WO1999055828A1 - Method for actively and selectively eliminating small molecules by enzymatic pumping: active dialysis - Google Patents

Abstract

The invention concerns a dialysis process assisted by enzymatic pumping, for transferring a solute S = Glucose from a liquid compartment CI to a liquid compartment CII by passage through a biologically active porous membrane (M). M carries on its surface FII opposite CII an enzyme E alpha = glucose kinase (GK), capable of transforming the glucose contained in CII (Sn) into glucose-6-phosphate (G-6-P<->). The enzyme E beta inverse to GK, namely alkaline phosphatase (PA), is immobilised on the surface FI of M opposite to FII and arranged opposite CI. PA transforms into glucose the G-6-P<-> which has migrated from CII into CI by diffusion through M. Glucose (SI) thus produced by E beta can be re-transferred from CI to CII. Preferably, an enzyme E delta = PA is also provided for transforming into glucose, the G-6-P<-> contained in CII. The enzymatic reactions catalysed by E alpha and E beta take place in the non-turbulent diffusion layers dI and dII respectively adjacent to FI and FII. Advantageously, M is negatively charged as the G-6-P<->. Thus is produced a variation in concentration between CI and CII: DELTA S = [SII]-[SI] > 0, which is maintained until the end of the dialysis and which leads to a depletion of CI in glucose which benefits CII. The invention is applicable to industrial dialysis and hemodialysis.

Description

ACTIVE AND SELECTIVE REMOVAL OF SMALL MOLECULES BY ENZYMATIC PUMPING: ACTIVE DIALYSIS

TECHNICAL FIELD: The field of the present invention is that of the separation and elimination / purification of small molecules (solutes) in the liquid phase, by passage through a porous bioactive membrane. This is aimed at the depollution of a given liquid compartment by elimination of undesirable solutes or, more generally, the isolation of certain solutes which are not necessarily polluting but which may have high added value. These techniques for transporting ionic solutes or not by diffusion through a membrane, the role of which is to prevent convective transport of the solvent, are commonly known by the term dialysis.

The invention therefore relates to a method of active and selective dialysis, assisted by enzymatic pumping of at least one solute S through at least one porous membrane separating at least partially two liquid compartments C, and C _π , the transfer s operant of C, which one impoverishes in S towards C ,, which one consequently enriches in S.

The invention also relates to a dialysis device assisted by enzymatic pumping, useful in particular for the implementation of the above-mentioned process.

PRIOR TECHNIQUE:

Passive transmembrane diffusion dialysis has been known for a long time. It has been the subject of numerous ad hoc applications in the chemical industry and in the food and pharmaceutical fields. It is essentially a slow operation which is conditioned by the existence of a concentration gradient on either side of the membrane for the solute (s) to be separated and / or eliminated. Dialysis has therefore proven to be relatively expensive on an industrial scale. This is how more radical processes, based on external driving forces (mechanical or electrical), have been much more studied and exploited, despite their higher energy cost. In fact, industrial dialysis only becomes competitive when these energy-consuming processes are ineffective, when they risk damaging the solute to be isolated or when the slowness of the dialysis operation does not constitute a handicap. These three conditions apply to a technique which is not specifically industrial, but which at present constitutes the most important application of dialysis: hemodialysis. Hemodialysis is a medical technique intended for subjects suffering from partial or total renal insufficiency. It consists of an extracorporeal treatment of the blood, ensuring the same functions as the kidney, thanks to a membrane process based on both dialysis and ultrafiltration. A less elegant solution than organ transplantation, it is essential given the disproportion between the number of potential donors and that of applicants. The small molecules that we seek to eliminate from blood on hemodialysis are urea, uric acid, creatinine and other metabolic wastes of low molecular weight. This elimination must be done selectively by allowing the treated blood to conserve the macromolecules and metabolites essential to these functions and by preserving its electrolytic and aqueous balance sheets.

In addition to hemodialysis, hemofiltration is also known as a method of removing certain undesirable solutes from the blood. The principle of hemofiltration consists in using filtration as a motor for the exchange of solutes. Hemofiltration allows good purification of medium molecules.

Generally, in these two processes, the blood is purified passively (hemodialysis) or by creating a slight pressure difference between the blood circulating in the lumen of hollow fibers and a liquid called dialysate circulating outside the fibers (hemofiltration). In both cases, passive or induced diffusion does not differentiate between them the small molecules contained in the blood, depending on whether they are harmful and therefore to be eliminated or essential and therefore to be preserved.

The selective elimination of certain small molecules is therefore not possible by known techniques of hemodialysis or hemofiltration. To overcome this, it would be interesting to have a new technique to quickly and selectively eliminate a given small molecule.

Furthermore, it is known that for dialysis techniques to be effective, it is imperative that there is a concentration gradient on either side of the membrane, the solute concerned circulating from the most concentrated compartment to the least compartment. concentrated in solute. This requires the use of large volumes of dialysate to reduce the concentrations outside the compartment to be decontaminated. In addition to the handling and bulk difficulties which they entail, these large volumes of dialysate also have the drawback of causing the leakage of molecules or of essential blood solutes. SHORT STATEMENT OF THE INVENTION:

In such a state of the art, one of the essential objectives of the present invention is to improve the known techniques of separation or elimination of certain solutes by transmembrane diffusion, by providing a method of forced dialysis making it possible to lower consequently quickly and selectively the level of concentration of a solute in a given liquid compartment, by transferring it through a membrane into another liquid compartment, whatever the level of concentration of the solute considered in this latter compartment, c that is to say even in the absence of a concentration gradient favorable to a diffusion flow from the most concentrated compartment to the least concentrated compartment.

