DE10019377A1 - Immobilization of biologically active substances, especially enzymes, comprises contacting a foamed polymeric material with the substance - Google Patents

Abstract

Process (I) for immobilizing a biologically active substance comprising contacting a foamed polymeric material with the substance, is new. Independent claims are also included for the following: (1) products (II) obtainable by process (I); and (2) chiral syntheses performed using (II) at 5-80 deg C.

Description

The invention relates to a method for immobilizing biologically active substances on carrier materials, the manufacture of a largely hydrophobic, non-polar carrier material here for and the use of those produced by this method supported immobilized biologically active substances for chiral Syntheses.

Due to their selectivity and high catalytic enzymes Activity increasing in food, pharmaceutical and chemical industry used. Technological disadvantages in the The use of enzymes consists in particular in that often because of their substrate solubility from the reaction medium cannot be separated satisfactorily. Besides that associated loss of these expensive biocatalysts ent there are additional expenses due to necessary Cleaning processes.

A number of advantageous effects can be achieved by suitable immobilization of the water-soluble enzymes:

• - Easy separation of the enzyme from the reaction solution;
• - Recyclability of the supported enzyme (lowering the specific catalyst costs);
• - no post-treatment (deactivation, cleaning) of the process solution required;
• - continuous process control possible (better process control)

Numerous attempts have been made in the past to to make biologically active substances as permanent as possible, in particular Enzymes, while maintaining their range of properties on solid Immobilize carrier materials.

EP-A-0035883 describes a transesterification process. there it is recommended to immobilize the enzyme on a support. As Carrier material are u. a. called: diatomaceous earth, kaolinite, Pearlite, silica gel, cellulose powder, polyvinyl alcohol and calcium carbonate. The enzyme is used as an aqueous solution only with this Stirred material. In WO-A-9936964 an enzyme on carriers such as e.g. B. silica, alumina and. a., immobilized, whereby  these carriers are coated with organic substances on the surface are to adjust a hydrophobic surface character. In EP-A-0140542 uses an anion exchanger as a carrier medium set. WO-A-9426883 describes an enzyme on porous materials, such as As soda and silica adsorbed, these Materials are covered with a protective layer. In DE-A-27 37 745 and US-A-0718549 discloses a method for producing such microporous, non-cellular body described, the Base material is a thermoplastic polymer. Reason The location of this procedure is the fact that you can first a homogeneous solution of this polymer in a suitable solution produces medium. This solution is then cooled down so quickly that thermodynamic non-equilibrium conditions are set which results in a liquid-liquid phase separation. Then there is at least one to form the microporous structure Remove part of the liquid. Pore sizes of 0.5 up to 100 µm. US-A-4247498 operates in an analogous manner. To In both writings, the micropore volume formed is partial still filled with functional liquids. In US-A-4250080 a solid microporous support is used (e.g. aluminum oxide), which is coated with a prepolymerized polystyrene. In the following, amino groups are still generated.

In EP-A-0044052 and EP-A-0044051 a technically demanding full process described. This is the Production of a microporous polypropylene. Expressly describes the fact that it is only under extreme Difficulties are possible, porous polymer materials such. B. Polypropylene, to be ground and converted into a powder fraction. A method is therefore proposed in which polypropylene dissolved in a hot pentaerythritol ester and slowly dissolved Room temperature is cooled. The solidified mass then becomes crushed and the ester released. The so made Material is manufactured by the Akzo company under the name ACCUREL® expelled.

According to US-A-3607793 a porous polypropylene carrier material by producing a dispersion of the polypropylene in a hot hydrocarbon. From the down cool resulting gel, the solvent is then extracted. Particles with diameters <0.5 µm are used.

According to US-A-4539294 (EP-A-0105736) proteins are on polymer carriers, u. a. also polypropylene, immobilized. This will be Polymers beforehand with a long chain cationic surfactant wetted. EP-A-0104542 describes a weak anion exchanger claims to which a lipase is applied. For improvement  post-treatment is also used for immobilization Glutaraldehyde. As exchanger base material u. a. Products, such as phenol formaldehyde resins and polystyrene resins. In DE-A-32 05 289 describes porous bodies with an adjustable pore wall described. These are in turn thermoplastic Polymers that have a system of solvents and non-solvents and a control of the phase separation processes is produced in a controlled manner become. In DE-A-33 11 889 attempts are made to biological liability active substances on non-polar polystyrene surfaces through a To improve surface pretreatment with diazonium compounds.

