DD285370A5 - PROCESS FOR IMMOBILIZING MICROORGANISMS OR COIMMOBILIZATION OF MICROORGANISMS AND ENZYMES - Google Patents

Abstract

The invention relates to a process for the immobilization of microorganisms or coimmobilization of microorganisms and enzymes, which provides very stable biocatalysts, which can also be used in long-term processes and material-converting processes in biotechnology, chemical, pharmaceutical and food industries and in medicine. The microorganisms are initially preimmobilized by inclusion in gel networks. In the case of coimmobilization of microorganisms and enzymes, the enzymes are bound to a carrier material and the microorganisms and carrier-bound enzymes are included in gel networks for preimmobilization. After separation and sedimentation of the pre-immobilized microorganisms or microorganisms and carrier-bound enzymes in the case of coimmobilization, these are enclosed in microcapsules and, after conversion to a suitable environment for the cultivation of the immobilized or coimmobilized biocatalysts, the desired use is brought {immobilization of microorganisms; Coimmobilization of microorganisms and enzymes; Vorimmobilisierung; encapsulation; polyelectrolyte complexes; Material conversion}

Description

Title of the invention

Process for the immobilization of microorganisms or coimmobilization of microorganisms and enzymes

Field of application of the invention

The invention relates to a process for the immobilization of microorganisms or coimmobilization of microorganisms and enzymes for the production of biocatalysts, which are suitable for carrying out stoffwandelnder processes in biotechnology, in the pharmaceutical, chemical and food industries and for use in medicine. You will find z. B. Use for the biosynthesis of drugs, high quality chemicals, enzymes, etc.

Characteristic of the known state of the art

To carry out biochemical reactions in the biotechnology, pharmaceutical, chemical and food industries, microorganisms are increasingly used. Increasingly, the microorganisms are used in immobilized form to make their use economical and effective. Immobilization allows for increased microbial productivity relative to reactor volume, easier separability of the biocatalyst from the product, and thus a reduction in the cost of isolating the desired products.

For the immobilization of microorganisms, a number of methods have been described, such as. As the inclusion in networks and in foams, the fixation on porous substrates and the encapsulation. In addition, some methods and materials have been used in combination.

When included in networks of gels, in addition to a relatively low mechanical strength, washing out of the cells from the gels is frequently observed (Stöcklein, Eisgruber, Schmidt, Biotechnol., Lett., 5 (10), 703 (1983)).

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Urn the danger of outgrowth. To reduce cells in the medium and to avoid the resulting disturbances of the process, a method is described in DE OS 34 32 932, which is intended to prevent the outgrowth of cells, even in Tiang-time experiments safely. This object is achieved in that the surface of the biocatalyst is formed by a cell-free protective layer of a cross-linked, no permeability to the cells having the cell-containing core enclosing gel. This process provides gel bodies that generally have limited mechanical durability. However, for the described purpose, the sparkling, the mechanical strength is of minor importance, since the biocatalyst is not heavily stressed, in other material-converting processes, the mechanical strength plays an important role.

This patent also states that the method of microencapsulation is not suitable for the immobilization of cells due to the toxicity of the solvents to be used and the resulting low catalyst activity, and that also in this method the outgrowth of cells is not reliably prevented can.

Mosbach (Phil Trans. R. Soc.Lond.B300, 355 (1933)) also states that in the encapsulation of microorganisms as compared to inclusion in networks, the number of cells that can grow into the medium is further reduced, but that nevertheless the cells incorporated in the membrane can deliver biomass to the outside.

DE-OS 30 12 233 describes a process for the immobilization of chemically or biologically active substances, living tissue and single cells, in which first the materials are enclosed in a gel and subsequently the acidic groups on the surface of the gel matrix are reacted with an amino group-containing polymer , se that forms a membrane. A disadvantage is the low resistance of the membrane thus formed to proteolytic attack, which requires a limited [life of the capsules, and the presence of a large number of free amino groups on the surface, in addition to the risk of clogging of the capsules when using anionic substrates to an accumulation thereof at the

Surface leads.

In recent years, microorganisms of various species and / or enzymes have been increasingly immobilized together (co-immobilization) after initially immobilizing only one particular microorganism or enzyme at or in a matrix, some of which are in a more advanced form were used in a reactor. The co-immobilization of microorganisms and enzymes makes it possible to broaden the scope of use of immobilized microorganisms by converting other substrates, which are not normally utilized by the microorganism under consideration, into a form which can be converted by the microorganism concerned with the aid of the coimmobilized enzymes. By choosing an appropriate sequence of enzymes, multi-step reactions can be catalyzed.

