HK1031237B - Method for producing l-carnitine from crotonobetaine - Google Patents

Description

The invention relates to a process for the production of L-carnitine, crotonobetaine, from salts of crotonobetaine, other derivatives of crotonobetaine or the like.

L-carnitine, a ubiquitous compound, is known to play an important role in metabolism, especially in the transport of long-chain fatty acids across the inner mitochondrial membrane. The function of carnitine in eukaryotic metabolism has led to numerous clinical applications, e.g. in the treatment of patients with carnitine deficiency syndromes, in the prevention and treatment of various diseases and in the treatment of haemodialysis patients. In addition, beta-L-carnitine is of importance as a supplementary nutrient and also promotes the growth of yeast and bacteria as a supplement to fermentation media. The growing demand for this biologically active carnitine-E has led to the use of L-carnitine-E in other carnitine transporters worldwide and to the synthesis of this carnitine-E inhibitor, which cannot be used as a synthetic form of carnitine.

The L-isomer is isolated by methods based on the splitting of racemate by fractional crystallization using optically active acids (e.g. US Patent 4, 254,053.1981), where D(+) carnitine is produced as a waste product. This problem can be overcome by various biological methods based on cheap achiral precursors (Adv. Biotechnol., 1993, 50, 21-44).

Chemical Abstract, vol. 115, no. 1, 8 July 1991 Columbus, Ohio, US; abstract no. 6956, JUNG, H. ET AL: 'Production of L-carnitine by biotransformation' XP002096268 & DECHEMA BIOTECHNOL. CONF. (1990), 4 (PT. B, LECT. DECHEMA ANNU. MEET. BIOTECHNOL. 8TH, 1990), 1041-4 CODEN: DBCOE reveals a method for the production of L-carnitine from crotonobetaine by use of immobilized Escherichia coli 00K74 cells which are then used to produce the L-carnitine.

The following is a list of the substances which are to be used in the preparation of L-carnitine: L-carnitine synthesis by stereoselective hydration of crotonobetaine XP002096269 & PROC. - EUR. CONGR. BIOTECHNOL., 5TH (1990), VOLUME 1, 251-4.

The numerous methods described in the literature involving immobilized microorganisms in a continuously operating reactor system have the advantage of: The use of purer reaction media, which facilitates extraction and purification,increases productivity by using higher concentrations of the biocatalyst in the reaction medium,while reducing the possibility of contamination,reducing sensitivity to inhibitors or nutrient deficiencies,and improving the stability of the biocatalyst.

The above advantages can also be used in a commercially exploited process.

A continuous-operating reactor, in which microorganisms are contained by micro- or ultrafiltration membranes, is an immobilization process which, in addition to the above advantages, has the low cost of immobilization and at the same time allows very easy scale expansion.

Accordingly, the invention relates to a process for the production of L-carnitine from crotonobetaine, crotonobetaine salts or other crotonobetaine derivatives in a continuous reactor with free or immobilized cells, growing or dormant cells of Escherichia coli 044K74 (DSM 8828) contained by micro- or ultrafiltration membranes in ceramics, glass beads or polymethane drives.

E. coli is kept in the above-mentioned reactor at temperatures between 20 and 40 °C, pH between 6.0 and 8.0 and under anaerobic conditions necessary for the induction of the carnitine metabolizing enzymes. A minimum or complex medium is used as the reaction medium. In both cases, Crotonobetaine, Crotonobetaine salts or other Crotonobetaine derivatives are added in concentrations between 25 mmol and 1 M. The minimum medium contains different concentrations of casein nitrolysate and derivatives (NH4) 2SO4, KH2PO4, K2HPO4, MgSO4 x 7H2O, MnSO4 x 4H2O, MnSO4 x 7H2O), while the complex medium contains different concentrations of carnitine, Crotonobetaine salts or other derivatives such as Crotonobetaine, Crotonobetaine, Crotonobetaine, Crotonobetaine, Crotonobetaine, Crotonobetaine, Crotonobetaine, Crotonobetaine, Croton, Croton, Croton, Croton, Croton, Croton, Croton, Croton, Croton, Croton, Croton, Croton, Croton, Croton, Croton, Croton, Croton, Croton, Croton, Croton, Croton, Croton, Croton, Croton, Croton, Croton, Croton, Croton, Croton, Croton, Croton, Croton, Croton, Croton, Croton, Croton, Croton, Croton, Croton, Croton, Croton, Croton, Croton, Croton, Croton, Croton, Croton, Croton, Croton, Croton, Croton, Croton, Croton, Croton, Croton, Croton, Croton, Croton, Croton, Croton, Croton, Croton, Croton, Croton, Croton,