Another essential objective of the invention is to provide a method of forced dialysis making it possible to extract, for example for the purpose of depollution, purification or isolation, a given solute by implementing a transmembrane diffusion having significantly kinetic superior to that of a conventional passive transmembrane diffusion process.

Another essential objective of the present invention is to provide a method of differential and therefore selective forced dialysis for one or more solutes.

Another essential objective of the present invention is to provide a simple, rapid and economical dialysis process. Another essential objective of the present invention is to provide a selective and rapid dialysis process which makes it possible to dispense with the use of large quantities of dialysates hitherto useful for generating concentration gradients necessary for passive diffusion.

Another essential objective of the present invention is to provide a simple, selective, rapid, economical dialysis process which can be applied in hemodialysis in an efficient manner.

Another essential objective of the present invention is to provide a dialysis device capable of allowing in particular the implementation of the abovementioned process. Another essential objective of the invention is to provide a simple, economical, selective and rapid dialysis device.

Having set these objectives, the inventors had the merit of highlighting, after long and laborious research and experiments, that it was possible to achieve these objectives by stimulating and optimizing the phenomenon of passive diffusion, by putting using an auxiliary enzymatic pumping system. This original auxiliary mechanism, discovered by the inventors, is based on the intervention of at least one enzyme specific to the solute considered and placed on the face of a porous membrane opposite that which is opposite the liquid compartment which it is sought to deplete in solute by transmembrane diffusion. Such an enzymatic system can comprise at least one pair of reverse enzymes, one of the enzymes of the pair being fixed on one side of the membrane, while the other enzyme is immobilized on the other side of this membrane, these enzymes being, in addition, possibly contained in the liquid medium of the compartments, with the exclusion of the porous enzymatic diffusion membrane. This system behaves like a pump which generates and maintains a difference in concentration of solute (s) to be transferred between the two compartments.

As a result, the present invention relates first of all to a dialysis process assisted by enzymatic pumping of at least one solute S through at least one porous membrane separating at least partially two liquid compartments C, and C ,, , (S being referenced S, in C, and S ,, in C ,,), this dialysis taking place from the liquid compartment C, which we aim to deplete in S, towards the liquid compartment C _π intended consequently for s '' enrich in S, characterized in that it consists essentially and successively or not:

• to use at least one porous membrane comprising on and / or in its face F _π opposite Cπ at least one enzyme E _α capable of catalyzing the reaction of transformation of the solute SU into solute- primary product Sound;

• to set in motion at least the liquid of C _π (dialysate), and preferably also that of C ,, by providing at least one non-turbulent d _π diffusion layer, adjoining the face F _π of the membrane carrying E _α and opposite C _π , and by adjusting the thickness ed _π of this diffusion layer d _π so that

- e ^m <ed ι „<10. em, d, e pré, f <* é, rence - em <ed„ <3.em;

10 2 em being the thickness of the membrane;

• and to ensure that the operating conditions are such that:

* the enzymatic transformation by E _α of S ,, into S _αII ,

* and correlatively the diffusion of S ,, through the membrane of compartment C _j towards compartment C, in particular under the effect of an enzymatic pumping, induced by the reaction

E α

S ^ S

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 solute S in a compartment (I) starting liquid, to a compartment (II) arriving liquid, by active transport through an porous enzymatic membrane. The solute S thus accumulates and concentrates in the arrival compartment, even if the concentration of the solute in the latter is higher than that of S in the departure compartment. What is more, the enzymatic pumping according to the invention has the effect of maintaining a concentration difference ΔS = [S _π ] - [S,]> 0.

Such an enzymatic pumping process is particularly advantageous since it allows rapid and selective elimination of one or more solutes from a liquid compartment Ci, for example blood (hemodialysis), without using large volumes of dialysate. Cπ without excessive energy consumption. 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).

It should be noted that the kinetic gain provided by the invention remains compatible with the duration threshold below which a blood stress is generated in the hemodialysis application.

This is linked to the fact that the non-turbulent diffusion layers specific to the method according to the invention somewhat limit the speeds of circulation of the blood and of the dialysate during hemodialysis. 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 medical or industrial separation specifications / elimination of chemical compounds, particularly for the purposes of elimination and isolation of toxic compounds, or for the recovery of molecules with high added value: chemical, pharmaceutical, cosmetic or others.

In accordance with a preferred characteristic of the invention, the enzyme E _α which governs the production of S _α ,, is selected on the one hand and, on the other hand, the porous membrane, so that the latter is impermeable to S _αII and thus prevents, at least partially, the diffusion of S _αI , from C _π to C _,; S _αII and the membrane thus being preferably entities of electric charges of the same sign.

The electrically charged nature of S _αII results from the action of the enzyme E _α on the neutral substrate S _π .

The electrical charge of the faces of the porous membrane can be obtained, for example, by immobilizing, on the membrane, the enzymes E _α and E _p and 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, membranes can be chosen whose zeta potential at the optimum pH is adapted to given conditions of the process of the invention. In the absence of means to prevent pollution of the liquid compartment C, by the product S _αII of the reaction involving S _π as a substrate and E _α as an enzyme or to complete the action of these means, a first variant is provided for. process according to the invention in which:

• at least one porous membrane is used, comprising on and / or in its face F, opposite C ,, at least one enzyme E _p capable of catalyzing the reaction of transformation of the solute-primary product S _αI resulting from the diffusion through the membrane of S _αII from C „to C, in solute-secondary product S _β ,,

Ep preferably being the inverse enzyme of E _α so that S _pι = S ,, "provision is made for a movement of the liquids of C, and C„ by providing a diffusion layer d ,, d _π connected non-turbulent on each face F ,, F ,,, and by adjusting the thicknesses ed, and ed _π of these layers so that: * 0.1. em <ed./ ed _rj ≤ 10. em, * preferably 0, 5. em <ed, / ed _π <3. em; • operating conditions favorable to the enzymatic transformation by E _p of S _αI into S _{pι are fixed} , and, correlatively, in the case where S _pι = S ,, the diffusion of S _pι from C, towards C _π , through the membrane, under the effect, in particular, of an enzymatic pumping

E α s „- S resulting induced by the reaction ^απ .

