Abstract:
The invention relates to a process for the L(-)-carnitine biochemical production, in an economically favorable way. 
     This new process is technically achievable with easily available raw, materials in simple reaction conditions and with not complicated substance separation techniques. 
     This process is carried out with bacteria that stereo-specifically hydrates crotonobetaine to L(-)-carnitine.

Description:
This is a continuation of co-pending application Ser. No. 06/665,765, filed on Oct. 29, 1984, now abandoned. 
    
    
     The present invention relates to a process for the production of optically active L(-)-carnitine(R-configuration) from an optically inactive precursor. 
     L-carnitine (3-hydroxy -4-trimethylaminobutyrate) is normally present in the organism where it exerts the role of activated long-chain free fatty acids&#39; carrier, through the mitochondrial membrane. Since the mitochondrial membrane is impermeable to the acyl CoA&#39;s derivatives, the long-chain free fatty acids can cross it only when the esterification with L-carnitine has taken place. The importance of using L(-)-carnitine for therapeutical use has recently been underlined. (&#34;Carnitine biosynthesis. metabolism, and functions&#34; Editors: R. A. Frenkel and J. D. Mc Garry Academic Press 1980). 
     Since the needed quantity of L-carnitine was not available, it was often substituted with the DL-carnitine racemate, obtainable by total chemical synthesis. But the DL-carnitine, which contains the D(+) isomer, caused certain side effects which had not taken place using L(-)-carnitine (Curr. Ther. Res. 28 (1980), 195-198). 
     It is also known that the transferases necessary for the L(-)-carnitine formation, the carnitine acetyl-transferase (EC 2.3.1.7) and the carnitine palmitoyl-transferase (EC 2.3.1.21), are specific for the L(-) form, whereas the D(+) isomer is a competitive inhibitor of these two transferases. 
     Administration of D-carnitine can, moreover, create a decrease of L(-)-carnitine in the cardiac muscle or in the skeletal muscle, as has been demonstrated on laboratory animals (guinea-pigs) (Life Sciences 28 (1981) 2931-2938). The symptoms of human hyperthyrosis which had improved after L-carnitine treatment, worsened drastically with D-carnitine (Endokrinologie, 38, (1959) 218-225), thus for patients&#39; treatment only L(-)-carnitine can presently be used. This is also and especially valid in the case of patients having chronical kidney failure who have no possibility of actively eliminating the D(+) isomer. 
     Hemodialysis induces an L-carnitine secondary deficiency since it is eliminated in the dialysate, given its low molecular weight (161.2). The oral administration or the addition of L(-)-carnitine in the dialysis solution has avoided carnitine decrease in the patients. 
     In the case of hyperlipoproteinemia, widespread in industrialised countries, a significative decrease of the levels of plasma risk factors, triglycerides and cholesterol, has been reached with L(-)-carnitine (Lancet II (1978) 805-807). Similar effects could have been obtained using acylcarnitine (DE-OS 2903579). 
     DESCRIPTION OF THE PRIOR ART 
     L(-)-carnitine has been isolated from meat extracts with a complicated purification procedure. 
     In the &#39;50&#39;s chemical synthesis were perfected, though only DL-carnitine could be obtained. 
     Up till now,the isomer L(-)-carnitine has been obtained by the racmate&#39;s resolution with fractional crystallisation of carnitine&#39;s salts with optically active acids. Various carnitine derivatives have been used, such as carnitine-nitrile, carnitinamide or even carnitine inner salt itself. 
     As optically active acids, tartaric, camphoric and camphor-sulphonic acids were used (p.e. DD-PS 23217; DD-PS 93 347; DE-OS 2927672). 
     DL-carnitine has also been resolved with specific L(-)-transferases. 
     But, since for the production of one mole of L(-)-carnitine, at least one mole of acyl CoA was needed, this procedure turned out to be too expensive to apply industrially. 
