JPH0638737A - Method for preparing l-carnitine by microbiological means - Google Patents

Abstract

PURPOSE: To produce L-carnitine by utilizing a microorganism.

CONSTITUTION: This method for producing L-carnitine by a microbiological means is to culture a microorganism belonging to the Genus Agrobacterium/ Rhizobium, having a function of producing L-carnitine from crotonobetaine and/or γ-butyrobetaine and not catabolizing the former, representatively DSM-2903, DSM-3225 and DSM-2938 in the presence of a growth substrate.

COPYRIGHT: (C)1994,JPO

Description

Detailed Description of the Invention

[0001]

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

[0002]

It is known to produce L-carnitine from γ-butyrobetaine. γ-Butyrobetaine is liberated from Neurospora crassa spores by a hydroxylase enzyme 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. Was reported (US Pat. No. 4,371,618). This method has the drawback of requiring a large number of cofactors. In the reaction, a stoichiometric amount of 2-oxoglutar hydrochloric acid is oxidatively decarboxylated to the succinate salt. 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.

Lindstedt et al. [Biochemistry, 6 , 1262-12]
70 (1967)] "Production and alteration of carnitine in Pseudomonas" (The Formation and Degrodation of
Carnitin in Pseudomonas)
-And Gattung Pseudomonas microorganisms that grow as N-sources were isolated. The first reaction in the metabolic process is the hydroxylation of γ-butyrobetaine to L-carnitine, whereby any L-carnitine produced as an intermediate is CO ₂ , H _2.
It is further decomposed and metabolized into O and NH ₃ .

Thus, if there is a hydroxylase obtained from a bacterium, when it is added for the production of L-carnitine, the necessity of the above-mentioned disadvantageous cofactor [Lindstedt et al. Biochemist
ry 16 , 2181-2188 (1977)] “Purification and Proposition of γ-Butyrobetaine Hydroxylase from Pseudomonas sp. AK1” (Purification and Pro.
perties of γ−Butyrobetaine Hydroxylase from Pseu
domonas sp. AK1) can be resolved.

[0005]

The object of the present invention is, on the basis of this object, of the enantioselective L-carnitine rather than the racemate, crotonobetaine, butyrobetaine, in a simple manner, without the disadvantages of the known methods. Another object of the present invention is to provide a method for producing L-carnitine by discovering a new type of microorganism, which enables production from a mixture thereof.

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

[0007]

The method for producing L-carnitine by the microbiological means of the present invention belongs to the genus Agrobacterium / Rhizobium and produces L-carnitine from crotonobetaine and / or γ-butyrobetaine. A microorganism that has the ability to produce, and that does not catabolize 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 demonstrated by comparative studies on soil samples from four continents, a wide range of microorganisms exist that degrade and metabolize butyrobetaine and crotonobetaine via L-carnitine. It has also been successfully isolated from activated sludge in sewage purification equipment. All of these strains have the potential as L-carnitine producing strains of interest when mutated according to the principles of the invention described below.

Such mutants can be obtained by the following classification method.

A) mutating a microorganism that grows with betaine, γ-butyrobetaine, crotonobetaine and L-carnitine as C- and N-sources according to a conventional method, and b) a culture obtained by inoculation of the mutated microorganism. , Stable, does not catabolize L-carnitine, does not grow with L-carnitine, crotonobetaine, γ-butyrobetaine, but grows with betaine.

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

It is expedient to further cultivate the mutated microorganism in a betaine medium and inoculate this cultivated microorganism in the sorting step b), preferably on the L-carnitine medium. Inoculation of a strain that grows with betaine, γ-butyrobetaine, crotonobetaine and L-carnitine as the C- and N-sources may be performed as follows. That is, a mixed culture is produced by inoculating a crotonobetaine nutrient solution from a bacterial mixture, from which a pure culture of a crotonobetaine-producing microorganism is started with the aid of conventional microbial engineering techniques.

Such mutation of the culture which 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] A suitable method for producing a stable mutant is a frame shift method, a deletion method or a transposon-insertion method. The microorganisms so mutated are further cultivated on betaine medium and transferred to L-carnitine medium and treated in the classification step b), where the known "cross-classification" (Counter-Selektionierenden Agenzi) is carried out.
en) [P. Gerhardt et al., Manual of Methods for General Bacteriol
ogy), American Society for Microbiology, 1981]
By classifying each microorganism according to, stable L-carnitine is not catabolized, L-carnitine, crotonobetaine, γ-
Obtain one that does not grow on butyrobetaine but grows on betaine.

Suitable microorganisms which grow using betaine, γ-butyrobetaine, crotonobetaine, L-carnitine as C- and N-sources are HK4 (DSM No. 293).
8) Strains and their progeny and variants.

These strains were purchased from Deutschen Sammlung von Mikroorganismen, DS on March 3, 1984.
M), Gesellschaftfuer Biotechnsche Forschung (Gesellschaftfuer Biofechonllgisc
he Forschung) mbH, Göttingen-4300-Griesebachstrasse 8, Germany, No.
It has been deposited with the DSM2938 number (international deposit, the same applies hereinafter).

