EP0942966A1

EP0942966A1 - Polymer membrane with enzymes localised in the membrane and process for preparing products by reactions that take place in polymer membranes

Info

Publication number: EP0942966A1
Application number: EP97951084A
Authority: EP
Inventors: Dieter Paul; Margot Becker; Annett Oechel; Alexander Papra; Heinz Nedelmann; Thomas Weigel; Mathias Ulbricht; Hans-Georg Hicke; Lothar Willmitzer
Original assignee: Max Planck Gesellschaft zur Foerderung der Wissenschaften eV; GKSS Forshungszentrum Geesthacht GmbH
Current assignee: GKSS Forshungszentrum Geesthacht GmbH
Priority date: 1996-11-26
Filing date: 1997-11-22
Publication date: 1999-09-22
Also published as: DE19648881A1; WO1998023734A1; DE19648881C2

Abstract

A polymer membrane (10) in whose pores are localised enzymes for biocatalytic purposes is formed by open pores (11) that extend through the membrane (10) with cylindrical to conical pores (10) in the cross-section of the membrane (10). At least the pore length (13) and/or pore cross-section (16) are adjusted depending on the type of a substrate (15) supplied to the membrane (10) and on a product (16) formed from the substrate (15) by the enzymes (12). Also disclosed is a process in which the enzyme charge density in the pores of the membrane is adjusted depending on pore diameter and pore length, so that the transverse diffusion of the substrate that flows through the pores up to the enzymes arranged in a side wall area of the pores be an adjustable parameter of the desired chain length and/or ramification of the thus obtained macromolecular product.

Description

Polymer membrane with enzymes localized in the membrane and process for the production of products by means of reactions taking place in polymer membranes

Description

The invention relates to a polymer membrane in which enzymes for biocatalytic purposes are located in the pores, and to a process for the production of products by means of bioreactions occurring in polymer membranes.

Bioreactions in artificial membranes have been known for a long time (DE-OS 44 20 086). Biocatalytic processes are localized in polymer membranes in order to make the enzymes, which are often very costly to produce or isolate, immobilized within the membrane for constant reuse, for example for carrying out continuous processes for the production of certain products. Previous enzyme immobilisers (particles, gels) are distinguished by the fact that pore diffusion, in particular, is speed-determining there, with the result that there are limits to rapid mass transfer and, in the case of high conversions of a substrate, the removal of potentially inhibiting products, for example by pH shift, there are clear limits.

In a known polymer membrane, the so-called S-layer UF membrane from SLEYTR et. al, it seems to be fundamentally possible to also be able to produce large membrane areas with which biocatalysts of the generic type can be built up. A disadvantage of this known membrane is that it is not possible to vary the pore diameter outside a range of 6 nm or to vary the pore shape. When enzymes are immobilized within these pores, the pores can easily become blocked, with the result that adequate substrate access is no longer ensured, that is to say that the membrane as a whole becomes unusable.

In the case of another type of membrane, the so-called nuclear trace filter membrane, very different pore diameters with a diameter> 30 nm can already be formed. However, the cylindrical capillary shape formed with this membrane cannot be changed, so that a desired convective mass transfer can only be achieved to a very limited extent in certain processes.

It is therefore an object of the present invention to create a polymer membrane of the type mentioned at the outset and to provide a method by which controllable, controlled biocatalysis is made possible in the membrane, adequate access to the substrates located in the pores being possible should be ensured and the resulting product can pass through the membrane convectively, the residence time for the substrate and product in the membrane should be adjustable, thus chen chen to achieve high conversions of the substrate and rapid removal of the product formed. The object according to the membrane according to the invention is achieved in that the open pores crossing the membrane have a substantially cylindrical to conical pore shape in cross-section of the membrane, at least the pore length and / or the pore cross-section depending on the type of substrate supplied to the membrane and of the product formed from the substrate by means of the enzymes is adjustable.

