IL95196A - Method of discontinuous production of L-carnitine by a microbiological process - Google Patents

Abstract

The process starts from gamma -butyrobetaine and/or crotonobetaine.

Description

A METHOD OF DISCONTINUOUS PRODUCTION OF L-CARNITINE BY A MICROBIOLOGICAL PROCESS A method of discontinuous production of L-carnitlne by a microbiological process Description The invention relates to a novel method of producing L-carnitine by a microbiological process.

L-carnitine is an essential substance for human metabolism, e.g. for breaking down fatty acids. Artifically produced L-carnitine is therefore used in pharmaceutical preparations as an active principle against corresponding deficiency d seases.

It is known to produce L-carnitine from γ-butyrobetaine. The Y -butyrobetaine, in the presence of sodium-2-oxoglutarate, a reducing agent, a source of iron ions and atmospheric oxygen as a hydroxyl-group donor, is brought into contact with a hydroxylase enzyme, released from spores of Neurospora crassa (US-PS 4 371 618). This process has the disadvantage of requiring a number of co-factors, which have to be externally supplied. In the reaction, for example, stoichiometric quantities of 2-oxoglutarate are oxidatively decarboxylated to succinate. Fe2* is required as an O2 activator, ascorbate is needed to keep the iron ion in the reduced form, and catalase is needed to destroy the traces of harmful H202 which are produced.

Lindstedt et al , [Biochemistry 6, 1262-1270 (1967) "The Formation and Degradation of Carnitin in Pseudomonas"] isolated a microorganism of the genus Pseudomonas, which is grown by using γ-butyrobetai ne as a source of C and N. The first reaction in the breakdown process was the hydrpxylation of γ -butyrobetaine to L-carnitine, which was produced as an intermediate and then completely catabolised to C02# H20 and H^ .

The hydroxylase involved, obtained from the bacteria, also has the aforementioned disadvantageous need for co-factors when used for production of L-carnitine [Lindstedt et al.

Biochemistry 1 , 2181-2188 (1977) "Purification and Properties of y -Butyrobetaine Hydroxylase from Pseudomonas sp. AK 1" ] - It is also known from US-PS 4 708 936 to produce L-carnitine by a continuous microbiological process. Disadvantages have been found, however, in that in this continuous process the stability of the strain has to be very high (more than 1000 h), and relatively low limits are set to the concentration of product in the medium. The product/educt ratio is also relatively low.

An object of the invention is to avoid the disadvantages of known methods and provide a method of enantioselective microbiological production of high yields of L-carnitine from crotonobetaine and/or y -butyrobetaine.

According to the present invention, there is provided a method of discontinuous production of L-carnitine by microbiological processing of crotonobetaine and/or f -butyrobetaine, wherein the Y -butyrobetaine and/or crotonobetaine, together with betaine as a source 6f C and N and an additional carbon source, are added to a culture medium containing a microorganism of the . genus Pseudomonas, Rhizobium, Agrobacterium, E.coli or a yeast of the genus Saccharomyces, and L-carnitine is separated after the maximum L-carnitine concentration has been obtained.

Advantageously, use is made of microorganisms of the genus Rhizobium, preferably the strain HK 1331b deposited on 8.2.1985 at the Deutsche Sammlung von Mikroorganismen (DSMJ , Gesellschaft fUr Biotechnologische Forschung mbH, Griesebachstr . 8, D-3400 Gfittingen, No. DSM 3225, and the strain HK 13 deposited on 23.1.1984 at the same place, no. DSM 2903.

Strains of microorganisms altered by genetic engineering are also suitable for the process.

In contrast to the systems known in the prior art, the microorganisms according to the invention use H2O instead of O2 as a hydroxy 1 group donor, as we have found from our own nvestigations using Η21βΟ and 1802.

The selection and characterisation of these preferred microorganisms are described in EP-A 0 158 194 t the contents which are incorporated herein by reference.

