WO1998018904A1

WO1998018904A1 - Method for separating and concentrating small molecules using enzymatic membrane pumps: separator/concentrator reactor

Info

Publication number: WO1998018904A1
Application number: PCT/FR1997/001936
Authority: WO
Inventors: Bernard Maisterrena
Original assignee: Universite Claude Bernard Lyon I
Priority date: 1996-10-31
Filing date: 1997-10-29
Publication date: 1998-05-07
Also published as: AU4952697A; FR2755036A1; FR2755036B1

Abstract

The invention concerns a method for separating and superconcentrating of small molecules in liquid phase, by passing them through a biologically active porous membrane (5). This method consists in the following steps: producing enzymatic pumping of the product to be concentrated (Pac) through the membrane (5), delimiting two basins one for starting liquid CI and for final liquid for CII. A couple of reversing enzymes E1/E2 are provided, E1 being fixed on the face of the membrane (5) opposite CI and E2 on the other face; causing E1 or E2 to transform the Pac into an intermediate migrant (IM) capable of passing through the membrane (5) to be subsequently transformed into Pac by the reversing enzyme CII. Liquids CI/CII are moved to produce non-turbulent diffusion layers adjacent to the two faces of the membrane (5); in the event the IM is electrically charged, a current of a kind opposite to that of this IM is provided for the membrane, whereas in the event the IM is neutral, the membrane (5) is provided with the same electric charge as that of the Pac. Finally the accumulated and superconcentrated Pac is collected in CII. The invention also concerns the separator/concentrator reactor (1) for implementing said method.

Description

PROCESS FOR SEPARATION AND CONCENTRATION OF SMALL MOLECULES USING MEMBRANE ENZYMATIC PUMPS: SEPARATOR / CONCENTRATOR REACTOR

TECHNICAL AREA :

The field of the present invention is that of the separation and over-concentration of small molecules in the liquid phase, by passage through a porous bioactive membrane. In other words, it is the active transport of certain solutes that one seeks to isolate and / or to over-concentrate.

More specifically, the invention refers to the techniques of purification / separation / concentration of molecules - generally small - and present in liquid media in the trace state. Such highly diluted molecules may be advantageous to purify or to isolate either because they have a high added value, for example in the pharmaceutical industry, or because they constitute an undesirable polluting load in these liquid media, or because they are below the analytical detection threshold.

The invention thus relates specifically to a process and a device for separating and concentrating at least one chemical compound, hereinafter referred to as the product to be concentrated = Pac, present in a liquid medium in dissolved form.

STATE OF THE ART:

The techniques of over-concentration known to date and implemented industrially, hardly and imperfectly meet the objectives mentioned above.

These known techniques involve passive, porous membranes, through which the molecules diffuse under the effect of an overpressure. Such forced filtration technology is not very selective and ineffective in terms of over-concentration ratios, especially for small molecules. The techniques of overconcentration forced by overpressure are difficult to implement, restrictive for the material including in particular the membranes and extremely energy consuming, therefore costly.

Dialysis is also known by passive diffusion of solute through a porous membrane. Dialysis is only effective for molecules present in significant quantities on one side of the membrane and capable of crossing the latter under the effect of a concentration gradient. It is clear that dialysis does not allow molecules that are present in trace amounts to be over-concentrated in a liquid on the other side of the membrane. It is therefore necessary to note the deficiency, in the state of the art, in means of over-concentration of small molecules (Molecular Weight <1000 Daltons) present in liquid media - more particularly in very small quantities - and whose over-concentration is exploitable: in recovery when it comes to molecules with high added value (chemical and / or pharmaceutical),

- in depollution when toxic and / or undesirable molecules are involved)

- And in the detection of said molecules present in trace amounts.

BRIEF DESCRIPTION OF THE INVENTION

In this state of affairs, one of the essential objectives of the present invention is to remedy this deficiency, by providing a process for the separation and concentration of at least one Pac concentrating product, present in a liquid medium in dissolved form. More specifically, the aim of this process would be to migrate the Pac Product from a starting compartment of the liquid medium weakly concentrated in Pac, to another compartment arriving from the liquid medium, in which it would accumulate in such a way that its concentration in said arrival compartment increases. Another essential objective of the invention is to provide a process of separation / over-concentration of one or more solutes, which is selective and which makes it possible to obtain a pure or substantially pure “over-concentrate”.

Another essential objective of the present invention is to provide a method of separation / over-concentration of at least one Pac solute, leading to over-concentration ratios from a departure compartment to an arrival compartment, clearly greater than 1.

Another essential objective of the invention is to provide a separation / over-concentration process for a Pac solute, which is simple to implement and economical.

Another essential objective of the present invention is to provide a device for separating and concentrating at least one Pac concentrating product, present in a liquid medium in dissolved form, in particular for implementing the method as mentioned above. Having set these objectives, the inventor had the merit of highlighting, after long and painstaking research and experiments, the fact that it was possible to implement an active transport system of a Pac solute, from a starting liquid compartment to another incoming liquid compartment, by enzymatic pumping through a porous membrane. This principle highlighted by the inventor is based on the intervention of a pair of reversible enzymes, one of the pair's enzymes being fixed on one side of the porous membrane and the other enzyme on the opposite side of said membrane. The product or the substrate linked to this pair of enzymes is loaded with a sign that is either opposite or identical to that of at least one of the faces of the membrane. The transfer of a Pac compound is carried out in fact through its enzymatic transformation product by one of the enzymes of the couple used. Throughout this presentation, this transformation product will be referred to as a “migratory intermediary”. If the migratory intermediate is charged, then the membrane will be of an opposite sign so as to favor the transmembrane diffusion of this charged product. If the migrating intermediate is not loaded, then the membrane is loaded with the same sign as the Pac compound which one seeks to over-concentrate, so as to produce a valve effect opposing the return of the Pac product in the departure compartment. Thus, the present invention relates to a simultaneous process for the separation and concentration of at least one Concentrated Product (PAC), present in a liquid medium in solubilized form, characterized in that it consists essentially and successively or not : - to select at least one pair of reversible enzymes E _j / E ₂ ,

E _j being able to catalyze the reaction of transformation of at least one substrate A into at least one product B and E ₂ the reverse reaction of transformation of the substrate (s) B into at least one product A:

A —B ^E2 so

* whether A or B corresponds to Pac

* and that at least one of the compounds A or B is electrically charged, - to provide at least one porous membrane comprising E, and E ₂ , respectively, on and / or in one and the other of its faces ,

- immersing the porous membrane (s) in the liquid medium, each membrane delimiting at least in part at least one liquid compartment C, and at least one liquid compartment C _π ,

