DE10164022A1 - Polymer membrane with biomolecules for bioaffine interactions localized in the pores and process for their production - Google Patents

Abstract

A polymer membrane (10) is proposed, in which biomolecules (12) for bioaffine interactions are located in the pores (11), the pores (11) being essentially in the form of open channels crossing the membrane (10), and wherein the membrane (10) on the supply side (16) is acted upon by a solution containing a substance or a mixture of substances, and on the outlet side (17) from a mixture (19) of the product (21) modified by means of the biomolecules (12) and possibly unchanged substance (15) is removed, and a method for producing such a polymer membrane (10). Means (20) for generating a turbulent flow are provided in the pores (11). In one possible type of production process for the polymer membrane (10), the solution supplied to the membrane (10) is loaded with corpuscular elements (20) and then passed into the pores (11) of the membrane (10). The corpuscular elements (20) are then left in the pores (11) for a predetermined time at a predeterminable temperature for reaction. Finally, the primary membrane (10) is then washed with at least one solvent, so that the non-adhesive corpuscular elements (20) are removed from the polymer membrane (10). The biomolecules then bind to the corpuscular elements adhering to the pore wall.

Description

The invention relates to a polymer membrane in which the pores biomolecules for bioaffine interactions are localized, the pores essentially in Form of open channels crossing the membranes are formed and with the membrane on the supply side a solution is applied that contains a substance or contains a mixture of substances and on the output side a mixture of which by means of the biomolecules modified product and possibly unchanged substance, and a process for their manufacture.

A polymer membrane of this type is known (DE-OS 196 48 881). By means of this known membrane controllable, controlled biocatalysis in the membrane basically possible, and it is at least one sufficient substrate accessibility to those in the pores localized enzymes.

In further investigations of the known membrane and also fundamental investigations in this area it has been found that the formation of the following particularly interesting macromolecular Product molecules not only of the average pore size the membrane and its pore size distribution, but very significantly from those present in the pore Depend on the flow conditions. The Tortuosity of the entire transmembrane transport path affects the Flow resistance and thus the process performance. In the Combination of material conversion and material separation within the pore structure of the generic membrane, which are viewed as an ensemble of microreactors may have been found to be significant Influencing variable also the type of Flow conditions in the membrane pores. For an effective Implementation of enzymatic polymer synthesis Reactions in such microreactors is one fastest possible removal of the resulting product from of great importance.

It has been found that otherwise, e.g. B. with only diffuse mass transport through the pores, quickly to catalyst poisoning due to product inhibition can come. Associated with this is the serious one Disadvantage that this also means that with the means of in the membrane embedded biomolecules in the form of Enzyme-linked enzymatic growth reaction is blocked. Experiments on enzymatic production, for example for the production of a polysaccharide, have shown that geometrically defined, ideally continuous sufficiently large pores, as in Nuclear trace membranes can be formed, are to be provided for To avoid constipation. Still has pointed out that after much too short a response time Enzyme activity more measurable and thus the desired continuous litigation is questioned.

From model tests it could be concluded that in a laminar flow in the cylindrical pores forms so that on the pore wall on which the enzymes were immobilized, just a purely diffusive one Mass transfer is possible, which is much too slow and thereby "suffocating" the enzymes in the product.

It is therefore an object of the present invention To improve membrane of the type mentioned above and to further develop that a required convective Mass transfer at least close to the immobilized biomolecules, for example in the form of enzymes, which are bound to the pore walls of the membrane, is made possible, the membrane with simple Means, starting from the generic membrane, can be made and a use of the like manufactured membrane for a continuous Process control should also be possible on a technical scale.

This object is achieved according to the invention Polymer membrane dissolved in that in the pores means for Generation of a turbulent flow of the substance and / or the mixture of substance solution and product are provided.

The advantage of the solution according to the invention, regarding the Polymer membrane, that is a significant Stabilizing the activity of the immobilized biomolecules is possible d. that is, the interaction forces between permeand and membrane polymer are reinforced or the contact of substrate molecules with active ligands, d. H. here the biomolecules, relieved or at all to a significant extent. Would like in the prior art, when flowing through the substrate through the pores, as before, essentially one form laminar flow, this would have the consequence that there is a quiescent zone on the pore wall would, with the disadvantageous consequence that only a diffuse Mass transport could take place. The invention Solution significantly eliminates these quiet zones, if not completely.