The method according to the invention of producing L-carnitine is preferably carried out as follows. Productive biomass is produced in a first or "batch" phase. To this end, one of the aforementioned strains is cultivated in the manner set out in EP-A 0 158 194, in a sterilized mineral medium, preferably containing vitamins [Kulla et al , Arch. Microbiol. 135, 1 (1983)] at 20 to 40eC, preferably at 30eC, advantageously at a pH between 6 to 8, preferably 7, and for 5 to 80 hours, preferably 15 to 40 hours. The medium advantageously contains 0.01 to 10 wt.X, preferably 0.01 to 5 wt.X of choline, glutamate, acetate, dimethylglycine or betaine as a growth substrate. It is particularly preferable to use betaine together with glutamate or another carbon source in proportions of 0.02 to 5 wt.X each.

The starting compounds to be converted, i.e. -butyrobetaine, crotonobetaine or mixtures thereof, are also supplied in the batch phase in proportions of 0.01 to 10 wt.X, preferably 0.1 to 5 wt.X, of the reaction medium.

The γ-buty.robetaine or crotonobetaine can be present partly in the form of the hydrochloride salt or as a free internal sal t .

The biomass cultivated in the batch phase can be used to inoculate other cultures. The other cultures advantageousl have the same composition as the pre-cultures.

In the method according to the invention the biomass cultivated in the batch phase is the starting point for L-carnitine production in the "fed-batch" phase.

The culture medium in the "fed-batch" phase is substantially the same as in the batch phase.

The "fed-batch" phase is characterised in that the educt crotonobetaine and/or -butyrobetaine, betaine and an additional carbon source are added in measured quantities to the culture solution.

The carbon sources can be compounds conventionally used by the experts and known in the literature e.g. for Rhizobium strains [The prokaryotes, Chapter 67, The genus Rhizobium, Springer Verlag 1981, page 825].

The carbon sources are advantageousl 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 (C to N) is advantageously chosen between more than 5 mo1 C to 1 mol N and 10000 mol C to 1 mol N, preferably between 10 mol C to 1 mol N and 100 mol C to 1 mol N.

At very high carbon-n trogen ratios, it may be necessary to add a nitrogen source in addition to betaine. The additional nitrogen source can be a known substance conventionally used by the experts, e.g. ammonium. Additional nutrients such as a sulphur source, a phosphorus source, trace elements, vitamins or complex nutrients such as meat extracts, yeast extracts or swollen maize water, can be added in measured amounts.

The educts γ-butyrobetaine or crotonobetaine are advantageously added in amounts such that ¾heir concentration in the culture medium is advantageously between 0.005 and 5X, preferably between 0.02 and 2%.

The carbon/nitrogen source is added at a rate depending on the amount of biomass in the bioreactor. One known method of determining the concentration of biomass is to measure the dry weight of the culture solution. Depending on the dry weight and the desired carbon/nitrogen ratio, the carbon source and betaine are added to the fermenter at a rate of advantageously 0.01 to 100, preferably 0.1 to 10 mol carbon per kg dry weight in the fermenter per hour.

It is advantageous to operate at a temperature range of 20 to 40°C and a pH between 6 and 8.

After cultivation in the "fed-batch" phase, usually for 25 to 250 hours, a concentation of L-carnitine of more than 6X can be obtained in the culture.

In an alternative modification of the process, part of the culture solution can be drawn off after fermentation and additional "fed-batch" cultivation can be started with the remaining part, once additional culture medium has been added.

This "repeated fed-batch" process can increase the volumetric productivity of fermentation.

The culture can be lightened by temporarily- demineralizing and purifying the y-butyrobetaine and/or crotonobetaine, using ion exchangers or electrodialysis.

Production of L-carnitine from a culture solution is known e.g. from EP-A 195 944 and can be carried out by freeing the solution from charged particles (cations and anions) after separating the biomass e.g. by centrifuging, ultrafiltration or microfi 1 tration in . a laboratory electrodialysis plant. The end point of demineralization can be determined by measurements of conductivity. In the process the salts migrate into the flow of concentrate, whereas the L-carnitine remains as an internal salt ("betaine") in the flow of diluate. The resulting yields of L-carnitine in the diluate after demineralization can be more than 95%.