- moving at least one of the liquids - preferably the liquids - of the compartments C, / C _π , by providing at least one non-turbulent diffusion layer, adjoining one of the faces of the membrane (s) , preferably a diffusion layer adjoining each face of the membrane (s),

- to set the operating conditions so that:

.1. in compartment C, or starting C _π , at least one enzymatic transformation with E, or E ₂ , of the compound Pac start corresponding to A or B, in metabolite B or A respectively as appropriate:

E ₂ .2. the migration of Pac metabolized by E, or E ₂ migrating intermediate through the pores of the membrane to pass from a compartment C _j or C _π to the other C _1I or C, this migration intervening in particular under the effect of a concentration gradient, with the conditions according to which:

If the migrating intermediary is an electrically charged compound A or B, then each membrane is charged with the opposite sign on at least one of its faces, preferably this adjacent to the face carrying the enzyme having transformed the Pac,

• and if the migrating intermediary is an electrically neutral compound A or B then each membrane is electrically charged with the same sign as the other non-metabolized and non-migrating compound A or B (Pac),

.3. then at least one reverse enzymatic transformation, by E ₂ or Ei, of the migrating intermediate (B or A), into starting Pac, in such a way that the latter accumulates and is concentrated in the compartment C _π or C _j of arrival, - to recover the Pac thus accumulated and over-concentrated. The innovative technical principle which governs the process according to the invention can be assimilated to an enzymatic pumping mechanism making it possible to transfer a diluted Pac solute in a starting liquid compartment, to a liquid arrival compartment, by active transport through of an enzymatic porous membrane. The Pac solute thus accumulates and over-concentrates in the arrival compartment.

Such an enzymatic pumping process is particularly advantageous since it allows a selective over-concentration of one or more Pac solutes, and this without excessive energy consumption, unlike the system of over-concentration by overpressure. Indeed, this enzymatic pump essentially only requires chemical energy, which can be provided for example by molecules with an energy-rich bond, such as adenosine triphosphate (ATP). Another advantage of the method according to the invention is to lead to an over-concentration of Pac in relatively pure form, that is to say unpolluted by the migratory intermediate.

The development of the method according to the invention finds its root, in particular, in understanding the phenomenon of active transport specific to biological membranes. This new scientific theory on biological transmembrane transport brings new blood to the state of scientific knowledge on the subject. The latter can be summarized in the theory based on pores and in the theory based on the conformational transformation of proteins of biological membranes. The new and innovative approach according to the invention therefore consisted, firstly, of understanding and explaining the biological phenomenon and, secondly, of reproducing it (mimicry) by adapting it to the industrial specifications of separation / over-concentration of chemical compounds, in particular for recovery of molecules with high added value: chemical, pharmaceutical, cosmetic or other, for the purpose of elimination and isolation of toxic compounds, or even for the purpose of lowering thresholds analytical detection so as to make analyzes possible.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the invention, for a given compound to concentrate Pac, it is first of all necessary to choose a pair of reversible enzymes E _X IE ₂ , capable of forming the enzymatic pump allowing the transfer of Pac, via its enzymatic transformation product with E, or E ₂ . The electrically charged nature of at least one of the product / substrate A and B of E, / E ₂ is another essential condition of the invention, knowing that - Compound A or B, useful as a transmembrane migratory intermediate, is neutral or has a charge opposite to that existing on the faces of the membrane; and that the Pac compound is neutral or has an electrical charge of the same sign as that existing on at least one of the faces of the membrane.

The electrical charge of the faces of the porous membrane can be obtained for example, by immobilizing with E _j and E ₂ polyamino acids, the pK of which give the membrane, for a given pH, negative or positive charges. Thus, in the case where the polyamino acid is eg polylysine, then the membrane is positively charged at pH 9, while with a polyamino acid corresponding eg to a polyglutamate, the membrane is negatively charged at pH 9.

According to an alternative for loading the membrane, it is possible to choose membranes whose zeta potential at optimum pH is adapted to given conditions of the process of the invention. This porous membrane must be arranged in such a way that it delimits at least in part, once immersed in the liquid medium, at least one compartment C, - preferably one - and at least one compartment C _π - preferably one.

The fact of providing a non-turbulent diffusion layer adjoining each face of the membrane (s) is an important provision of the method according to the invention.

Preferably, the compartment C _x or C _π from the start of the PAC has a volume greater than that of the compartment C „or C, for arrival. Advantageously, the ratio

Volume of C, or II of arrival / V of C _π or, of departure must be as low as possible, this ratio playing on the kinetics of transport (but not on the final report of over-concentration).

In order for the enzymatic transformations E _j E _{2 to take place} in the vicinity of each of the faces of the membrane (s), it is important to provide not only the diffusion layers mentioned above but also to set up optimal operating conditions, in particular to pH and temperature.

It will be understood that the active transmembrane transport according to the invention, passes by the exploitation of a first effect which one can call “flapper effect” and / or a second effect which can be called the effect of promoting the dissemination of the migratory intermediary.

The valve effect is obtained by ensuring that the Pac, the migration of which it is desired to limit through the membrane, is of a sign opposite to the latter, so as to be repelled by it and thus not having tendency to cross it.

On the other hand, in the promotion of diffusion effect, we play on the opposite character of the signs of the migratory intermediary which crosses the membrane and of the membrane itself, which thus tends to attract the migratory intermediary.

The invention is not limited to the implementation of only one of these effects. It is entirely possible to predict their cumulation.

The steps - 1 - of enzymatic transformation in one direction, - 2 - of transmembrane migration and - 3 - of enzymatic transformation in the other direction, respectively, lead to an over-concentration of Pac in the arrival compartment. It is then easy either to recover the Pac for recovery purposes (evaporation of the solvent) or elimination, or to detect and / or measure the Pac which now titer beyond its analytical detection threshold.

According to a first embodiment of the method according to the invention (which can be described as "discontinuous"), the enzymatic membrane is in the form of a monolayer or multilayer film, preferably substantially planar.

This discontinuous mode corresponds for example to the case in which there is a reactor formed by a first container containing the liquid medium in which a second container is at least partially immersed. The submerged part of this second container being constituted at least in part by the porous enzymatic membrane. These two containers define compartments C, or „.

The membrane can form all or part of the wall of the container. Preferably, this membrane constitutes a substantially planar part of the partition wall between the two compartments C _j and C _π .

According to a second embodiment of the method according to the invention (which will be called "continuous"), the enzymatic membrane is in the form tubular and more precisely constitutes all or part of the wall of a tube, the lumen of which forms one of the compartments C _j / C _u .

Advantageously, the enzymatic membrane can be made of a plurality of hollow fibers or tubular membranes, preferably united in a bundle (x). In accordance with this second continuous mode, at least part of this (or these) tubular membrane (s) is immersed in the liquid medium forming the other compartment Ci C, and a liquid is circulated to be enriched or deplete the Pac compound in this (or these) tubular enzymatic membrane (s).