Basically, it is possible to generate these funds a turbulent flow of the substrate in the pores train in any suitable way. That's the way it is for example conceivable and also possible in principle, the pore wall in the manufacture of the membrane itself if this the membrane in the shortest way traverse, train in a structured manner so that when flowing through of the substrate through the pores the structures as Baffles for the flowing substrate solution act with the Consequence that the desired turbulent flow in forms the pores.

In a particularly advantageous embodiment of the Membrane is the remedy in the form of corpuscular Formed elements that fed into the membrane Substance solution itself are included. That’s it for example, advantageously the middle one Size of the corpuscular elements directly to the Substrate, the membrane itself and the type of in adapt the biomolecules bound to the pores of the membrane. It is also possible with this configuration that Membrane, for example, one after the other with different to load large corpuscular elements, for example alternately and / or with increasing or decreasing size.

In another very advantageous embodiment of the Membrane in which the means in turn corpuscular Are elements, these are attached to the pore wall of the membrane themselves deposited and remain there continuously.

The means are advantageously basic chemically inert, d. H. participate in the interaction between Substance and the biomolecules not part. I.e. With in other words, that the chemically inert Turbulence as it provides mechanical resistance to the Form substance solution, only physically conditioned for the turbulent flow of the substance solution or the mixture of substance solution and in the Pore-generated product.

But it has also proven to be advantageous turbulence-generating agents from a mixture of chemically inert and chemically reactive corpuscular elements train, so that it is possible, for example, that at appropriate chemically reactive adaptation of the corpuscular Elements on the membrane material in the pores Attachment to the pore walls enables or improves becomes.

It is advantageous to use the reactive corpuscular Elements from a chemically inert core and one to form chemically reactive coating. With the additional chemically reactive coating can cover the entire Surface of the pore wall can be cleverly enlarged, with the result that significantly more biomolecules per Volumes can be bound on the surface making the performance of the membrane essential and can be increased in a targeted manner.

The corpuscular elements responsible for the turbulent Flow of the substance in the pores have any suitable structure per se. It has proven to be advantageous, however corpuscular elements essentially with a to provide spherical structure, the manufacture of which in Comparison to other structures may be easier.

The corpuscular elements can basically consist of any suitable materials, where has shown to be advantageous, the corpuscular Elements made of polystyrene (latex), for example train. This material points for the here applications of the polymer membrane described a good chemically inert behavior.

The chemically reactive corpuscular described above Elements consist at least partially Polyglyceride metachrylate, but there are also others chemically reactive materials conceivable.

The average diameter of the corpuscular elements is largely depending on the membrane-forming Material, the substance solution, and the pore diameter and possibly also depending on the type of in the Pore wall selected biomolecules. It has exposed the average diameter of the corpuscular elements preferably in the range of 50 to 1000 nm depending on the given pore diameter, whereby a quarter of the Diameter of the average pore size is selected.

It should also be noted that the middle one Diameter of the corpuscular elements in the chemical inert and chemically reactive corpuscular elements can be chosen the same or different.

• a) zunächst mit korpuskularen Elementen beladen wird,
• b) anschließend in die Poren der Membran geleitet wird,
• c) anschließend an den Orten gemäß Merkmal b. eine vorbestimmte Zeit bei vorbestimmter Temperatur belassen und
• d) schließlich mit wenigstens einem Lösemittel gewaschen wird.

The process according to the invention for producing a polymer membrane is characterized in that the substance solution supplied to the membrane

• a) is first loaded with corpuscular elements,
• b) is then passed into the pores of the membrane,
• c) then at the locations according to characteristic b. leave at a predetermined temperature for a predetermined time and
• d) finally washing with at least one solvent.

The advantage of solving the method according to the invention is that a membrane made in this way The prerequisite for this is that continuous bioaffins, for example biocatalytic, Manufacturing processes of products much more economical can be designed as before, d. H. also in are technically feasible, for example Manufacture of medicines, food additives, which have certain compatibility properties need, and other products.