In the hydrolysis described in US-A-4629742 (EP-A-0232933) of fats is u. a. Lipase, immobilized on ACCUREL®, described. The lipase is transferred from the aqueous solution to the Polymer applied, optionally with a pretreatment is carried out with a polar organic solvent. The best results are obtained when the porous polymer pretreated with a metal salt solution before immobilization becomes. EP-A-322213 describes the production of fatty acid esters described, wherein the enzyme is immobilized on a support. In addition to ion exchangers as carriers, lipase is immobilized on Polystyrene or ACCUREL® described. According to WO-A-9015868 Lipase directly onto a porous support from aqueous solution brought, with a special pretreatment of the carrier (including ACCUREL®). To do this, the backing material previously with a polar, water-miscible solvent - mostly an alcohol - pretreated. In WO-A-9103565 the hydrolysis of phospholipids using immobilized lipase. Acrylic resins, polystyrene, Phenol formaldehyde resins and ACCUREL®. According to WO-A-9428118 a Enzyme (lipase) immobilized on a hydrophobic carrier material, previously pretreated with a nonionic surfactant becomes. As a carrier material u. a. Polystyrene, polyethylene and Called polypropylene. WO-A-9533047 describes a method for Reprocessing of immobilized on hydrophobic carriers Enzymes such as B. Lipase. Here again the alcohol treatment of ACCUREL® underlined. According to WO-A-9815572 small peptides are adsorbed on polystyrene. There simple adsorption of these peptides on solid supports leads to unsatisfactory results, therefore becomes a bond linkage via an acyl group proposed.

In addition to these adsorptive processes for the immobilization of Biologically active substances are still known processes to which a covalent bond is made. With the covalent Binding via reactive groups can occur in addition to the desired one  Fixation of the enzymes to influence the catalytic Activity coming.

DE-A-41 31 546 describes lipase on a commercially available Carrier (Eupergit C - Röhm Pharma Germany) over existing Epoxy groups covalently linked. With the help of so immobili chiral alcohols are to be produced. In US-A-4342834 becomes an enzyme if maintained or increased its catalytic activity bound to a polyurethane. there preferably the enzyme from aqueous solution with the poly urethane contacted before the polyurethane foamed becomes. When foaming, care is then taken to ensure that the heat of reaction does not denature the enzyme.

In WO-A-9746590 and WO-A-9746267 various methods for Immobilization of bioactive materials called. On one The carrier is first a layer of a polymeric surface active material applied. This applied material will networked in-situ with the help of a crosslinker. Become a crosslinker u. a. Vinyl compounds, imidazoles, epoxides, isocyanates, anhydrides etc. called. A second layer is ultimately added to this layer Layer of a hydrophilic polymer applied and so with the Base layer connected. The bioactive molecule is made via the second layer attached.

EP-A-0571748 discusses the fact that matrices are made of PUR hydrogels have good mechanical properties that existing free isocyanate groups, however, high toxicity possess biological materials. This is one considerable property damage to be expected. That is why suggested initially capping the isocyanate groups to generate by a bisulfite adduct. To the aqueous solution this bisulfite adduct is given the cell mass. This The mixture is dropped into a sodium alginate solution. The out forming alginate shell has a stabilizing effect and can subsequently be removed in a phosphate buffer.

Allow the proposals corresponding to the state of the art immobilization is biologically more active in individual cases Species, however, often with a drop in catalytic Activity. To make this necessary Carriers with a hydrophobic surface are technically applied agile procedures used. There is therefore a sufficient of improvement potential. In particular, it is necessary Develop solutions that allow both in anhydrous Media as well as in particular from aqueous solutions and / or Suspensions of biologically active species, especially on non-polar ones  Matrices permanently while maintaining the catalytic activity to tie. It must be ensured that the biological active species are sufficiently firmly attached to the surface and that it doesn't affect their catalytic activity becomes.

The object of the invention was therefore to develop a method wrap that a permanent fixation of biologically active Substances, especially enzymes, on non-polar matrices, ins special of foams, allowing for sufficient mechanical strength of the carrier material can be guaranteed should. The biocatalyst-support composite produced in this way should with high selectivity in chiral syntheses, especially in Transesterification processes can be used.