In practice, in this continuous mode, use is made of a reactor forming one of the compartments C, or C _π containing the liquid medium and the tubular membrane (s) are immersed in this reactor, the circulation of the liquid in the or the tubular membranes constituting the other or the other compartments C _π or ,, being provided by any suitable means such as a pump.

On reading the above, one can easily perceive the importance of the liquid medium of the compartments C _t and C „, and in particular their physicochemical characteristics, for the conduct of the process according to the invention.

Thus, according to another advantageous modality of the method according to the invention, the pH and temperature parameters of the liquid media are adjusted, so as to obtain an optimum in kinetics and in yield for the enzymatic transformations by E, and E ₂ .

In practice, the liquid medium of C and / or Cn is therefore prepared by using a solvent - preferably essentially aqueous - optionally comprising solutes chosen from the following group: o pH buffers adapted to the optimum operating pH of E, and E ₂ , o molecules with energy-rich bonds - advantageously ATP or any molecule capable of transferring a phosphate group onto a solute (for example: phosphoenol pyruvate, carbamyl phosphate, phosphocreatine ...) o and their mixtures, the compositions starting from the liquid media of C _j and C, _j being preferably substantially the same or not of the Pac concentration. In addition to adjusting the pH and supplying the chemical energy source necessary for the enzymatic transformations E, and E ₂ ; the temperature of the liquid media is also regulated by adding or removing calories, eg thermostatically controlled reactor with double wall for circulation of temperature-regulating fluid. Advantageously, it is ensured that the compartment C, / Cι, receiver of the migratory intermediate through the membrane, initially comprises a concentration of this intermediate, lower than that existing in the transmitting compartment.

According to the invention, the diffusion layers are obtained by setting in motion the liquid in each compartment C, / C _π , this setting in motion being effected by stirring - preferably using a rotor - in the first mode of implementation according to the invention and / or by circulation of said liquid according to the second mode of implementation of the method according to the invention.

By means of these, the stirring and / or circulation conditions are adjusted so that the non-turbulent diffusion layer or layers have an identical or different thickness on either side of the membrane and between 1 and 10 times the thickness of the membrane.

In order to improve the kinetics and the transfer performance of the migrating metabolized intermediate, it is possible, in accordance with the invention, to provide for the “flap” and / or “diffusion promotion” effects, auxiliary means of assistance for transmembrane migration of (or of) migratory intermediary (ies), said means preferably being constituted by a gradient of ionic and / or electrical force, on either side of the membrane, and more preferably still by pH gradients (protomotor force). According to a variant of the process of the invention, the overconcentration of the Pac is supplemented by enzymatic pumping of the said Pac through the membrane (s) from a compartment C, or C „containing Pac to a compartment Cn or C, to be enriched with Pac, by implementing a translocation of Pac, the latter consisting in introducing into the starting compartment C _] or C _π loaded with Pac, the migrating intermediate A or B of enzymatic transformation - 1 - of the Pac by E _j or E ₂ , so that this added migratory intermediary migrates cumulatively to that from stage - 1 -, under the same conditions as those of stage - 2 -, to be finally subjected to stage 3 after having crossed the membrane, stages - 1 -, - 2 - , and - 3 - at issue being those as defined above.

Such an association between the enzymatic pumping in accordance with the invention (active transport) and the translocation makes it possible to notably improve the over-concentration of Pac.

The present invention also relates to a device for separating and concentrating at least one Concentrated Product (PAC), present in a liquid medium in solubilized form, in particular for implementing the method as defined above, characterized in what it basically includes:

- at least one porous enzymatic membrane, comprising at least a pair of reversible enzymes E _l IE ₂ , E, being able to catalyze the reaction of transformation of at least one substrate A into at least one product B and E ₂ the reaction reverse of transformation of the substrate (s) B into at least one product

AT :

E ₂ so

* that A or B corresponds to Pac * and that at least one of the compounds A or B is electrically charged, Ei and E ₂ being respectively supported by one and the other of the faces of the membrane,

at least one container of the liquid medium in which the enzyme membrane is bathed or capable of bathing in order to at least partially delimit at least one liquid compartment C, and at least one liquid compartment C _π ,

means for setting in motion at least one of the liquids, preferably liquids from the compartments C _r , C _π ,

- Possibly means for adjusting the temperature of the liquid medium. Advantageously, each membrane is electrically charged, on at least one of its faces, preferably on that adjacent to the face carrying the enzyme which has transformed the Pac, and more preferably still on both.

According to a preferred characteristic of the invention, this enzymatic membrane is a porous matrix, in and / or on the two faces of which are included and immobilized the enzymes Ei, E ₂ respectively, this matrix being formed by at least one macromolecular compound , preferably chosen from proteins, polysaccharides, synthetic (co) polymers and their mixtures and / or alloys, etc., these compounds being chosen for their porosity, thickness, zeta potential (charges) and the ease of grafting of E, / E ₂ , cellulose and its derivatives (eg cellulose acetate, regenerated cellulose) as well as (co) polyamides being particularly preferred.

According to a first embodiment of the device according to the invention, the enzymatic membrane is in the form of a monolayer or multilayer film, preferably substantially planar, once mounted in the device.

According to a second embodiment of the device according to the invention, the enzymatic membrane constitutes all or part of the wall of at least one tube whose lumen forms one of the compartments C, / C _π .

There are many pairs of enzymes that can be used in the process and the device according to the invention. To fix the ideas one can indicate that these pairs Eι / E ₂ are mainly chosen from the pairs of enzymes E _j / E ₂ allowing the addition / removal of a charged chemical group on a metabolite, preferably among the pairs d enzymes E, / E ₂ of phosphorylation / dephosphorylation, and even more preferably among kinases / phosphatases.

INDUSTRIAL APPLICATION:

The method according to the invention can be applied for the purpose of recovering molecules with high added value: chemical, pharmaceutical, cosmetic or other, for the purpose of elimination and isolation of toxic compounds, or else to the purpose of lowering the analytical detection thresholds so as to make analyzes possible.

The invention will be better understood and its advantages or other alternative embodiments will become apparent from the description which follows, of two exemplary embodiments of the device which is the subject of it. This description of device will be supplemented by tests of implementation of the method according to the invention using the two previously exemplified devices each corresponding to a particular mode of implementation of the method of the invention.

DESCRIPTION OF THE FIGURES:

The description of the two embodiments of the device according to the invention will be made with reference to the accompanying drawings in which: Figure 1 shows a simplified diagram of the first embodiment of the separation and concentration device according to the invention (discontinuous mode ).