The method is advantageously thereby further developed that a mixture of chemically inert and chemically reactive corpuscular elements used is, the density of the loading of the pore wall with chemically reactive corpuscular elements due to their predetermined, desired interaction with the material of the membranes in the pores too can be adjusted.

The dwell time according to characteristic c. can be dependent the material of the membrane, the material of the corpuscular elements, the chemically reactive material of corpuscular elements and also the mechanical ones Dimensions of the membrane can be selected. But it turned out to be the residence time was found to be advantageous to be provided, for example, in the range of 12 h, the Temperature is preferably in the range of room temperature lies, so that no special for the production Measures related to tempering the Storage process must be taken. This closes however, it does not mean that it is used for certain purposes temperature control is also conceivable and possible to be provided in the area of higher temperature.

The process is described as follows completed that with the corpuscular elements finally filled membrane with an aqueous solution is washed out, so that all not firmly bound Remove particles and turn a high one To achieve membrane permeability as it did before loading has passed. The washing process can preferably be carried out with an aqueous solution, for example only with water without additives or, for example, with water and at least one surfactant.

The invention will now be described with reference to the following schematic drawings using a Described embodiment. In it show:

Fig. 1 shows schematically a starting substrate in the form of saccharose, which is produced by means of the membrane in inulin and glucose by an FTF-based biocatalytic conversion,

Fig. 2A is a schematic section through a pore of the polymer membrane with the laminar flow pattern of the substrate solution and the blocking of the enzyme molecules through the resulting product as the starting situation,

FIG. 2B, an increase in flow gradient of the enzyme molecules by incorporation of particles on the pore walls, which act as flow baffles and thus bring about a rapid removal of product,

Fig. 3 is a section through the membrane, taken with a scanning electron microscope, the corpuscular elements which are embedded in the pores in the form of latex beads,

Fig. 4 is a graphical representation of the rapidly increasing clogging behavior as a significant decrease in the flux of not loaded with particulate elements membranes in the form of nuclear track membranes and formed therein cylindrical pores, depending on their diameter,

Fig. 5 shows the result of an investigation of a planar model system of a loaded with enzyme-porous film, the extent to-cell cross-flow changed at a high flow velocity in the activity of the system in dependence on the reaction time,

Fig. 6 shows the result of an experiment with the membrane of the invention in the form of a nuclear track membrane with a Zylinderkapillardurchmesser of 1000 nm and the same flow of 320 l / hm ² with and without added corpuscular elements (latex beads),

Fig. 7 is a diagram in which the dependence of the enzyme activity measured for glucose and fructose, a membrane in the form of nm of a nuclear track membrane with cylindrical pores diameter of 400, the enzyme is directly attached to the pore walls, it can be seen from the reaction time. Enzyme activity can no longer be recovered from the higher flow rate during the

Fig. 8 membrane according to the invention in the same scaled diagram according to Fig. 7, in which by the vortex generated by the corpuscular elements at the same initial speed of 32 l / hm ² a higher activity with higher initial effectiveness can be seen, which is due to a stronger enzyme loading is. A higher flow rate can even increase the enzyme activity again.

Reference is first made to the illustration of FIG. 2A. A polymer membrane 10 , of which only a pore 11 or a channel 11 is shown in cross section, for example, is produced in the usual way, for example. In the present case, the polymer membrane 10 can be a so-called core track membrane with very uniform pores 11 , for example in the form of continuous cylindrical channels. When the membrane 10 functions as intended, a substance or substance solution 15 is fed to the membrane 10 on the inlet side 16 . Biomolecules in the form of enzymes 12 have previously been incorporated into the pores or channels 11 of the membrane 10 , cf. also the DE-PS 196 48 881. From the outlet 17 shown in the illustration of FIG. 2A below, after the biocatalytic reaction, the product 21 and the substrate or substance solution 15 flowing through it emerge in the form of a mixture 19 (permeand).