The advantage of the membrane according to the invention lies essentially in the fact that by forming a predetermined pore shape in relation to the cross section and the length of the pores, i.e. by the geometrically predeterminable pore shape depending on the type of substrate and the product, an optimal convective transme braner mass transfer can be realized, i.e. a so-called tortuosity factor with a small amount is possible, which is decisive for a convective transmembrane mass transfer. Otherwise, as is known from particulate or gel-like enzyme immobilized seeds, the preblend infusion would increase, which would reduce the possibilities of controlled product formation.

The demand for the highest possible proportion of convective mass transport could be achieved by an increasing mean pore diameter, but only with the disadvantageous consequence that more and more substrate molecules would pass through the membrane without using the enzymes that are localized or immobilized on the pore wall to be able to interact.

In the formation or manufacture of polymer membranes, there are various ways of producing the Pores, if they are pronounced pore membranes, known.

In an advantageous embodiment of the polymer membrane, the pores are produced as such by subsequent chemical and / or physical influence after the actual manufacture of the membrane, i.e. for example, after the formation or curing of the polymer film by contact with appropriate chemical agents.

Not only the pores as such, but also the density of the pores can accordingly be adjusted after the manufacture of the membrane as such by chemical and / or physical influencing, with the time and the means for forming the pores ultimately determining parameters for the pore density in this of the membrane.

As mentioned at the beginning, the pore length and the pore shape determine the behavior of the membrane as a bioreactor. It has been shown that in macromolecular products it is advantageously not the cylindrical pore shape for the low molecular weight substrates that is the optimal pore shape, but such a pore shape in which the opening of the pores facing the substrate is preferably smaller than the opening of the pores on the product side.

With a high molecular weight substrate and a low molecular weight product, optimal product results have been achieved in that the opening of the pores facing the substrate is larger than the opening of the pores on the product side. The polymer membrane itself is advantageously constructed in the manner of a nuclear trace filter membrane, which advantageously ensures a uniform pore size distribution, which is of great importance for a uniform transport of the substrate acting on the membrane to the enzymes located in the pores.

The core track filter membrane can advantageously consist of polycarbonate or of polyethylene enterephthalate, which enable a geometrically very uniform, cylindrical formation of the capillaries and also a very narrow pore size distribution.

It is also advantageous to provide the substrate-loaded side of the membrane with a coating and, if necessary, also to subject it to a surface modification, which can advantageously be done by the surface modification being capable of being formed with monomers by photopolymerization or by photo-initiated graft polymerisation or in the case of one another advantageous embodiment of the polymer membrane by plasma polymerization or by plasma-initiated graft polymerization with monomers. Acrylic acid is preferably used as the monomer, for example. Instead of the carboxyl groups generated in this way, other functional groups which are directly or indirectly suitable for enzyme formation, such as e.g. preferably primary amino groups are used.

Finally, it is advantageous to subject the polymer membrane to a hydrothermal aftertreatment, the composition of the aftertreatment medium and the pH either being a sole chemical surface modification without changing the membrane structure or else being reproducible asymmetrical swelling is possible with one-sided and short-term exposure.

A process for the production of products by means of bioreactions taking place in polymer membranes of the aforementioned type is characterized in that the density of the enzyme charge in the pores of the membrane is adjusted as a function of the pore diameter and the pore length in such a way that the transverse diffusion of the substrate crossing the pores is increased the enzymes arranged essentially in the side wall region of the pores is an adjustable parameter of the desired chain length of the macromolecular product to be achieved.

These proposed measures have a direct influence on the quality of the product produced by the process.

In an advantageous embodiment of the method, the rate at which the product is discharged from the pores of the membrane can be adjusted, specifically by means of the pore size and / or pore shape and / or the pore size distribution, the adjustability of the rate also being a further adjustable parameter for the achieving desired chain length and / or branching of the macromolecular product. The rate at which the product is discharged from the pores of the membrane is in turn linked to the residence time of the product in the membrane, which in turn is a parameter influencing the quality of the product.

For an optimal function of the polymer membrane according to the invention, it is not only the shape of the pores in the The cross-section and the pore length are decisive, rather the type of overflow of the polymer membrane is decisive for the formation of an optimal mass transfer. It has been found that an optimal mass transfer is achieved by the substrate flowing tangentially over the polymer membrane (cross-flow process control), with the result that no process-determining concentration polarization can develop when there is a strong tangential flow over the membrane surface.