The reaction process in the continuous cell recycling reactor used here can be divided into two sections. One section consists of a reactor tank in which E. coli cells, together with the reaction medium, convert the largest part of the crotonobetaine into L-carnitine. This reactor tank has controls for pH, temperature and stirring rate, and for controlling and correcting oxygen concentration. The reaction medium is fed into the reactor by a dosing pump. If necessary, a re-loading of the reactor tank from excess medium must be carried out. The second section consists of an external return pump, which is used at different rates of concentration and is used to reduce the concentration of the reaction product.

The term free E. coli cells refers to the state in which whole cells are suspended in the reaction medium without being prevented from leaking by the leakage solution, while immobilised cells refers to the state in which whole lines are bound to soluble polymers or insoluble media or are enclosed in membrane systems (In Methods in Enzymol, 1987, vol. 135, 3-30).

Err1:Expecting ',' delimiter: line 1 column 348 (char 347)

The procedure is explained in the following examples:

Example 1:

Escherichia coli 044 K74 is cultured in a fully filled, airtight, sponge flask at 37 °C under anaerobic conditions on a rotary shaker (150 rpm). The complex medium used has the following composition: 50 mM Crotonobetaine, 50 mM Fumarat, 5 g/l NaCI and different concentrations (between 0.5 and 10 g/l) of pancreatic peptone. The pH is adjusted to 7.5 by KOH. The specific growth rates at different peptone concentrations are summarised in Table 1. Tab. 1.

Peptone (g/l)	0.5	1.0	2.5	5.0	10.0
	0.224	0.296	0.351	0.372	0.325

Under the above conditions, growing E. coli cells are able to produce 20 to 30 mM L-carnitine until the end of the test.

Concentrations above 5 g/l peptone give similar growth and kinetic parameters and biomass levels (OD 600 nm), whereas lower peptone concentrations give lower growth parameters.

Example 2:

Escherichia coli 044 K74 is cultured in a fully filled, airtight Erlenmeyer flask at 37 °C under anaerobic conditions on a rotary shaker (150 rpm). The complex medium used has the following composition: 50 mM Crotonobetaine, 5 g/l pancreatic peptone, 5 g/l NaCI and graduated concentrations (between 0 and 75 mM) of fumarate.

Table 2 shows that the addition of fumarate results in higher growth rates of E. coli 044 K74 and an OD at 600 nm of almost 1.0 at steady state. Tabelle 2.

Fumarat(mM)	0	25	50	75
	0.21	0.37	0.38	0.39
Biomasse OD (600 nm)	0.980	0.910	1.00	0.950

Example 3:

The ability of Escherichia coli 044 K74 to form L-carnitine from crotonobetaine is induced by crotonobetaine. The induction studies were conducted with crotonobetaine between 5 and 75 mM using resting gels. At high crotonobetaine concentrations, conversion rates of over 60% L-carnitine are achieved (Table 3). Other Tabelle 3.

Crotonobetain (mM)	5	25	50	75
L-Carnitin produktion (%)	55	60	62	65

Example 4:

Escherichia coli 044 K74 is cultured in an airtight, filled to the brim flask at 37 °C under anaerobic conditions on a rotary shaker (150 rpm). The complex medium used has the following composition: 50 mM Crotonobetaine, 5 g/l pancreatic peptone, 5 g/l NaCl and 50 mM fumarate. The pH is adjusted to 7.5 with KOH.