According to a method of B. Hägerdal and K. Mosbach (US 4,524,137 for the coimmobilization of microorganisms and enzymes, the enzyme is covalently bound to the carrier material which serves to enclose the microorganisms Gel beads and the problem of washing out cells from the gels when used in long-term experiments.

In Ε]? O 041 610 is proposed by C. H. Bochringer son, Ingelheim the binding of the enzyme to yeast cells. However, this method is not applicable to all species of microorganisms, as most microorganisms are inactivated by the immobilization protein of the enzyme. In addition, the size of the cells is not significantly changed, so that the retention of coimmobilisate in continuous fermentation processes is very difficult.

From V/. Hartmeier and A. Heinrichs DE 35 34 983 a method for Coimrncbilisierung of microorganisms and enzymes is described in which the microorganisms and enzymes are included at the same time in a polymeric matrix, in situ with an oppositely charged, semipermeable! Membrane is coated. In this method, the number of cells which can grow into the medium in the case of long-term fermentation is reduced compared to the sole inclusion in a gel network. However, it can not be prevented that when dripping the

Microorganisms / enzyme / alginate solution in the calcium chloride / methacrylate / acrylate copolymer solution microorganisms are incorporated into the membrane, which may be the cause of the outgrowth of microorganisms in the outer medium. In the case of the methods described herein for the immobilization of microorganisms or microorganisms and enzymes is to be noted that in the case of Geleinschlussos from the outset have a low mechanical strength and that in the known methods for encapsulation of microorganisms or microorganisms and enzymes in the same Capsule membrane are incorporated and caused by growth and cell division processes of vital objects, a significant reduction in capsule strength.

Object of the invention

The aim of the invention are stably encapsulated biocatalysts whose stability, even in the case of the encapsulation of proliferating systems, is not diminished by growth and cell division processes of the same and which can be used in biotechnological processes.

Explanation of the essence of the invention

The invention has for its object to find a method for producing immobilized or coimir.obilisiertor biocatalysts with high operational stability by way of encapsulation of microorganisms or microorganisms and enzymes that prevents the incorporation of immobilized biocatalysts in the capsule wall and capsules with high membrane strength generated.

According to the invention, this is achieved by binding the enzyme (s) in the case of coimmobilization of microorganisms and enzymes to a support material containing microorganisms to be immobilized, for example yeasts and bacteria, or microorganisms and carrier-bound enzymes for preimmobilization in an aqueous solution be suspended from gellable materials, this suspension is made to gel, then the sedimented and separated gel particles (Vorirnmobilisate) are suspended in an aqueous solution of cellulose sulfate, the suspension is dropped into an aqueous polydimethyldiallylammonium chloride solution, and the immobilized or coimmobilisierten biocatalysts after completion of the encapsulation process separated in a known manner and introduced into a suitable for the cultivation of microorganisms environment.

In the case of coimmobilization of microorganisms and enzymes, the binding of the enzyme (s) is either adsorptive or covalent to a support material, preferably an insoluble material, in an aqueous solution at pHs between 3.5 and 8, at temperatures of 277 K up to the stability limit of the enzymes and at a reaction time of

min to 24 hours. The coupling reagent is preferably glutarclialdehyde with a concentration of between 0.1 and 10%, but preferably 2.5%. M is insoluble support material preferably amino-containing Polystyrencerivate with a particle diameter of 5 to 200 yum vorwärts.

For the pre-immobilization of the microorganisms or carrier-bound enzymes in the case of coimmobilization, both GeIo formed by lowering the temperature, for example agar, agarose, gelatin, and gels produced by ionotropic gelation, for example alginate, carrageenan, pullulan, pectinate , be formed with the aid of a crosslinking agent used.

The concentration of gellable material is between 1 and 100 g / l, and when using agar, agarose and alginate, preferably at 30 g / l.

When ionotropic gelling materials are used, a crosslinker solution containing from 1 to 100 g of prewetting agent / l is used. In the case of alginate, alkaline earth salt solutions, preferably calcium chloride, and in the case of carrageenan, alkali metal salt solutions, preferably potassium chloride, are used as crosslinking agents.

