FR2621325A1 - Improved process for preparing L-carnitine - Google Patents

Abstract

Process for preparing L-carnitine by reducing dehydrocarnitine by means of nicotinamide adenine dinucleotide (NADH), in the presence of carnitine dehydrogenase (CDH), the NAD<+> formed being reduced by a formate under the effect of a formate dehydrogenase (FDH); the aqueous solution which constitutes the reaction medium has an ionic strength of at least 0.5 M.

Description

Improved process for preparing L-carnitine.

The invention relates to an improved process for the preparation of carnitine, i.e., 4-trimethylamino-3-hydroxy-butyric acid, and in particular of
L-carnitine, biochemically.

Carnitine is mainly used for biological applications, in particular pharmacological, cosmetic and food, in particular as stimulant of gastric and pancreatic secretion, or as antihyperlipo protein. As the chemical methods of its preparation lead to racemic mixtures, while only the L stereoisomer is biologically active, it is above all the biochemical preparation process which is of interest, because it provides the L isomerism. It is therefore important to have a preparation process which makes it possible to obtain this compound economically. This is the advantageous advantage afforded by the process according to the invention; it allows the biochemical production of L-carnitine in good yields, practically without contamination by microorganisms.

A known process for the preparation of L-carnitine consists of reducing dehydrocarnitine by means of the reduced nicotinamide adenine dinucleotide, NADH, in the presence of the enzyme carnitine dehydrogenase (CDH); the resulting NAD + is in turn reduced to the state of NADH by a suitable reducing agent, for example glucose, alcohol, aldehyde, alkali metal dithionate or a formate, in the presence of a dehydrogenase cor-e ~ - -Idante . Such a process is in particular described in I. and French published under No. 2 398 046.

In particular, the preparation of L-carnitine is effected by the action of the reduced nicotinamide adenine dinucleotide, NADH, on dehydrocarnitine, and reduction of the NAD formed, by means of the formic group with formate dehydrogenase FDH as catalyst.

The synthesis scheme is then

(L-carnitine) CDH = carnitine dehydrogenase
The CDH used is generally obtained from an extract of a bacterial culture, for example from Pseudomona putida. It is precipitated with ammonium sulfate at 250 g per liter of crude extract (55% of saturation).

However, the prior art did not lend itself well to industrial applications, above all because of the very high quantities of enzymes which it required per mole of L-carnitine to be produced, and the low productivities per unit capacity of the reactors, per hour. The use of formate as a reducing agent for NAD, industrially tempting, has so far led to very low yields, as can be seen in Example 5 of the patent cited above.

The present invention results from the observation that the above-mentioned biochemical process can be carried out with considerably reduced quantities of enzymes, brought to industrially acceptable proportions, if the reaction medium contains dissolved salts, the concentration of which exceeds a certain minimum.

The presence of such an ionic force exerts a stabilizing effect on the enzymatic system present, avoiding its contamination and destruction during the reduction of dehydrocarnitine.

Thus, the process according to the invention is characterized by the fact that the aqueous medium, in which the biâconversion reaction (1) takes place, has / an ionic strength of at least 0.5 M and preferably 0.6 to 3M, ionic strengths of 0.8 to 2.5 M being particularly favorable; in the cited prior art, they did not exceed 0.3 M.

In the preferred form of the invention, where a solution of dehydrocarnitine is gradually poured into a solution of the required enzymes, and - consequently - the volume of the reaction medium varies from the start to the end of the operation, the ionic strength, varying at the same time, must reach the limits indicated above at least at the end of the reaction. As for the beginning, there may be, in the solution of the enzymes, only 0.1 to 0.4 M of ionizable salts.

Despite the beneficial effect, on the stability of the components of the reaction medium, exerted by the electrolytes, it is preferable that the concentration of the latter does not deprive certain limits, in particular the value of approximately 3M.

The adequate ionic strength can be obtained by dissolving in the liquids of the reaction medium various salts of organic or inorganic acids and bases.

The choice is conditioned by the pH of the medium, which must be between 6 and 8 and, preferably, between 6.8 and 7.8. By way of nonlimiting examples of salts which can be used, mention may be made of chlorides, sulphates and phosphates of alkali metals, of rium or of amines, or of salts of such cations with efferent organic acids, such as formates, tartrates, etc. alkyl-sulfonates, aryl-sulfonates, phthalates, citrates, maleates, malonates or others.

The introduction of the electrolyte into the reaction medium can take place in different ways: initially, in the solution of the enzymes; in the dXhydro-carnitine solution which is gradually poured into the previous one; directly in the reaction medium, separately from the dehydrocarnitine solution. Two or all three of these iodines can be used together.

