ES2220219B1 - PROCEDURE FOR THE PRODUCTION OF L-CARNITINE FROM CROTONOBETAINA, D-CARNITINE, ITS SALTS AND DERIVATIVES, BY PERMEABILIZED CELLS OF PROTEUS SP. OR ESCHERICHIA COLI. - Google Patents

Abstract

Procedure for the production of L-carnitine from crotonobetaine, D-carnitine, its salts and derivatives, by permeabilized cells of Proteus sp . or Escherichia coli .

The process for the production of L-carnitine, from a biotransformation substrate selected from crotonobetaine, a crotonobetaine salt, a crotonobetaine derivative, D-carnitine, a D-carnitine salt, a D-carnitine derivative, and mixtures thereof, includes growing free or immobilized Escherichia coli or Proteus sp . permeabilized cells, in a culture medium containing said biotransformation substrate and the nutrients necessary to maintain the viability of said cells, under conditions that allow L-carnitine production. Said culture medium may contain growth activating compounds of said cells and / or inducers of the enzymes involved in the metabolism of L-carnitine and / or inhibitors of the transformation of crotonobetaine into γ-butyrobetaine.

Description

Procedure for the production of L-carnitine from crotonobetaine, D-carnitine, its salts and derivatives, by permeabilized cells of Proteus sp . or Escherichia coli .

Field of the Invention

The present invention relates to a new process for the production of L-carnitine from crotonobetaine, D-carnitine, its salts and derivatives, in a reactor in which permeabilized cells of Proteus sp . o Escherichia coli act as biocatalysts of the hydration and racemization reaction.

Background of the invention

It is well known that the L-carnitine [R - (-) - 3-hydroxy-4-N-trimethylaminobutyrate]  it is a ubiquitous substance that plays different roles in the metabolism, especially in the transport of fatty acids from long chain through the inner mitochondrial membrane. The role of L-carnitine in the metabolism of eukaryotic cells has led to a number of applications clinics, for example, in the treatment of patients with syndromes of carnitine deficiency, in the prophylaxis and treatment of various heart diseases and as replacement therapy in hemodialysis patients. Additionally, the L-carnitine as an additive in energy drinks or has been added to fermentation media to increase the speed of Yeast and bacteria growth. The increase in demand for biologically active enantiomer, L-carnitine, for these and other purposes, it has caused the development of different procedures to synthesize this betaine in a way optically pure, since the chemically synthesized racemate does not it can be administered because it inhibits important enzymes such as carnitine transport proteins and the carnitine acetyltransferase.

Chemical procedures for obtaining L-carnitine based on the resolution of racemic carnitine, or its precursors, have been described through its diastereomers by means of optimally active acids (US 4,254,053), in which D-carnitine is the Residual product of the resolution process. This problem can be solved by biological procedures that allow large-scale production of L-carnitine from its low-cost aquatic precursors (Adv. Biochem. Eng. Biotechnol., 1993, 50: 21-44). Of special interest is the stereospecific hydration of trans- crotonobetaine to L-carnitine using strains belonging to the genus Escherichia (EP 121444; DD 221905; EP 320460) or Proteus (Agric. Biol. Chem., 1988, 52: 2415-2421; US 5,300,430). The advantage of this method is that the aquiral precursor can be obtained by chemical dehydration of the product D-carnitine that would otherwise be worthless.

The biotransformation of crotonobetaine in L-carnitine with poor performance (35-45%) using the enzyme L-carnitine dehydratase (WO99 / 24597; FEMS Microbiol Lett., 2001, 196: 1-6).

Gram-negative bacteria, for example Escherichia coli , Proteus sp ., Etc., possess an extra, mainly protective outer membrane, which is capable of expelling hydrophilic macromolecules and hydrophobic substances. The integrity of the outer membrane in gram-negative bacteria can be altered by permeabilizing agents (Microbiol. Rev., 1992, 56: 395-411; Lett. Appl. Microbiol., 1999, 1, 13-16). By way of illustration, it has been described that detergents such as Triton X-100 (Enz. Microbiol. Technol., 1996, 18: 18-22; Enz. Microbiol. Technol., 1998, 22: 621-628) and Tween (Enz Microbiol. Technol., 1996, 19: 145-149), as well as organic solvents, such as toluene (Biotechnol. Lett., 1994, 16: 345-348), act as cellular permeabilizing agents. However, additional research on the permeabilization of gram-negative bacteria is needed as there is little information available due to its outer membrane and the special characteristics of its cell wall and effective methods need to be developed to reduce the barrier-like action of the shell cell towards substrates and products in biological processes (bioprocesses).

Permeability tests have been performed on different bacteria. However, apparently, no permeability tests have been performed on Proteus sp . or E. coli as a means to increase the bioprocess of conversion into L-carnitine.

On the other hand, it is known that E. coli and Proteus sp . they are capable of biotransforming trimethylammonium compounds (FEMS Microbiol. Lett., 2001, 196: 1-6; Biochem. Biophys. Acta, 1989, 1003: 270-276; Enzyme Microbiol. Technol., 2001, 28: 785-791) , so the generation of secondary products of the biotransformation of crotonobetaine in L-carnitine, such as γ-butyrobetaine, supposes a lower production of L-carnitine as well as serious downstream problems that significantly affect the industrial applicability of process.

Compendium of the invention

There is a need to find a procedure alternative microbiological to produce L-carnitine from a substrate of appropriate biotransformation, such as crotonobetaine, that exceeds the all or part of the inconveniences associated with the microbiological production processes of L-carnitine known.