The invention will now be described in detail with reference to the following schematic drawings using an exemplary embodiment. In it show:

1 is a sectional view of a membrane in a greatly enlarged and simplified form for illustration purposes, in which the pores are influenced by the formation of a monomer pl as a to form conical pore shapes,

2, depending on the molecular size of a substrate and a product, three basic types of pore shapes to illustrate the principle according to the invention,

3 pores similar to FIG. 1, in which defined enzyme molecules are incorporated,

Fig. 4 in section a pore of a membrane, in which a cross flow of the substrate is schematically shown, which interacts with the enzymes localized in the pore in functi onel 1 he interaction, wherein in the supply area of the pore (schematically) an oligomer arises and with the formation of intermediate steps in the direction of the substrate-side output of the pore polymers in functi onel 1 he interaction with the enzymes, which are then discharged as a defined product, and

5 shows the intensity ratio of characteristic IR absorption bands from ATR spectra for the exposed upper side (d = 0) and the non-directly exposed lower side (d = 120 μm) of PP-MFM functionalized with graft polyacrylic acid (dp _Q = 200 nm).

The relationship according to the invention is illustrated with reference to FIG. 2. 2 a there shows several ideal pores 11 which are cylindrical in the polymer membrane 10, as are known, for example, from known nuclear trace filter membranes. A substrate 15 and a product 16 have approximately the same size molecules 19 (low molecular weight substances). A funnel or conical pore 11 is shown in FIG. 2 b, in which the molecules of the substrate 15 are substantially larger than the molecules of the product 16. In the case of a pore 11 of this type, a substrate jam is fundamentally possible.

2 c shows the structure of a polymer membrane 10 according to the invention in which the pore has a conical structure with which a polymeric product 16 according to the invention can be produced from a monomeric substrate 15.

3 shows the polymer membrane 10 as it is possible with regard to the pores 11 designed according to the invention to create a functioning bioreactor. To the Enzymes 12 are localized on the walls of the pores 11, the enzyme loading density being adjustable in that the pores 11 are equipped either a priori with reactive membrane polymers or with subsequent chemical or physical modification with the desired density of such functionalities which enable covalent enzyme binding.

In the case of a membrane 10 according to FIG. 3, the diameter of the opening 18 of the pores 11 corresponds to the product diameter of the resulting macromolecule. 3 c, the diameter of the opening 18 on the product outlet side is very much larger than the macromolecule diameter. Here, for example, an emerging substrate cross flow, which must be avoided as far as possible, can be reduced by the incorporation of defined molecular associations of enzymes 12.

In Fig. 3 a, finally, the product diameter of the opening 18 is smaller than the desired macromolecule diameter. Depending on the substrate concentration and pore size, pore clogging occurs more or less quickly, so that after a corresponding start-up phase, such macromolecular fractions are mutually exclusive in this way. Thus, in the pores 11, which have a diameter approximately the same size as the desired macromolecule, clogging can also quickly occur with very large amounts of substrate. Depending on the distribution of the resulting molecular fractions, one can either pass the filtrates contained again over the same polymer membrane 10 or combine several layers of membranes 10 of different pore diameters according to the invention in a corresponding cascade. In such a cascade of membranes according to the invention, in which the distance between the Membranes 10 is defined, for example, by spacers, one can optionally apply a corresponding action to the individual membranes 10 after prior filtration of the filtrate.

To prevent so-called fouling, one can either sterilize all substrates 15 immediately before the first biocatalytical membrane, e.g. by using a Mi krofi 1 trati onsme bran, or by appropriately modifying the membrane top, e.g. achieve constant degradation of germ deposits by coupling proteolytic enzymes (anti-fouling).

The polymer membrane 10 according to the invention can in principle be designed in the form of flat membranes but also hollow fiber membranes, wherein the flat membranes and the hollow thread membranes can be constructed as so-called composite membranes. With the solution proposed according to the invention, multi-enzyme systems or coenzyme bindings can be realized, it being possible to start from mixtures and to couple the enzymes in a statistically distributed manner, or to use different binding affinities and thus to determine a topochemical sequence or distribution.