As it is convenient to use dehydrocarnitine in the form of a salt, in particular of hydrochloride, the achievement of the appropriate ionic strength can be effected by the introduction of a base solution, into the reaction medium, simultaneously with that of the dehydrocarnitine solution, which conne takes place in the formation of a salt, chloride for example. It should be noted in this connection that the concentration of ions in the reaction medium naturally depends on that of the hydrochloride or other salt of dehydrocarnitine in the solution of the latter. So we can act on the ionic strength by adjusting this concentration.

The remarkable result of the adequate adjustment of the ionic strength, according to the invention, is first of all the possibility of considerably reducing the proportion of enzymes; thus, instead of 3200 to 8700 units of carnitine dehydrogenase (CDH) used per mole of dehydrocarnxtine treated, in the examples of the French patent cited above, only 200 to 1000 units, and most often 400 to 900 U, are sufficient, therefore about 5 to 10 times less, which constitutes a very important technical progress. In addition, thanks to the stability of the enzymes provided by the ionic strength, the reaction time can be greatly prolonged and thus achieve much higher yields than would be possible in the prior art. Hourly production, per unit of reactor volume, is thus greatly increased.

While it was hardly possible to allow the reaction to continue for more than 40 hours, the present invention makes it possible to go well beyond 48 hours and to reach, for example, around one hundred hours. You can usually work 45 to 80 hours.

Another interesting result is that the enzymes used still retain most of their initial activity, after preparation of L-carnitine; they can thus be reused for a new operation.

Preferably, the temperature does not exceed 3O0C, it can vary between 10 "and 3O0C.

The enzymes used in the process of the invention are well known in the art, there is therefore no need to describe their preparation here. It will only be noted that carnitine dehydrogenase (CDH) can be obtained from the extract of the bacterial cultures of Pseudomona of the fluorescent, seruginosa and especially putida groups (collection of the Pasteur institute no. CIP 52191).

Formate dehydrogenase (FDH) can be obtained from various microorganisms, in the known manner, and in particular from yeasts of the genera Candida,
Kloeckera, Pichia, Torulopsis etc. and especially from
Candida bodiini.

Given the fragility of aqueous solutions of dehydrocarnitine (DHC) at temperatures above 100C, it is not advisable to immediately put all of the
DHC in the reactor containing a solution of CDH enzyme.

The preferred procedure consists in keeping the DRC solution in a separate tank, at a sufficiently low temperature, for example between 0 and 4iC, and gradually introducing into the reactor only a certain current compatible with the temperature. reaction rate, so that DHC is practically converted into L-carnitine as it enters the reactor.

This leads to the preferred form of the process of the following invention. In a reactor, equipped with means for stirring and regulating the temperature, a determined volume of a solution of NADH and the two enzymes, CDR and
FDH, suitably buffered to a pH in the region of 7.

A salt is also added, according to the invention, so that the ionic strength of the enzyme solution is at least 0.2 M.

To this solution maintained between 10 and 30 ° C., in particular at 200 ° C., is gradually added, with stirring, an aqueous solution of dehydrocarnitine, coming from a special tank where this DRC solution is stored at low temperature. , the reaction rate (2) DRC + NADH

on fait couler simultanément, dans le réacteur, un débit de solution basique, juste pour neutraliserl'acidité de la DRC introduite ; la solution basique peut etre par exemple de NaOH, KOH, CH3COONa etc. et, plus particulièrement de l'ammoniaque qui donne du NR4Cl bien favorable à la force ionique du milieu. L-carnitine + NAD the flow rate of the solution is adjusted so that the DRC is, at least for the most part, converted as it arrives in the solution
Preferably, the DRC being in the form of its hydrochloride DHC. HCl, or

a flow of basic solution is simultaneously run into the reactor, just to neutralize the acidity of the DRC introduced; the basic solution can be for example NaOH, KOH, CH3COONa etc. and, more particularly ammonia which gives NR4Cl which is very favorable to the ionic strength of the medium.

The DHC concentration of the aqueous solution used can vary between wide limits, but is most often of the order of 0.2 to 2 M; one of the consequences of this factor is explained above with regard to the adjustment of the ionic strength.

When all of the DRC has been introduced into the reactor, the content of the medium in the L-carnitine formed is determined; the latter is then separated and purified according to the known technique.

The invention is illustrated by the nonlimiting examples which follow.

EXAMPLE 1
In a reactor of 250 ml capacity was placed 50 ml of an aqueous solution containing the following components
40 U of CDR carnitine dehydrogenase enzyme
with a specific activity of 55U / ml
from Pseudomona putida
2mM NAD +;
40 U of formate dehydrogenase, FDH, from
the yeast Candida bodiini (EC 1.2.1.2 .; fri
due by BOEHRINGER MANNHEIM)
50mM of HNa2P04 / KH2P04 phosphates (pH 7.5)
150mM ammonium formate HCOONH4
6mg of chloramphenicol
Into this starting solution was poured, with stirring, at a rate of 1.2 ml / h, an aqueous solution
0.8 M DRC hydrochloride and
0.8 M formic acid from a tank in which this solution was stored at 40C.