The solution provided by this invention is based on a new method for increasing the biotransformation of crotonobetaine, D-carnitine, its salts and derivatives, into L-carnitine or its derivatives by using permeabilized cells of gram-negative bacteria, such as Proteus sp . and E. coli . Cell permeabilization improves substrate entry and product flow, factors that are controlled by the outer membrane and diffusion of the cell wall, and the transport system itself, leading to an increase in the conversion of the substrate. up to double (80-90%). In addition to obtaining L-carnitine with high purity along with high productivity and yield in L-carnitine, the production of biotransformation by-products, such as γ-butyrobetaine, is very low or virtually nil since L-levels Carnitine inside the cell are lower due to permeabilization, thus reducing treatment costs downstream of the process.

Therefore, in one aspect, the invention relates to a process for the production of L-carnitine, from a biotransformation substrate, which comprises culturing permeabilized cells of E. coli or Proteus sp ., In a culture medium containing said biotransformation substrate and the nutrients necessary to maintain the viability of said cells, under conditions that allow the production of L-carnitine.

Because the induction of carnitine-related enzymes requires aerobic conditions for Proteus sp ., Or anaerobic for in a particular embodiment, said method comprises culturing permeabilized E. coli cells at a pH between 4.5 and 8.5 , in a culture medium comprising the biotransformation substrate under aerobic conditions, while in another particular embodiment, said method comprises culturing permeabilized cells of Proteus sp . at a pH between 4.5 and 8.5, in a culture medium comprising the biotransformation substrate under anaerobic conditions.

Brief description of the figures

Figure 1 is a graph showing the cell growth curves of Proteus sp . in the presence of different concentrations of yeast extract, namely 1 gL -1 (black), 5 gL -1 (iangle), 10 gL -1 (black), 20 gL ^ {-1} (black) and 30 gL -1 (-).

Figure 2 is a graph showing the specific growth rate (µ) in h -1 of Proteus sp . in the presence of a concentration of yeast extract of 30 gL -1 ()).

Figure 3 is a graph showing the growth curves (Figure 3A) and the specific growth rate (µ), in h -1 (Figure 3B), of Proteus sp . at different pH values, specifically at pH 6.6 (zen), 6.8 (down), 7.1 (black) and 7.8 (black).

Detailed description of the invention

The invention provides a process for the production of L-carnitine, from a biotransformation substrate selected from crotonobetaine, a salt of crotonobetaine, a derivative of crotonobetaine, D-carnitine, a salt of D-carnitine, a derivative of D- carnitine, and mixtures thereof, hereinafter method of the invention, comprising culturing cells permeabilized from Escherichia coli or Proteus sp ., in a culture medium containing said biotransformation substrate and the nutrients necessary to maintain the viability of said cells, under conditions that allow the production of L-carnitine.

The process of the invention comprises the use of permeabilized cells of E. coli or Proteus sp . Said permeabilized cells can be obtained by conventional methods comprising contacting E. coli or Proteus sp . with a permeabilizing agent under conditions that allow the permeabilization of such cells. Virtually any cell permeabilizing agent, in particular, of gram-negative bacteria, can be used. However, in a particular embodiment, said permeabilizing agent is selected from detergents, organic solvents and mixtures thereof. Examples of detergents that can be used for permeabilization of said cells include octoxinols, for example, octoxynol-9, a non-ionic detergent sold under the brand name Triton X-100; the polysorbates, for example, a polyethoxylated sorbitan ester marketed under the Tween 20 brand, etc .; polyethyleneimine (PEI), etc. Examples of organic solvents that can be used in the permeabilization of said cells include alcohols, for example, ethanol, isopropanol, etc .; ketones, for example, acetone, etc .; or aromatic hydrocarbons, for example, toluene, etc. The permeabilization of said E. coli or Proteus sp . It can be done using one or more detergents and / or one or more organic solvents.

In a particular embodiment, the process for permeabilizing E. coli or Proteus sp . it comprises incubating said cells in the presence of a permeabilizing agent, in a concentration between 1 and 50% (weight / volume), under aerobic conditions, at a pH between 4.5 and 8.5, at a temperature between 4 ° C and 40 ° C, for a period of time sufficient to guarantee cell permeabilization, for example, between 1 minute and 24 hours. The pH value between 4.5 and 8.5 can be obtained, for example, using a phosphate buffer, for example, potassium phosphate, with the appropriate concentration (20-80 mM). The resulting permeabilized cells can be used directly in the process of the invention by adding the biotransformation substrate to the reactor in which the permeabilized cells are found. Alternatively, said permeabilized cells can be separated by conventional methods and washed before being used in the process of the invention. Virtually any method of cell separation can be used; however, in a particular embodiment, the permeabilized cells are separated by centrifugation, for example, at 1,000-20,000 xg, for a period of time between 1 and 60 minutes, and washed, for example, with the buffer used in the permeabilization process. Examples 6 and 7 illustrate particular embodiments of permeabilization of E. coli and Proteus sp .

Permeabilized cells of E. coli or Proteus sp . Are cultured for the implementation of the process of the invention. in a culture medium containing said biotransformation substrate and the nutrients necessary to maintain the viability of said cells, under conditions that allow the production of L-carnitine. Said culture medium may also contain, if desired, one or more growth-activating compounds of said cells and / or one or more inducers of the enzymes involved in the metabolism of L-carnitine and / or one or more inhibitors of the transformation of crotonobetaine into γ-butyrobetaine.

The biotransformation substrate for L-carnitine production according to e]. process of the invention may be crotonobetaine, a salt of crotonobetaine, a derivative of crotonobetaine, D-carnitine, a salt of D-carnitine, a derivative of D-carnitine, or mixtures thereof. Virtually any Salt. or derived from crotonobetaine or D-carnitine, for example, its alkali metal or alkaline earth metal salts, its Acyl derivatives, their esters, etc., can be used as a substrate of biotransformation in the process of the invention. Examples illustrative of said biotransformation substrates include crotonobetaine or D-carnitine, its metal salts alkaline or alkaline earth, for example, its sodium salts, potassium, etc., its acyl derivatives, for example, acetyl-crotonobetaine, propionyl-crotonobetaine, etc., or its esters, by example, crotonobetainyl palmitate, crotonobetainyl isovalerate, etc. In a particular embodiment, said biotransformation substrate is crotonobetaine The biotransformation substrate may be present in the culture medium in virtually any concentration, preferably, in. a concentration included between 25 mM and 1 M.

