DD285372A5 - 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 first preimmobilized by encapsulation in ultramicrocapsules of polyelectrolyte complexes. In the case of coimmobilization of microorganisms and enzymes, the enzymes are bound to a carrier material that encapsulates microorganisms for preimmobilization in ultramicrocapsules. After separation and sedimentation of the pre-immobilized microorganisms or microorganisms and carrier-bound enzymes in the case of coimmobilization of microorganisms and enzymes, these are enclosed in microcapsules and, after conversion to a suitable environment for the growth of the immobilized or coimmobilized biocatalysts, the desired use is achieved {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 material-converting 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 in proportion to reactor volume, easier separability of the biocatalyst from the product, and thus a significant 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 of porous Trägcrmaterialien and the encapsulation. In addition, some methods and materials have been tested 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)).

In order to reduce the risk of outgrowth of cells in the MeJium and to avoid resulting disturbances of the process, a method is described in DE OS 34 32 932, which should prevent c! As outgrowth of cells, even in long-term experiments. This object is achieved in that the surface of the biocatalyst is formed by a cell-free protective layer of a crosslinked, no permeability to the cells having the zallenhaltigen core enclosing gel. This process provides gel beads which 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.

In this patent also is festcnste ¹ It, that the process of microencapsulation for the immobilization of cells is not suitable due to toxicity of the solvent to be used and the resulting low catalyst activity, and that the outgrowth of cells are not securely prevented even with this method can.

Mosbach (Phil. Trans. R. Soc. Lond. B300, 355 (1983)) also notes that in the encapsulation of microorganisms 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.

In DE-OS 30 12 233 a method for the immobilization of chemically or biologically active substances, living tissue and single cells is described, in which first the materials enclosed in a gel and then reacted the acidic groups on the surface of the gel matrix with a aminocjruppenhal term polymer become, so that forms a membrane. A disadvantage is the low resistance of the membrane thus formed against 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 Verklurnpungsgefahr the capsules when using anionic substrates to an accumulation of the same the

Surface leads.

In WO 83 / O3C61 a process for Doppelkapsalung is described, which does not serve the immobilization of microorganisms, but is directed to a targeted release of drugs. This is a complex or a reaction product of an active ingredient in the mini-microcapsules. By diffusion, said material from the mini-microcapsules enters the same surrounding fluid within the double capsule, reacts there with an enzyme, and the active ingredient is released. The active ingredient passes by diffusion through the outer membrane in the Empfängerorqan. The advantage consists in a better meterability of the active ingredient.

In DD 219 795 similar or different biological objects are encapsulated together with certain substrates in multiple capsules for the purpose of compartmentalization. In recent years, microorganisms of various species and / or enzymes have been increasingly immobilized together (coimmobilization), after initially immobilizing only one particular microorganism or enzyme at or in a matrix, some of which are combined in a more advanced form were used in a reactor. The coimmobilization of microorganisms and enzymes makes it possible to broaden the scope of use of immobilized microorganisms by converting other subsets normally not exploited by the microorganism under consideration with the aid of the coimmobilized enzymes into a form which can be converted by the microorganism concerned. By choosing an appropriate sequence of enzymes, multi-step reactions can be catalyzed in this way,

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 support material which serves to enclose the microorganisms.) The disadvantage of this method is the relatively low mechanical strength of the gel beads and to look at the problem of washing out cells from the gels when used in long-term experiments.

EP 0 041 610 proposes binding of the enzyme to yeast cells by C.H. Doehringer Sohn, Ingelheim. However, this method is not applicable to all microorganism species, since the most microorganisms by the Immobilisierungsprozedur

of the enzyme are inactivated. In addition, the size of the cells is not significantly changed, so that the retention dos coimmobilisate in continuous fermentation processes is very difficult.

In DE 35 34 983, W. Hartmeier and A. Heinrichs describe a process for the coimmobilization of microorganisms and enzymes in which the microorganisms and enzymes are simultaneously enclosed in a polymeric matrix which is coated in situ with an oppositely charged, semipermeable membrane , In this method, the number of cells that can grow into the medium during long-term fermentations is reduced compared to the sole inclusion in a gel network. However, it can not be prevented that in the calcium chloride / methacrylate / acrylate copolymer solution microorganisms are incorporated into the membrane during the dripping of microorganisms / enzyme / alginate solution, 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 Geleinschlusses from the outset have a low mechanical strength and that in the known methods for encapsulating microorganisms or microorganisms and enzymes the same in the 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 stable encapsulated biocatalysts whose stability, even in the case of the encapsulation of proliferating systems, is not reduced 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 coirnmobilisierter biocatalysts with high operational stability by way of encapsulation of microorganisms or microorganisms and enzymes, which prevents the incorporation of the biocatalysts to be immobilized in the capsule wall and produces capsules with high membrane strength.