So here we are in the presence of a porous membrane separating C _! and C „and carrying on each of its faces F, and F _π enzymes, preferably reversible, E _p and E _α respectively. The enzymatic pump thus equipped with additional enzymatic means, allows the retransformation of the metabolites S _αI resulting from the transfer of the metabolites S _α „from C ,, to C ,. The metabolite S _αI retransformed into S, in C, can therefore join the transfer flow of S from C, to C ,, through the membrane.

According to the invention, it could be envisaged to fix E _p on a support other than the face F, of the membrane. E _p could thus be carried by an annex membrane and / or on any other solid support dispersed or not in C, according to a second variant of the method according to the invention:

_• at least one enzyme E is used in C _π capable of catalyzing the reaction for transforming the solute S ^ into the solute S ,,,

• and operational conditions favorable to the development of

^E δ

SS this transformation In this second variant, the metabolite S _αII is eliminated as it is formed, to further enrich C ,, in solute S ,,. In doing so, the concentration difference ΔS = [S _ι ,] - [S,] is further increased. This confirms whether it was necessary for the enzymatic pump according to the invention to operate by generating this ΔS and maintaining it.

It emerges from the above description that it is important according to the invention to provide a non-turbulent diffusion layer di, dπ adjoining each face Fi, Fπ of the membrane or membranes, especially when the face considered is carrying an enzyme constituting the enzyme pump.

The differences between the rates of appearance and disappearance of the solute S in the compartments C ,, C „, on either side of the membrane, depends in particular on the proper functioning of the enzymatic pump and therefore in part on the thicknesses of the layers non-turbulent diffusion, but also for another part of the volumes of compartments C, and C ,,. This is how the volume V _π of C _π is preferably greater than the volume V, of C ,. Advantageously, the ratio Vπ / Vi is as large as possible so as to facilitate the kinetics of the transport.

ES, ^α

So that the enzymatic transformations * ^• S au and

^E P

^{Sa l S} P 'occur in the vicinity of each of the faces Fi, F _π of the membrane, it is not only important to provide the diffusion layers di and d _π mentioned above, but also to set up optimal operating conditions , in particular pH and temperature, which will be detailed below. With regard to the diffusion layers, the adjustment of the stirring conditions, therefore of the thicknesses Edi and Ed _π of the non-turbulent diffusion layers di and dπ, can be carried out on the basis of the article by BARDELETTI, G, B. MAISTERRENA and PR COULET, 1985 "Boundary rent effect on product flux-splitting in an immobilized ensyme membrane System"; J. Sci membrane. 24; 185-296. According to a preferred characteristic of the invention, Edi, Ed _π are between 50 and 500 micrometers, depending on the hydrodynamic conditions imposed. The second variant corresponds to the preferred embodiment of the invention, which combines the advantages of an enzymatic pumping system comprising two reversible enzymes fixed, respectively, one and the other on the opposite faces of a membrane porous diffusion and a third active enzyme in compartment C „intended to enrich in solute S diffusing through the membrane. This third enzyme E _δ is preferably (just like E _p ) the inverse enzyme of the enzyme E _α of C ,,.

The enzyme E _δ is advantageously contained in C _π on a support other than the face F _π of the membrane. E _δ can thus be, for example, fixed on an annex membrane or any other suitable support immersed in C ,, or even be dispersed in C _π .

The enzymatic pump according to the invention has an action of promoting the diffusion of the solute S through the membrane. This action involves creating and maintaining a concentration gradient ΔS = [S _π ] - [S,]> 0 on either side of the membrane. The retransformation in C, and C _π of the enzymatic metabolites resulting from the enzymatic reactions with E _α and Ep, in solutes S, and / or S ,,, optimizes the pumping effect. We can further complete this by giving the membrane a valve effect compared to the S _αI1 metabolites produced in C ,,, to prevent the latter from re- _diffusing in C ,. The result of the enzymatic pumping according to the invention is the transfer of S, from C, into C ,,. Once this result has been achieved, it is easy to recover S ,, for the purpose of elimination or recovery.

According to the first mode of operation 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, and C _π . The membrane can form all or part of the wall of the container. Preferably, this membrane constitutes a substantially flat part of the partition wall between the two compartments C, and C ,,. According to the second mode of operation of the method according to the invention,

(which will be called "continuous"), the enzymatic membrane is in tubular form and more precisely constitutes all or part of the wall of a tube, the lumen of which forms one of the compartments C,. Advantageously, the enzymatic membrane can be made of a plurality of hollow fibers or tubular membranes, preferably united in bundle (x).

In accordance with this second continuous mode of operation, at least part of this (or these) tubular membrane (s) is immersed in the liquid medium forming the other compartment C _π and a liquid to be depleted is circulated in solute S, in this (or these) tubular enzymatic membrane (s). The circulation of the liquid in the tubular membrane or membranes constituting the other compartments C ,, being ensured by any suitable means such as a pump.