     All the other chemical synthesis with subsequent racemate resolution have the ulterior disadvantage that from synthesised DL-carnitine not more than 50% of the wanted isomer can be isolated. 
     This problem has been overcome with enzymatic stereospecific synthesis with achiralic precursors. 
     The U.S. Pat. No. 4, 221,869 describes the production of L(-)-carnitine from dehydrocarnitine with carnitine dehydrogenase (EC 1.1.1.108) isolated from &#34;pseudomonas fluorescens&#34; using NAD as coenzyme,.though other enzymes are necessary, such as glucose dehydrogenase or alcohol dehydrogenase for the regeneration of NADH. Moreover, dehydrocarnitine is very instable and it spontaneously decomposes in acetonyltrimethylammonium and carbon dioxide. 
     The Patent application DE-OS 3123975 describes L(-)-carnitine production from γ-butyrobetaine with γ-butyrobetaine hydroxylase (EC 1.14.11.1) isolated from &#34;Neurospora crassa&#34; mould. During this hydroxylase reaction α-ketoglutaric acid and a reducing agent (i.e. ascorbate) must be added to the incubation medium. 
     To have the maximum yield of L(-)-carnitine,catalase is also needed. γ-butyrobetaine hydroxylase is obtained after mould growth, isolation and purification of its spores with detergents and mechanical or ultrasonic treatment. The precursors of the L(-)-carnitine biosynthesis are L(-)-methionine and L-lysine. Through the ε-N,N,N trimethyl lysine, ε-N,N,N trimethyl-β-hydroxy-lysine and N,N,N trimethylamino butyraldehyde intermediates, γ-butyrobetaine is formed. It is hydroxylated to L-carnitine by γ-butyrobetaine hydroxylase in presence of molecolar oxigen, α-ketoglutaric acid, ferrous ions and a reducing agent. Crotonobetaine is not an intermediate in the biosynthesis. 
     The invention&#39;s purpose is that of overcoming the disadvanages of the known methods of L(-)-carnitine production and to indicate a procedure that allows, in a favorably economical way, the biochemical production of the compound. 
     DESCRIPTION OF THE INVENTION 
     The invention describes the technically realisable procedures to produce L(-)-carnitine from easily available raw materials under simple reaction conditions and with not complicated intermediate separation techniques. 
     Until now the only notions were: 
     (1) L(-)-carnitine, D(+)-carnitine and crotonobetaine are metabolized to γ-butyrobetaine. 
     (2) the enterobacter growth stimulation depends on the γ-butyrobetaine formation, obtained by the crotonobetaine reduction. (Arch. Microbiol. 132 (1982), 91-95). 
     (3) The &#34;clostridia-bacteria&#34; are capable of reducing crotonic acid to butyric acid in anaerobic conditions. (FEBS Lett. 109, (1980) 244-246). 
     Surprisingly it was found that even some bacteria, in certain particular conditions, had been capable of stereospecifically hydrating crotonobetaine to L(-)-carnitine; a process which had not been observed in presence of γ-butyrobetaine in the incubation medium. 
     These bacterial strains were: 
     Escherichia coli (E. coil 044 K74; 055 K 59; 0 111 K58; 0 114 K90) Salmonella (S. typhimurium LT 2  ; cottbus; anatum; newington) 
     Proteus (P. vulgaris; mirabilis) 
     Shigella (S. flexneri 1a) . 
     Hafnia (H. alvei Biotyp A and B) 
     Clostridium (C. kluyveri; sporogenes) 
     Citrobacter (C. freundii). 
     According to the invention, to the bacteria growing on complex or minimal mediums, or to resting cells, crotonobetaine (4-N,N,N-triethylamino crotonic acid) or one of its salts, such as for example chloride, iodide, perchlorate, nitrate phosphate, or crotonobetaine amide, nitrile and aryl or alkyl ester is added. 
     After a certain period of incubation the L(-)-carnitine formed is isolated from the reaction medium. 
     The crotonobetaine concentration in the incubation medium was included between 10 μ moles and 5 moles/1. As described in tables V, VI, VII the quantity of L(-)-carnitine formed increased with the increase of crotonobetaine conoentration, but the percentual yield refered to the raw material decreased. 