The temperature during the pre-immobilization process by ionotropic gelation ranges from 273 K to the stability limit of the microorganisms and enzymes to be immobilized. When using materials which form gels by lowering the temperature, the gelable material is dissolved at a temperature of 29'3 to 373 K, the solution is cooled to 313 K when using temperatures greater than 313 K and with a suspension of the microorganisms or microorganisms and carrier-bound enzymes. By cooling the suspension to 273 to 283 K v / ird the Vorimmobilisat obtained.

The temperature during the subsequent encapsulation of the sedimented and separated preimmobilisates is also chosen between 273 K and the stability limit of the biocatalysts to be immobilized.

The cellulose sulfate concentration for the encapsulation of the Vorimrnobilisate is between 5 and 100 g / l, the concentration of Polydimethyldiallylammoniumchlorid-fjösung also between 5 and 100 g / l.

The residence time of the immobilized microorganisms or microorganisms and Cnzymc is between 30 seconds and 12 hours. To. Separation of the orally prepared immobilized Diokatalysatorcn of the Pclydimethyldiallylammoniumchlorid solution they are converted into a suitable for the cultivation of the immobilized biocatalyst environment and then fed to the desired use.

Thus, the inventive method provides immobilized biocatalysts which are characterized by a high vitality, activity and Oporationsstabilität, which is also i: i case of encapsulation of vital, proliferiendor systems in long-term experiments not by growth and Zeil'ceilungssbense the same or washing is reduced. The binding of the enzyme (s) protects it from degradation and inactivation. Preimmobilization ensures that no microorganisms or carrier-bound enzymes are incorporated into the capsule wall and the surface of the immobilized biocatalyst is free of microorganisms and carrier-bound enzymes. Thus, the risk of outgrowth of microorganisms in the culture medium, which can not be prevented by the inclusion of microorganisms in Golnetwerke and leads to Störuncjen the process is excluded. Another advantage is an increased mechanical stability of the biocatalysts over that of gel networks.

In the case of the coimmobilization of invertase and Yarrowia lipolytica cells, it becomes possible to use sucrose as a substrate, which normally can not be utilized by these cells, but represents a substantially less expensive substrate than the actual substrate glucose.

The process according to the invention will be explained by means of the following examples.

Exemplary embodiments Example 1

0.1 y agar are under. Heating to 353 K dissolved in 10 ml V / asser. After cooling to 313 K, 1 ml of a Zc.llsuspension of the yeast Yarrowia lipol tica containing 1 mg HTM / mi is added, quickly suspended and dripped into water having a temperature of 283 K. The gel obtained in coarse lumps is placed in 10 ml of water at a temperature of 283 K and comminuted by known methods to a particle size of 100 to 200 ρ. The particulate gel is sedimented and used as described in Example 4.

Example 2

0.5 ml of a cell suspension of the yeast Yarrowia lipolytica containing 1 mg HTM / ml v / earth suspended in 5 ml of 3% Na-Alginöt solution. By dropping the thus obtained cell alginate suspension by means of conventional methods in a 2% calcium chloride solution with gentle stirring gel beads are obtained, the stirred for a further 30 min in this solution v / earth. The gel beads will sediment.! he t and, as described in Example 4, used.

Example 3

By dissolving 0.3 g of Agaroso in 10 ml of Kasser at 373 K, a 3% agarose solution is prepared. After cooling the solution to 313 K, 1 ml of a suspension of the yeast Yarrowia lipolytica containing 1 mg HTM / ml is added, rapidly suspended and dripped into water at a temperature of 2S3K. The gel obtained in coarse lumps is dissolved in 10 ml of water at a temperature of 283 K.

give and crushed by known methods to a particle size of 100 to 200 yiim. The particulate gel is sedimented and used as described in Example 4.

He is playing 4

0.5 ml sediment sediments of vorimmobilisierton yeast Yarrowia Iipolytica according to Example 1, 2 and 3 are suspended in 5 ml of 2.2% Celluloscsulfatlösung (cellulose sulfate with a DS of 0.43) and distributed as homogeneously as possible.

Then this suspension is dissolved in 50 ml of a solution of l, 2 ^? Dropped polydimethyldiallylammonium chloride with a molecular weight of 40,000 with gentle stirring and stirred slowly for at least 5 min.

After separation of the precipitation bath from the microcapsules formed, they are washed twice with 50 ml of sterile distilled ton of water.

For cultivation, 20 g of the capsules thus obtained are used in a 500 ml formenter; After culturing for 24 h, the culture medium is consumed and cell growth is complete. The cell mass in the capsule interior has grown to a concentration of 15 mg HTM / ml immobilizate. The external medium is not clouded, d. H. free of yeast cells. These capsules are used in a continuous fermentation process for the production of citric acid.