Permeabilized cells of E. coli or Proteus sp . they can be in a state of growth or in a state of rest. The terms "growth state or resting state" define a situation in which whole cells carry out their life cycle by consuming substrates and producing products. In that situation, whole cells in a minimal medium are in a latent state or their membrane and cell wall show an increased permeability towards certain substrates, due to the physical-chemical changes that take place externally within the medium. Such changes do not prevent certain biochemical processes directed to the production of the desired product (Anal. Biochem, 1180, 1982, 211-234; in "Biotechnology"), (Kieslich K. vol. Ed. Rehm HJ & Reed G. Ed) Verlag Chemie, Weinhein, Germany, 1984, vol. 6a. 5-30).

As used in this description, the expression "culture medium containing the nutrients necessary to maintain the viability of said permeabilized cells of E. coli or Proteus sp ." It is intended to include the culture media, both minimal and complex, that contain the necessary nutrients (carbon source, nitrogen source, micronutrients, etc.) suitable for the culture of said cells either in a growing state or in a resting state. In general, said culture media are known to those skilled in the art. Illustrative examples of culture media in which the process of the invention can be carried out include:

(i)

a minimal culture medium for growing E. coli cells in a growth state comprising casein hydrolyzate (0.1-1 gL -1), crotonobetaine (1-5 gL -1) and inorganic salts such as (NH 4) 2 SO 4 (1-5 gL -1), KH 2 PO 4 (1-5 gL -1), K_ 2} HPO4 (1-10 gL <-1>), MgSO_4 \ O2 (0-0.5 gL <-1>), MnSO_ {4} \ cdotH_ {2 O (0-0.5 gL -1) and FeSO 4 · H 2 O (0-0.001 gL -1) 1); the pH is adjusted between 4.5 and 8.5, for example, by adding a base, such as NaOH or KOH, before sterilization;

(ii)

a complex culture medium for growing E. coli cells in a growth state comprising pancreatic peptone (1-30 gL -1), crotonobetaine (1-100 gL -1) and NaCl (1-10 gL -1); the pH is adjusted between 4.5 and 8.5, for example, by adding a base, such as NaOH or KOH, before sterilization;

(iii)

a culture medium for culturing permeabilized E. coli cells in a resting state containing only phosphate buffer at a concentration between 10 and 1,000 mM, pH 4.5-8.5, to which crotonobetaine is added at a concentration comprised between 25 and 1,000 mM (depending on the case);

(iv)

a culture medium for culturing Proteus sp . in a growth state comprising yeast extract (1-30 gL -1), K 2 HPO 4 (1-10 gL -1), KH 2 PO 4 ( 1-10 gL -1) and crotonobetaine (25-1,000 mM); Y

(v)

a culture medium for culturing permeabilized cells of Proteus sp . at rest, it contains crotonobetaine (1-150 gL -1) and glycerol (1-10 gL -1).

Also, as used in this description, the expression "under conditions that allow L-carnitine production "aims to include generically the necessary conditions (temperature, pH, agitation, oxygen, etc.) for the production of L-carnitine according to the process of the invention. In general, these conditions can be determined by the subject matter experts through trial and error tests. Do not However, in a particular embodiment:

(i)

The production of L-carnitine with permeabilized E. coli cells in a growth state comprises incubating in complex medium for E. coli cells in a growth state, under anaerobic conditions, at a temperature between 35 ° C and 40 ° C, at a pH comprised between 5.5 and 8.5 and with stirring (100-200 rpm);

(ii)

the production of L-carnitine with permeabilized cells of E. coli at rest comprises incubating said cells in a culture medium for E. coli cells at rest, under aerobic conditions, with oxygen supply (air or nitrogen) , at a temperature between 4 ° C and 40 ° C, at a pH between 5.5 and 8.5 (for example, with 30-80 mM phosphate buffer) and with stirring (100-200 rpm);

(iii)

L-carnitine production with permeabilized cells of Proteus sp . under growth conditions it comprises incubating said cells in a culture medium for Proteus sp . in a state of growth, under aerobic conditions with oxygen supply (air or nitrogen), at a temperature between 35ºC and 40ºC, at a pH between 5.5 and 8.5 (for example, with 30-80 mM phosphate buffer ) and with stirring (100-200 rpm); Y

(iv)

L-carnitine production with permeabilized cells of Proteus sp . at rest, it comprises incubating said cells in a culture medium for Proteus sp . at rest, under aerobic conditions, with oxygen supply (air or nitrogen), at a temperature between 35ºC and 40ºC, at a pH between 5.5 and 8.5 (30-80 mM phosphate buffer) and with stirring (100-200 rpm).

The culture medium used for putting in practice of the process of the invention may also contain, if desired, one or more growth activating compounds of said cells and / or one or more inducers of the enzymes involved in the metabolism of L-carnitine and / or one or more crotonobetaine transformation inhibitors in γ-butyrobetaine.

In a particular embodiment, said culture medium further comprises a growth activating compound of said cells. Virtually any growth-activating compound of said cells can be used. However, in a specific embodiment, said growth-activating compound of permeabilized cells of E. coli or Proteus sp . it is selected from glycerin, glucose, ribose, sucrose, lactose and mixtures thereof. In case the culture medium contains said growth activating compound, it may be present in a concentration between 20 mM and 150 mM.