Scientific description of HK4 (DSM No. 2938) strain Cell morphology Bacillus, partly deformed Length 1-2 μm Width 0.5-0.8 μm Motility + flagella peripheral gram reaction-spore-poly-β- Hydroxybutyrate formation-oxidase + catalase + growth Anaerobic-Maconkey agar + 37 ° C + SS agar-41 ° C-cetrimide agar-pH5.6-pigmentation non-diffusible-diffusible-fluorescent-acid formed (OF test) ) From glucose aerobic-anaerobic-from fructose aerobic-ASS glucose + xylose + trehalose + ethanol-gas formation from glucose-ONPG + arginine dihydrolase-lysine decarboxylase-phenylalanine deamylase-ornithine decarboxylase-H ₂ S-Forges Roscauer-Indole-nitrate to nitrite + denitrification + levan formation-lecithinase-urease + growing 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 Thus, 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.

The above-mentioned strain was prepared on January 23, 1984,
Deutsche Zammulung von Microorganismen (DSM), Gesellschaft Hür Biotechnsche Forschung mbH, Göttingen-4300-Griesebachstrasse 8, Germany, No. Deposited under the number DSM 2903.

Scientific description of HK13 (DSM No. 2903) strain Cell morphology Bacillus, partly deformed Length 1-2 μm Width 0.5-0.8 μm Motility + peripheral flagella gram reaction-spore-poly-β- Hydroxybutyrate formation-oxidase + catalase + growth Anaerobic-Maconkey agar + 37 ° C + SS agar-41 ° C-cetrimide agar-pH5.6-pigmentation non-diffusible-diffusible-fluorescent-acid formed (OF test) ) From glucose aerobic-anaerobic-from fructose aerobic-ASS glucose + xylose + trehalose + ethanol-gas formation from glucose-ONPG + arginine dihydrolase-lysine decarboxylase-phenylalanine deamylase-ornithine decarboxylase-H ₂ S-Forges Proscour-Indole-nitrate to nitrite + denitrification + levan formation-lecithinase-urease + growing 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, L-carnitine, crotonobetaine and γ-butyro Does not grow on betaine, but betaine, L-glutamate and crotonobetaine, L-glutamate and butyrobetaine, L
-The strain that grows on glutamate and L-carnitine is strain HK1331b. It was isolated as a well-growing mutant colony due to spontaneous mutation on the surface of nutrient medium containing L-glutamic acid and γ-butyrobetaine solidified in agar.

The above-mentioned strains were obtained on February 8, 1985 by Deutschen Zammulung Hua Microorganisms (DSM), Gesellschaft Hüh Biotechnique Forschung mbH, Göttingen-4300-Germany. Griesebachstrasse 8 No. Deposited under the number DSM3225.

HK1331b (DSM No. 3225)
Scientific description of the strain Cell morphology Bacillus, partly deformed Length 1-2 μm Width 0.5-0.8 μm Motility + flagellar peripheral Gram reaction-spores-poly-β-hydroxybutyrate formation-oxidase + catalase + growth Anaerobic-Maconkey agar + 37 ° C + SS agar-41 ° C-cetrimide agar-pH 5.6-Dye formation non-diffusible-diffusible-fluorescent-acid formed glucose aerobic-anaerobic-fructose-aerobic manner - ASS glucose + xylose + trehalose + ethanol - gas formation from glucose - ONPG + arginine dihydrolase - lysine decarboxylase - phenylalanine deaminase - ornithine decarboxylase - H ₂ S - Fogesu & Purosukaua - indol - nitrite + de nitrates + Levan formation- Lecithinase- Urease + Growing starch- Gelatin- Casein- Tyrosine- Tween 80- DNA + Esculin + Substrate conversion Acetate- Citrate- Malonate- Glycine- Norleucine- Xylose + Fructose + Glucose + Autotrophic growth In the presence of H ₂ -3-ketolactose-development betaine + L-carnitine-γ-butyrobetaine-crotonovetaine-L-glutamate and crotonobetaine + L-glutamate and butyrobetaine + L-glutamate and L-carnitine +. The method for producing L-carnitine is preferably carried out as follows. That is, a microorganism, preferably number HK1
The microorganisms of 3 were sterilized and pre-cultured, preferably vitamin-containing mineral medium [Kulla et al., Arch. Microbiol., 135, 1 (1
983)] at 20-40 ° C., preferably 30 ° C., at a suitable pH of 6-8, preferably pH 7, 20-20.
Incubate for 50 hours, preferably 30-40 hours. This preculture is typically 0.1-10% by weight, especially 0.
It contains 5-5% by weight choline, glutamate, acetate, dimethylglycine or betaine as growth substrate. Especially preferred is the addition of 0.5 to 5% by weight betaine.