- Figure 2 is a symbolic representation of the enzymatic membrane separating the two compartments C _j and C _π of the device of Figure 1 or 4, said representation corresponding to a first enzymatic pathway of active transfer through the membrane and overconcentration d '' A compound, from compartment II to compartment I

_Λ E. Membrane _, ^E ι. A - * B>B> A

- Figure 3 is a symbolic representation of the same nature as Figure 2, with the difference that it concerns a second enzymatic pathway for transfer through the membrane and over-concentration of a compound B, from compartment II to compartment I

_, E. _v Membrane,. ^E 2. B - ^ A>A> B - Figure 4 is a simplified diagram illustrating the second embodiment of the separation and concentration device according to the invention (continuous mode).

- Figure 5a shows a simplified view in straight cross section along the line V-V in Figure 4.

- Figure 5b is a magnified view of one of the elements of the tubular membrane seen in section in Figure 5a.

The device for separating and concentrating at least one compound to be concentrated Pac, according to the first embodiment of the invention, is designated in FIG. 1 by the general reference 1. This device or reactor 1 is constituted by a tank 2 thermostatically controlled with a double wall, intended for the circulation of a thermoregulating fluid, between inlet 2 and outlet 2 ₂ .

This tank 2 has, for example, a generally substantially hollow cylindrical shape and can be produced from any suitable material, metallic, plastic (polymer e.g. of the polymethacrylate type).

This tank contains a liquid medium not referenced in the drawing and is also equipped with stirring means 3 symbolically represented and which are advantageously constituted by a rotor, which can be, for example, a magnetic bar operating with a magnetic stirrer, or still a classic stirring propeller.

A tubular body 4 also containing liquid medium, partially bathes in the liquid medium contained in the tank 2. This tubular body 4 advantageously has a circular cross section. The submerged lower end of this tubular body 4 is closed by an enzymatic membrane 5 inserted between two annular peripheral seals 6, made - for example - of paraffinic plastic. The membrane 5 is secured to the tubular body 4 via a lip 7 or an annular rim formed at the free immersed end of said tubular body. The fixing means used are for example screws and nuts 8, eg in Teflon. The tubular body 4 is equipped with stirring means 9, of the paddle type agitator. The liquid levels in the tank 2 (1st container) and in the tubular body 4 (2nd container) are adjusted so that they are identical.

The end enzymatic membrane 5, with the wall of the tube 4, delimits the compartment referenced by C, in Figure 1. This membrane 5 also separates said compartment C, from compartment C _π constituted by the tank 2 filled with liquid medium.

In this example, the internal volume V, of compartment C, = 4 cm ³ , while the volume V „of compartment C _π = 300 cm ³ . According to the invention, the membrane 5 is made of natural polymer, modified or not, or of synthetic polymer. In this case, it is a polyamide membrane sold by Pall Europe limited, Portsmouth England under the reference PallNAZ, in the form of nonwoven material, having a thickness of 100 μm eg The porosity of this membrane is defined by the cut-off threshold, the latter being naturally chosen as a function of the size of the molecules which it is desired to migrate through the membrane in order to over-concentrate them in a compartment. Expressed in maximum molecular weight for the molecules likely to migrate through the pores, this cut-off threshold is advantageously between 500 and 10,000 D, depending on the size of the molecules that one wants to over-concentrate.

According to a variant, this membrane can also be made of regenerated cellulose and is in the form of a nonwoven film with a thickness of between 10 and 100 μm.

Each of the faces of the membrane 5 opposite, respectively, of compartment C, and of compartment C _π , was subjected to a grafting of enzymes E _j E ₂ , forming a pair of reversible enzymes in which the enzyme E _j is able to catalyze the transformation of A into B and the enzyme E ₂ the transformation of B into A. The grafting of the enzyme can be of a covalent nature or even a simple inclusion of the enzyme by absorption within the fibrous polymer matrix. Covalent grafting can be carried out according to conventional techniques. The examples which follow give an illustration of the enzymatic grafting. The symbolic representation of FIG. 2 shows the membrane 5 supporting in its face opposite compartment C _π the enzyme E ₂ and in its facing face of compartment C ,, the enzyme E, of the pair of reversible enzymes E, / E ₂ . This membrane has a thickness d and is adjoined by its carrier face of E ₂ with a diffusion layer δ of thickness δ ₂ , and by its carrier face of E, with another diffusion layer δ of thickness δ,. In the first pathway of enzymatic transformation and transmembrane migration A - B - »B → A illustrated by FIG. 2, component A plays the role of substrate in compartment C„ and therefore of compound to be concentrated Pac in compartment C, . In this Figure 2, the substrate and product A and B are assigned an exponent δ or b depending on whether they are in a diffusion layer δ or in the rest of the compartment (b for bulk in English), respectively. A and B are also identified by a first index in Roman numeral corresponding to the compartment in which it is located, followed by a second index t designating time. In this first way at time t = 0 we have: A ^b _{I1; t} ≠ 0; A ^b , _t = B ^b _{1 t} =

In this first route, it is therefore compound B which serves as a transmembrane migratory intermediary

Regarding the second path shown in Figure 3, the goal is to over-concentrate compartment C, in compound B by taking it from compartment Cn and making it pass through the membrane. In this case, it is therefore compound A, which serves as a transmembrane migratory intermediary. The enzyme E, is this time in the face of the membrane facing compartment C _π and the enzyme E ₂ is included in the face of the membrane facing compartment C ,. The path of transformation

^E 1 E enzymatic and transmembrane migration is therefore: * °.

The implementation of the method according to the invention using the device above exemplified and shown in Figure 1, operates by selecting the pair of reversible enzymes, for example E _t = alkaline phosphatase and E ₂ = glycerol kinase with A = glycerol-3-phosphate ^2- and B = glycerol. After immobilization of these enzymes on the faces of the membrane 5, the stirring conditions of the means 3 and 9 are adjusted. This adjustment can be carried out on the basis of the article by BARDELETTI et al., 1985, whose references are as follows: Bardeletti, G; Maïsterrena, B. Coulet, PR. (1985) J. Membr. Sci 24, 285-296). According to a preferred characteristic of the invention, the thickness of the diffusion layers δ, and δ ₂ is between 50 and 500 μm, according to the hyrodynamic conditions.

According to another arrangement of the process of the invention, the volume ratio between the compartments C 1 and C ₁₁ is chosen (C, playing the role of inlet compartment (or receiver) in which the over-concentration in Pac occurs) , so that this ratio is as low as possible if you want to over-concentrate the Pac in a short time.

Preferably, the composition of the liquid medium in compartments C, and C _π at time t = 0, is substantially the same, including as regards compound A or B to be over-concentrated (Pac).