     After the separation of the L(-)-carnitine from crotono betaine the latter may be used again. 
     The resting cells were incubated in a minimal culture medium with salt solutions, organic and inorganic buffer mixture which did not contain any C and/or N sources or of which the C and contents could not be used by the bacteria. 
     The incubation times were comprised between 3 hrs and 5 days. A preferred interval of time is 12 hrs-48 hrs. 
     The bacteria capable of hydrating crotonobetaine can grow on the more diverse complex culture mediums, solid or liquid, thus commercial nutritive mediums containing meat, yeast, malt and starch water extracts can be used, adding, in partially anaerobic conditions, C and/or N sources such as ammonium hydroxide,urea, alcohol, carbohydrates, organic acids including fatty acids. The cultures are grown in calibrated tubes, in anaerobic condition and without stirring. 
     The reduction of crotonobetaine to γ-butyrobetaine leads to a loss of substance for L(-)-carnitine synthesis, thus this reduction reaction is inhibited by adding electron acceptors of the anaerobic respiration and other substances. Non limiting examples of the invention are oxygen, nitrates, trimethylamine N-oxide and other N-oxides, dimethyl sulphoxide, glucose, fructose, saccarose, maltose and electron acceptors such as fumarate, crotonate and acrylate. (Tables II, III, IV). 
     The bacteria grown on commercial nutritive mediums, supplemented with meat broth or pancreatic peptone were able to hydrate crotonobetaine to L(-)-carnitine (table IX). The rate of L(-)-carnitine synthesis is increased, though, by inducing crotonobetaine hydroxylase, adding to the culture medium of growing bacteria,crotonobetaine,DL-carnitine, fractions of DL-carnitine enriched with D(+) (obtained by the chemical resolution of the racemate) or its derivatives (i.e. carboxylic esters, O-acylcarnitine or O-acylcarnitine esters). 
     Crotonobetaine, its salts or its derivatives. were prepared according to the following known proceedings: 
     1) Dehydration with sulphuric acid or acetic anhydride of DL-carnitine, D(+)-carnitine or D(+) enriched DL-carnitine, or alternatively elimination of the acid from the corresponding O-acyl DL-carnitine or O-acyl D carnitine. 
     2) Exhaustive methylation of the 4-ammino crotonate with methyl halogenides. 
     3) Reaction of trimethylamine with 4-halogen substituted crotonate. 
     A good separation of the L(-)-carnitine from non reacted crotonobetaine or from the formed γ-butyrobetaine. by ion-exchange cromatography (&#34;Recent research on carnitine&#34; Ed. G. Wolf, pg 11-21 (1965), US-PS 4,221,869; DE-OS 3123975) can only be carried out for small quantities. 
     L(-)-carnitine in larger quantities has been separated after reaction of the hydroxy group with the acyl chlorides of medium and long chain fatty acids. 
     The O-acyl carnitines formed can be extracted from the water phase with n-butanol or isobutanol (Biochim. Biophys. Acta 280, (1972), 422-433). 
     The O-acyl carnitines are obtained by evaporation of the organic phase. They can be used directly in the biochemical research, or hydrolyzed with ammonium hydroxide to L(-)-carnitine which is then utilized for therapeutical application. 
     The crotonobetaine which remains in the water phase is added back to the incubation medium. 
     The L(-)-carnitine formed was measured using the carnitine acetyltransferase after the addition of acetyl CoA with the DTNB method. 
     The excess presence of crotonobetaine from 10 to 100 does not disturb the L(-)-carnitine determination. 
     Some of the advantages of this procedure are: 
     1) By dehydration of the D(+)-carnitine or of the D(+) enriched DL-carnitine, obtained by the chemical resolution of the racemate, crotonobetaine, which is a cheap raw material for the L(-)-carnitine synthesis, is formed. 
     2) In this process no biochemical substances or other enzymes are to be employed. 
     3) The cost of incubation and of the raw materials are very low. 
    