Furthermore, as is customary in the microbiological arts, in addition to the starting compounds added to this preculture, .gamma.-butyrobetaine, crotonobetaine or mixtures thereof are added in an amount of 0.1, based on the reaction medium. -10% by weight, preferably 0.5-5% by weight. γ-Butyrobetaine or crotonobetaine may be in the form of a hydrochloride salt, a free inner salt, or a derivative thereof. The preculture produced by the method described above can be inoculated for another culture. For this other culture, it is preferable to use the same composition as the preculture.

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

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

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

The production of L-carnitine thus carried out reaches a stop 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 used as inoculum for new cultures. L-carnitine can be prepared by known methods [J. P. Vande Castile
casteele), Applied Environmental 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 case the cells are advantageously diluted in the chemistry at a rate of 0.04 to 0.2 / h, preferably 0.
At 06 to 0.08 / h, the cells can be grown under the same conditions as in batch culture.

[0027]

【Example】

[Example 1] Isolation of crotonobetaine-growing microorganisms Microorganisms were extracted from soil with stirring using a neutral phosphate buffer solution, and finally, large components were separated by a filter paper to obtain crotonobetaine nutrition. The solution was inoculated with the bacterial mixture obtained as described above until it became slightly cloudy.
After 9 days, turbidity had increased to a population with 90-fold cell concentration. Crotonobetaine disappeared completely from this solution,
Ammonia was detected as a production byproduct.

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

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

Example 2 Isolation of Stable Carnitine-Dehydrogenase Negative Mutant Such a mutant does not further decompose and metabolize L-carnitine produced from γ-butyrobetaine or crotonobetaine. In the ideal case, it is secreted.

A culture of HK4 was incubated with 5 μg / ml of "Acridine Mutagen ICR 191" in a succinate medium [J. H. Miller, "Experimental Molecular Biology" Cold Spring Harbor Laboratory,
(1972)] and then cells were stimulated to develop mutations in "nutrient broth" according to standards, followed by betaine media. This matured 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-cyclozelin (0.5 mg / ml) were added [Ornston et al., Biochim. Biophys. Res. Comm.
un. 36, 179 (1969)]. This "countersorted" reagent killed only grown bacteria. Can no longer grow on L-carnitine,
Variants of interest to us were left over and relatively increased.

After 30 hours, the number of living cells is 100
Retreated to one-half. Antibiotics were washed out and the culture was carried out in betaine medium. After growth, the corresponding culture dilutions on solidified nutrient agar were distributed. The cells so individualized were grown into colonies and tested individually. The mutant HK13 was selected. It is stable and no longer contains carnitine dehydrogenase and therefore does not grow on L-carnitine, γ-butyrobetaine or crotonobetaine but on betaine.
Upon growth with betaine, dimethylglycine, choline, glutamate or acetate, this strain converted crotonobetaine or γ-butyrobetaine to L-carnitine and secreted it.

[Example 3] 5 l of a preculture of HK13 was
Vitamin-containing mineral medium [Kura et al., Arch. Microbio
l. 135 , 1 (1983)] and betaine 1% by weight
And those containing 0.5% by weight of crotonobetaine chloride, at 30 ° C. and pH 7.0, 32
Cultured for a period of time. This was inoculated into 15 liters of the same composition and the same as in the preculture (30 ° C., pH 7.0, pO ₂
= Ppm) for 24 hours. When the production stopped, the cells were centrifuged and used as an inoculum for a new batch. The concentration of L-carnitine in the supernatant (19.8 l) was measured by enzymatic analysis.

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

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

As described [J. P. Van de 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
It was cultivated in a vitamin-containing mineral medium (according to Example 1) containing 1% choline and 0.6% γ-butyrobetaine at pH 7.0 at a temperature of 30 ° C. for 32 hours. This preculture was inoculated into a medium of 15 l of medium of the same composition and cultivated under the same conditions as in feasibility 1.

Production stopped after about 30 hours and cells were separated by microfiltration (Amicon hollow fiber cartridge). This cell population could be used for subsequent L-carnitine production. The concentration of L-carnitine in the filtrate (19.6 l) was measured 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 distinct organic by-products were detected in the filtrate by extended NMR analysis either. L-Carnitine could be isolated from this solution by known techniques, for example azeotropic distillation (German Patent 2300492).

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

This is an analytically detected yield of 99.% 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 a microorganism has been improved, and it has become possible to produce enantioselective L-carnitine by a simple method that does not require a cofactor.

Continuation of front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display area C12R 1:01)

Claims (4)

[Claims] 1. A 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 which does not catabolize the latter. Were cultured in the presence of growth substrate with crotonobetaine and / or γ-butyrobetaine to accumulate accumulated L
-A method for producing L-carnitine, which comprises collecting carnitine. 2. Crotonobetaine, γ-butyrobetaine or a mixture thereof in an amount of 0.1 to 10% by weight based on the medium.
The manufacturing method according to claim 1, wherein the amount is used. 3. The method 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 culture medium.