Surprisingly, this task was solved by that non-polar matrices with the largest possible surface, ins special polymer foams that have no or only very small proportions have reactive groups, preferably comminuted, used as carrier materials and these with aqueous Solutions and / or suspensions or with enzyme suspension in suitable solvents are brought into contact. This the resulting inhomogeneous mixture is adhered to Gentle temperature at which the enzyme is not destroyed evaporated and can then be used in the synthesis process.

The invention accordingly relates to a method for Immobilization of biologically active substances on carriers materials, which is characterized as a carrier foamed polymer materials used and with the biologically active substances are brought into contact.

Objects of the invention are also those according to this Processed supported immobilized biologically active substances themselves, their use for chiral syntheses as well as these chiral syntheses themselves.

Surprisingly, it has been shown that it is quite possible foamed polymers, such as polypropylene or polystyrene, to shred with comparatively little effort. We found Surprisingly, our investigations continue to show that when using largely hydrophobic, non-polar carriers materials, preferably in a shredded state, organic Ver active materials permanently on the foam surface can be anchored and thus immobilized without this lose their activity. The result of the invention was achieved without further special measures being necessary  were to add an additional surface area to the polymer to generate particles. It was just the Her the existing surface, made accessible available through the shredding process. It was surprisingly, that already a good adsorptive fixation could be achieved.

The special shredding technology is that during and / or before comminution, for example by Grinding or grating, the foam particles are cooled. This is preferably done in the presence of dry ice.

Furthermore, you can with little technical effort from ver foamed polymer particles, especially foamed polystyrene types, produce a porous powder by molding made of this material or expanded foam balls and rubs it in with a rasping device. This can then cooling is usually not required. The particle size of the polymer particles obtained in this way is more advantageous as 0.5 to 5 mm, preferably 0.5 to 3.0 mm.

If necessary, but not absolutely necessary, one can be tightened after the following sieving process to classify the foam powder. This crushing technology produces porous polymer particles with a partially roughened surface, whereby the foam skins, which themselves represent a large surface, are at least partially preserved (see Fig. 1).

The polymer powder thus obtained is particularly suitable for Immobilization of biologically active materials directly from aqueous solutions, with some solvent can be included.

A shredding of Neopolen® foam beads with a analog technology succeeds even with a corresponding one Cooling with great difficulty. Here, however, offers itself a modified grinding process. First, the Neopolen® Foam beads pre-cooled with dry ice and in one optionally multi-stage, preferably two-stage, meal process using a mill, preferably a cutting mill, crushed. It is of particular advantage if one during the Add some crushed dry ice to the grinding process. That so Porous foam powder obtained based on Neopolen® can may be classified by screening, however, is also directly for the immobilization of biologically active compounds suitable. This process becomes a porous polypropylene powder  receive. The grain size range is from selected grinding technology.

However, without further cooling, a molded body consisting of foamed polypropylene, over a rasp device to shred. Such moldings can be Filling a mold with, for example, Neopolen® foam beads and weld them under the influence of steam getting produced.

Coarse are well suited as carriers for biologically active substances granular particles. These materials can, for example, in a reactor tube can be introduced. By an appropriate Varying the particle size can change the pump pressure required be optimized when using these materials.

The foams of the invention should be as large as possible Surface for the contact between the foam surface and the biologically active materials that come from the liquid medium to be fixed on the foam surface.

In addition, it can be advantageous to use the foams adjust hydrophilic, so that an optimal wetting of the Foam is ensured with the reaction medium. The Hydrophilicity of the foams can be achieved, for example, by Use surfactants during foaming process or subsequently incorporated into the foam will be increased. If necessary, solvents with can be used to improve particle wettability.

As a rule, the foams according to the invention are hydrophobic. The foams have good structural stability.

The preparation of the polymer used according to the invention materials can be done in the usual way. The Process conditions and the necessary input products as well as auxiliaries and / or additives are in the specialist literature extensively described (compare e.g. A. Echte, manual der Technische Polymerchemie, Verlag Chemie, 1993).

Preferred blowing agents for the production of the foams are Water and / or hydrocarbons used. Physically too active blowing agents, for example hydrocarbons, such as n-, iso- or cyclopentane, or halogenated hydrocarbons, such as Tetrafluoroethane, pentafluoropropane, heptafluoropropane, pentafluor butane, hexafluorobutane or dichloromonofluoroethane can be set. About the amount of physical  Blowing agent can change the bulk density of the foam and given if the porosity is affected.