On reading the above, one can easily perceive the importance of the liquid medium of compartments C, and C _π , and in particular their physicochemical characteristics, for carrying out 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 _α and E _p , or even E _δ . In practice, the liquid medium of C, and / or C _{π is therefore produced} by using a solvent - preferably essentially aqueous -, optionally comprising solutes chosen from the following group: 10

• pH buffers adapted to the optimum operating pH of E _α and E _p , or even E _δ ;

• 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, etc.)

• and their mixtures.

In addition to adjusting the pH and supplying the chemical energy source necessary for the enzymatic transformations E _α and E _p , or even E _δ ; 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.

In accordance with the invention, the diffusion layers di, dπ, are obtained by setting the liquid in motion in each compartment C, / C _π , this setting in motion taking place by stirring - preferably with using a rotor - in the first operating mode according to the invention and / or by circulation of said liquid according to the second operating mode 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 d, and d _u have, in practice, thicknesses of at least 100 μm. In order to improve the kinetics and the transfer performance of the migrating solute S, it is possible, in accordance with the invention, to provide auxiliary means for assisting the transmembrane migration, preferably consisting of an ionic and / or electrical force gradient, on either side of the membrane, and more preferably still by pH gradients (protomotor force). According to a preferred arrangement of the invention:

• S = S, = S i = S _π = glucose,

• S _α = glucose-6-phosphate,

• E _α = glucose kinase,

• E _p = E _δ alkaline phosphatase.

POSSIBILITY OF INDUSTRIAL APPLICATION: Among the interesting applications of the method according to the invention, there are those in which the purification of physiological liquids, preferably blood, is carried out, this liquid then constituting the liquid contained in C,. It follows that the present invention also relates to a hemodialysis process. 11

According to another of its aspects, the present invention relates to a dialysis device assisted by enzymatic pumping of at least one solute S characterized in that it essentially comprises:

• at least one container of a liquid medium; "At least one porous membrane of thickness em, bathing or capable of bathing in the liquid medium, to at least partially delimit a liquid compartment C, intended to be depleted in solute S and at least partially a liquid compartment C„ intended to be receiver of the solute S _j coming from C ,, C ,, C _π containing respectively volumes V, and V ,,;

At least one enzyme E _α capable of catalyzing, in C ,,, the reaction of transformation of S into solute-product S _αII primary, E _α being carried by the membrane on and / or in its face opposite C _π ,

• preferably at least one enzyme E _p able to catalyze, in C ,, the transformation reaction of the primary solute S _αI = S _αI1 into the solute- secondary product S _pι , E _p being carried by the membrane on and / or in its face F _j opposite C ,,

E _p being, even more preferably, the reverse enzyme E _α , so that S _pι = S _,; • preferably at least one enzyme E _δ capable of catalyzing in C ,,, the reaction of transformation of the primary solute S _αII into solute S ,,, E _δ being dispersed in C _π in immobilized form;

• means for setting in motion at least the liquid of C _rι and, preferably, also of the liquid of C _r ; • and possibly means for adjusting the temperature of the liquid medium. This device is, in particular, suitable for implementing the method as described above.

Advantageously S _αI1 and the membrane are entities of opposite electrical charges, the membrane (s), being charged, on at least one of its faces, preferably on F „carrying E _α and more. preferably still on both.

In practice, it is preferable that each membrane is electrically charged, on at least one of its faces, preferably on that adjacent to the face carrying the enzyme E _p and more preferably still on both. 12

According to a preferred characteristic of the invention, this enzymatic membrane is a porous matrix, in and / or on the two faces F _π , F, from which are included and immobilized the enzymes E _α E _p 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 ease grafting of E _α / E _p ; 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, the lumen of which forms one of the compartments C,.

There are many pairs of enzymes that can be used in the process and the device according to the invention. To fix the ideas, it may be indicated that these pairs E _α / E _p are mainly chosen from pairs of enzymes allowing the addition / removal of a charged chemical group on a metabolite, preferably from pairs of phosphorylation enzymes. / dephosphorylation, and more preferably still among kinases / phosphatases.

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 three modes of implementing the method according to the invention using the two devices exemplified before.

BRIEF DESCRIPTION OF THE DRAWINGS:

The description of the two embodiments of the device according to the invention will be made with reference to the accompanying drawings in which;

- fig. 1 represents a simplified diagram of the first embodiment of the dialysis device assisted by enzymatic pumping, in accordance with the invention (discontinuous mode);

- fig. 2 is a simplified diagram illustrating the second embodiment of the device according to the invention (continuous mode); 13

- fig. 3 is a symbolic representation of the enzymatic membrane separating the two compartments C, and C „of the device of FIG. 1 or 2, said representation corresponding to an embodiment of the dialysis process for a solute S, from compartment C, to compartment C _π :

Membrane E S, Su —e → ^

- fig. 4 is a symbolic representation of the same nature as FIG. 2, with the difference that it relates to a 1st variant implementation of the dialysis process of the solute S from compartment Ci to compartment Cπ:

Membrane E Membrane E _R

S,> S _π ° → S li »^ T - Ê → Spi = S, - fig. 5 is a symbolic representation of the same nature as FIG. 2, with the difference that it relates to a second variant of implementation of the dialysis process for the solute S from compartment Ci to compartment Cπ:

Membrane E _α Membrane E _β

S, ¹ > • S „" + S αïï ”- ^û ςα _τ l 's ^a ι _β ι s

^E δ

- fig. 6 is a simplified view in right cross section along the line VI-VI of FIG. 2;

- fig. 6 bis is an enlarged view of one of the elements of the tubular membrane seen in section in FIG. 6;

- fig. 7 represents a graph of the concentrations in Sκ _D ), Sπ („), Scj ( ₀ ), Sαπ ( _# ) in Ci, C _π , as a function of time t in min in the test carried out according to the topography of FIG. 3

- fig. 8 shows a graph of the concentrations SK), Sπc ,,), Sαi ( ₀ ), Sαπ _(# ), in mM, as a function of time t in min, in the test carried out according to the topography of FIG. 4;

- Fig. 9 represents a graph of the experimental concentrations Si (), Sπ ( _B ), S ^ _o ), Sont.) And theoretical Sι _th (curve a) in mM as a function of time t in min, in the test carried out according to the topography of fig. 5.