To increase the biocatalyst concentration in the reactor and to allow L-carnitine production at dilution rates higher than the maximum specific growth rate, a membrane reactor was used. The cells were restrained and reused by a polysulfone microfiltration membrane with an exclusion limit of 0.1 μm. The membranes were arranged in a plate module. Biomass content is summarized in Table 4 and carnitine production, crotonobetaine conversion and productivity in this system in Table 5. Other Tabelle 4.

	0.0	0.2	1.0	2.0
	0.5	2.1	9.4	27.0

Tabelle 5.

	0.0	0.5	1.0	1.5	2.0
L-Carnitin-Produktion (%)	0.0	38	42	42	38
Crotonobetainumwandlung (%)	0.0	24	26	26	30
	0.0	1.75	3.5	5.5	6.5

The tables show that immobilized cells of Escherichia coli 044 K74 in a cell recycling reactor produce L-carnitine from crotonobetaine at a rate of 6.5 l/h and a rate of metabolism of almost 40%.

Claims (13)

1. Process for producing L-camitine from crotonobetaine, characterised in that an immobilised microorganism converts the starting material crotonobetaine as such or in the form of its salts or derivatives to L-carnitine in a continuously operating cell reactor, wherein ceramics, glass beads, polyurethane discs are used as substrate for the microorganism.
2. Process according to claim 1, characterised in that the microorganism is E. coli 044 K74 (DSM 8828).
3. Process according to claim 1 and 2, characterised in that E. coli is immobilised on substrate which does not impair its viability.
4. Process according to claim 1 to 3, characterised in that the reaction mixture is filtered by ceramics which support the microorganism, the L-carnitine is separated off in a manner known per se and the crotonobetaine is returned to the circuit.
5. Process according to claim 1 to 4, characterised in that the crotonobetaine concentration in the reaction medium is between 25 mM and 1 M.
6. Process according to claim 1 to 5, characterised in that electron acceptors for respiration of the inhibitors, such as fumarate, nitrate, oxygen, N-oxides or glucose, which prevent hydrogenation of the crotonobetaine to form γ-butyrobetaine, are added to conventional commercial complex medium flowing to the reactor or well defined E. coli minimal medium.
7. Process according to claim 1 to 6, characterised in that inductors of carnitine-metabolising enzymes, such as L-carnitine, D-carnitine, or DL-carnitine, derivatives and salts as well as crotonobetaine, derivatives and salts, are added to the minimal medium.
8. Process according to claim 1, characterised in that E. coli 044 K74 is cultivated on a complex medium anaerobically or partially anaerobically, wherein a fumarate concentration of 50 mM is maintained.
9. Process according to claim 1, characterised in that E. coli 044 K74 is cultivated on a complex medium anaerobically or partially anaerobically, wherein the complex medium contains 50 mM of crotonobetaine, 5 g/ of pancreatic peptone, 5 g/l of NaCl and 50 mM of fumarate, has a pH of 7.5, and the reaction proceeds in a continuously operating membrane reactor.
10. Process according to claim 1 to 7, characterised in that the return of bacteria to the reactor takes place continuously using commercial cross-flow filtration modules or hollow-fibre modules, which comprise ultrafiltration or microfiltration membranes of different chemical composition known per se, such as cellulose, polysulphone or polysalphonated polysulphone having an exclusion limit of 300 kDa or 0.211.
11. Process according to claim 1 to 9, characterised in that the synthesis is carried out under anaerobic or partially anaerobic conditions.
12. Process according to claim 1, characterised in that the carnitine production is carried out in a continuously operating reactor at different dilution rates, which are set by two pumps, the metering pump and filtration pump, and at different stirring rates and biomass concentrations.
13. Process according to claim 12, characterised in that the discharge rate of the filtration stream is controlled to manage the process.