Auxiliaries and / or additives are, for example surface-active substances, foam stabilizers, cell regulators, external and internal release agents, fillers, pigments, hydrolysis protective agents as well as fungistatic and bacteristically acting Substances used.

The foams can be in open or closed metallic molds or through the continuous Application of the reaction mixture to production lines made of foam blocks or stand after the Foaming process available as expanded polymer particles.

The inventive immobilization of biologically active Substances on the carrier materials described are made by Bringing the carrier materials into contact with the biologically active ones Links.

Biologically active compounds are understood to mean products from the protein group of antibodies, antigens, protein Conjugates, lectins and preferably the enzymes originate. A in particular cell cultures, cell suspensions, Microorganisms and especially enzymes. These materials ver like to activate biological processes. You can for that Application of the invention both as a solid material or preferably as a solution or optionally as a suspension in are present in an aqueous and / or organic medium.

Biologically active substances have proven to be particularly advantageous in addition to cell suspensions also a protruding fermenter solution the fermentation of cell aggregates and enzymes in pure form proven. In particular, Lipase has opted for transesterification Enzyme proven. Often a lipase is used, which by Fermentation from Pseudomonas Plantarii is obtained. After a appropriate freeze drying, this lipase is a solid Powder available, this preparation step costs is intense. It is therefore of particular interest to have one Fermenter solution after centrifuging the cell material remains and the enzyme to be fixed in an aqueous Contains suspension, with the help of the carrier according to the invention work up materials.

The contacting of carrier material and biologically active Substance and the subsequent subsequent evaporation of the water or solvent must be done under conditions where  the biologically active material is not damaged. This is guaranteed at temperatures below 45 ° C. Furthermore, should a pH range of 4 to 7 during immobilization being held.

It is advantageous to carry out the method according to the invention adheres to the biologically active substances in solution or suspension to bring the porous polymer particles into direct contact. For example, a fermenter solution or a cell suspension sion containing lipase directly with an appropriate amount of the porous polymer are stirred. After a contact time, which depends on the respective biologically active substance using a rotary evaporator excess water and any solvent shares. This process is with reaching one free-flowing polymer material completed. In this case lipase is fixed to the surface via adsorptive bonds and can then be used to generate chiral reagents become.

It is also possible and customary to use the inventive porous foam particles into a reaction column to fill. The fermenter solution can then be loaded over this for loading Column. A subsequent drying step is possible.

An enzyme loading of 1 to 20% is advantageous preferably 1 to 10%. The control takes place at Known enzyme activity via an appropriate weight. It is still possible to measure the enzyme charge reached. To the activity is determined in a suitable test reaction (see also test reaction example 1).

The supported produced according to the methods described immobilized biologically active substances are for use suitable for chiral syntheses. Enzymes are especially too chiral syntheses. A common synthesis used is the transesterification of suitable raw materials if possible selective yield of the desired chiral esters.

The chiral syntheses are preferably in one temperature range from 5 to 80 ° C.

The present invention is intended to be illustrated by the examples below are explained in more detail.  

Test reaction

As a test reaction to check the effectiveness of the lipase served the enzyme-catalyzed esterification of 1-phenylethanol with vinyl propionate.

The procedure is as follows:

The immobilized lipase is treated with a substrate solution (see model reaction below) and at room temperature shaken. Sales are tracked using gas chromatography. The maximum turnover is 50%. After 24 hours it is filtered the recovered lipase added new substrate and again shaken.

A lipase obtained from Pseudomonas Plantarii served as the enzyme.

example 1 comparison

Polypropylene granules (not expanded) were ground using a mill shredded under cooling. Similar efforts were made using a rasp. This could be done using both technologies Only crush material with extreme difficulty. It came to glue the moving parts.

Example 2

Foamed polypropylene beads (Neopolen®) were initially included Chilled dry ice for 30 minutes. Then they became like this cooled foam beads using a high-performance granulator crushed. Thereby, the Neopolen® during the shredding added some dry ice.

The particles thus obtained had a grain size of <3 mm.  

Example 3

Neopolen® molded body (manufactured by filling a mold with Neopolen® foam beads and welding them together under one effect of steam) have been cut so that the shape body into the feed opening of a household rasp (Quelle AG) fit in. By using a suitable rasp insert foam particles could be easily produced without cooling.

Example 4

With the rasp technology used in Example 3 in an analogous manner, foamed polystyrene types are crushed. A The material was cooled with this shredding technology not mandatory.

Both Styrodur® and Styropor® have been powdered Group received. The grain size was 0.8 to 3 mm.