DETAILED DESCRIPTION OF THE INVENTION:

The device for separating and concentrating at least one solute S to be dialyzed, 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 14

thermostatically controlled having a double wall, provided for the circulation of a thermoregulating fluid, between the inlet 2 _d and the 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 2 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 even a conventional 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, e.g. 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 FIG. 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, = 1.5 cm ³ , while the volume V ,, of compartment C _u = 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 Pall NAZ, in the form of nonwoven material, having a thickness of 100 μm eg The porosity of this membrane is defined by the cutoff threshold, the latter being naturally chosen as a function of the size of the molecules which it is desired to migrate through the membrane to overconcentrate them in a compartment. Expressed in maximum molecular weight for molecules 15

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.

Alternatively, this membrane can also be made of regenerated cellulose and is in the form of a nonwoven film of thickness between

10 and 100 μm.

Each of the faces F _π , Fi of the membrane 5 opposite, respectively, of compartment C ,, and of compartment C ,, has been subjected to a grafting of enzymes

E _α / E _p , forming a pair of reversible enzymes in which the enzyme E _α is able to catalyze the transformation of S _π into S _α π and the enzyme E _p the transformation of S _α , into Si.

The grafting of the enzyme can be of covalent nature or even a simple inclusion of the enzyme by adsorption 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 second embodiment of the device according to the invention is shown in FIG. 2, on which it is designated by the general reference 10. This device 10 for dialysis of Ci- »Cπ of a solute S is capable of operating in a continuous mode.

This device 10 comprises a thermostatically controlled tank 11 of the same type as that (2) described in the first embodiment of FIG. 1. References ll _j and

11 ₂ respectively designate the inlet and the outlet of the circulation circuit of a thermoregulatory fluid in the jacket of the tank 11.

This tank 11 contains the liquid medium initially containing the solute S 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 FIG. 1 and designated by the 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 shown in fig. 1 in the bundle 13 gives the direction of flow of the flow of the compartments C ,.

The section in fig. 6 shows the bundle 14 of tubular enzymatic membranes 13. 16

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 depleted in solute S by active transmembrane migration . To this end, the bundle 14 is equipped with circulating means 15 constituted, for example, by a pump.

Fig. 6 bis 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 on the other hand, with regard to 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 internal and external faces an enzyme E _α and E _p respectively, constituting a pair of reversible enzymes. 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 the membrane 5 of FIG. 1. In practice, this will, for example, be regenerated cellulose of a porous nature, having a cut-off threshold which may vary from 500 to 10,000 D.

The stirring means 12 of the tank 11 as well as the setting in motion of the liquid of C, in the hollow fibers 13 by means of the pump 15, make it possible to define diffusion layers d _j and d _π , shown on fig. 6 bis. The latter also shows the internal diameter D of the hollow fiber 13, as well as the thickness em of the membrane wall charged with enzymes E _α / E _p .

The sum of the volumes of compartments C, immersed in C ,, is less than the volume V _π of compartment C _π . The VV ratio is as low as possible.

The tubular membranes 13 can be electrically charged in positive or in negative, so as to avoid the parasitic diffusion of Sound in Ci.

This second embodiment of the device according to the invention is adapted to the second continuous mode of implementation of the dialysis process. We find the same methodology used as for the first discontinuous mode of implementation, with the difference that the liquid medium of compartments C circulates in the hollow enzymatic fibers 13, the active transmembrane transport of S 'occurring naturally at the submerged part of the bundle 14 in the liquid compartment C _n of the tank 11. The speed of circulation of the liquid medium in the compartments C is adapted to the total surface of the exchange zone in the submerged part, 17

kinetics of enzymatic transformation E _α / E _p , as well as transmembrane diffusion rates of solute S.

The symbolic representation of fig. 3 shows the membrane 5 carrying in its face Fπ opposite Cπ the enzyme E _α . This membrane has a thickness em. It is adjoined by its face Fπ with a diffusion layer d _π and by its face Fi opposite Ci with another diffusion layer di, of thickness edi. Depending on whether the solute S is in Ci or in Cπ, the reference S includes the index I or II separated by a comma from the parameter t corresponding to time. Depending on whether the solute S is in a diffusion layer or in the rest of the compartment Ci or C _π , the reference S includes by exposing the letters d and b (b for "bulk" in English) respectively. The second exponent of the reference S corresponds to the negative (-) or neutral (o) charge of the solute.

At time t = 0 we have: S ^ _t ≠ o; S ^b _π , t = S ^b απ, t = S ^b αi, t = 0. At time t, the solute Si has migrated through the membrane to pass from Ci to C _π . And it is in the diffusion layer dπ of Cπ that S _π is transformed into Sound by E _α . The part of S _π which is not metabolized by E _α diffuses into the Cπ compartment. Part of the bran passes through the membrane to pass into the compartment Ci, so that the latter comprises self-solute in its diffusion layer and in the rest of the compartment. Part of the Sound also plays in the entire C _π compartment.