According to the invention this is achieved in that the microorganisms to be immobilized, for example yeasts and bacteria, for preimmobilization in Ultramikr '. be encapsulated that the enzyme / enzymes are bound in the case of coimmobilization of microorganisms and enzymes to a support material, the sedimented and separated Vorimmobilisate or the sedimented and separated Vorimmobilisate and carrier-bound enzymes are suspended in an aqueous solution of cellulose sulfate, the suspension is dropped into an aqueous polydimethyldiallylammonium chloride solution, and the immobilized biocatalysts after the completion of the encapsulation process in a known manner and introduced into a suitable for the cultivation of microorganisms environment '- / ground. 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 with a reaction duration of 30

min to 24 hours. The coupling reagent is preferably glutaric dialdehyde having a concentration between 0.1 to 10%, but preferably 2.5%. As the insoluble support material, aminomethyl polystyrene having a particle diameter of 5 to 200 μm is preferably used.

The microorganisms are encapsulated for pre-immobilization in ultramicrocapsules, preferably formed from polydimethyldiallyl ammonium chloride and cellulose sulfate. The concentration of cellulose sulfate for the preparation of the ultramicrocapsules (Vorinmobilisate) is 5 to 100 g / l, that of the polyoimethyldiallyl ammonium chloride solution between 5 and 100 g / l. The residence time of the microorganisms preimmobilized in ultramicrocapsules in the polydimethyldiallylamine chloride solution is chosen to be between 30 seconds and 60 minutes. The temperature during the preimmobilization process ranges from 273 K to the stability limit of the microorganisms that are obilizing. For the encapsulation of the microorganisms enclosed in ultramicrocapsules or of the microorganisms and carrier-bound enzymes enclosed in ultramicrocapsules, a concentration of the cellulose sulfate of 5 to 100 g / l is used. The concentration of polydimethyldiaHylammoniumchlorid solution is also between 5 and 100 g / l.

The temperature during the subsequent encapsulation of the sedimented and separated preimmobilizates is likewise selected between 273 K and the stability limit of the biocatalysts to be immobilized.

The residence time of the immobilized microorganisms or microorganisms and enzymes is between 30 seconds and 12 hours. After separating the iminobilisicrtsn Riokatalysatoren of the polydimethyldiallylammonium chloride solution produced according to the invention, these are converted into a mixture suitable for the growth of the immobilized hiocatalysts and then supplied to the desired use.

Thus, the method of the present invention provides immobilized biocatalysts characterized by high vitality, activity, and surgical stability , even if , in the case of encapsulation of vital, proliferating systems in long-term experiments, it is not diminished by growth and cell culture processes thereof or washing out. The binding of the enzyme (s) protects it from degradation and inactivation. The

microfiltration 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 prevent boim inclusion of microorganisms in the gel network and leads to process disruption, is excluded. Another advantage is an increased oil mechanical stability of biocatalysts over that of gel networks.

In the case of 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 is a much cheaper substrate than the actual substrate glucose.

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The process according to the invention will be explained with reference to the following examples.

Ausführungsbeispielc

example 1

0.8 ml of a cell suspension of yeast Yarrow.ia .lipolytica containing 1 mg HTtl / ml are homogeneously mixed with 5 ml of a 2.2% cellulose sulfate solution (cellulose sulfate with a DS of 0.43). This suspension is added dropwise, with the aid of a blow-off device (according to Dautzenberg) with stirring, to a 1.5% solution of polydimethyldiallylamonium chloride having a molecular weight of 40,000, so that ultramicrocapsules with a maximum diameter of 200 μm are formed. After a maximum of 5 min residence time of the capsules in the precipitation bath they are sedimented and separated. Subsequently, the ultramicro capsules are washed three times with water. After sedimentation of the ultramicrocapsule η, they are used, as described in Example 2.

Example 2

0.5 ml of the sediment of the ultramicrocapsules containing yeast cells of the species Yarrowia lipolytica are suspended in 5 ml of a 2.2% solution of cellulose sulfate (cellulose sulfate with a DS of 0.43) and distributed as homogeneously as possible. Thereafter, this suspension of the ultramicrocapsules is added dropwise to a solution of 1.2% polydimethyldiallylammonium chloride having a molecular weight of 40,000 and slowly stirred for at least 5 minutes. '

Separate the precipitation bath from the microcapsules formed by washing twice with 30 ml sterile distilled haters.

For cultivation, 20 g of the capsules thus obtained are used in a 500 ml fermenter. Make a cultivation cure of 24 h, the culture medium is eaten and cell growth completed. The cell mass in the capsule interior is

to a concentration of 15 mg HTM / rnl 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 Pseudomonas aeruginosa.

Example 3

0.2 g of polystyrene Wofatit UF 93 (particle diameter 5 to 100 μl) 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 washed with water until complete removal of the excess .Giutardialdehyds. Subsequently, the activated polystyrene is stirred with 300 U invertase in 3 ml of SÖREHSEK phosphate buffer pH 7.0 for 4 h at room temperature and then with 25 mM sodium acetate buffer pH 5.0 / 1 M NaCl and 25 ml -1 Ud trium acetate buffer pH 5.0 washed until no protein is detectable in the wash water.