The polymer membrane 10 according to the invention also includes the possibility of adjusting controllable biocatalysis distribution equilibrium and one can either carry out resynthesis in the aqueous medium or possibly synthesize new products in the organic medium.

1) Plasma treatment Process data:

A. Duration of activation by argon plasma treatment from 1 to 360 s, preferably 5 s;

Argon flow rate from 100 to 1000 sccm, preferably 500 sccm; additionally oxygen flow rate from 50 to 500 sccm, preferably no oxygen;

Pressure of 10 to 70 Pa, preferably 30 Pa;

Microwave field continuous;

Microwave power from 200 to 1500 W, preferably 340

W.

B. Plasma polymerisation with non-equilibrium plasma-poly eri sat ion system in the remote process at:

2.45 Ghz microwave field for plasma excitation;

Argon as a plasma carrier gas;

Acrylic acid as a monomer, taken in downstream in the

Afterglow area;

Polymerization time of 10 to 500 min, preferably 20 min;

Argon flow rate from 100 to 500 sccm, preferably 500 sccm;

Acrylic acid flow rate from 30 to 200 sccm, preferably 50 sccm;

Pressure of 10 to 70 Pa, preferably 35 Pa; Mi krowell power from 200 to 1500 W, preferably 280 W;

Microwave field continuously or pulsed, preferably pulsed with:

Pulse frequency of 500 to 10,000 Hz, preferably 10,000 Hz; Duty cycle of 30 to 70%, preferably 50%. Example 1 :

A polyester core trace filter membrane with a capillary diameter d = 100 nm was coated with an acrylic acid plasapolymer layer after 5 min of cleaning in ethanol by means of a non-equilibrium plasma polymerisation system using the remote method.

For this purpose, the membrane was exposed for 5 s at a process pressure of 30 Pa, an argon flow rate of 500 sccm and a continuous microwave field with an output of

340 W (microwave input power P = 400 W with a reflected power ³ P_re.t _PT I = 60 W) activated.

Subsequently, it was let into the afterglow range at a pressure of 35 Pa, with an argon flow rate of 500 sccm and an acrylic acid flow rate of 50 sccm, in a pulsed microwave field with a pulse frequency of 10 kHz, a duty cycle of 50% and with a power of 280 W (microwave input power P = 360 W at a reflected power P _fl = 80 W) plasma-treated or coated. Fluorescence spectroscopy of 900 pmol / cm carboxyl groups was measured on the membrane.

Using the carbodimide method, the enzyme inulin insucrase was covalently coupled to the carboxyl group-containing polyester core trace filter membrane coated in this way.

For this purpose, the membranes were first activated with a 1% strength aqueous solution of N-ethyl -N'-3-dimethyl-aanopropyl-carbodiimide at pH = 4.75 for 0.5 h and then immediately with the enzyme from the Load 0.1% solution in phosphate buffer at pH = 7 and T = 25 ° C for 3 h. The initial water permeability of J _Q = 2400 1 / hm bar decreased due to the plasma treatment

J = 650 l / hm ² bar. w

From a sucrose solution (600 mM), a transmembrane flow at 40 ° C. and a filter were used.

2 vibration v ° = 500 1 / hm an inulin with the molecular weight

M = 10,000 kDa obtained. A continuous process was possible, the membrane did not become blocked.

2) Photo graft polymerization

Example 2:

Polypropylene and membrane coatings (MFM) with a sponge structure, symmetrical pore size distribution and average effective pore diameters (dp _Q ) of 100, 200 and 450 nm were formed by photoinitiated heterogeneous graft copolymers of carboxyl and ami no-acrylic data on the entire surface functional i si ert.

