MXPA00004356A - Method for producing l-carnitine from crotonobetaine - Google Patents

Abstract

The invention relates to a method for producing L(-)-carnitine. The aim of the invention is to depict a method which permits the synthesis of L(-)-carnitine from crotonobetaine, crotonobetaine salts or derivatives in an ecologically advantageous manner by immobilizing cells of i(Escherichia coli) 044 K74 in a continuously operating cell recycle reactor. Growing or resting cells of i(E. coli) are retained in a continuously operating cell recycle reactor by micro or ultrafiltration membranes which are arranged as a flat membrane module or hollow chamfer module.

Description

PROCEDURE FOR PRODUCING L-CARNITINE FROM CROTONOBETAIN DESCRIPTION OF THE INVENTION The invention relates to a process for the preparation of L-carnitine from crotonobetaine, from crotonobetaine salts, other derivatives of crotonobetaine or the like. It is known that L-carnitine, a compound that occurs ubiquitármente, has an important role in metabolism, especially in the transport of fatty acids long chains through the inner membrane of the mitrocondrias. Many clinical uses arise from the role of carnitine in the metabolism of eukaryotes, for example in the treatment of patients with carnitine deficiency syndromes, in the prophylaxis and therapy of different cardiac diseases, as well as in the treatment of hemodialysis patients. . This is why L-carnitine is important as a food supplement and as an additive to fermentation media it promotes the growth of yeasts and bacteria. This growing need for this enantiomer of biologically active L-carnitine for these and other uses has led to a worldwide search for synthetic routes for that betaine in an optically pure form, since the chemically synthesized racemate can not be used due to the inhibition of the carnitine acetyltransferase as well as the carnitine carrier protein.