It also provides the conditions of pH, temperature and chemical energy source, necessary for enzymes E, E ₂ to carry out enzymatic transformations. Thus, in the case of the pair of alkaline phosphatase / glycerol kinase enzymes, the pH is between 8 and 10 (buffer), the temperature is between 20 and 30 ° C, preferably of the order of 25 ° C and a source of ATP is introduced into the medium in the form of an aqueous saline solution (ATP / MgCl ₂ ).

As soon as the membrane has been immersed in the liquid medium, by fixing the other parameters set out above, the enzymatic pumping according to the invention takes place and in the end, recovery is made in compartment C ,, of the compound A = ² "glycerol-3-phosphate or of compound B = glycerol.

In the case where the Pac compound is loaded, it is appropriate that at least one of the faces of the membrane, preferably both, are loaded with the same sign. This is the case for Pac = A = glycerol-3 -phosphate ^2- , the membrane then also being negatively charged so as to have a valve effect and thus to avoid the parasitic backscattering of A through the membrane. In the case where the migratory intermediate is loaded, then an opposite charge is provided on at least one of the faces of the membrane, preferably so as to promote the transmembrane diffusion of this migratory intermediate. It is thus for Pac = B = glycerol and A = glycerol-3-phosphate ^2- . In accordance with the invention, it cannot be excluded that the two scenarios referred to above are cumulative, that is to say that A and B are of opposite signs and that that constituting the migratory intermediary is of sign opposite to the membrane, while the compound corresponding to Pac has the same sign as the membrane.

As already indicated above, according to an advantageous variant of the method according to the invention, it is possible to associate or combine the enzymatic pumping of a Pac A or B compound by providing for a translocation. This manipulation consists in incorporating the migrating intermediate in compartment C, or C _n of the pumping start. In the first path Figure 2, we will therefore provide an initial non-zero concentration of compound B in compartment C _π and in the second path Figure 3, the initial concentration of compound A in compartment C ,, will likewise be greater than 0.

It will be noted that in the two enzymatic pathways 1 and 2 according to FIGS. 2 and 3, the Pac also crosses the membrane by direct diffusion to a much lesser extent (dotted arrows in Fig. 2 and 3). The second embodiment of the device according to the invention is shown in Figure 4, in which it is designated by the general reference 10. This device 10 for separation and concentration of at least one compound to be concentrated Pac, present in a liquid medium in solubilized form, can be assimilated to a separator / concentrator reactor, capable of operating in a continuous mode. This reactor 10 comprises a thermostated tank 11 of the same type as that (2) described in the first embodiment of Figure 1. The references ll _j and 11 ₂ respectively denote the inlet and the outlet of the circulation circuit of a thermoregulating fluid in the jacket of the tank 11.

This tank 11 contains the liquid medium initially containing the compound Pac and constituting the liquid compartment C _π . Means 12 of mechanical agitation are provided within this liquid medium forming C ,,. These stirring means 12 are of the same type as those described for the first embodiment shown in the

Figure 1 and designated by reference 9 in this figure.

In this second embodiment, the enzymatic membrane consists of a plurality of tubes 13 with porous walls and grouped together to form a bundle 14, part of which is immersed in the liquid medium of the tank.

11 (Cπ).

In fact, the lights of the tubes 13 constitute as many compartments C, liquid, involved in the method according to the invention.

The arrows indicated in Figure 4 in the beam 13 gives the direction of flow of the flow of the compartments C _τ .

The section in FIG. 5a shows the bundle 14 of tubular enzymatic membranes 13.

This bundle 14 has a curved U-shaped part, the base of which dips into the liquid compartment C _π defined by the tank 11. This tubular bundle 14 is intended to allow the circulation of liquid medium to be over-concentrated in Pac by active transmembrane migration. To this end, the bundle 14 is equipped with circulating means 15 constituted, for example, by a pump.

Figure 5b is a right cross-sectional view of a tubular enzymatic membrane 13 constituting the bundle 14 and delimiting through its wall, on the one hand, an internal compartment C (tube lumen), and on the other hand, as regards the submerged part of the bundle 14, an external compartment C _π , defined by the tank 11. Like the membrane 5 of FIG. 1, the wall of the tube 13 comprises on each of these faces internal and external an enzyme E, and E ₂ respectively, constituting a pair of reversible enzymes E _l IE ₂ . The methods of fixing and immobilizing the enzymes are the same as those described above. In addition, the constituent materials of these tubular membranes are also of the same nature as those mentioned for membrane 5 of FIG. 1. In practice, this will be, for example, regenerated cellulose of porous nature, having a cutoff threshold which can vary from 500 to 10,000 D. The agitating means 12 of the tank 11 as well as the setting in motion of the liquid of C _! in the hollow fibers 13 using the pump 15, allow define diffusion layers δl and δ2, shown in Figure 5b. The latter also shows the internal diameter D of the hollow fiber 13, as well as the thickness d of the membrane wall charged with enzymes E _j E ^

Preferably, the multiple compartments C _r constituted by the holes in the hollow fibers 13 serve as the seat for the over-concentration in Pac originating from the compartment C _π .

The sum of the volumes of the compartments C _τ immersed in C _π is less than the volume V _π of the compartment C _π . The ratio VV _π is as low as possible. The tubular membranes 13 can be electrically charged in positive or in negative, so as to ensure the valve effect and the promoter effect of transmembrane diffusion of the substrates and of the products A and B charged.

According to a variant, the liquid medium of over-concentration of the Pac. could be C _π , that is to say the liquid medium contained in the tank 11, the lights C, tubes 13 forming the starting compartment of the Pac.

This second embodiment of the device according to the invention is suitable for the second form of continuous implementation of the separation / over-concentration process. We find the methodology used for the first discontinuous mode of implementation, with the difference that the liquid medium of the compartments C, circulates in the hollow enzymatic fibers 13, the active transmembrane transport of Pac occurring naturally at the level of the part immersed in the beam 14 in the liquid compartment C _π of the tank 11.

The speed of circulation of the liquid medium of the compartments C, is adapted to the total surface of the exchange zone in the submerged part, the kinetics of enzymatic transformation E, / E ₂ , as well as the rates of transmembrane diffusion of the products A or B loaded or not.

The examples which follow illustrate the device and the method according to the invention in their two preferred but not exclusive modes of implementation. These examples will make it possible to better understand the invention and to grasp all of its advantages and variant embodiments. EXAMPLES

EXAMPLE I: Separation / over-concentration of glycerolSP ² - in a compartment C „by enzymatic pumping from a compartment c _u , according to the 1st embodiment of the process of the invention and using the device of the invention in its 1st embodiment - 1. 1. Material

The device used is that described above and shown in FIG. 1.