The temperature in the reactor was set at 300C.

Simultaneously with the introduction of the DHC solution, the pH of the reaction medium was adjusted to the value of 7, by the injection of a 2N solution of NH4OH
After 70 hours, the volume of solution in the reactor is 150 ml and its L-carnitine concentration 428 mM.

The results of the operation are therefore as follows
0.0672 mole of DRC used,
0.0642 "of L-carnitine formed (10.3g)
or yield of 96 X
This result is obtained with
ionic strength at the start 0.20M
I 'He at the end 0.96M
number of CDR units
per mole of DRC 595
EXAMPLE 2
Seeking to limit the increase in volume during the bioconversion, reagents were used in more concentrated solutions of dehydrocarnitine, formic acid and ammonia. On the other hand, the concentration of NAD + has been reduced.

50 ml of solution containing
HNa2PO4 / KH2PO4 50 mM pH: 7.5
NAD + 0.2 mM HCOONH4 150mM
6 mg chloramphenicol
40 U of CDR summarily purified as described
below
25U of FDH BOEHRINGER-tiANNHEIM from
Candida bodiini.

CDH, extracted from P. Putida, had been precipitated with ammonium sulfate (55 X saturation), centrifuged and the pellet redissolved in 50 mM phosphate buffer pH: 7.5; the solution obtained dialyzed for 16 h against this same buffer.

The reactor was fed with a solution maintained at 40 ° C., containing dehydrocarnitine and formic acid at a concentration, both of them, of 1.6 M. The flow rate was 0.6 ml / h. The temperature of the reactor maintained at 300C and the pH 7.5 by controlled addition of 8N ammonia. After 48 hours, a final volume of 90 ml and a L-Carnitine concentration of 500 mM is reached, ie a yield of 98 ×.

The ionic strength at the start was 0.20
The ionic strength at the end was 1.13 CDH Units / mole DRC ,, 863
EXAMPLE 3
The synthesis of L-carnitine was carried out with, initially, 1 liter of solution, under the following initial conditions
sa2HPO4 / KH22o4 50 mM pH: 7.5
RCO2NR4 150 mM
NAD + 0.6 mM
120 mg of chloramphenicol
CDR 800 U summarily purified as
in example 2
FDH 500 U or 750 mg of the lyophilisate
marketed by Boehringer-Mannheim.

The reactor was fed with the same 0.8 M solution as in Example 1, with a flow rate of 0.4 ml / min. The pH was maintained at 7.5, by a regulatory system suitable for the reactor, with 2N ammonia; the temperature was set at 300C. Mechanical stirring ensured the homogenization of the whole.

After 67 h, a volume of 3.2 1 was reached and an L-carnitine concentration of 390 mM, ie a yield of 97%.

In this example we worked with
an initial ionic strength of 0.20
a final ionic strength of ........... 0.86
a ratio of CDH units / moles DRC 622
The determination of the CDH present in the reactor gives 704 U or 88 X of residual activity. For FDH, the assay gives 384 U, ie 80 X of residual activity. The NAD + was completely transformed into NADH by the enzymatic route, then assayed spectrophotometrically at 340 mm. 0.2 mM is obtained which, taking into account the dilution, corresponds to 100 × activity.

EXAMPLE 4
Starting from one liter of solution, the synthesis of L-carnitine was carried out with the following initial conditions
50 mM phosphate buffer pH 7.5
HCO2NH4 0.5 M
Read
NAD 0.4 mM
CDH 820 U purified as in example 2
FDH 503 U extracted from a Pichia culture
Pastoris, precipitated with ammonium sulphate
(50 X saturation), centrifuged and cu
batch redissolved in 50 mM phosphate buffer
pH 7.5, the solution obtained dialyzed for 16 hours con
be this same buffer.

The reactor was fed with the same DRC solution as in Example 2, with a flow rate of 12 ml / h.

After 49 h, a volume of 1.75 liters is reached and an L-carnitine concentration of 526 mM, ie a yield of 98%. The determination of the residual activities in CDH and FDH gave respectively 459 U and 111 U.

During this preparation the ionic strength of the reaction medium was
at the start 0.505
at the end - 1.363 and the CDH / mol DRC ratio = 872 units / mol
The production is 1.72 g of L-carnitine per hour, per liter of final liquid volume.

The ionic strength, imposed by the salts (formate, phosphate, ammonium chloride) has proved sufficient to delay any bacterial contamination and allow work without antibacterials such as chloramphenicol.