In another particular embodiment, said culture medium further comprises an inducer of the enzymes involved in the metabolism of L-carnitine. Practically, any inducer of the enzymes involved in the metabolism of L-carnitine can be used. However, in a specific embodiment, said inducer of the enzymes involved in the metabolism of L-carnitine is selected from crotonobetaine, a salt of crotonobetaine, a derivative of crotonobetaine, D-carnitine, a salt of D-carnitine, a derivative of D-carnitine, DL-carnitine, a salt of DL-carnitine, a derivative of DL-carnitine, and mixtures thereof. If the culture medium contains said inducer of the enzymes involved in the metabolism of L-carnitine, it may be present in a concentration between 1 mM and 20 mM. In general, when the process of the invention is carried out using permeabilized cells of E. coli or Proteus sp . It is advantageous or convenient to include said inducer of the enzymes involved in the metabolism of L-carnitine.

In another particular embodiment, said culture medium further comprises an inhibitor of the transformation of crotonobetaine into γ-butyrobetaine. Practically, any inhibitor of the transformation of crotonobetaine into γ-butyrobetaine can be used. However, in a specific embodiment said inhibitor of the transformation of crotonobetaine into γ-butyrobetaine is selected from fumarate, oxygen, carbon dioxide, nitrate, glucose and mixtures thereof. Such inhibitors may be present in the culture medium in the following amounts: fumarate (1-50 mM), oxygen (1-100%), carbon dioxide (1-10%), nitrate (1-50 mM), glucose (1-10 gL -1). In general, when the process of the invention is carried out using permeabilized cells of Proteus sp . it is not necessary to include in the culture medium said inhibitor of the transformation of crotonobetaine into γ-butyrobetaine.

The process of the invention can be carried out with the permeabilized cells of E. coli or Proteus sp . free or, alternatively, immobilized on a support. The term "free cells" refers mainly to a situation in which whole cells are suspended within the liquid medium of the reactor without anything preventing them from flowing out through the reactor outlet (Methods in Enzymology, 135, 1987 , 3-30). On the contrary, the term "cells immobilized on a support" refers to a situation in which whole cells are fixed to a support by any type of interaction, physical or chemical. Virtually any support commonly used to immobilize cells can be used. Permeabilized cells of E. coli or Proteus sp . they can be immobilized on said support by conventional methods (Enz. Microbiol Technol. 21, 1997, 531-536; J. Appl. Microbiol. 85, 1998, 883-890; Appl. Microbiol. Biotechnol. 51, 1999, 760-766 ).

The process of the invention can be carried out. conducted in a reactor selected from a reactor operating in continuous, a reactor that operates in semi-continuous and a reactor that operates in batch (lots).

In a particular embodiment, the procedure of the invention is carried out in reactors operating in discontinuous.

In another particular embodiment, the process of the invention is performed in a reactor that operates continuously. As used herein, the term "continuously operating reactor" refers to a reactor, for example, a stirred tank type reactor, wherein the cells of E. coli or Proteus sp . they are contacted with the culture medium to transform all or most of the biotransformation substrate into L-carnitine. In general, said reactor comprises monitors for controlling the pH, temperature, stirring or oxygen concentration and is provided with means for checking and correcting the values of said parameters, as well as dosing pumps and filtration pumps. The culture media are introduced into the reactor through the corresponding inlets by dosing pumps. When necessary, the reactor undergoes a bleeding operation. In a particular embodiment, the process of the invention is carried out in a reactor that operates continuously, at different dilution rates, which are adjusted by means of two pumps, the metering pump and the filtration pump, at different proportions of recycling, agitation speeds and biomass concentration, where the output speed of the filtration current is controlled for optimum reactor operation.

In general, the most appropriate conditions for the culture of permeabilized cells of Proteus sp . or E. coli in a continuously operating reactor comprise culturing the microorganism at a temperature between 2 ° C and 50 ° C and a pH between 4.5 and 8.5. To induce carnitine-related enzymes, anaerobic conditions are required for E. coli or aerobic for Proteus sp .

The process of the invention can be carried out. carried out by performing one or more biotransformation cycles. In a particular embodiment, the process of the invention is carried out carried out through two or more biotransformation cycles. Operating from this way it is possible to obtain in the second cycle of biotransformation a conversion greater than 50% and a Productivity greater than 2.5 g.L-l .h-l .g-1 (Example 8).

The following examples illustrate the production of L-carnitine using permeabilized cells of Proteus sp . and E. coli and should not be considered limiting its scope.

Examples

Examples 1-5 were performed with non-permeabilized cells of Proteus sp . and served to establish the optimal conditions for the use of said bacteria. The optimal growth and biotransformation media for Proteus sp . they were established in experiments in which the cells were in a state of growth or rest. Example 6 illustrates the permeabilization of E. coli cells while Examples 7-9 illustrate the permeabilization of Proteus sp . and its use in the biotransformation of crotonobetaine in L-carnitine.

The procedures with the cells at rest were always performed in 10-1,000 mM phosphate buffer, pH 4.5-8.5 and adding crotonobetaine (50-1,000 mM) well as a biotransformation substrate (depending on the experiment) in case using E. coli or crotonobetaine (50-1,000 mM) and glycerol (1-10 gL -1) if Proteus sp . The pH of the growth and resting media was adjusted to 4.5-8.5 with 1 M NaOH or KOH before sterilizing. In general, the cells were grown in a medium with an initial concentration of 0.5-20% (v / v) of liquid culture stored at -20 ° C in 20% (v / v) glycerol at 20-40 ° C for 8-10 hours in a 1 L flask filled with 0.2 L of growth medium and stirred at 150-300 rpm by an orbital shaker ( Proteus sp .) or in a 1 L flask filled to the neck and closed with a stopper rubber ( E. coli ). During continuous experiments, the medium was oxygenated or maintained anaerobic, as described in the examples. Crotonobetaine and γ-butyrobetaine were determined by high performance liquid chromatography (HPLC) with a Tracer Spherisorb-NH2 3 µm column, 25 x 0.46 cm, supplied by Teknokroma (Spain) under isocratic conditions with a mobile phase composed of acetonitrile / H 3 PO 4 0.05 M pH 5.5 (65/35) and a flow rate of 1 ml / min. L-carnitine was determined by the carnitine acetyltransferase assay (Enzyme Microbiol. Technol., 2001, 28: 785-791.