Example 5 comparison

10 g polypropylene granules (uncrushed) were placed in one Reaction flask and with a fermenter solution (enzyme activity 25000 U / ml) from Pseudomonas plantarii (enzyme activity 10000 U / ml) brought into contact. This mix was made for Stirred slowly for 15 minutes. After that, the excess became Carefully use water in a rotary evaporator at temperatures <45 ° C evaporated in vacuo.

The enzyme-carrier composite produced in this way was: its activity in chiral transesterification (see test reaction) used.

There was significant activity after 2 runs waste.

Example 6

Analogously to Example 5, 10 g of those obtained in Example 2 were porous foam particles with an aqueous fermenter solution (Enzyme activity 25000 U / ml) treated and in the same way as given in Example 5, worked up.

The enzyme-carrier composite produced in this way was: its activity in chiral transesterification (see test reaction) used.  

There was significant activity after 15 runs waste.

Example 7

10 g of the porous foam produced according to Example 3 particles were washed with an aqueous fermenter solution (enzyme activity 50000 U / ml) from Pseudomonas plantarii (enzyme activity 10000 U / ml) brought into contact. This mix was made for Stirred slowly for 15 minutes. After that, the excess became Carefully use water in a rotary evaporator at temperatures <45 ° C evaporated in vacuo until a free-flowing powder was formed.

The enzyme-carrier composite produced in this way was: its activity in chiral transesterification (see test reaction) used.

There was significant activity after 18 runs waste.

Example 8

10 g of a porous polypropylene powder according to Example 2 were classified by means of sieves before the immobilization process. For Immobilization analogous to Example 5 became foam particles the size 0.5 to 1.8 mm used. The fermenter solution are pro rata 10% methyl tertiary butyl ether was added.

The enzyme-carrier composite produced in this way was: its activity in chiral transesterification (see test reaction) used.

There was significant activity after 16 runs waste.

Claims (19)

1. A process for immobilizing biologically active substances on carrier materials, characterized in that foamed polymer materials are used as carrier material and are brought into contact with the biologically active substances. 2. The method according to claim 1, characterized in that the foamed polymer materials are mechanically crushed. 3. The method according to claim 1 or 2, characterized in that a largely hydrophobic, non-polar polymer is used. 4. The method according to any one of claims 1 to 3, characterized records that as polymer materials polypropylene, poly ethylene and polystyrene using blowing agents have been foamed. 5. The method according to any one of claims 1 to 4, characterized characterized in that the foam particles thus obtained by Grind or grate. 6. The method according to any one of claims 1 to 5, characterized records that during and / or before shredding the Foam particles are cooled. 7. The method according to any one of claims 1 to 6, characterized records that the grinding or crushing process in Presence of dry ice is carried out. 8. The method according to any one of claims 1 to 7, characterized records that made from foamed polymer particles compact molding materials through a shredding process into one Be converted into particle form. 9. The method according to any one of claims 1 to 8, characterized records that the particle size of the polymer particles 0.5 to 5 mm.   10. The method according to any one of claims 1 to 9, characterized records that the immobilization of biologically active Substances in aqueous solution or in the presence of solution middle components is carried out. 11. The method according to any one of claims 1 to 10, characterized records that a pH range of 4 to 7 is observed. 12. The method according to any one of claims 1 to 11, characterized records that the contacting of carrier material and biologically active substance and if necessary after subsequent evaporation of water or solvent Temperatures below 45 ° C. 13. The method according to any one of claims 1 to 12, characterized records that the process of immobilizing the biologically active substances takes place directly in the reaction vessel, whereby if necessary, a drying step can be added can. 14. The method according to any one of claims 1 to 13, characterized records that the biologically active substances from the protein group of antibodies, antigens, protein conjugates, lectins and preferably derive from the enzymes. 15. The method according to any one of claims 1 to 14, characterized records that lipase is used as the enzyme. 16. The method according to any one of claims 1 to 15, characterized records that an enzyme loading of 1 to 20%, preferred wise 1 to 10% is set. 17. Polymer materials supported with biologically active substances, producible according to one of claims 1 to 16. 18. Use of those supported with biologically active substances Polymer materials according to claim 17 for chiral syntheses. 19. Chiral syntheses, characterized in that supported immobilized biologically active substances, producible after a method according to any one of claims 1 to 16 be set and the syntheses in a temperature range run from 5 to 80 ° C.