Fig. 4 schematically represents the topography specific to the first variant of implementation of the method according to the invention. The Fπ and Fi faces of the membrane respectively carry the reversible enzymes E _α / Ep. The solute Si crosses the membrane to pass from Ci into C _π and to be partly metabolized in the diffusion layer dπ adjoining the face F _π of Cπ, in solute Son, by the enzyme Eαi. A part of its diffuse through the membrane of Cπ towards Ci and the compound Soi having thus diffused and transformed into Si in the diffusion layer di adjoining Si, by the enzyme Ep. Sπ and Son diffuse in Cπ, similarly that Self diffuses in Ci. The membrane is preferably electrically charged with the same sign as Sound, in this case negatively, so as to limit the diffusion of Sound in Ci.

Fig. 5 corresponds to the second variant of implementation of the method according to the invention. We find the same diagram as that of fig. 4, with the difference that the compartment Cπ comprises an enzyme E _δ capable of metabolizing Sound to S _π , this transformation taking place preferably in the part of Cπ different from the non-turbulent diffusion layer dπ. 18

In the implementations corresponding to the diagram of FIGS. 3 to 5, the enzymatic pumping, provided by E _α , Ep and E _δ , causes the formation of a concentration gradient Δ [S] = [S ₂ ] - [Si]> 0.

The examples which follow illustrate the process according to the invention in its three preferred modes of implementation. These examples will make it possible to better understand the meanings of the invention and to grasp all of its advantages and implementation variants.

EXAMPLES EXAMPLE 1: ASSISTED DIALYSIS BY ENZYMATIC PUMPING OF A

SOLUTE S = GLUCOSE, FROM A LIQUID COMPARTMENT Cj, TOWARDS A COMPARTMENT CJJ ACCORDING TO THE 1 ^st MODE OF IMPLEMENTING THE PROCESS OF THE INVENTION AND USING THE DEVICE OF THE INVENTION IN ITS 1 ^ST EMBODIMENT

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

The dialysis membranes used are membranes of the Pall NAZ type sold by the company PALL EUROP LIMITED. These are polyamide membranes with a thickness equal to 100 μm of active transfer area (Aw) of 12% and referenced under the number 09025. We also use regenerated cellulose dialysis membranes, with a thickness of 40 μm, sold under the name spectra / Por. 3500 MWCO - ref 132723 by spectrum. These dialysis membranes are those that do not carry enzymes. The enzyme E _α = glycerolkinase - GK - (EC2.7.1.30) from the species cellulomonas (52 units. Mg ^-1 , ref.g. 6142).

E _p = E _δ alkaline phosphatase -PA- (EC3.1.3.1.) Extracted from bovine intestinal mucosa (1200 Units.mg " ¹ , ref. P 6672).

The enzymes E _α , E _p and E _δ 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 5 cm in diameter which are activated using a solution of sulfuric acid in methanol under reflux for 6 hours. They are then subjected to the action of a 2% V / V glutaraldehyde solution, as described in the article by Michalon, P., Couturier, R., Hacques, MF., Favre-Bonvin, G, Ville , A. & Marion, C. (1990) Biochem. Biophys. Res. Common, 167, 9-15. 19

The grafting of the enzymes E _α = GK and Ep = E _δ = PA 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 a 0.1 M borate buffer, pH 8.5. The volume of C _f = 1.5 cm ³ and the volume of C _π = 300 cm ³ .

1.2. Operating conditions

The liquid media in C and C _π have the same composition with the exception of the concentrations of compound A = ² -glycerol-3-phosphate 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 according to the topography of fig. 3

In this test, the liquid media C _[ / C _π contain 0.1 M borate buffer in an amount such that the pH is of the order of 9 as well as ATP Mg Cl ₂ - 7 mM. S = Si = Sπ = glucose. Son = Soi = glucose -6-phosphate (G-6-P ^" ). E _α = GK. The enzymatic reactions used in the examples are as follows:

MgCl ₂ / ATP + glucose - ^ - ^ glucose-6-phosphate + ADP / MgCl ₂

PA G-6-P "- glucose + phosphate The glucose concentrations are determined from samples taken in the compartments Ci and Cπ, using a sensor formed by an enzymatic electrode composed by an amperometric platinum probe associated with a membrane nylon loaded with glucose oxidase, this probe being coupled to a PRGE type polarograph from the company RADIOMETER (VILLEURBANNE, FRANCE), (THEVENOT et al), 1979. This biosensor allows glucose concentration determinations according to the following reaction: glucose oxidase D-glucose + O ₂ ^ _ac ide gluconic + H ₂ O ₂

Pt / anode (650mV) VsAg / AgCl

^H 2 ° 2 O ₂ + 2H ⁺ + 2e ^"

The anode current thus obtained is proportional to the H ₂ O ₂ concentrations, which are themselves proportional to the glucose concentrations.

The thickness em of the dialysis membrane loaded with GK opposite Cπ is 240 μm.

The stirring conditions of this test are respectively 180 revolutions / min and 100 revolutions / min in Ci and C _π . 20

These conditions make it possible to obtain the thicknesses of diffusion layer di and dπ which are:

- edi = 100 μm.

- edπ = 180 μm. During the course of the tests, samples of G-6-P "are taken from the two compartments Ci and Cπ and analyzed using G-6-P" dehydrogenase by measuring the increase in absorption at 340 nm associated with reduction of NADP ⁺ (Lang and Michal, 1974). The determination of the enzymatic activities of the NAZ membranes loaded with GK and PA is carried out in the following manner.