Example 1 Media optimization

In this example, the growth of Proteus sp . in different culture media containing different concentrations of yeast extract (1-30 gL -1) while the rest of the components [crotonobetaine (15 g.
L -1), K 2 HPO 4 (4.4 gL -1) and KH 2 PO 4 (6.8 gL -1)] were kept constant. Proteus sp . grown aerobically at 37 ° C in shake flasks with an orbital shaker set at 150-250 rpm.

Figure 1 shows the growth curves (biomass) at different concentrations of yeast extract rehearsed The highest levels of biomass were obtained with the highest concentration of yeast extract (30 g.L -1), per what the concentration of yeast extract selected for the following experiments was 30 g.L -1; however, in all cases a production of 50% L-carnitine.

Figure 2 shows that the speed of maximum specific growth for an extract concentration of Yeast 30 g.L -1 was 0.31 h -1.

The effect of pH was checked within range from 5.5 to 8.5, specifically between 6.6 and 7.8, varying the phosphate concentration The results obtained are collected in the Figure 3, where it can be seen that the highest concentrations of biomass were obtained at the highest pH values tested, between 7.1 and 7.8 (Figure 3A). It is also observed that the speed of highest specific growth was obtained at pH values tested higher (Figure 3B). For such reasons, a pH was chosen of 7.5 to perform the following studies.

Example 2 Production of L-carnitine by Proteus sp . in a state of growth

The production of L-carnitine by Proteus sp . Cells has been studied. in a growth state using 2 culture media containing 10 and 30 gL -1, respectively, of yeast extract, and different concentrations of crotonobetaine (5.10 and 15 g. L-1). To do this, Proteus sp . in said culture media under aerobic conditions, at 37 ° C, in shaken flasks at 100-200 rpm and at a pH between 5.5 and 8.5 with 50 mM phosphate buffer.

The production of L-carnitine was faster in the culture containing 30 gL -1 of yeast extract than in the culture containing 10 gL -1 of yeast extract due, among other causes, to the fact that the specific growth rate of Proteus sp . It was also higher at that concentration of yeast extract (30 gL -1). Qcarnitine, which represents the rate of production of L-carnitine, was also higher as the concentration of crotonobetaine increased (Table 1). The production yield of L-carnitine was not affected, with values between 45% and 50% in all cases.

TABLE 1 Biotransformation with Proteus sp . in a state of growth in the presence of different concentrations of yeast extract and crotonobetaine

Qcarnitine (g.L -1 - .h -1) Crotonobetaine (g.L -1) Yeast Extract (10 g.L -1) Yeast Extract (30 g.L -1) 5 0.210 0.296 10 0.378 0.418 fifteen 0.614 0.850

Therefore, the growth medium for Proteus sp . Selected for use in the following examples contained yeast extract (30 gL -1), K 2 HPO 4 (4.4 gL -1), KH 2 PO 4 (6.8 gL -1) and crotonobetaine (15 gL -1).

Example 3 Production of L-carnitine by Proteus sp . at rest

The production of L-carnitine by Proteus sp . Cells has been studied. in a state of rest. To do this, Proteus sp . they were grown under aerobic conditions, at 37 ° C, in shake flasks in an orbital shaker at 150-250 rpm or in a continuous stirred reactor. The complex medium had the following composition: crotonobetaine (50-150 g.
L -1), yeast extract (0-75 gL -1), K 2 HPO 4 and KH 2 PO 4. The pH was adjusted to 7.5 by the addition of KOH. It was concluded, after the studies carried out, that the cells grew with a higher growth rate in the presence of oxygen, obtaining similar values of A 600 in all cases (close to 1.0-4.0 units in steady state).

In addition, after growth in the medium optimally, the cells were collected or kept in the reactor, to which the biotransformation medium was added. He studied the L-carnitine production changing the concentrations of crotonobetaine, biomass and glycerol (5-10 g.L -1) in that medium. The production of L-carnitine, its production speed and its yield, were calculated with different concentrations of crotonobetaine (50-150 g.L -1). Although at one crotonobetaine concentration of 150 g.L -1 - the speed of L-carnitine production was higher than the obtained with other concentrations of crotonobetaine, the performance could not be considered satisfactory. Concentration of 100 g crotonobetaine -1 L seemed to be the best option, as shown in Table 2.

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TABLE 2 Biotransformation with Proteus sp . at rest in the presence of different concentrations of crotonobetaine

Crotonobetaine (g.L -1) Qcarnitine (g.L -1 - .h -1) Production of L-carnitine (%) fifty 1.76 40 75 2.44 42 100 2.90 55 150 2.98 36

Example 4 Crotonobetaine induction of L-carnitine production in Proteus sp cells

The cell capacity of Proteus sp . To produce L-camitin from crotonobetaine it must be induced within the growth medium and, for that reason, induction studies were carried out with different concentrations of crotonobetaine in the medium between 1 and 20 gL -1. The experiments were performed with different concentrations of resting cells (biomass measured as A 600 between 1.0 and 4.0) and the concentrations of crotonobetaine mentioned in Table 2 (50-150 gL -1). . Briefly, Proteus sp . were cultured under aerobic conditions, at 37 ° C, with stirring (100-150 rpm), in a medium containing crotonobetaine (100 gL-1), yeast extract (20 gL-1) and adjusted pH at 7.5 with 50 mM phosphate buffer.