    
     The following non-limiting examples illustrate the present invention. 
     EXAMPLE 1 
     A 500 ml reaction vessel containing a complex culture medium with crotonobetaine: 
     
         ______________________________________pancreatic peptone         20     g/l        bidistilled H.sub.2 Osodium chloride         5      g/l        bidistilled H.sub.2 Ocrotonobetaine         50     m moles/l  bidistilled H.sub.2 OpH7 (ΔE.sub.540 = 0.050)______________________________________ 
    
     was inoculated with a suspension of E. coli 044 K74, previously grown in 5% blood agar, in physiological solution. 
     The reaction flask was subsequently covered with paraffin oil and incubated at 37° C. 
     Table 1 shows the L(-)-carnitine formation in relation with the incubation time and the bacteria growth. 
     
                       TABLE 1______________________________________relation between L(-)-carnitine synthesis, incubationtime and bacteria growth.      L(-)-carnitine synthesis time [h]Incubation    [Δ E.sub.540 ]Growth             [m moles/l]                       ##STR1##______________________________________ 0      0.0      --        -- 3      0.100    --        -- 6      0.120    0.5        0.912      0.190    2.0        3.924      0.200    6.7       13.448      0.175    6.6       13.3______________________________________ 
    
     EXAMPLE 2 
     The complex culture medium containing crotonobetaine, inoculated with E. coli (example 1), after 24 hrs of incubation was centrifugated at 6000 rounds per minute to separate the grown bacteria. The obtained centrifugate was washed with phosphate buffer according to Sorensen 0.01 M at pH 7.5 (0.20 g/1 KH 2  PO 4  and 3.05 g/1 Na 2  HPO 4  ·12 H 2  O), suspended in a 1 l Sorensen phosphate buffer containinq 5 g/1 crotonobetaine (34.9 m moles/1) (ΔE 540  =0.080) and maintained for 24 hrs at 37° C. Subsequently it was again centrifugated and 9.08 m moles/1 of L(-)-carnitine were found in the solution. A yield of 26% in relation with crotonobetaine. 
     EXAMPLE 3 
     To a complex culture medium containing L(-)-carnitine (31 m moles/1 of L-carnitine corresponding to 5 g/1) and inoculated, as in example 1, with several strains of enterobacteria, electron acceptors of the anaerobic respiration and other substances (sodium succinate, sodium fumarate and glucose 10 g/1, potassium nitrate, trimethylamine N-oxide (TMO) 5 g/1) were added. 
     Table II expresses the yield in moles of γ-butyrobetaine in relation with the L(-)-carnitine utilized. 
     
                                           TABLE II__________________________________________________________________________Influence of the electron acceptors and of othersubstances on the L(-)-carnitine reduction toγ-butyrobetaine         γ-butyrobetaine formation [% moles]Bacteria strains         - succinate                fumarate                     KNO.sub.3                         TMO glucose__________________________________________________________________________Escherichia coli 044 K74         71           77   13   0   0   0Escherichia coli K12 Hfr H         45           43    5   0   0   0Salmonella typhimurium LT.sub.2         87           89   68   0   8   71Proteus vulgaris         79           81   17   0   17  0__________________________________________________________________________ 
    
     EXAMPLE 4 
     The complex culture medium (KM) described in example 1, or the minimal culture medium (MM) containing: 
     
         ______________________________________Na.sub.2 HPO.sub.4.12H.sub.2 O                   15.0    gKH.sub.2 PO.sub.4       3.0     gNH.sub.4 Cl             1.0     gMgSO.sub.4.7 H.sub.2 O  0.15    gCaCl.sub.2              0.014   gFeCl.sub.3              0.0002  gD-ribose                7.5     gdistilled H.sub.2 O sufficient for 1 l______________________________________ 
    
     were additioned with 50 m moles of crotonobetaine and with sodium fumarate (10 g/l), potassium nitrate (5 g/l), and trimethylammine N-oxide (5 g/l). They were then incubated with a layer of paraffin (anaerobic conditions) or without paraffin (aerobic conditions), at 37° C. with E. coil 044 K74 for 48 hrs. 
     
                       TABLE III______________________________________Influence of electron acceptors on the L-carnitineformation in the growing bacteriaCulture  L(-)-carnitine synthesis [m moles/l]medium O.sub.2 absence            O.sub.2 presence                       fumarate                              KNO.sub.3                                    TMO______________________________________KM     4.5       2.6        10.7   0.9   5.1MM     2.7       1.5        10.8   0     5.9______________________________________ 
    
     EXAMPLE 5 
     A complex culture medium (KM) or a minimal culture medium (MM) containing 50 m moles/1 of crotonobetaine were inoculated with E. coil 044 K74 and incubated for 48 hrs. The cells, collected and washed twice (example 2), were incubated for 48 hrs in Sorensen buffer containing 50 m moles/1 of crotonobetaine to which the electron acceptors were added as in example 4 
     
                       TABLE IV______________________________________Influence of the electron acceptors on the L-carnitineformation with resting cellsCulture  L(-)-carnitine synthesis [m moles/l]medium O.sub.2 absence            O.sub.2 presence                       fumarate                              KNO.sub.3                                    TMO______________________________________KM     7.5        8.3       0      7.8    8.4MM     9.1       12.1       0      9.8   10.4______________________________________ 
    
     EXAMPLE 6 
     Minimal culture mediums (MM), with various crotonobetaine concentrations, supplemented with D-ribose as C source (example 4) were inoculated with E. coli 044 K74 (example 1) and incubated at 37° C. for 48 hrs. 
     