The NAZ discs loaded with GK are immersed in a ml of 0.1 M borate buffer pH 8.5 containing 7 mM ATP / MgCl ₂ in the presence of a saturated concentration of glucose, i.e. 0.2 M, at 25 ° C. Samples are taken every 5 min for 20 min. In these samples, the G-6-P "concentration is measured, so as to determine the GK activity.

Similarly, NAZ disks loaded with PA are immersed in 5 ml of 0.1 M borate buffer, pH 8.5 in the presence of 33 mM G-6-P "(saturation concentration). Samples are sampled every 5 min for 20 min and the glucose concentration is measured in these to deduce the PA activity. The liquid medium of Ci, Cπ consists of 0.1 M phosphate buffer containing 7 mM ATP / MgCl ₂ , at pH 8.5 and at 25 ° C. S _u ^b '° = S, ^» ≈ S ₂ - ° = S _2> ^tb - ° = 2 mM.

Fig. 7 clearly shows the decrease in the glucose concentration Si in the compartment which is much more marked than the decrease in Sπ, although the enzymatic reaction promoted by E _α = GK occurs only in Cπ. Furthermore, the concentration of Son in G-6-P "in Cπ increases more slowly than the concentration of Self in G-6-P" in the non-enzymatic compartment Ci.

EXAMPLE 2: TESTS ACCORDING TO THE TOPOGRAPHY OF FIG. 4 In these tests, the dialysis membrane comprises a membrane NAZ charged with E _α = GK opposite C _π , a membrane NAZ charged with Ep = PA opposite Ci and a spectra / Por membrane interposed between these two enzymatic membranes. The dialysis membrane has a thickness em = of 240 μm. The stirring conditions are 180 revolutions / min and 100 revolutions / min in Ci and Cπ respectively.

Such conditions determine thicknesses for dr and dπ which are: 21

- edι = 100 μm.

- d _π = 180 μm.

The other experimental conditions are identical to those described above in Example 1. FIG. 8 clearly shows that the G-6-P ^" produced in Cπ (Son) does not pollute C,; S _o i is constant and is equal to 0. Furthermore, the concentration Si of glucose in Ci decreases rapidly in 500 min. S _π glucose concentration in Cπ decreases less rapidly than Si while Son increases regularly.

EXAMPLE 3: GLUCOSE DIALYSIS TESTS ACCORDING TO THE TOPOGRAPHY OF FIG. 5

In this test, we find the same topography as in fig. 4, with the difference that compartment Cπ contains an enzyme E _s = PA, which is supplied via 4 NAZ membrane disks loaded with PA. The latter is intended to transform the glucose-6-phosphate present in Cπ. The experimental conditions are the same as in Examples 1 and 2 above.

The stirring conditions are the same as in Examples 1 and 2 above. These conditions determine a thickness edi = lOOμrn for the diffusion layer d _π and edπ = 180 μm for the diffusion layer dπ. The thickness of the dialysis membrane is em = 240 μm. Fig. 9 shows the results obtained and it appears that no pollution of compartment I by the G-6-P "produced in Cπ occurs. Likewise, the concentration S _o n remains constantly zero in Cπ. Furthermore from t = 500 min, we observe a constant ΔS = [Sπ] - [Sτ] deviation between 0.5 and 1 mM. The comparison of the curves Sι _th and Si = f (t), shows a good correlation between the theoretical model and the experimental model.

It appears from the above that a glucose dialysis assisted by an enzymatic pumping resulting from an enzymatic and cyclic mechanism of phosphorylation / dephosphorylation, on either side of the membrane in the compartments Cπ of arrival and Ci of departure , is quite effective, selective and offers good yields without requiring the use of large volumes of dialysates. This mechanism is further optimized when G-6-P "dephosphorylation is predicted outside of the diffusion layer dπ of the compartment Cπ. Phosphorylation / dephosphorylation take place in d _π and di respectively. In all these tests, the membrane is negatively charged like G-6-P "so as to limit its diffusion from C _π to Ci.