The membranes used are membranes of the Pall NAZ type sold by the company PALL EUROP LIMITED. These are polyamide membranes with a thickness = 100 μm of active transfer area (Aw) of 12% and referenced under the number 09025.

The enzyme E _j = glycerolkinase - GK - (EC2.7.1.30) coming from the species celhdomonas (52 units.mg ^-1 , refg. 6142). E ₂ = alkaline phosphatase (EC3.1.3.1.) Extracted from bovine intestinal mucosa (1200

Units.mg ^-1 , ref. P 6672).

The enzymes E and E ₂ defined above are marketed by the company SIGMA.

The immobilization of the enzymes on the membrane operates as follows:

The membranes are in the form of discs of 5 cm in diameter which are activated using a solution of sulfliric acid in methanol under reflux for

6 hours. They are then subjected to the action of a 2% glutaraldehyde solution

V / V, as described in the article by Michalon, P., Couturier, R., Hacques, M-F.,

Favre-Bonvin, G, Ville, A. & Marion, C. (1990) Biochem. Biophys. Res. Common,

167, 9-15. The grafting of enzymes E, = GK and E ₂ = alkaline phosphatase is carried out by immersing the discs at room temperature for 3 hours in 5 cm ³ of an enzyme solution titrating 2 mg. cm ^-3 and 0.2 mg. cm ^-3 for GK and phosphatase respectively, in 0.1 M borate buffer, pH 8.5.

The volume of C _j = 4 cm3 and the volume of C _π = 300 cm3. 1.2. Operating conditions

The liquid media in C _j and C _π have the same composition with the exception of the concentrations of compound A = glycerol-3-phosphate ^2- and of compound B = glycerol. The temperature of the liquid medium is thermostatically controlled by the tank at 25 ° C.

The other operating conditions vary according to the tests carried out.

1.3. Tests 1 to 3 according to the 1st enzymatic route of FIG. 2:

E membrane E, _(Cι) A → B »B> A (C„) For these tests 1 to 3, the liquid media C, / C _π contain 0.1 M borate buffer in an amount such that the pH is l '' of 9 and ATP Mg Cl ₂ - 7 mM. At pH 9, the electrical resistance of the membrane vis-à-vis the substrate A (r ψ A), which is added to the passive resistance of the membrane vis-à-vis the diffusion of the substrate A in compartment C _π (RmA), is around 300 min.cm ^-3 . The stirring conditions are as follows: W _j = 70 rpm (compartment I) W ₂ = 80 rpm (compartment II).

Under these conditions, the diffusion layers δ, and δ ₂ are respectively 100 μm and 150 μm.

The initial concentrations are as follows: Test 1:

= 0.6 mM, B ^b „ ₀ = A ^b , ₀ = B ^b , _t = 0 enzymatic pumping channel 1 Test 2:

= 0.7 mM, A ^b „ ₀ = B ^b , ₀ = A ^b , _t = 0 Translocation of B = glycerol from C, to C _π with transformation

B ^E 1 v 2 A = G3P ² Test 3: A _{l 0} ≈ 0.6 mM, B ^b _I10 = 0.7 mM,

enzymatic pumping channel 1 + translocation similar to that of test 2. In these tests, the concentrations A ^b _{π 0} , B ^b _{π 0} , B ^b , ₀ → A ^b _nt , B ^b , _jt , B are continuously measured ^b ,, using analyzers with amperometric electrodes.

Figures 6, 7 and 8 appended are graphs showing the evolution of the concentrations of G3P ^2- and Glycerol in compartments C, and C ,,, for tests 1 to 3 respectively.

Figure 6 clearly shows the over-concentration of G3P ^2- (A) in C ,, obtained by active transmembrane transport of Glycerol (B), acting as an intermediary E migratory and resulting from the transformation ^A ' ^B in C _π . The translocation (test 2) combined with the enzymatic pumping (test 1) makes it possible to further improve the over-concentration in A (G3P ^2- ) in C, (test 3).

EXAMPLE II: Separation / over-concentration of glycerol 3-phosphate ^2- and glycerol in C ,, by using the enzymatic pathways 1 and 2, (FIGS. 2 and 3) respectively, with the same methodology as that used in example 1.

2. 1. Material

The device used is that described above and shown in FIG. 1. For the description of membranes and enzymes, reference is made to the point

1.1. supra.

2.2. Operating conditions

The liquid media in C and C _π have the same composition, with the exception of the concentrations of compound A = glycerol-3-phosphate ^2- and of compound B = glycerol. The temperature of the liquid medium is thermostatically controlled by the tank at 25 ° C. The other operating conditions vary depending on the tests carried out.

2.3. Tests 4 to 6: Channel 1 Transport of A from C _n into C, membrane E,

(Ci) ^A → ^BB - * ^{A (C} ">

The configuration of the enzymatic membrane is the same as that of Example 1, the membrane is electrically charged so that: r ψ A = electrical resistance of the membrane with respect to A = + 300 min. cm ^-3 . r ψ B = electrical resistance of the membrane against B = 0.

The diffusion coefficients of A and B across the membrane are as follows:

D _A = 4.09 10 ^-4 cm .min ^-1 . D _B = 5.05.10 ^-4 cm ² .min- ¹ .

The 1st order speed constants with respect to A for EE ₂ , are: k, = 3.4 cm ³ , min ^-1 k ₂ = 0.6 cm ³ . min ^-1 the thickness of the membrane d = 2,4.10 ^-2 cm The pore surface of the membrane is Aw = 0.38 cm ² . The surface of the membrane is A = 3.14 cm ² .

The volumes of C, / C _π are V _t = 4 cm ³ and V ₂ = 300 cm ³ . δl = 1.10 ^-2 cm δ2 = l, 5.10- cm.

In the. tests 4 to 6, the initial and final concentrations (mol. cm ^-3 ) in A and B therefore C ,, C _π are given in Table 1 below: Table 1

5 = translocation of B from C ,, to A in C ,.

6 = 4 + 5.

-> Tests 7 to 9: transport of B from C _π into C,: Path 2

These are the same conditions as for tests 4 to 6 except with regard to r ψ A and r ψ B. c membrane E „

- A - B (C,)

(Cn) B - A

All other things being equal, the configuration of channel 2 differs from that according to channel 1 (tests 4 to 6), in that:

E, is in the face of the membrane opposite C _π and E ₂ in that facing C ,. The initial and final concentrations (mol. Cm ^-3 ) measured in B and A in C _π and C, are given in Table 2: Table 2

7 = enzymatic pumping of B from C _π into C,

8 = translocation of A from C „to B in C ,. 9 = 7 + 8.

EXAMPLE III Separation / over-concentration of 3-p ^2- glycerol from a compartment C, by enzymatic pumping according to the second embodiment of the method of the invention and using the device of the invention in its second embodiment

3. 1. Material

The device used is that described above and shown in FIG. 4.