                       TABLE V______________________________________L(-)-cartinine synthesis with bacteria grown on minimalculture medium with various crotonobetaine concentrationsL-cartinine synthesis [m moles/l]betainecrotono-    [m moles/l]              ##STR2##          [E.sub.540 ]Growth______________________________________500     5.3       1.1               0.12050      4.3       8.6               0.4505       0.3       6.0               0.3850.5     0.09      18.0              0.220______________________________________ 
    
     EXAMPLE 7 
     A complex culture medium as described in example 1 (A) and a minimal culture medium supplemented with D-ribose, as described in example 4 (B), each containing 8 g/1 of L(-)-carnitine, were inoculated with E. coli 044 K74. After 48 hrs the collected bacteria, washed twice, were suspended in Sorensen buffer, containing various crotonobetaine concentrations and incubated for 48 hrs a 37° C. 
     
                       TABLE VI______________________________________L(-)-carnitine synthesis with induced resting cellsby L(-)-carnitine at various crotonobetaine concentration     L-carnitine synthesis [m moles/l]betainecrotono-        [m moles/l]                   ##STR3##______________________________________(A)  500        9.0        1.8 50        7.5        15.1 5         2.9        58.4(B)  500        16.2       3.2 50        8.3        16.5 5         3.7        74.0______________________________________ 
    
     EXAMPLE 8 
     A complex culture medium containing crotonobetaine as in example 1 inoculated with E. coli 044 K74 was centrifugated after 48 hrs. The centrifugate, washed twice, was dispersed in a minimal culture medium, without C and N sources, containing various concentrations of crotonobetaine and incubated for 48 hrs. 
     
                       TABLE VII______________________________________L-carnitine synthesis with resting cells induced bycrotonobetaine at various crotonobetaine concentrations    L-carnitine synthesis [m moles]crotonobetaine       [m moles/l]                 ##STR4##______________________________________500        24.3      4.950         12.2      24.55          2.4       48.80.5        0.47      94.0______________________________________ 
    
     EXAMPLE 9 
     A minimal culture medium as described in example 4 which contains, instead of crotonobetaine, 50 m moles/1 of L(-)-carnitine was inoculated with E. coli 044 K74 and incubated for hrs. Subsequently the collected and twice washed bacteria were incubated at 37° C. in a minimal culture medium without C and N sources but with 50 m moles/1 of crotonobetaine. The optical density at the beginning of the incubation was ΔE 540  =0.210. 
     
                       TABLE VIII______________________________________L(-)-carnitine formation with resting cells in function of time    L-carnitine synthesis [h]Incubation time       [moles/l]                 ##STR5##______________________________________ 3         2.5       5.1 6         3.7       7.512         6.6       13.224         9.0       18.048         10.1      20.3______________________________________ 
    
     EXAMPLE 10 
     A complex culture medium (KM) or a minimal culture medium (MM) without C sources, additioned with the substances indicated in table IX, at the concentrations of 50 m moles/1, was inoculated with E. coil 044 K74 and incubated for 48 hrs. Subsequently, the collected bacteria, washed twice with Sorensen buffer, were incubated for 48 hrs in Sorensen buffer with 50 m moles/1 crotonobetaine. 
     
                       TABLE IX______________________________________L-carnitine formation with resting cells in functionof the various substances&#39; addition in the growth mediumGrowing cells       Resting cells                                L-carnitine  added      ΔE.sub.540 after                       ΔE.sub.540 at the                                systhesisMedium substances 48 hrs    beginning                                m moles/l______________________________________KM     --         0.315     0.225    1.4MM     D-Ribose   0.196     0.136    0.3MM     D-Glucose  0.325     0.260    0.6MM     D-Ribose   0.165     0.100    0  FumarateMM     D-Ribose,  0.395     0.220    0.3  GABOB.sup.+MM     D-Ribose   0.255     0.190    6.6  L-carnitine______________________________________ .sup.+ DL- γ amino β-hydroxybutyric acid.