REF. : 119752 To isolate the L-isomers, methods have been used up to now. they are based on the separation of racemate by means of fractional crystallization using optically active acids (for example US Pat. No. 4,254,053, 1981) wherein D (+) - carnitine is present as a waste product. This problem can be overcome by means of different biological procedures from inexpensive achiral precursors (Adv Biochem, Eng. Biotechnol., 1993, 50, 21-44). Of special interest is the stereospecific hydration of trans-crotonobetaine to L-carnitine using strains of the genera Escherichia (ED 0211444, 1984; DD 221 905, 1987; EP0320460, 1989) or Proteus (Agrie. Biol. Chem.1988, 52, 24152421; US Patent 5,300,430, 1994). The advantage of these methods is that these achiral precursors can be obtained by chemical dehydration of the D-carnitine waste product. The numerous procedures described in the literature with immobilized microorganisms in a continuously operating reactor system have the advantage that: • pure reaction media can be used and thus facilitate extraction as well as the purification process • by using higher concentrations of the biocatalyst in the reaction medium, higher productivities can be obtained and simultaneously avoiding possibility of contamination, • a reduction in sensitivity to inhibitors or a lack of nutrients is obtained, • a greater stability of the biocatalyst is obtained. The mentioned advantages can also be used in a commercial process. A continuously operating reactor, in which microorganisms are retained by means of micro- or ultrafiltrate membranes, makes possible an immobilization procedure which has the aforementioned advantage and also low immobilization costs and at the same time allows a slight increase in the scale. The corresponding object of the invention is 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 resting cells of Escherichia coli 044 74 ( DSM 8828), which are retained by means of micro- or ultrafiltration membranes in flat membranes or hollow fiber modules. E. coli is maintained in the aforementioned reactor at temperatures between 20 and 40 ° C and pH values between 6.0-8.0 and anaerobic conditions, which are necessary for the induction of carnitine metabolizing enzymes. A minimal or complex medium is used as the reaction medium. In both cases crotonobetaina, crotonobetaina salts and other derivatives of crotonobetaina are added in concentrations between 25 mmol and 1 M. The minimum means contains different concentrations of casein hydrolyzate and salts of (NH4) 2qS04, KH2P04, K2HP04, MgS04 x 7H20, MnS? 4 x 4H20, FeS04 x 7H20), while the complex medium contains different concentrations of pancreatic peptone and NaCl. To improve the growth of E. coli, glycerin, glucose, ribose, sucrose or lactose are added. Additionally, inhibitors that prevent the transformation of crotonobetaine to β-butyrobetaine (fumarate, glucose or nitrate) and inducers of metabolizing enzymes such as D-, L-, DL-carnitine, its salts and derivatives or crotonobetaine, its salts or derivatives are added to the medium. . The reaction in the continuous cell recycling reactor used here can be divided into two parts, that part consists of a reactor tank in which the E. coli cells together with the reaction medium transforms most of the crotonobetaine into L-carnitine . This reactor tank presents control elements for the pH value, temperature and agitation speed as well as for the control and correction of the oxygen concentration. The feed of the reaction medium to the reactor is carried out by means of a dosing pump. When necessary, the reactor tank discharge of excess medium must be carried out. The second section consists of a feedback elbow, which is connected to the reactor tank and through a pump drives the contents of the reactor through a filtering unit. While the filtrate is collected, to isolate L-carnitine as the reaction product, the residue is again conducted from the filtrate zone to the reactor. To filter the cell suspension, commercial filtering systems of different origin can be used, as long as they have a pore size, which is below the cell size of E. coli. The speed of the feedback pump remains unchanged, to obtain the best possible filtration rates and to minimize the formation of a polarization membrane during the filtering process. The expression of E.coli free cells refers to the state in which the whole cells are suspended in the reaction medium, without the cellular solution being inhibited by the outgoing solution. The expression of immobilized cells describes the state in which the cells are bound to soluble polymers or insoluble carriers, or are encased in a membrane system (in Methods in Enzymol, 1987, vol 135, 3-30). The concept of growth conditions is defined as the situation in which whole cells during their life cycle use substrates and produce products. Under resting cells is understood intact but not growing cells, which under certain conditions show certain metabolic properties (in "Biotechnology" (Kieslich, K .; Eds. Rehm, H.J. and Reed, G.) Verlag Chemie, Weinheim, Germany, 1984, vol. 6a, 5-30). The procedure will be illustrated below with some examples of embodiment: Example 1: Escherichia coli 044 74 is grown in a flask Hermetically sealed Erlenmeyer filled to the brim 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 fumarate, 5 g / 1 NaCl and different concentrations (between 0.5 and 10 g / 1) pancreatic peptone. The pH is adjusted with KOH to 7.5. Table 1 summarizes different specific growth rates with different concentrations of peptone. Table 1. Maximum specific growth rate of Escherichia coli 044 K74 Peptone (g / 1) 0.5 1.0 2.5 5.0 10.0 μmax (Ir1) 0.224 0.296 0.351 0.372 0.325 Under the conditions mentioned, the growing E. coli cells are in a position to produce 20-30 mM L-Carnitine until the end of the test. Concentrations greater than 5g / l peptone gave as much similar parameters of growth and kinetics as well as biomass contents (OD 600 nm). Contrary to this with low peptone concentrations, reduced growth parameters are obtained. Example 2: Escherichia coli 044 K74 is grown in a flask Hermetically sealed Erlenmeyer, filled to the brim at 37 ° C under anaerobic conditions in a rotating shaker (150 rpm). The complex medium used has the following composition: 50 mM crotonobetaine, 5 g / 1 pancreatic peptone, 5 g / 1 NaCl as well as different concentrations (between 0 and 75 mM) of fumarate. The pH is adjusted with KOH to 7.5. Table 2 shows that a fumarate addition leads to higher growth rates of E. coli 044 K74 and an OD at 600 nm of almost 1.0 at steady state. Thus the fumarate produces an L-carnitine formation of 20-30 mM until the end of the test. In the absence of fumarate a carnitine concentration of only 5 mM is obtained. Table 2 - Biomass (OD600 nm) with different concentrations of fumarate after a test with a duration of 10 hours Fumarate (mM) 0 25 50 75 μmax (ir1) 0 21 0.37 0.38 0.39 biomass 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. Induction studies are performed with crotonobetaine between 5 and 75 mM using resting cells. At high crotonobetaine concentrations, transformation rates greater than 60% L-carnitine are obtained (Table 3). Table 3. Dependence of the production of L-carnitine by resting cells of Escherichia coli 044 K74 depending on different concentrations of crotonobetin Crotonobetanine (mM) 25 50 75 fl Production of L-carnitine C%) 55 60 62 65 Example 4: Escherichia csli 044K74 is grown in a tightly closed Erlenmeyer flask filled to the brim 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 / 1 pancreatic peptone, 5 g / 1 NaCl and 50 mM fumarate. The pH is adjusted with KOH to 7.5. To increase the concentration of the biocatalyst and to allow an L-carnitine production with dilution rates higher than the specific growth rate maximum, a membrane reactor was used. The cells were retained through a polysulfone microfiltration membrane and an exclusion limit of 0.1 μm and reused. The membranes were placed in a plate module. Table 4 summarizes the biomass content and table 5 shows the production of carnitine, the transformation of crotonobetaine and the productivity in that system. Table 4. Biomass content of E. coli 044 K74 in a continuously operating membrane reactor Dilution rate (Ir1) 0. 0 0. twenty-one . 0 2. 0 Biomass (gpeBO aeco / l) 0. 5 2. 1 9 .4 27. 0 Table 5: Production of L-carnitine, transformation of crotonobetine and productivity in a continuous-functioning cellular reactor with Escherichia coli 044 K74 Dilution rate (h'1) 0.0 0.5 1.0 1.5 2.0 Production of L-carnitine (%) 0.0 38 42 42 38 Transfor.de crotobetaina (%) 0. 0 24 26 26 30 Productivity (g / lre! LcCor / h) 0.0 1.75 3.5 5.5 6.5 It can be seen from the table that with immobilized cells of Escherichia coli 044 K74 in a reactor of Cellular recycling results in a formation of L-carnitine from crotonobetaine of 6.5 / 1 / h with a metabolization rate of approximately 40%. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (14)

