DE8520835U1 - Biocatalyst - Google Patents

Description

Prof. Dr. Klein, Dr. Vorlop* Dr. Steihert Attorney file

360-6 EN"2

Date

19 Feb 1988

Biocatalyst

The invention relates to a biocatalyst with immobilized, enzymatically active substances which are immersed in a liquid and are immobilized by a material layer which is permeable to the liquid but impermeable to the enzymatically active substances.* Such enzymatically active substances can be formed by cells, organelles and enzymes.

Biocatalysts are used for a wide range of applications. They can be used, for example, to produce medicines such as antibiotics or ethanol, to purify waste water, etc. The use of corresponding cells is well known in the production of sparkling wine, where a second fermentation is initiated by adding yeast. This fermentation is also carried out in the sparkling wine bottle itself. The known processes are particularly complex because the free, active cells have to be removed from the liquid after fermentation has taken place. In bottle fermentation for sparkling wine, the bottles are shaken and gradually turned upside down so that the cells settle in the neck of the bottle. By freezing the neck of the bottle, the cells form a removable plug.

Theodor-Heuss-Strasse2 :*":"": .** i '::':.

D-3300 Braunschweig Federal Republic of Germany

Telephone 0531-80079
Telex 0-952620 gramm d Fax 0531-81297 (CCnT2+3)

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It has been known for some time that immobilized cells can be used for biocatalysts for such applications. These are bound to one place by suitable measures, but remain viable and active. After the microbiological reaction has been carried out, they can generally be easily removed together with the immobilization mass. It is known that such immobilization can be achieved using gels that crosslink in the aqueous phase (cf. Klein, Wagner "Methods for the Immobilization of Microbial Cells" in Applied Biochemistry and Bioengineering 1983, Vol. 4, pages 11 - 51).

In practice, however, it has been shown that for longer-lasting processes, such as sparkling wine fermentation, the effects aimed for by immobilization are not achieved. It is gradually becoming apparent that the cells "grow out" of the immobilization material. Although the gels prevent the enclosed cells from migrating due to their network structure, it is unavoidable that cells also sit on the surface of the biocatalyst during the production of the gels. These find more favorable nutritional conditions than the enclosed cells and multiply much more than the enclosed cells. These cells take part in the fermentation process as free cells, enter the liquid and lead to considerable turbidity.

A process known as "microencapsulation" is not suitable for immobilizing cells because it requires a polymer to be dissolved in an organic solvent, which kills the cells almost completely. The remaining activity of the catalyst would not be sufficient in most cases. In addition, even with microencapsulation, it is not possible to reliably prevent the cells from growing out.

The invention is based on the object of developing a biocatalyst

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which is easy to produce and which reliably prevents the outgrowth of the enzymatically active substances even in long-term processes.

This object is achieved according to the invention in a biocatalyst of the type mentioned at the outset in that the substances in a above γ?δθθθθθθθ ?δθθθ!·δθ1=δθ3θθθθθθθθθθθθ sincL- sizes V?c>n.clvm£t at the lower end is at least partially formed by the material layer.

Claims 2 to 8 mention embodiments of the invention.

While the known immobilization processes are based on a complete coating of cells with a material that is permeable to the liquid but impermeable to the cells, the structure of the biocatalyst according to the invention differs fundamentally from this. In a very simple way, a container that is open at the top is prefabricated, the lower end of which is intended to be immersed in the liquid. In this lower end area, the wall of the container is at least partially formed by the layer of material that is permeable to the liquid but impermeable to the cells.

The advantage of this arrangement is that the container with the material layer can be prefabricated completely free of cells and that the cells can be expelled into the prefabricated container via the upper open part of the container, whereby the outer wall of the container and the material layer can be kept completely free of cells. There is therefore no danger of overgrowth.

The walls of the container not formed by the material layer will generally be impermeable to both the liquid and the cells. However, it is also possible.

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to form a container entirely from such a material.

In a simple and inexpensive way, the container can be formed by a piece of pipe that is open at both ends and that is closed with the material layer at the end intended for immersion in the liquid. The piece of pipe can be a piece of glass pipe, a piece of plastic pipe or even a normal drinking straw. The material layer can be a glass frit insert that is inserted into the piece of pipe, for example. A simple closure with a suitable material can preferably be made with a gel layer that can be formed on the piece of pipe by immersion. This method is generally simpler to implement than inserting a piece of glass frit into the container.