Proteus sp . were able to express enzymes dependent on L-carnitine metabolism to similar levels, reaching 50% L-carnitine production accompanied by a 10-15 mM γ-butyrobetaine production. The production rate of L-carnitine and the yield in carnitine production increased at increasing concentrations of biomass.

TABLE 3 Biotransformation using Proteus sp . at rest at different concentrations of biomass

Biomass A_ {600} γ-butyrobetaine (g.L -1) Qcarnitine (g.L -1 - .h -1) Carnitine production (%) 1.20 1.9 2.16 40 2.05 2.5 2.66 fifty 3.35 3.1 3.51 55

Example 5 Production of L-carnitine by Proteus sp . at rest in the presence of different concentrations of glycerol

The production of L-carnitine by Proteus sp . Cells has been studied. in the state of) resting in the presence of different concentrations of glycerol. To do this, Proteus sp . under aerobic conditions, at 37 ° C, in a continuous stirred tank type reactor. The complex medium had the following composition: crotonobetaine (100 gL-1), yeast extract (10-20 gL-1) and pH adjusted to 7.5 with KOH. Glycerol was added at different concentrations (0-10 gL -1) to observe its effect on the biotransformation medium at rest used.

TABLE 4 Biotransformation using Proteus sp . at rest at different concentrations of glycerol

Glycerol (g.L -1) γ-butyrobetaine (g.L -1) Qcarnitine (g.L -1 - .h -1) Carnitine production (%) 0 2.5 2.86 43 5 2.1 2.56 44 10 2.3 2.79 Four. Five

Crotonobetaine and γ-butyrobetaine were determined by high performance liquid chromatography (HPLC) with a column Tracer Spherisorb-NH2 3 µm, 25 x 0.46 cm, 17 cm, supplied by Teknokroma (Spain) in isocratic conditions with a mobile phase composed of acetonitrile / H 3 PO 4 0.05 M pH 5.5 (65/35) and a flow rate of 1 mUmin. The L-carnitine was determined by testing the carnitine acetyltransferase (Enzyme Microbiol. Technol., 2001, 28: 785-791).

Example 6 Permeabilization of E. coli cells

E. coli cells underwent a permeabilization process consisting of incubating said cells in the presence of a permeabilizing agent, such as Triton X-100, Tween 20 and polyethyleneimine (PEI), at different concentrations, under aerobic conditions, at a temperature between 4ºC and 40ºC, after a period of time between 12 and 24 hours of cell culture. The biotransformation assays of crotonobetaine in L-carnitine were performed with the resulting E. coli permeabilized cells using a concentration of crotonobetaine between 1 and 150 gL -1 in growth medium and 1-100 gL - 1} in the middle of rest.

Table 5 shows the results obtained in a biotransformation test with permeabilized E. coli cells by incubation with Triton X-100, Tween 20 or PEI for 10 minutes, at 4 ° C. Before being subjected to the permeabilization process, said E. coli cells were cultured under aerobic conditions, for a period of time between 12 and 24 hours. The biotransformation tests of crotonobetaine in L-carnitine were performed in the presence of crotonobetaine (80 gL -1) under aerobic conditions at 37 ° C with 80 mM phosphate buffer, pH 7.5.

TABLE 5 Biotransformation with permeabilized cells of E. coli after permeabilization with Triton X-100, Tween 20 or PEI (10 minutes incubation at 4 ° C); The biotransformation was performed in the presence of crotonobetaine (80 gL -1) under aerobic conditions at 37 ° C with 80 mM phosphate buffer, pH 7.5

Concentration (%, v / v) L (-) - carnitine (g.L <-1> .h <-1> .g <-1>) Conversion L-carnitine (%) Control 0 0.294 42 one 0.455 65 Triton X-100 2 0.469 70 5 0.483 75 Tween 20 2 0.500 75 5 0.503 75 PEI 0.05 0.510 80 0.5 0.511 85 Triton X-100, Tween 20 and PEI are commercial products (Sigma-Aldrich)

Example 7 Permeabilization of Proteus sp .

Proteus sp . Cells they were permeabilized by incubating said cells in the presence of a permeabilizing agent, under aerobic conditions, at a temperature between 4 ° C and 40 ° C, for a period of time between 5 and 30 minutes. Biotransformation assays with Proteus sp . The resulting permeabilized were performed in the presence of a concentration of crotonobetaine between 1 and 150 gL -1 in growth medium and 1-100 gL -1 in resting medium. The results are collected in the following examples.

7.1 Permeabilization of Proteus sp . with Triton X-100, isopropanol and ethanol

The results of 3 simultaneous experiments performed with Proteus sp . Cells are described. using as permeabilizing agents Triton X-100 1%, 20% isopropanol and 20% ethanol. After 12 h of cell culture, the agents were added and the cultures were maintained at 35 until reaching the stationary phase. Next, the culture broth was centrifuged (or maintained as it was) at 16,000 xg and the pellet (cells) was resuspended for 48 hours in a biotransformation medium at rest containing crotonobetaine (50-100 gL -1). and glycerol (1-10 gL -1). Biotransformation assays with Proteus sp . Permeabilized were performed at a temperature between 35 ° C and 40 ° C, at a pH between 5.5 and 8.5 with 30-80 mM phosphate buffer, conagitation (100-200 rpm), oxygen supply (air or nitrogen), in discontinuous reactors (48 hours and in two phases) or in continuous. During the experiments, samples were removed to determine the biomass content and the concentration of L-carnitine. The experiments were performed in triplicate. The conversion and productivity values of L-carnitine shown in Table 6 correspond to the average values of the analyzes.