For this purpose, the membranes were first coated from a methanolic solution (0.01 ... 0.2 mol / 1) with the photoinitiator benzophenone (BP) for 1 ... 16 h. Subsequently, the membranes in aqueous, with BP saturated solutions of acrylic acid (AA; 5 ... 100 g / 1) or 2-amino methyl methacrylate (A EMA, 10 ... 50 g / 1) with a UV light Irradiated excitation wavelength of λ> 300 nm or 290 nm> λ> 320 nm between 15 and 120 min. The membranes were then extracted thoroughly with water, possibly also with methanol. In this way, the functional level (DG) between 0 and

2 3000 μg / cm can be varied. By varying coating, exposure and polymerization conditions, different functionalization gradients could be set in the functionalized membranes, which correspond to the gradients of the average pore size. This was derived from values for local degrees of modification obtained by means of ATR-IR spectroscopy (see FIG. 5).

According to FIG. 5, the following functional conditions are present:

1: BP loading 0.05 mol / 1, 1 h; AA 5 g / 1, 60 min UV (λ> 300 nm); DG = 100 µg / cm ² ;

2: BP loading 0.2 mol / 1, 16 h; AA 10 g / 1, 30 min UV (λ

> 300 nm); DG = 320 µg / cm ² ;

3: BP loading 0.2 mol / 1, 16 h; AA 100 g / 1, 30 min UV (λ

> 300 nm); DG = 2100 µg / cm ² ;

4: BP loading 0.2 mol / 1, 16 h; AA 100 g / 1, 30 min UV (290>λ> 320 nm); DG = 210 µg / cm ² ;

Example 3:

Polyester core trace filter membrane (KSM) with capillary diameters (dp _Q ) between 30 and 3000 nm were functionalized on the entire surface by photoinitiated heterogeneous graft copolymers of carboxyl or amino acrylates.

For this purpose, the membranes were first coated from a methanolic solution (0.1 mol / 1) with the photoinitiator benzophenone. Then the membranes in aqueous solutions saturated with benzophenone from

Acrylic acid (AA; 5 ... 100 g / 1) or 2-aminoethyl ethacrylate (AmEMA, 10 ... 50 g / 1) with UV light

Irradiation excitation wavelength of λ> 300 nm between 15 and 120 min. After that, the membranes were thoroughly cleaned

Water - if necessary additionally extracted with methanol. In this way, the functionalization level (DG) could be varied between 0 and 100 μg / cm.

In the case of the graft polyacrylic acid-functional membranes ("graft tentacles"), particularly significant changes in the effective pore diameter - with consequence for the membrane permeability - 1 i tat - are achieved for medium degrees of functionalization and depending on the pH value .

A KSM (dp _Q = 450 nm) with DG = 30 μg / cm ²

(Membrane 1: 100 g / 1 AA; 45 min UV) at pH = 3 a

2 permeability of 6300 1 / m hbar, at pH = 7 a permeability of 50 1 / m hbar (the water permeability of the

2 unmodified KSM is 45000 1 / m hbar).

A KSM (dp _Q = 3000 nm) with DG = 18 μg / cm ² (membrane 2:

100 g / 1 AA; 60 min UV) has a permeability at pH = 3

2 of 195,000 1 / m hbar, at pH = 7 a permeability of

2 3800 1 / m hbar (the water permeability of the unmodi ﬁ ed KSM is 210000 l / m ² hbar).

In the case of the graft poly (2-aminoethyl methacrylate) - unfunctionalized membranes (graft layer), the changes in the effective pore diameter - with consequence for the membrane permeability - are significantly smaller and not pH-dependent. A KSM (dp _Q = 450 nm) with DG = 16.5 μg / cm ² (membrane 3: 40 g / 1 A EMA; 60 min UV) has a permeability of 42000 1 / m hbar (the water permeability of the unmodified KSM is 45000 l / m ² hbar).

The enzyme inulin sucrase (FTF) was covalently coupled to carboxyl group-containing membranes using the carbodiimide method. For this purpose, the membranes were first activated with a 15% strength aqueous solution of N-ethyl -N'-3-dimethylamino propyl carbodi id at pH = 4.75 for 0.5 h and then immediately with the enzyme the 0.1% solution in phosphate buffer at pH = 7 and T = 25 ° C for 4 h.