Before applying the gel layer, a paper filter insert, a filter piece or a cotton plug can be inserted into the pipe section to prevent the gel from penetrating into the interior of the pipe section.

To increase the exchange surface for the penetrating liquid, it can be advantageous if the filter piece is designed as a hollow tube and continues the pipe piece. The hollow tube is then also surrounded by the gel layer. This arrangement promotes the diffusion of fresh liquid into the area of the enzymatically active substances.

To carry out bottle fermentation, it is advisable to use the pipe piece. This is inserted into the bottle and the bottle is closed. In order to be able to easily remove the pipe piece after the fermentation process has ended, it is advantageous if the pipe piece is provided with a buoyancy body that lifts the pipe out of the liquid so far that it can be grasped outside the bottle.

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can. In another embodiment, the same purpose is served by a ring-shaped gas collecting chamber that is open at the bottom and is arranged on the inner wall of the pipe section. During fermentation, CO2# is produced, which rises upwards in the pipe section. Part of the carbon dioxide is caught in the gas collecting chamber and thus provides the buoyancy that lifts the filled pipe out of the liquid. §

I The invention will be explained in more detail below using embodiments shown in the drawing. It shows:

Figure 1 - a filled glass tube piece, which at the lower end

is finished with a glass frit insert;

Figure 2 - a piece of pipe closed off by a gel layer at the lower end, with a paper filter inserted into the lower opening and which contains a gas

has a collecting space;

a filled piece of pipe with a gel layer at the bottom and into which a filter piece is inserted;

figure 3 25 figure 4 30 figure 5

a filled piece of pipe, sealed with a layer of gel/ which continues at the lower end with a hollow tube;

a device for producing the gel layer on a piece of pipe.

The drawing shows tubular containers 1, which are primarily used for bottle fermentation in the production of sparkling wine.

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are suitable. Of course, such containers can also be used for other purposes and may have a different shape for this purpose.

Figure 1 shows a piece of pipe as the container 1, which is open at the top and bottom. The lower opening is closed with a glass frit insert 2, which is made with such a density that it allows the diffusion of a liquid, but not the passage of yeast cells. The arrangement of the piece of pipe and glass frit insert 2 is filled with a yeast suspension 3, which is suitable for the second fermentation of a cuvée.

In the embodiment shown in Figure 2, the pipe section is provided with a paper filter insert 2' at the lower end. A gel layer 4 is applied around this arrangement, the production of which is explained in more detail below. This arrangement is then filled with a yeast suspension 3. Within the filling level of the yeast suspension 3 there is an annular gas collection chamber 5 that is open at the bottom and in which the carbon dioxide produced during fermentation collects. After the bottle is opened after fermentation has ended, the CO2 collected in the gas collection chamber 5 ensures that the pipe section protrudes far enough from the liquid that it can be grasped above the bottle opening and pulled out of the bottle.

In the embodiment shown in Figure 3, a prefabricated filter piece 6 in the style of a cigarette filter is inserted into the lower end of the pipe section. This porous filter essentially serves to mechanically stabilize the subsequently applied gel layer 4. To mechanically attach the gel layer 4 to the outer wall of the pipe section, the latter can be provided with toothed grooves 7.

Figure 4 shows an embodiment in which a

A hollow tube 8 made of filter paper is inserted, which extends the tube piece by a considerable distance. A cotton plug 9 is inserted into the hollow tube 8 at the lower end, which, like the filter piece 6 or the paper filter insert 2', has the task of contributing to the mechanical stabilization during the production of the gel layer 4 and preventing the gel layer from penetrating into the interior of the hollow tube 8 or the tube piece. 10

The production of the gel layer 4 will be explained using examples in Figure 5.

To produce silica gels, the silica is prepared from an 8.5% Na2SiC>3 solution using an acidic ion exchange resin (Lewatit 100 S). The Na^SiC^ solution is passed through the ion exchange resin column, which has been washed with distilled water until free of chloride. The pH of the silica flowing out is constantly monitored using a pH electrode. The fraction with a pH below 3.0 is collected and this silica is adjusted to pH 2.0 using dilute hydrochloric acid.