Bundle 14 is made up of 88 hollow fibers 13 made of regenerated cellulose

(spectra / Por ^® ) 25 cm long, totaling 2200 cm in fiber length. The cutoff threshold of the wall of these fibers 13 which forms the enzymatic membrane is 6,000 D (90% molecules of PM = 6000 D are retained) D diameter of a fiber =

240 μm, d = 40 μm.

The total internal volume of the beam = 1 ml. A (total area) = 166 cm ² . The porosity is 12%, i.e. an active transfer surface of Aw = 20 cm ² . Enzymes: E ₂ = alkaline phosphatase

E, = glycerol kinase. These enzymes are of the same nature as those of Examples I and II. The grafting of E _j on the internal face of the wall of the fibers 13 takes place as follows: The interior of the fibers is hydrolyzed by filling with 0.1 M KOH for 24 hours. The KOH is used to de-esterify. The fibers are then rinsed with distilled water. Then they are filled with 0.25 M sodium periodate, in the dark, for varying periods of time. At the end of the incubation, they are rinsed with distilled water, to remove the excess periodate. The fibers are then filled with 15% (w / v) urea to which is added 0.9% (v / v) sulfliric acid. The whole is incubated in a water bath at 45 ° C. The fibers are then washed until the rinsing water is neutral and then incubated for 16 hours in a water bath at 45 ° C with constant shaking. in formaldehyde 12.5% (v / v) with phosphate buffer 0, IM pH7.5. The excess formaldehyde is removed by rinsing with distilled water.

E is then fixed inside the fibers, the latter being brought to a concentration of 1 mg.ml ^-1 , in 0.1 M phosphate buffer pH7.5. The incubation lasts about 24 hours, with gentle shaking. The fibers are then rinsed with distilled water and then put in KCl IM for 15 minutes with gentle stirring. They are rinsed again, then put back for 15 minutes in KCl IM and rinsed one last time.

The same steps are reproduced for grafting E ₂ outside the fibers, which are no longer filled but submerged. The characteristics of fibers grafted with E and E ₂ are as follows: E _j and E ₂ activity of 25 n mol. min ^-1 cm ^-1 of fiber length, i.e. 25.10 ^-9 mol. min ^-1 x 2200 cm = 55.10 ⁶ mol.min ^-1

* E ₂ = alkaline phosphatase (Km = 6.10 ^-6 mol.cm ^-3 ) (k = Vm / Km) k ₂ = 55.10- ⁶ /6.10 ^-6 = 9, l cm ³ min ^-1 .

* E, = glycerol kinase (Km = 7.10 ^-8 mol.cm ^-3 ) (k _! = 55.10 ^-6 /7.10 ^-8 = 785 cm ³ .min ^-1 ).

D _A = 5.05.10 ^-4 cm ² .min ¹ . D _B = 4,09.10- ⁴ cm ² .min ^-1 . * δ ₂ = 150 μm = l, 5.10 ^-2 cm. * δ, = 10 μm = 1.10- cm. * V, = 0.83 ml (by subtracting the volume corresponding to δ,). 3.2. Tests 10 to 12: Variation in the tubular enzymatic membrane load active transport of:

(n) (I) membrane E ₁

,. -. A - → BBA (C „). . . „, Outside fibers"'inside fibers. For these tests, the experimental parameters are as follows: k, = 7.85 x 10 ² cm ³ x min ^-1 k ₂ = 9, l cm ^3. Min ^-1 . D = 4.10- ³ cm

Aw = 20 cm ² A = 166 cm ² δ, = 1.10 ^-3 cm

D _A = 4.09 x 10 ^-4 cm ² .min ^-1 .

D _B = 5.05 .10- ⁴ cm .min ^-1 .

V, = 0.83 cm ³ δ ₂ = l, 5.10 ^-2 cm rΨ _A = 2 min. cm ^-3 test 10 5 min. cm ^-3 test 10 10 min. cm ^-3 test 12 rΨ _B = 0 min. cm ^-3 The concentrations (mol x cm ^-3 ) of A (G3P ^-2 ) and B (Glycerol) in C _π (tank 11) and C, (light immersed fibers) are given in Table 3 below. after p.27

Figure 9 attached also reports these results.

3.3. Tests 13 to 1 - Variation in membrane charge * active transport A (C „) → A (C,) * + translocation B (C _π ) → A (C,)

The parameters are identical to those of tests 10 to 12.

The results are given in Table 4 below on p.28.

Figure 10 attached also reports these results. EXAMPLE IV: Separation / over-concentration of glycerol (B) from a compartment C _π to a compartment C, by enzymatic pumping according to the second embodiment of the process of the invention and using the device of the invention in its second embodiment 4. 1. Material and experimental conditions

The device used is the same as that of Example 3, with the difference that, E ₂ is grafted inside the fibers 13 and E _{ outside. The experimental conditions are the same as in Example 3, with the exception of the following parameters: k, = 9.1 cm ³ min ^-1 .

7.85.10 ² cm ³ .min- ".

D _B = 5.05.10 ^-4 cm ² .min ^-1 . D _A = 4,09.10- ⁴ cm ² .min ^-1 . rΨ _B = 0 min.cm ^-3 rΨ _A ⁼ variable.

4.2. Tests 15 to 17: Variation in load and porosity of the tubular membrane active transport B from C _π to C,: test 15: Aw = 20cm ² and rΨ _A = - 0.4 min.cm ^-3 test 16: Aw = 20cm ² and rΨ _A = - 0.48 min.cm ³ test 17: Aw = 2cm ² and rΨ _A = - 0.48 min.cm- ³

The results are given in Table 5 below p.29.