The inventive method was carried out analogously with bacteria of the species Pseuciomonas aerucjinosa.

Example 5

0.2 g of Polystyron Wofatit UF 93 (particle diameter 5 to ICO um) are stirred with 3 ml of 2.5% glutaraldehyde solution in SÖRENSEN phosphate buffer pH 7.0 for 2 hours at room temperature. The activated carrier material is then mixed with water until complete removal of the excess GIu.

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tardialdohyds washed. The activated polysaccharene is then stirred with 800 U invertase in 3 ml of SÖRENSEH Phosphafc buffer pH 7.0 for 4 h at room temperature and then with 25 mM sodium acetate acetate buffer 5.0 / 1 M KaCl and 2 ml of sodium acetate buffer pH 5, 0 washed until no protein was detectable in Waschwasscr.

The invertase polystyrene Komolex is used as described in Examples 7 to 10.

Example G

0.2 g polystyrene Wofatit UF 93 (particle diameter 5 to 100 p) are stirred with 800 U invertase in 3 ml SÖREiISEN Phosphatpuff er pH 7.0 for 4 h at room temperature and then with 25 mM Matriumacetatpuffer pH 5.0 / 1 W NaCl and 25 mW sodium acetate buffer pH 5.0 until no more protein was detectable in the wash water. The invertase polystyrene complex is used as described in Examples 7-10.

Example 7

0.1 g of agar dissolved with warming to 353 K in 10 ml of water. After cooling to 313 K v / erden 1 ml of a cell suspension of the yeast Yarrowia lipolytica containing 1 mg HTM / ml and 0.5 ml invertase-polystyrene complex was added, quickly suspended and dripped into water, which has a temperature of 283 K. having. The gel obtained in coarse lumps is added to 10 ml of water at a temperature of 283 K and comminuted by known methods to a particle size of 100 to 200 pm . The particulate gel is sedimented and used as described in Example 10.

Example 8

0.5 ml of a cell suspension of the Yarrowia lipolytica yeast containing 1 mg HTM / ml and 0.25 ml invertase polystyrene.

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ren complex are suspended in 5 ml of 3% Na alginate solution. By dropping the thus obtained Zcll alginate iJuspension with lixlfe conventional methods in a 2% calcium chloride solution with gentle stirring v / erdenhen ge gelen, which are stirred for more than 30 min in this solution. The gel beads are sedimented and used as described in Example 10.

Example 9

By dissolving 0.3 g of agaroso in 10 ml of water at 373 K, a 3% agarose solution is prepared. After cooling the solution to 313 K, add 1 ml of a cell suspension of Yarrowia lipolytica yeast containing: 1 mg / ml and 0.5 ml of invertase-polystyrene complex, rapidly suspended and dissolved in water at 283 ° C K dripped. The gel obtained in coarse lumps is added to 10 ml of water at a temperature of 283 K and comminuted by known methods to a particle size of 100 to 200 urn. The particulate gel is sedimented and used as described in Example 10.

Example 10

0.5 ml of the sediment of Yarrowia lipolytica yeast preimmobilized together with invertase polystyrene complex according to Example 7, 8 and 9 are suspended in 5 ml of 2.2% strength cellulose sulfate solution (cellulose sulfate with a DS of 0.43) and distributed as homogeneously as possible , Then, this suspension is added dropwise in 50 ml of a solution of l, 2% polydimethyldiallylammonium chloride having a molecular weight of 40,000 with gentle stirring and stirred slowly for at least 5 min. After separation of the precipitation bath from the microcapsules formed, they are washed twice with 50 ml of sterile distilled water.

For cultivation, 20 g of the capsules thus obtained are used in a 500 ml formenter. After a culture period of 24 h, the culture medium is eaten and the cell culture

growth abcjeschlossen. The cell mass in the capsule interior has grown to a concentration of 15 mg HTM / ml immobilizate. The external mediuii is not clouded, d. H. free of yeast cells. These capsules are used in a continuous fermentation process for the production of citric acid.