As an alternative to electrodialysis, L-carnitine can be demxneralized by using a strongly acid cation exchanger in the form [compare J. P. Vandecasteele, Appl . Environ. Microbiol. 39 327 (1980)]. The solution is made to flow over an ion exchange column until the ion exchanger is exhausted and L-carnitine breaks through. The anions go as free acids into the flow, whereas the cations remain on the ion exchanger. After the ion exchanger has been washed to neutrality with water, the L-carnitine can be eluted with aqueous ammonia solution. The resulting yields of L-carnitine in the ammoniacal eluate can be more than 95%.

The dilute L-carnitine solutions obtained by electrodialysis or by ion exchange can be concentrated by evaporation or reverse osmosis and then dewatered azeotropicall y .

The L-carnitine thus obtained can be converted into pure white L-carnitine by subsequent recrystall i zation advantageously from isobutanol /acetone, methanol, ethanol or n-butanol ; or from a combination of solvents in which L-carnitine is only slightly soluble, e.g. acetone, ethyl acetate, -y--butyl acetate, isobutyl methyl ketone or acetoni tri le, preferably isobutanol, and additional treatment with active carbon. This method can be used to obtain L-carnitine with specific rotations of [« ]25ο -30.5 to -31.0°, c = 1 in H2O [the value in the literature is -30.9°; Strack et al , Hoppe-Seyler ' s Z.f. physiolog.Chem. , 318 (1960), 129], the content being more than 99X (HPLC).

Example 1 A 0.3 1 preculture of the strain HK 1331b was cultivated in the following nutrient medium at 30"C, pH 7.0 for 24 hours: Composition of nutrient medium L-glutamate 2 9 Betaine 2 9 γ-butyrobetai ne 2 9 Buffer solution 100 ml Mg-Ca-Fe solution 25 ml Solution of trace elements 1 ml Vitamin solution with water to 1 Buffer solution Na2 SO* 1 9 Na2HP04.2H20 25.08 g H2 PO 10 g NaCl 30 g with water to 1 1 M -Ca-Fe solution MgCl2.6H2O 16 g CaCl2.2H20 0.58 g FeC .6H2O 0.032 g with water to 1 1 Solution of trace elements H3BO3 300 mg C0CI2.6H2O 200 mg CUC12.2H2O 10 mg NiCl2.6H2O 22 mg Na2Mo04.2H20 30 mg with water to 1 1 Vitamin solution Pyridoxal .HC1 10 mg Riboflavin 5 mg Nicotinamide 5 mg Thiamin. HC1 5 mg Biotin 2 mg Sodium pantothenate 5 mg p-amine-benzoic acid 5 mg Folic acid 2 mg Vitamin B 12 5 mg with water to 1 1 Using the preculture, 5 litres of nutrient medium having the same composition were inoculated in the fermenter and cultivated at 30eC and pH 7 for 24 hours. The pH was kept constant at 7.0 by adding 8% phosphoric acid. a) "Fed-batch" operation was then started. Two solutions having the following composition were continuously added in measured amounts.

Composition of carbon-nitrogen feed Betaine 100 g Glucose 135 g w th water to 1 1 Carbon/nitrogen ratio 10.3 : 1 Composition of the -butyrobetaine feed γ-butyrobetaine 300 g . with water to 1 1 The solution containing the carbon and nitrogen source was added at a rate of 4.5 ml/h, corresponding to a specific feed rate of 4 mol C/kg dry weight/h at the beginning of the "fed-batch" phase. The dry weight was determined in the usual manner (e.g. Appl . Microbiol. Biotechnol 28 [1988] 109f.). The concentration of γ -butyrobetaine and L- carnitine was determined by HPLC. The γ-butyrobetaine solution was added at a rate such that the concentration of γ-butyrobetaine in the fermenter was between 0.05 and 0.5 wt.x. 150 hours after inoculating the fermenter, the L- carnitine concentration was 6.4% and the concentration of unreacted ^-butyrobetaine was 0.29%. This corresponded to 95% conversion of γ-butyrobetaine,. b) "Fed-batch" operation was carried out as per a) using the same composition of the carbon/nitrogen feed and the γ-butyrobetaine feed.