Claims

 22 CLAIMS: 1 - Dialysis process assisted by enzymatic pumping of at least one solute S through at least one porous membrane separating at least partly two liquid compartments C, and C _π , (S being referenced S, in C _j and S „In C _tI ), this dialysis taking place from the liquid compartment C, which is aimed at depleting in S, towards the liquid compartment C _π intended consequently to enrich in S, characterized in that it consists essentially and successively or not: • using at least one porous membrane comprising on and / or in its face F _π opposite Cπ at least one enzyme Eα capable of catalyzing the reaction of transformation of the solute SU into solute- primary product Sound; • to set in motion at least the liquid of C ,, (dialysate), and preferably also that of C _j , by providing at least one non-turbulent _π diffusion layer, adjoining the face Fn of the membrane carrying E _α and opposite C ,,, and by adjusting the thickness ed _π of this diffusion layer d _π so that em,,, „, em, - <ed „<10.em, preferably - <ed„ <3.em; 10 2 em being the thickness of the membrane; • and to ensure that the operating conditions are such that: * the enzymatic transformation by E _α of S _π into S _αII , * and correlatively the diffusion of S ,, through the membrane of compartment C, towards compartment C _π , in particular under the effect of an enzymatic pumping, induced by the reaction E α ^S π— "V 2 - Method according to claim 1, characterized in that one selects on the one hand, the enzyme E _α which governs the production of S _αI1 and, on the other hand, the porous membrane, so that this the latter is impermeable to S _αII and thus prevents, at least partially, the diffusion of S _αI , from C _π to C _j ; S _αI! and the membrane thus being preferably entities of electric charges of the same sign. 3 - Method according to claim 1 or claim 2 characterized in that: 23 • at least one porous membrane is used, comprising on and / or in its face F, opposite C ,, at least one enzyme E _p capable of catalyzing the reaction of transformation of the solute-primary product S _αI resulting from the diffusion through the membrane of S _αI1 from C _π to C _j as a secondary product-solute Sp ,, Ep preferably being the inverse enzyme of E _α so that S _pι = S ,, • provision is made for a movement of the liquids of C, and C _π by providing a non-turbulent diffusion layer d ,, d _π connected to each face F ,, F ,,, and by adjusting the thicknesses ed _t and ed _π of these layers so that: * 0, 1. em ≤ ed _j / edu ≤ lO.em, * preferably 0.5. em <ed, / ed _π <3.em; • operational conditions favorable to the enzymatic transformation by E _p of S _αI into Sp ,, are _fixed and, correlatively, in the case where Sp, = S ,, the diffusion of Sp, from C, to C _π , through the membrane, under the effect, in particular, of an enzymatic pumping E α S - * ^" S resulting induced by the reaction ^π . 4 - Process according to any one of claims 1 to 3, characterized in that: • C _{π is used} , at least one enzyme E _g capable of catalyzing the reaction for transforming the solute S _αII into the SJJ solute, • and operational conditions favorable to the development of SL * S this transformation '" ^{• II π} . 5 - Process according to any one of claims 1 to 4, characterized in that the enzymatic membrane is in the form of a monolayer or multilayer film, preferably substantially planar. 6 - Method according to any one of claims 1 to 4, 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 at least part of this tubular membrane is made to bathe in the liquid medium forming the other compartment C _π C _,; • and in that one circulates the liquid to be depleted in solute S ,, in this or these tubular enzymatic membranes, preferably united in at least one bundle. 24 7 - Method according to any one of claims 1 to 6, characterized in that one therefore develops the liquid medium of C, and / or C _π by using a solvent - preferably essentially aqueous -, optionally comprising solutes chosen from the following group: • pH buffers adapted to the optimum operating pH of E _α and Ep, or even E _δ ; • molecules with energy-rich bonds - advantageously ATP or any molecule capable of transferring a phosphate group onto a solute; "And their mixtures. 8 - Process according to any one of claims 1 to 7, 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 _α and / or E _p and / or E _g . 9 - Method according to any one of claims 1 to 8, characterized in that the setting in motion of the liquid in at least one of the compartment C ,, C „, is effected by stirring and / or by circulation of the liquid . 10 - Process according to any one of claims 1 to 9, characterized in that recourse is made to means for assisting the transmembrane migration of S, said means preferably being constituted by an ionic strength gradient and / or electric, on either side of the membrane, and more preferably still pH gradients (protomotor force). 11 - Method according to any one of claims 1 to 10, characterized in that: • S = S, = S _P ι = S „= glucose, • S _α = glucose-6-phosphate, • E _α = glucose kinase, • Ep = E _δ alkaline phosphatase. 12 - Process according to any one of claims 1 to 11 characterized in that the liquid of C _r is a physiological liquid, preferably blood. 13 - Dialysis device assisted by enzymatic pumping of at least one solute S characterized in that it essentially comprises: • at least one container of a liquid medium; • at least one porous membrane of thickness em, bathing or capable of bathing in the liquid medium, to at least partially define a liquid compartment C, intended to be 25 depleted in solute S and at least partially a liquid compartment C _π intended to be the receiver of solute S, coming from C ,, C ,, C „containing volumes V, and V _{π respectively} ; At least one enzyme E _α capable of catalyzing, in C „, the reaction for transforming S into solute-product S _αII primary, E _α being carried by the membrane on and / or in its face opposite C _π , • preferably at least one enzyme E _p capable of catalyzing, in C ,, the transformation reaction of the primary solute S _α] = S _αII into the solute- secondary product S _pι , E _p being carried by the membrane on and / or in its face F, opposite C ,, Ep being, even more preferably, the reverse enzyme E _α , so that S _pι = S _,; • preferably at least one enzyme E _δ capable of catalyzing in C _π , the reaction of transformation of the primary solute S _αII into solute S „, E _g being dispersed in C _π in immobilized form; • means for setting in motion at least the liquid C _H and, preferably, also the liquid C,; • and possibly means for adjusting the temperature of the liquid medium. 14 - Device according to claim 13, characterized in that S _αII and the membrane are entities of opposite electrical charges, the (or) membrane (s), being charged (s), on at least one of its faces , preferably on F _π carrying E _α and even more preferably on both. 15 - Device according to claim 13 or 14, characterized in that the enzymatic membrane is a porous matrix in and / or on the two faces F _π / Fι of which are included and immobilized the enzymes E _α , E _p 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. 16 - Device according to any one of claims 13 to 15, characterized in that the enzymatic membrane (5) is in the form of a single or multilayer film, preferably substantially planar. 26 17 - Device according to any one of claims 13 to 15, characterized in that the membrane constitutes all or part of the wall of at least one tube whose lumen forms one of the compartments C / C ,,. 18 - Device according to any one of claims 13 to 17, characterized in that the pair of reversible enzymes E _α / Ep is chosen mainly from the pairs of enzymes allowing the addition / removal of a chemical group loaded on a metabolite, preferably among the pairs of phosphorylation / dephosphorylation enzymes, and even more preferably among the kinases / phosphatases.