TABLE 6 Conversion and productivity values using 50 g.L -1 Crotonobetaine in biotransformation medium in state of growth and rest, respectively

20% ethanol Isopropanol 20% Triton X-100 1% Control Rep. Cto. Rep. Cto. Rep. Cto. Rep. Cto. Conversion (%) 31.2 45.8 20.5 10.6 48.1 65.2 28.5 45.2 Productivity 0.77 1.34 0.51 0.28 0.93 1.60 0.72 0.12 (g.L <-1> .h <-1> .g <-1> cell) Cto .: Growth; Rep .: Rest

The results obtained showed that Triton X-100 was the permeabilizing agent that provided the highest conversion values and productivity.

7.2 Permeabilization of Proteus sp . using different concentrations of Triton X-100

Since Triton X-100 was able to improve the production of L-carnitine, different concentrations, ranging from 0 to 0.5 gL -1, of Triton X-100 were tested. The cell permeabilization process of Proteus sp . said cells were incubated with Triton X-100 under aerobic conditions, at a temperature of 35 ° C, for 12 hours. Next, the culture broth was centrifuged at 16,000 xg and the cells were resuspended for 48 hours in biotransformation medium at rest containing crotonobetaine (50-100 gL -1) and glycerol (1-10 gL - one}). Biotransformation assays with Proteus sp . Permeabilized were performed at a temperature between 35 ° C and 40 ° C, at a pH between 5.5 and 8.5 with 30-80 mM phosphate buffer, with stirring (100-200 rpm), oxygen (air) supply, in reactors discontinuous (48 hours and in two phases) or in continuous. During the experiments samples were removed to determine the biomass content and the concentration of L-carnitine. The experiments were performed in triplicate. The conversion and productivity values of L-carnitine shown in Tables 7 and 8 correspond to the average values of the analyzes.

TABLE 7 Conversion and productivity values of L-carnitine in the state biotransformation medium of growth using different concentrations of Triton X-100

Triton X-100 (g.L -1) 0 0.05 0.1 0.2 0.3 0.5 Conversion (%) 44.4 71.6 70.6 69.5 85.9 75.3 Productivity 1.06 1.93 1.98 1.97 2.21 2.11 (g.L <-1> .h <-1> .g <-1> cell)

TABLE 8 Conversion and productivity values of L-carnitine in the state biotransformation medium resting using different concentrations of Triton X-100

Triton X-100 (g.L -1) 0 0.05 0.1 0.2 0.3 0.5 Conversion (%) 42.0 71.5 75.5 81.3 88.7 67.3 Productivity 1.38 1.99 2.05 2.03 2.37 2.04 (g.L <-1> .h <-1> .g <-1> cell)

In Tables 7 and 8 it can be seen that the biotransformation values of crotonobetaine in L-carnitine obtained using any of the permeabilized cells of Proteus sp ., At any of the concentrations of permeabilizing agent tested, were higher than those of the control. It is worth noting the concentration of Triton X-100 of 0.3 gL -1, a concentration that resulted in a substantial improvement in biotransformation with permeabilized cells of Proteus sp . both in a state of growth and in a state of rest.

Example 8 Biotransformation, in several cycles, of crotonobetaine in L-carnitine through the use of Proteus sp . Permeabilized using different concentrations of Triton X-100

The cell permeabilization process of Proteus sp . It was performed using varying concentrations of Triton X-100. Briefly, Proteus sp . they were grown under aerobic conditions, at a temperature of 35 ° C, for a period of 12 hours, and in the presence of a concentration of crotonobetaine of 50 gL -1 in the growth biotransformation medium and 100 gL1 -1} in the biotransformation medium at rest. After 12 h, Triton X-100 was added at concentrations between 0 and 0.5 gL -1 and the cultures were maintained at 35 ° C for 12 h. Next, the culture broth was centrifuged at 16,000 xg and the cells were resuspended for 48 hours in biotransformation medium at rest containing crotonobetaine (50-100 gL -1) and glycerol (1-10 gL - one}).

Biotransformation assays with Proteus sp . Permeabilized were performed at a temperature between 35ºC and 40ºC, at a pH between 5.5 and 8.5 with 30-80 mM phosphate buffer, with stirring (100-200 rpm), oxygen (air) supply, and in discontinuous reactors (48 hours and in two phases). After the first biotransformation cycle the final volumes of each experiment were divided into 2 equal samples that were centrifuged under the same conditions. The cells were resuspended in two different media, on the one hand, 60 mM phosphate buffer pH 7.5 at 4 ° C (storage) and, on the other hand, Proteus sp. constituted by yeast extract (1-30 gL -1), K 2 HPO 4 (1-10 gL -1), KH 2 PO 4 (1.40 gL 1) -1}) and crotonobetaine (10 mM). The cultures were centrifuged again under the same conditions and a new biotransformation cycle was performed under the previous conditions. After the second cycle of biotransformation, a third cycle of biotransformation was performed under the same conditions. The results obtained are shown in Tables 9 and 10.