The solution containing the carbon and nitrogen source was added at a rate of 4.5 ml/h, corresponding to a specific feed rate of 4 mol C/kg dry weight/h at the beginning of the "fed-batch" phase. The dry weight was determined in conventional manner (e.g. Appl . Microbiol. Biotechnol 28 [1988] 109f.). The concentration of γ-butyrobetaine and L-carnitine was determined by HPLC. The "γ-butyrobetai ne solution was added in a proportion such that the concentration of Ύ-butyrobetai ne in the fermenter was between 0.05 and 0.15 wt.%. 155 hours after inoculating the fermenter, the concentration of L-carnitine was 6.3% and the concentration of unreacted γ-butyrobetai ne was 0.13%. This corresponded to 98% conversion of γ-butyrobetaine.

Isolation of L-carnitine Pure L-carnitine could be isolated from the solution, which contained 64 g/1 L-carnitine, 2.9 g/1 -γ-butyrobetaine and inorganic salts, by a method as set out in EP-A 195 944.

The product after the purification stage by recrystal 1 i zation was 56.3 g (88%) of white L-carnitine, HPLC > 99%, specific rotation «*25o -30.9°, (c = 1, H2O).

Example 2 300 ml of the nutrient medium described in Example 1 but containing 2 g/1 crotonobetaine instead of γ-butyrobetaine, was inoculated with the strain HK 1331b and cultivated at 30°C, pH 7 for 24 hours.

Using the preculture, 5 litres of nutrient medium were inoculated as in Example 1 and cultivated at 30eC and pH 7.0 for 24 hours. The pH was kept constant at 7.0 by adding 8X phosphoric acid. "Fed-batch" operation was then started. As described in Example 1, the carbon and nitrogen source and a solution having the following composition were continuously added.

Composition of crotonobetaine feed Crotonobetai ne 300 g with water to 1 litre.

The solution containing the carbon and nitrogen source was added at a rate of 4.5 ml/h, corresponding to a specific feed rate of about 4 mol C/kg dr weight/h at the beginning of the "fed-batch" phase. The sample was treated and analysed in the same manner as described in Example 1. The crotonobetaine was added at a rate such that the concentration of crotonobetaine in the fermenter was between 0.05 and 0.5 wt.X. 150 hours after inoculation of the fermenter, the concentration of L-carnitine was 6.1X and the concentration of unreacted crotonobetaine was 0.17X. This corresponded to 95* conversion of crotonobetaine .

Isolation of L-carnitine Pure L-carnitine could be prepared from the solution, which contained 61 g/1 L-carnitine, 1.7 g/1 crotonobetaine and inorganic salts, by a method. as set out in £P-A 195 944. -— The product after recrystal 1 i zation was 52 g (86*) of white L-carnitine having the same specifications as in Example 1. • 1 -

Claims (7)

1. A method of discontinuous production of L-carnitine by microbiological processing of crotono-betaine and/or Y-butyrobetaine, wherein Y -butyro-betaine and/or crotonobetaine, together with betaine as a source of carbon and nitrogen and an additional carbon source, are added to a culture medium containing a microorganism of the genus Pseudomonas, Rhizobium, Agrobacterium, or E.coli or a yeast of the genus Saccharomyces, and L-carnitine is separated after a maximum L-camitine concentration has been obtained.

2. A method according to claim 1, wherein the crotonobetaine and/or T -butyrobetaine are added in proportions such that their concentration in the culture medium is between 0.005 and 5%.

3. A method according to claims 1 and 2, wherein the betaine and the additional carbon source are added in proportions such that the molar ratio of carbon to nitrogen is from more than 5 to 1 to 10000 to 1. - 2 -

4. A method according to claim 1, wherein a sugar, sugar alcohol or an organic acid is used as the additional carbon source.

5. A method according to claim 4, wherein glucose or glycerol is used as the additional carbon source.

6. A method according to claim 1, wherein betaine and the carbon source are added at a rate of between 0.01 and 100 mol of carbon per kg dry weight in the fermenter per hour.

7. A method of discontinuous production of L-camitine by microbiological processing of crotonobetaine and/or y -butyrobetaine, substantially as hereinbefore described in examples 1 and 2. E. ULFORO ATTORNEYS FOR APPLICANTS