4.3. Tests 18 to 20 - Load and porosity variation in enzymatic tubular membranes: active transport B (C _π ) → B (C _r ) + translocation A (Cn) → BA (C,)

These are the same conditions as in tests 15 to 17. B ^b _{I 0} = 0. The results are given in Table 6 below p.30. These results are also given in Figure 11. Table 3

Table 4

Table 5

Table 6

Claims

CLAIMS:

1 - Process for the separation and concentration of at least one Concentrated Product (PAC), present in a liquid medium in solubilized form, characterized in that it consists essentially and successively or not: - of selecting at least one pair d 'reversible enzymes E, / E ₂ ,

E, being capable of catalyzing the reaction of transformation of at least one substrate A into at least one product B and E ₂ the reverse reaction of transformation of the substrate (s) B into at least one product A:

A - B ^E 2 so

* whether A or B corresponds to Pac

* and that at least one of the compounds A or B is electrically charged, - to provide at least one porous membrane comprising E _j and E ₂ , respectively, on and / or in one and the other of its faces , immersing the porous membrane (s) in the liquid medium, each membrane defining at least in part at least one liquid compartment C, and at least one liquid compartment C _π , to set in motion at least one of the liquids - preferably liquids - from compartments C, / C _π , by providing at least one non-turbulent diffusion layer, adjoining one of the faces of the membrane (s), preferably a diffusion layer adjoining each side of the membrane (s), fix the operating conditions so that: .1. in compartment C, or starting C _π , at least one enzymatic transformation with E _l or E ₂ , of the compound Pac start corresponding to A or B, in metabolite B or A respectively as appropriate:

.2. the migration of the Pac metabolized by E, or E ₂ (migratory intermediate) through the pores of the membrane to pass from a compartment C, or C _π to the other C _π or C ,, this migration occurring in particular under the effect of a concentration gradient, with the conditions under which:

If the migrating intermediate is an electrically charged compound A or B, then each membrane is charged with the opposite sign on at least one of its faces, preferably the one adjacent to the face carrying the enzyme having transformed the Pac,

.3. then at least one reverse enzymatic transformation, by E ₂ or E ,, of the migrating intermediate (B or A), into the starting Pac, so that the latter accumulates and is concentrated in the compartment C _π or C , of arrival, to recover the Pac thus accumulated and over-concentrated.

2 - Process according to claim 1, characterized in that the enzymatic membrane is in the form of a monolayer or multilayer film, preferably substantially planar.

3 - Method according to claim 1, characterized in that the enzymatic membrane constitutes all or part of the wall of at least one tube whose lumen forms one of the compartments C, / C _π , in that one does bathe at least part of this tubular membrane in the liquid medium forming the other compartment C _π / C ,, in that a liquid to be enriched or depleted in compound Pac is circulated in this or these tubular enzymatic membranes, preferably combined in at least one bundle.

4 - Method according to any one of claims 1 to 3, characterized in that one therefore develops the liquid medium of C, and / or C _π using a solvent - preferably essentially aqueous -, optionally comprising solutes selected from the following group: o pH buffers adapted to the optimum operating pH of

o molecules with energy-rich bonds - advantageously ATP or any molecule capable of transferring a phosphate group onto a solute (for example: phosphoenol pyruvate, carbamyl phosphate, phosphocreatine ...) o and their mixtures, the starting compositions of liquid media of C, and C _π being, preferably, substantially the same or not of the Pac concentration. the starting compositions of the liquid media of C, and C ,, being preferably substantially the same, with the exception of the concentration of Pac.

5 - Method according to any one of claims 1 to 4, characterized in that one makes sure that the compartment C, / C _π receptor for the migratory intermediate through the membrane, initially includes a concentration of this intermediary, lower than that existing in the issuing compartment.

6 - Process according to any one of claims 1 to 5, characterized in that the pH and temperature parameters of the liquid media are adjusted so as to obtain an optimum in kinetics and in yield for the enzymatic transformations by E, and E ₂ .

7 - A method according to any one of claims 1 to 6, characterized in that one sets in motion the liquid of each compartment C, / C _π , this setting in motion operating by stirring and / or by circulation of liquid, in that the stirring and / or circulation conditions are adjusted so that the non-turbulent diffusion layer (s) have an identical or different thickness from on either side of the membrane and between 1 and 10 times the thickness of the membrane.

8 - Method according to any one of claims 1 to 7, characterized in that recourse is made to assistance means for transmembrane migration of (or of) migratory intermediary (ies), said means being preferably constituted by an ionic and / or electric force gradient, on either side of the membrane, and more preferably still pH gradients (protomotor force).

9 - Method according to any one of claims 1 to 8, characterized in that one supplements the over-concentration of Pac, by enzymatic pumping of said

Pac through the membrane (s), from a compartment C, or C _π containing Pac to a compartment C „or C, to be enriched in Pac, by implementing a translocation of the Pac, the latter consisting to be introduced into compartment C, or C _π of departure loaded with Pac, of the migratory intermediate A or B, so that this added migratory intermediate migrates cumulatively to that resulting from stage - 1 -, under the same conditions than those of step - 2 -, to be finally submitted, in step - 3 - after having crossed the membrane, the steps - 1 -, - 2 -, and - 3 - in question being those as defined in claim 1. 10 - Device for separation and concentration of at least one Product to

Concentrate (Pac), present in a liquid medium in dissolved form, in particular for the implementation of the method according to any one of claims 1 to 9, characterized in that it essentially comprises:

- at least one porous enzymatic membrane (5,13), comprising at least one pair of reversible enzymes E, / Ε ₂ , E, being able to catalyze the reaction of transformation of at least one substrate A into at least one product B and E ₂ the reverse reaction of transformation of the substrate (s) B into at least one product A: A -BE ₂ so

* whether A or B corresponds to Pac

* and that at least one of the compounds A or B is electrically charged, E, and E ₂ being respectively supported by one and the other of the faces of the membrane,

at least one container (2, 11) of the liquid medium in which bathes or is likely to bathe the enzymatic membrane (5, 13) to delimit at least partially at least one liquid compartment C, and at least one liquid compartment C _π ,

means for setting in motion (3, 9, 12, 15) of at least one of the liquids, preferably liquids from the compartments C, / C _π ,

- Possibly means (2 „2 ₂ , 11 ,, 11 ₂ ) for adjusting the temperature of the liquid medium.

11 - Device according to claim 10, characterized in that each membrane (5, 13) is electrically charged, on at least one of its faces, preferably on that adjacent to the face carrying the enzyme having transformed the Pac, and even more preferably on both. 12 - Device according to claim 10 or 11, characterized in that the enzymatic membrane (5, 13) is a porous matrix in and / or on the two faces of which are included and immobilized the enzymes E ,, E ₂ respectively, this matrix being formed by at least one macromolecular compound, preferably chosen from proteins, polysaccharides, synthetic (co) polymers and their mixtures and / or alloys, cellulose and its derivatives, as well as (co) polyamides being particularly preferred .

13 - Device according to any one of claims 10 to 12, characterized in that the enzymatic membrane (5) is in the form of a single or multilayer film, preferably substantially planar. 14 - Device according to any one of claims 10 to 12, characterized in that the membrane constitutes all or part of the wall of at least one tube (13) whose lumen forms one of the compartments C, / C _π .

15 - Device according to any one of claims 10 to 14, characterized in that the pair of reversible enzymes E, / E ₂ is chosen mainly from the pairs of enzymes E _l IE ₂ allowing the addition / removal of a chemical group loaded on a metabolite, preferably among the pairs of enzymes E, / E ₂ of phosphorylation / dephosphorylation, and more preferably still among kinases / phosphatases.