The enzyme inulin sucrase (FTF) was covalently coupled to membranes containing amino groups by means of glutaral dehyde. For this purpose, the membranes were first activated with a 10% solution of glutaraldehyde for 5 hours, then washed with water until the aldehyde had been completely removed and then immediately with the enzyme from a 0.1% solution in phosphate buffer at pH = 7 and Load T = 25 ° C for 16 hours.

With sucrose as the substrate (600 mM), a pH = 7.2 and an internal overflow flow rate of 1000 l / m ² h were obtained

for AGV membrane 1:

Inulin with M = 80,000 kD, but with rapid blocking of the membrane (high capacity for enzyme loading, but pore constriction due to tentacles);

for AGV membrane 2:

Inulin with M = 80,000 kD, continuous product extraction, as there is hardly any blocking due to larger matrix pores (high capacity for enzyme loading through tentacles); for AGV membrane 3:

Inulin with M = 1000 kD, continuous product extraction (high capacity for enzyme loading through graft layer, hardly any blocking due to layer structure, so that the use of smaller pores is possible and influencing the product structure).

Reference character list

10 polymer membrane (membrane)

11 pore

12 enzyme

13 pore length

14 pore cross section

15 substrate

16 product (polymer)

17 opening (substrate)

18 opening (product)

19 molecule

20 Monomer pl asma

21 coating

22 01 igomer

Claims

claims

1. Polymer membrane in which enzymes for biocatalytic purposes are located in the pores, characterized by open pores (11) which cross the membrane (10) and have essentially cylindrical to conical pores (10) in cross-section of the membrane, at least the pore length (13) and / or the pore cross section (16) can be set as a function of the type of substrate (15) supplied to the membrane (10) and the product (16) formed from the substrate (15) by means of the enzymes (12).

2. Polymer membrane according to claim 1, characterized in that the pores (11) are produced in the course of the manufacture of the membrane (10).

3. Polymer membrane according to one or both of claims 1 or 2, characterized in that the pores (11) after the manufacture of the membrane (10) as such are produced by chemical and / or physical influence.

4. Polymer membrane according to one or more of claims 1 to 3, characterized in that the density of the pores (11) after the manufacture of the membrane (10) as such is adjusted by chemical and / or physical influence.

5. A polymer membrane according to one or more of claims 1 to 4, characterized in that the opening (17) of the pores (11) directed towards the substrate (15) is smaller than the opening (18) of the pores (11) of the pores (11) i st .

6. Polymer membrane according to one or more of claims 1 to 5, characterized in that the membrane is constructed in the manner of a nuclear gauge filter membrane.

7. Polymer membrane according to claim 6, characterized in that the membrane consists essentially of polycarbonate.

8. Polymer membrane according to claim 6, characterized in that the membrane consists essentially of polyethylene terephthalate.

9. Polymer membrane according to one or more of claims 6 to 8, characterized in that the substrate-loaded side of the membrane is provided with a coating or is surface-modified.

10. Polymer membrane according to claim 9, characterized in that the surface modification by photopolymerization or photo-initiated graft polymerization with monomers.

11. Polymer membrane according to claim 9, characterized in that the surface modification can be formed by plasma polymerization or by plasma-initiated graft polymerization.

12. Polymer membrane according to one or more of claims 10 or 11, characterized in that acrylic acid is used as the monomer.

13. A polymer membrane according to one or more of claims 6 to 12, characterized in that it can be subjected to a hydrothermal aftertreatment.

14. A process for the production of products by means of bioreactions taking place in polymer membranes according to one or more of claims 1 to 6, characterized in that the density of the enzyme charge in the pores of the membrane is adjusted as a function of the pore diameter and the pore length in such a way that the achieved Transverse diffusion of the substrate crossing the pores to the enzymes arranged essentially in the side wall region of the pores is an adjustable parameter of the desired chain length and / or branches of the macromolecular product to be achieved.

15. The method according to claim 14, characterized in that the speed of discharge of the product from the pores of the membrane is adjustable.

16. The method according to one or both of claims 14 or 15, characterized in that the membrane is flowed tangentially over the feed side of the substrate.