a facility for continuous cultivation contains 1.5 l of a vitamin-containing mineral medium (according to Example 1) containing 1.5% betaine and γ-butyrobetaine chloride. And inoculated with 150 ml of HK13 preculture of the same medium. 30 ° C, pH
After 20 hours of aerobic cultivation at 7.0, the culture was fully matured, so continuous operation was started at a flow rate of 0.1 l / h. The culture solution flowing out of the fermenter was received in a container cooled to 4 ° C. Cells were removed by centrifugation. As a result of the enzymatic analysis, the supernatant was 1 l of the culture.
8.8 g of L-carnitine.

This is the analytically detected yield for the added concentration of γ-butyrobetaine chloride.
It was calculated to correspond to 2%.

Solid L-carnitine could be isolated from this solution by ion chromatography and water separation.

[0044]

According to the present invention, crotonobetaine, γ-
The method for producing L-carnitine from butyrobetaine or a mixture thereof using microorganisms has been improved, and it has become possible to produce enantioselective L-carnitine by a simple method that does not require a cofactor.

Claims (4)

(57) [Claims] An L-carnitine-producing bacterium belonging to the genus Agrobacterium / Rhizobium, which has the ability to produce L-carnitine from crotonobetaine and / or γ-butyrobetaine and does not catabolize the latter. Was cultured in the presence of a growth substrate using crotonobetaine and / or γ-butyrobetaine and the accumulated L
-A method for producing L-carnitine, which comprises collecting carnitine. 2. The amount of crotonobetaine, γ-butyrobetaine or a mixture thereof is 0.1 to 10% by weight based on the medium.
2. The method according to claim 1, wherein the amount is used. 3. The process according to claim 1, wherein dimethylglycine, choline, glutamate, acetate and / or betaine are used as growth substrates. 4. The method according to claim 1, wherein the growth substrate is used in an amount of 0.1 to 10% by weight based on the medium.