Example 11

50 ml of a 2.2% strength cellulose sulfate solution (cellulose sulfate with a DS of 0.43) are autoclaved at 121 ° C. for 10 minutes. Then 0.5 ml of the sediment according to Example 1, 2 and 3 or 7, 8 and 9 are suspended in 5 ml of the autoclave cellulose sulfate and homogeneously distributed. This suspension is added dropwise in 50 ml of a solution of 0.25% polydimethyldiallylammonium chloride with a molecular weight of 40,000 with gentle stirring and stirred for at least 40 min. Thereafter, the precipitating bath is diluted 1: 1 with water. The capsules are stirred for a further 80 minutes with gentle stirring in the precipitation bath.

If the precipitation bath is separated off, the capsules are immediately added to the cultivation process without further treatment, as described in Examples 4 and 10.

Claims (18)

claims 1. A method for the immobilization of microorganisms or coimmobilization of Mikroorcjonismon and enzymes by gel embedding and Polyolektrolytcinkapsolung, characterized in that the microorganisms or the microorganisms and the enzyme / enzymes which have been bonded to a support material for preimmobilization in an aqueous solution gellable materials are suspended, this suspension is gelled, then the sedimented and separated gel particles (pre-mixed) are suspended in an aqueous solution of cellulose sulfate, the suspension is dripped into an aqueous polydimethyldiallyl ammonium chloride solution, and the immobilized microorganisms or the obtained complex (coimuiobilisierte) biocatalysts are separated in a known manner and introduced into a suitable for the cultivation of the immobilized or coimmobilisiertcn biocatalyst environment. 2. The method according to claim 1, characterized in that the L'enzyme / enzymes adsorptively or kovalont on a support material in an aqueous solution at pH values between 3.5 and S, at temperatures of 277 K to the stability limit of the enzymes and in a reaction time of 30 minutes to 24 hours bound. 3. The method according to claim 1 and 2, characterized in that for the covalent binding of the enzymes to the support material preferably glutaric dialdehyde is used as a coupling reagent. 4. The method according to claim 1 to 3, characterized in that the concentration of glutaric dialdehyde is 0.1 to 10%, preferably 2.5%. 5. The method according to claim 1 to 4, characterized in that preferably an insoluble carrier material is used for binding of the enzyme. - / ν- 2 Ö 5 3 7 0 6. The method according to claim 1 to 5, characterized in that is preferably used as insoluble support material Arninomethylpolystyren. 7. The method according to claim 1 to 6, characterized in that the particle diameter of the polystyrene beads of 5 to 200 yiim is selected. 8. The method according to claim 1, characterized in that for pre-immobilization of the microorganisms or microorganisms and carrier-bound enzymes, a gellable material, such as alginate, carrageenan, pectinate, is used, which forms gels by the action of a crosslinking agent, for example, alkaline earth or alkali salt solutions. 9. The method according to claim 1 and 3, characterized in that the concentration of the gellable material is between 1 and 100 g / l. 10. The method of claim 1, S and'9 characterized in that the concentration of the crosslinking agent is 1 to 100 g / l. 11. The method of claim 1, and S to 10 characterized in that the temperature ranges during the Vorimmobil i sierungsprozesses from 273 K up to the stability limit to be immobilized microorganisms or microorganisms and carrier-bound enzymes. 12. The method according to claim 1, characterized in that for VoL-o-mmobilisierung the microorganisms or microorganisms and carrier-bound enzymes, a gelierfähiges material, such as agar, agarose, gelatin is used, which forms by lowering the temperature gels. 13. The method according to claim 1 and 12, characterized in that the gellable material is dissolved at a temperature of 293 to 373 K, the solution is cooled at temperatures of 320 to 373 K to 313 K, with a suspension of microorganisms or microorganisms and carrier-bound Enzymes is added and by cooling the suspension to 273 to 23? K is transferred to the vorirnmobilisier te form. 14. The method according to / claim 1, 12 and 13, characterized in that the concentration of gellable material is from 3 to 100 g / l. 15. The method according to claim 1, characterized in that a concentration of CeI-lulosesulfats of 5 to 100 g / l is used for encapsulation of Vorimmobilisate. 16 ancestors according to claim 1 and 15, characterized in that a concentration dee Polydirnethylallylammoniumchlorids between 5 and 100 g / l is used for encapsulation of Vorimmobilisate. 17. The method according to claim 1, 15 and 16, characterized in that the temperature in the encapsulation of Vorimmobilisate of 273 K to the stability limit of the encapsulated microorganisms or microorganisms and carrier-bound enzymes ranges. 18. The method of claim 1, 15 to 17, characterized in that the residence time of the immobilized or co-immobilized biocatalysts in the polydimethyldiallylammonium chloride solution from 30 seconds to 12 hours is selected.