TABLE 9 Conversion and productivity of L-carnitine in biotransformation media in presence of a crotonobetaine concentration of 100 g.L -1 [Storage of cells in 60 mM phosphate buffer pH 7.5 a 4 ° C]

Triton X-100 (g.L -1) No. of cycles 0 0.5 0.1 0.2 0.3 0.5 1st Conversion 45.5 73.9 69.2 73.9 89.3 65.0 Productivity 1.29 2.04 2.02 2.16 2.30 1.23 2nd Conversion 31.0 47.5 48.6 41.3 61.4 0 Productivity 2.26 3.49 3.02 3.01 2.99 0 3rd Conversion 11.1 10.4 13.5 8.4 8.4 0 Productivity 0.73 0.68 0.93 0.56 0.56 0 Conversion (%); Productivity (g.L <-1> .h <-1> .g <-1> cell)

TABLE 10 Conversion and productivity values of L-carnitine in biotransformation media in presence of a crotonobetaine concentration of 100 g.L -1 [Storage of cells in growth medium a 4 ° C]

Triton X-100 (g.L -1) No. of cycles 0 0.5 0.1 0.2 0.3 0.5 1st Conversion 44.5 73.9 69.2 85.9 88.3 65.0 Productivity 1.29 2.04 2.02 2.16 2.10 1.23 2nd Conversion 40.5 46.1 31.6 42.8 55.4 0 Productivity 2.67 3.22 2.32 2.59 2.99 0 3rd Conversion 28.1 33.4 15.8 26.1 27.3 0 Productivity 1.73 2.00 1.04 1.57 1.62 0 Conversion (%); Productivity (g.L <-1> .h <-1> .g <-1> cell)

In Tables 9 and 10 it can be seen that the conversion and productivity values reached in the second cycle for Triton X-100 concentrations of 0.05, 0.2 and 0.3 gil were greater than 40% and 2.5 g. L <-1> .h-1 .g <-1>, respectively. Also, in said Tables 9 and 10 it can be seen that even in the third cycle, it was possible to obtain a productivity of 2 g.L -1 - .h - 1. g - 1 for a Triton X-100 concentration of 0.05 g.L -1.

Example 9 Production of γ-butyrobetaine by Proteus sp . permeabilized at different concentrations of Triton X-100

Following the procedure described in Example 5 but using Proteus sp . Permeabilized with different concentrations of Triton X-100 (0-0.5 gL -1), the production of γ-butyrobetaine was analyzed. The results obtained are shown in Table 11.

TABLE 11 Production of γ-butyrobetaine by Proteus sp . Permeabilized with Triton X-100

Triton X-100 (g.L -1) 0 0.05 0.1 0.2 0.3 0.5 Conversion (%) 44.1 60.8 79.4 80.5 90.1 66.9 γ-butyrobetaine (g.L -1) 2.9 1.5 ND ND ND ND ND: No detected

The results show that Triton X-100 concentrations greater than 0.05 g.L -1 was not produced? -butyrobetaine, so which means superior performance in L-carnitine and a process "downstream" more easy since the product is practically clean after centrifuge cell pellets.

Crotonobetaine and γ-butyrobetaine were determined by high performance liquid chromatography (HPLC) with a column Tracer Spherisorb-NH2 3 µm, 25 x 0.46 cm, supplied by Teknokroma (Spain) in isocratic conditions with a mobile phase composed of acetonitrile / H 3 PO 4 0.05 M pH 5.5 (65/35) and a flow rate of 1 ml / min. The L-carnitine was determined by testing the carnitine acetyltransferase (Enzyme Microbiol. Technol., 2001, 28: 785-791).

Claims (16)

1. A process for the production of L-carnitine, from a biotransformation substrate selected from crotonobetaine, a crotonobetaine salt, a crotonobetaine derivative, D-carnitine, a D-carnitine salt, a D-carnitine derivative , and mixtures thereof, which comprises culturing cells permeabilized from Escherichia coli or Proteus sp ., in a culture medium containing said biotransformation substrate and the nutrients necessary to maintain the viability of said cells, under conditions that allow production of L-carnitine. 2. A method according to claim 1, wherein said permeabilized cells of E. coli or Proteus sp . they are grown in a culture medium comprising said biotransformation substrate at a concentration between 25 mM and 1 M. 3. The method according to claim 1, wherein said E. coli permeabilized cells are cultured at a pH between 4.5 and 8.5, in a culture medium comprising said biotransformation substrate under aerobic conditions. 4. The method according to claim 1, wherein said permeabilized cells of Proteus sp . they are grown at a pH between 4.5 and 8.5, in a culture medium comprising said biotransformation substrate under aerobic conditions. 5. The method according to claim 1, wherein said permeabilized cells of E. coli or Proteus sp . they are grown in a culture medium comprising a growth activating compound of said cells. 6. The method according to claim 5, wherein said growth activating compound of said permeabilized cells of E. coli or Proteus sp . it is selected from glycerin, glucose, ribose, sucrose, lactose and mixtures thereof. 7. Method according to claim 1, in the that said culture medium comprises an inhibitor of transformation of crotonobetaine in γ-butyrobetaine. 8. Method according to claim 7, in the that said crotonobetaine transformation inhibitor in γ-butyrobetaine is selected from fumarate, oxygen, carbon dioxide, nitrate, glucose and their mixtures 9. Method according to claim 1, in the that said culture medium comprises an enzyme inducer involved in the metabolism of L-carnitine. 10. Method according to claim 9, in which said inducer of the enzymes involved in metabolism of the L-carnitine is selected from crotonobetaine, a salt of crotonobetaine, a derivative of crotonobetaine, D-carnitine, a salt of D-carnitine, a derivative of D-carnitine, DL-carnitine, a salt of DL-carnitine, a derivative of DL-carnitine, and mixtures thereof. 11. A method according to claim 1, wherein said permeabilized cells of E. coli or Proteus sp . They are in a state of growth. 12. A method according to claim 1, wherein said permeabilized cells of E. coli or Proteus sp . They are in a state of rest. 13. A method according to claim 1, wherein said permeabilized cells of E. coli or Proteus sp . They are free. 14. A method according to claim 1, wherein said permeabilized cells of E. coli or Proteus sp . They are immobilized on a support. 15. The method according to claim 1, wherein the culture of said permeabilized cells of E. coli or Proteus sp . it is carried out in a reactor selected from a reactor that operates continuously, a reactor that operates in semi-continuous and a reactor that operates in discontinuous (lots). 16. Procedure according to. claim 1, that comprises performing one or more biotransformation cycles of biotransformation substrate in L-carnitine