In the enzymatic reaction with a fructosyl transferase (FTF) described here as an example, cf. Fig. 1 sucrose is cleaved into glucose substance 15 first 21 a and an active fructosyl. Immediately afterwards, these fructosyl residues combine with their binding energy to form polyfructane 21 in a coupled reaction. If the enzyme 12 is covalently immobilized in the pores 11 of the polymer membrane 10 , this reaction can be carried out continuously in the case of a pressure-driven transmembrane passage of the substance 15 . When using z. B. of inulin sucrase (FTF) from Streptokoccus mutans, inulin with high molecular weight M _W : 10. .80 million daltons with a very low polydispersity P: 1.1. (Hicke et al.)

Integral-asymmetrical membranes with pores of different geometry and size that have been customary up to now clog very quickly. According to the invention, therefore, so-called nuclear membrane membranes with very uniform pores in the form of continuous cylinder channels or capillaries are preferably used as polymer membranes 10 . From Fig. 4 it can be seen that only with a diameter of the pores 10 of 3000 nm, ie without loading of the corpuscular elements 20 , hardly any clogging occurs. However, only a low enzyme activity was measured after 12 hours. With the help of a flat model system of a pore-free film loaded with the enzyme 12 , cf. Fig. 5, it was investigated to what extent at high flow velocity in a cross-flow cell alters the activity as a function of the reaction time. It can be seen from FIG. 5 that in each individual case, even when comparing with a small overflow, the enzyme activity measured on the basis of glucose formation is retained over longer periods (440 h in total). However, there is little inulin activity after 140 h since the glucose / fructose ratio is almost 1: 1. This fact supports the assumption that no enzyme activity can be found in the pores 11 so quickly only because a diffusion zone forms on the pore wall 110 , cf. Fig. 2A, the product 21 is in the faster than it can diffuse out, whereby the enzyme 12 as it were "choking", see FIG. again Fig. 2.

According to the polymer membrane according to the invention FIG. 2B, the desired convective mass transfer can be significantly improved by adding corpuscular elements, here in the example in the form of polystyrene latex balls (diameter d _K : 100 nm) into the substrate solution 15 . In the case of enzyme-loaded membranes 10 in the form of nuclear track membranes with a cylindrical capillary diameter d _Z : 1000 nm and the same flow rate J: 320 l / hm ² , with the addition of corpuscular elements 20 in the form of polystyrene latex balls, not only significantly more product 21 was created, but above that also find a very favorable glucose / fructose ratio. The very low fructose content suggests a high proportion of the target product inulin. The corpuscular elements 20 in the form of latex balls are given a swirl (Dean vortex) before entering the pores 11 . As a result of this rotational speed, the corpuscular elements 20 are moved out of the laminar flow in the form of latex balls and then abut the pore wall 110 . The rebound creates further turbulence and thus the desired convective mass transfer. According to the invention, the effect which interferes with the formation of a laminar flow should also be used to fasten the corpuscular elements 20 to the pore wall 110 and, as it were, to create turbulence as baffles protruding into the flow of the substrate 15 or the substrate solution. Furthermore, the larger specific surface area associated with the binding of the corpuscular elements 20 is intended to immobilize a larger amount of enzyme per unit area or volume (see FIG. 2B).

With a mixture z. B. from 3 RT of a 2% latex solution, the balls (d _K : 250 nm) are chemically inert, ie consist only of polystyrene, and 1 RT is a 2% latex solution, the balls of a polystyrene core and a chemical reactive sheath made of polyglycidil metachrylate, the pores 11 of the polymer membrane 10 are filled and for example 12 h at z. B. room temperature. After a washing procedure with water and a 0.1% surfactant solution, the initial value was again measured when determining the water permeability of the polymer membrane 10 .

The immobilization of the fructosyl transferase FTF (inulin sucrase) takes place over 20 h at room temperature and pH 7.2 to these covalently bound corpuscular elements 20 (latices). After washing several times with phosphate buffer pH 7.2 and rinsing with surfactant solution, the enzymatic reaction was carried out exactly as above with 5% sucrose solution at pH 7.2 and 28 ° C. The result is shown in FIGS. 7 and 8.

The diagram according to FIG. 7 shows the dependence of the enzyme activity (measured for glucose and fructose) on a polymer membrane 10 formed as a nuclear trace membrane with cylindrical pores 11 with a diameter of D _Z : 400 nm, to the pore walls 110 of which the enzyme 12 was directly bound, recognizable by the response time. This polymer membrane 10 was selected for the comparison in order to be able to approximately take into account the pore distribution in the membrane according to the invention to 500 nm due to the bound latices with D _Z 250 nm. The polymer membrane is irreversibly blocked at a flow rate v: 32 l / hm ² after a reaction time t> 70 min.