1. CLAIMS Having described the invention as above, the contents of the following are claimed as property: 1. Procedure for the preparation of L-carnitine from crotonobetaine, characterized in that, a microorganism immobilized in a cellular reactor of continuous operation with the substance starting crotonobetaine as such or in the form of its salts or derivatives, is transformed to L-carnitine.
2. 2. Method according to claim 1, characterized in that the microorganism is E. coli 044 K74 (DSM 8828).
3. 3. Method according to claim 1 or 2, characterized in that E. coli is immobilized in a carrier material, which does not adversely affect its vitality.
4. 4. Method according to claim 3, characterized in that ceramic material, glass beads, polyurethane discs are used as the carrier material.
5. 5. Process according to claims 1 to 4, characterized in that the ceramic reaction mixture carrying the microorganism is filtered, L-carnitine is separated in a known manner and the crotonobetaine is recycled.
6. 6. Procedure according to claim 1 a 5, characterized in that the concentration of crotonobetaine in the reaction medium is between 25 m and 1 M.
7. 7. Method according to claim 1 a 6, characterized in that to the commercial complex medium or the minimal well-defined E. coli medium, which is added to the reactor, are added electron acceptors of the type of inhibitors such as fumarate, nitrate, oxygen, N-oxides or glucose, which avoid hydration of - crotonobetaine to give? -butirobetaine.
8. 8. Procedure according to claim 1 7, characterized in that carnitine-metabolizing enzymes are added to the minimal medium, such as L, D- or DL-carnitine, as well as their derivatives and salts, as well as crotonobetaine, its derivatives and salts.
9. 9. Process according to claim 1, characterized in that E. coli 044 K74 is cultured in a complex medium anaerobically or partially anaerobically, where a fumarate concentration of 50 mM is maintained.
10. 10. Process according to claim 1, characterized in that, E. coli 044 K74 is cultured in a complex anaerobic or partially anaerobic environment, wherein the complex medium contains 50 mM crotonobetaine, 5 g / 1 pancreatic peptone, g / 1 NaCl and 50 mm fumarate, has a pH of 7.5, and the reaction is carried out in a continuously operating membrane reactor.
11. 11. - Method according to claims 8, characterized in that the feedback of bacteria in the reactor is carried out continuously using modules of transversal current filtration or commercial hollow fibers, consisting of known membranes of ultra- or microfiltration with different chemical compositions as cellulose, polysulfone or polysulfone polysulfone with an exclusion limit of 300 kDa or 0.2 μ.
12. 12. Method according to claim 1 to 10, characterized in that the synthesis is carried out under anaerobic or partially anaerobic conditions.
13. 13. Process according to claim 1, characterized in that the production of carnitine is carried out in a continuously operating reactor at different dilution rates, which are adjusted by means of two pumps, the dosing pump and the filtration pump, and It is carried out with different agitation speeds and biomass concentrations.
14. 14. - Method according to claim 13, characterized in that the discharge rate of the filtering stream is controlled during the process.