The invertase polystyrene complex is used as described in Example 5.

Example 4

0.2 g Folystyrene Wofatit UF 93 (particle diameter 5 to 100 / am) are stirred with 800 U invertase in 3 ml SÖRENSEM Phosphatouffer pH 7.0 for 4 h at room temperature and then with 25 mM sodium acetate buffer pH 5.0 / 1 M NaCl and 2 5 mfi sodium acetate buffer pH 5.0 washed until no protein is detectable in the wash water. The invertase polystyrene complex was used as described in Example 5.

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Example 5

0,5 ml of sediment of ultramicrocapsules, yeast cells of a variety Yarrowia lipolyticc? contained, according to Example 1 and 0.2 g of the invertase Polystyren Kopplcxes according to Example 3 or 4 v / erden in 5 ml of 2.2 ⁹ öiger cellulose sulfate solution (cellulose sulfate with a DS of 0.43) suspended and distributed as homogeneously as possible.

Then this suspension is dissolved in 50 ml of a solution of 1, 2 ^? drip 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 water.

For cultivation, 20 g of the capsules thus obtained are used in a 500 ml fermenter. After a culture period of 24 h, the culture medium is consumed and the 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 with sucrose as substrate.

Example 6

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 2 or 5 is suspended in 5 ml of the autoclaved cellulose sulfate and distributed homogeneously. This suspension is added dropwise in 50 ml of a solution of 0.25% Polydimethyl.ldiallylammonium chloride with a molecular weight of 40,000 with gentle stirring and stirred for at least 40 minutes. 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. After separation of the precipitation bath, the capsules are immediately added to the cultivation process without further treatment, as described in Examples 2 and 5.

Claims (12)

- A1- claims Method for the immobilization of microorganisms or coimmobilization of microorganisms and enzymes by preimmobilization and encapsulation characterized in that the microorganisms encapsulated for preimmobilization in ultramicrocapsules, in the case of coimmobilization the enzyme (s) bound to a support material which sedimented and the separated Ultramikrokapseln (Vorimmobilisate) and the carrier-bound enzymes are suspended in an aqueous solution of cellulose sulfate, the suspension is grafted into an aqueous polydimethyldiallylammonium chloride solution, and the immobilized microorganisms or the obtained complex ( co-immobilized) biocatalysts are separated in a known manner and introduced into a suitable for the cultivation of immobilized or coimmobilized biocatalysts environment. 2. The method according to claim 1, characterized in that the enzyme / enzymes adsorptively or! -Covalent on a support material in an aqueous solution at pH-V.'erten between 3.5 and 8, at temperatures of 277 K to the stability limit of Enzymes and bound at a reaction time of 30 nin to 24 hours. 3. The method according to claim 1 and 2, characterized in that for covalent bonding 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 to connect the enzymes. G. The method of claim 1 to 5, characterized in that as insoluble Trägermateria] preferably Aminornethylpolystyren vorv / ends. 7. The method according to claim 1 to 6, characterized in that the particle diameter of the polystyrene beads of 5 to 200 Ajm is selected. S. The method of claim 1, characterized in that for the production of the ultraricrocapsules preferably Polyeloktrolytkomplexe consisting of ^ olydirne thyldiallylammoniumchlorid and cellulose sulfate use. 9. The method according to claim 1 and 8, characterized in that the concentration of the cellulose sulfate for the preparation of the ultramicrocapsules is 5 to 100 g / l. 10. The method of claim 1, 0 and 9 characterized in that the concentration of the polydimethyldiallyl ammonium chloride solution for the preparation of the ultramicrocapsules is 5 to 100 g / l. 11. The method of claim 1 and S to 10, characterized in that the residence time of pre-immobilized in ultramicrocapsules microorganisms in the polydimethyldiallyl ammonium chloride solution is selected from 30 seconds to GO minutes. 12: ancestors according to claim 1 and 8 to 11, characterized in that the temperature during the Vorimmobilisierungsprozesses the microorganisms of 273 K to the stability limit of the microorganisms to be immobilized ranges. 13. The method according to claim 1, characterized in that a concentration of the cellulose sulfate in the encapsulation of the ultramicrocapsules or ultramicrocapsules and carrier-bound enzymes (Vorirninobilisate) of 5 to 100 g / l is used. 14. The method according to claim 1 and 13, characterized in that for the encapsulation of Vorimnobilisate a concentration of Polydimethylallylaminoniumchlorids between 5 and 100 g / l is used. 15. The method of claim 1, 13 and 14, 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. IG. Process according to claim 1 and 13 to 15, characterized in that the residence time of the immobilized bzv /. co-immobilized eggcatalysts in the polydimetyldial.lylammonium chloride solution are chosen from 30 seconds to 12 hours.