The polymer membrane 10 according to the invention in the same scaled diagram according to FIG. 9 shows, due to the flow vortices generated at the same initial speed v: 32 l / hm ^2, a higher activity with a higher initial activity, which results from the stronger enzyme loading.

In contrast to the comparison membrane, the constipation is reversible. This means that with increasing overflow velocity v: 320 and 1000 l / hm ^2, the polymer membrane 10 is flushed out again, the activity measured in terms of glucose then also increasing again. Legend: 10 polymer membrane
11 pore / channel
110 pore wall
12 biomolecule (enzyme)
13 pore length
14 pore cross section
15 Substance solution / substance
16 entrance
17 exit
18 -
19 Mixture (product / substance solution or substance)
20 middle / corpuscular element
21 product

Claims (16)

1. Polymer membrane in which biomolecules for bioaffine interactions are located in the pores, the pores being essentially in the form of open channels crossing the membrane and the membrane being acted upon on the supply side with a solution which contains a substance or a mixture of substances , and on the output side consists of a mixture of the product modified by means of the biomolecules and possibly unchanged substance, characterized in that means ( 20 ) in the pores ( 11 ) for generating a turbulent flow of the substance solution ( 15 ) and / or the mixture ( 19 ) of substance solution ( 15 ) and product ( 21 ) are provided. 2. Polymer membrane according to claim 1, characterized in that the means ( 20 ) are corpuscular elements which are contained in the membrane ( 10 ) supplied substance solution ( 15 ). 3. Polymer membrane according to claim 1, characterized in that the means ( 20 ) are corpuscular elements which are attached to the pore walls ( 110 ) of the membrane ( 10 ). 4. Polymer membrane according to one or both of claims 1 or 2, characterized in that the means ( 20 ) are chemically inert. 5. Polymer membrane according to one or more of claims 1 to 4, characterized in that the means ( 20 ) consist of a mixture of chemically inert and chemically reactive corpuscular elements. 6. Polymer membrane according to claim 5, characterized characterized in that the reactive corpuscular elements from one chemically inert core and a chemically reactive Wrapping exist. 7. Polymer membrane according to one or more of the claims 2 to 6, characterized in that the corpuscular Elements have a substantially spherical structure exhibit. 8. Polymer membrane according to one or more of the claims 2 to 7, characterized in that the corpuscular Elements made of polystyrene (latex). 9. polymer membrane according to one or more of the claims 5 to 8, characterized in that the chemically reactive corpuscular elements at least partially consist of polyglycide methacrylate. 10. Polymer membrane according to one or more of the Claims 2 to 9, characterized in that the middle Diameter of the corpuscular elements in the range of 50 up to 1000 nm. 11. Polymer membrane according to claim 10, characterized characterized in that the average diameter in the range of is a quarter of the pore diameter of the membrane. 12. A method for producing a polymer membrane in which biomolecules for bioaffine interactions are localized in the pores, the pores being essentially in the form of open channels crossing the membrane and the membrane being acted upon on the supply side with a solution which is a substance or contains a substance mixture, and on the output side consists of a mixture of the product modified by means of the biomolecules and possibly unchanged substance, characterized in that the substance solution supplied to the membrane a) is loaded with corpuscular elements, b) is then passed into the pores of the membrane, c) then at the locations according to characteristic b. leave at a predetermined temperature for a predetermined time and d) finally washing with at least one solvent. 13. The method according to claim 12, characterized in that that the solution supplied is a mixture of chemically inert and chemically reactive corpuscular elements contains. 14. The method according to one or both of claims 12 or 13, characterized in that the residence time in Range of 12 h, with the temperature in the range the room temperature is. 15. The method according to one or more of claims 12 to 14, characterized in that the solvent Mixture of water and at least one surfactant. 16. The method according to one or more of claims 12 to 15, characterized in that the biomolecule directly to the reactive surface of the corpuscular Elements is covalently bound.