EXAMPLE 5
In order to achieve a higher final L-carnitine concentration, a reactor was initially charged with 50 ml of solution.
50 mM phosphate buffer pH 7.5
0.4 mM NAD
HCO2NH4 0.5 M
CDR: 80 U purified as in example 2
FDH: 50 U purified as in example 4
The reactor was fed with an aqueous solution of 3.2 M dehydrocarnitine DRC, mixed with 3.2 M formic acid, with a flow rate of 0.55 ml / h, the pH in the reactor being maintained at 7.5. by means of ammonia.

After 47 h, a final volume of 82 ml is reached and an L-carnitine concentration of 880 mM, ie a yield of 87 ×. CDR and FDH activities were 21 U and 10 U respectively.

The ionic forces were in this test: initially 0.505 M, at the end 2.3 M.

Ratio of CDH units / moles DRC = 967 and production 3 g / h per liter of final volume; it can be seen that this increased productivity was obtained at the cost of a slight decrease in yield.

As in Example 4, the absence of antibacterial is justified by the even higher ionic strength of the medium, which delays any bacterial contamination.

EXAMPLE 6
The procedure is the same as in Example 3, but the mixture solution of DRC + formic acid is only 0.1
M. The ionic strength of the medium goes from 0.20 M at the start to 0.16 M after 67 hours of reaction, because the dilution largely compensates for the addition of salts during the bioconversion.

The yield of L-carnitine is no longer than 60%, and the residual enzyme activities are zero due to contamination of the medium. The production is 0.07 g / h, per liter of final volume.

EXAMPLE 7
The reaction is started as in Example 1, but with an initial formate concentration of 3.0 M instead of 0.15 M. The DRC solution then does not contain HCOOH.

The ionic strength of 3.05 M at the start increases to 3.45 M at the end. The yield is 40%. No contamination has occurred, but the CDR activity drops to 15% of its initial value. The production is 1.03 g / h x 1 final volume.

Raritulation of the examples.

The main results of the above examples are shown in the table below. The following abbreviations are adopted
Fii ..... initial ionic strength,
in moles per liter Fif "Il" final, in M.

CDH / DHC .... number of CDH em units
folded per mole of DHC.

Rdt. X yield of L-carnitine on
the DHC consumed.

Prod. ... g of L-carnitine obtained
per hour, per liter of solu
final tion.

Example n F.i.i F.i.f CDH / DHC Yield% Prod.

1 0.20 0.96 595 96 0.98
2 0.20 1.13 868 98 1.67
3 0.20 0.86 622 97 0.93
4 0.50 1.36 872 98 1.72
5 0.50 2.30 967 87 3.0
6 0.20 0.16 4975 60 0.07
7 3, s5 3.45 595 40 1.03
Examples 1 to 5 visibly represent optimal cases, while Examples 6 and 7 show a drop in yield when the ionic strength is too low (e.g. 6) or too high (e.g. 7).

On the other hand, Example 5 indicates a certain limit to the speed of the operation: a fairly high production (3 g / l.h.) Is obtained at the cost of a drop in yield (87 X).

Claims (8)

Claims 1. Process for preparing L-carnitine by reduction of dehydrocarnitine by means of nicotinamide adenine dinucleotide (NADH), in the presence of carnitine dehydrogenase (CDH), the NAD + formed being reduced by a formate under the effect of a formate dehydrogenase (FDH), charac- terized in that the aqueous solution, constituting the reaction medium, has, during bioconversion, an ionic strength of at least 0.5 M. 2. Method according to claim 1, characterized in that the ionic strength of the reaction medium is 0.6 to 3 M, at least at the end of the operation. 3. Method according to claim 1, wherein an aqueous solution of dehydrocarnitine is gradually poured into a solution of the enzymes, characterized in that the reaction medium has an ionic strength of 0.1 to 0.4 M at the start and 0.8 to 2.5 M at the end of the operation. 4. Method according to one of claims 1 to 3, characterized in that the ionic strength is formed mainly by ammonium formate. 5. Method according to one of claims 1 to 4, wherein the dehydrocarnitine is taken in the form of hydrochloride, the reaction medium being neutralized with ammonia, characterized in that a solution of formic acid is poured into this medium. together with the dehydrocarnitine solution. 6. Method according to one of the preceding claims, characterized in that the reaction medium contains 200 to 1000 units of CDR enzyme per mole of dehydrocarnitine employed in total. 7. Method according to one of the preceding claims, characterized in that the dehydrocarnitine solution has a concentration of 0.2 to 2 M. 8. Method according to claim 7, characterized in that this solution is poured into the solution of the enzymes with a speed such that the dehydrocarnitine is converted into L-carnitine as it arrives in the enzymatic solution.