The silica is poured into a small cylinder 10 and immersed in a Na 2HPC»4 buffer solution with a pH of 9.0.

After about 60 seconds the gel has formed.

The cylinder 10 containing the gel contains another, smaller cylinder 11 with holes, which is connected to the pipe section at the top. A cotton plug 9 is inserted into the cylinder 11 at the lower end. This arrangement, which only serves to provide mechanical stabilization, is firmly surrounded by the gel layer 4 when the yeast suspension 3 made up of yeast and the cuvee is filled up to about half the height of the pipe section. The pipe filled with the yeast suspension 3 is then placed in

= a champagne bottle filled with Cuvee and the

Bottle closed. There will then be approx. 0.14 g of dry yeast in the piece of pipe.

■ For the embodiment shown in Figure 4, the pipe section, which has a hollow tube 8 made of filter paper about 6 cm long and closed at the bottom, is placed in a silica

i 10 acid solution to be placed on the paper tube;:-,* Silicic acid-

i layer. The silica solution was previously mixed with a

j Na2HP04 solution with pH 5.0. Three parts of Kie-

- Seiic acid is added to a part buffer. By mixing with

In the buffer, the silica gels faster, so that the

\ 15 Silica is easier to apply.

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i The tube is then immersed in a buffer solution pH 9.0.

I This procedure is repeated to form a second gel layer 4 of

J silica gel. The pipe section is then filled up to halfway with yeast suspended in Cuvee (approx. 0.14 g I dry yeast per pipe section). The surface-sterile pipe I can now be used for sparkling wine fermentation.

In a completely analogous manner, the embodiment according to Figure 3

25 with the filter piece 6 or according to Figure 2 with the paper filter insert 2'.

To form an agar gel as gel layer 4, a 4% agar solution is cooled to about 45 ⁰ C until shortly before the gel point. In

30 The piece of pipe, which has the approximately 6 cm long hollow paper tube 8 at the bottom, is dipped into this solution. The arrangement is dipped into cold water with the agar adhering to it. The agar solution gels when it cools. This procedure is repeated to produce a second agar layer.

35 The pipe section is then half filled with a yeast suspension 3 and is then ready for use.

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The gel layers 4 can be formed in the same way from carrageenan, pectin, alginate, gelatin, etc. Silica gels and agar are particularly suitable for making sparkling wine, because these substances are recognized as being safe for food use.

Claims (7)

1. Biocatalyst with immobilized, enzymatically active substances which are immersed in a liquid and are immobilized by a layer of material which is permeable to the liquid but impermeable to the substances, characterized in that the substances (3) are filled into a container (1) which is open at the top and whose wall at the lower end is at least partially formed by the layer of material. 10 2. Biocatalyst according to claim 1, characterized in that the container (1) is formed by a pipe section that is open at both ends and is closed with the material layer at the end intended for immersion in the liquid. 3. Biocatalyst according to claim 1 or 2, characterized in that the material layer is formed by a glass frit insert (2). 4. Biocatalyst according to claim 1 or 2, characterized in that the material layer is formed by a gel layer (4). Theodor^HeUss-Straße 2 i _it '- _ttt i ,.], &iacgr;,,&iacgr; !,/· P»3300 Braunschweig Federal Republic of Germany Telephone 0531-80079 Telex 0-9 52620 gramm d Fax 0531-81297(COnT 24-3) >■ 11 ' · " !111 1 1 > &igr; 1 > 1 · ' I 1 Δ 111 5. Biocatalyst according to claim 4, characterized in that a paper filter insert (2'), a filter piece (6) or a cotton plug (9) is inserted into the pipe section before the gel layer (4) is applied. 6. Biocatalyst according to claim 5, characterized in that the filter piece is designed as a hollow tube (8) and the pipe section continues. 7. Biocatalyst according to one of claims 2 to 6, characterized in that the pipe section is provided with a buoyancy body. Biocatalyst according to one of claims 2 to 7, characterized in that the tube has an annular gas collecting chamber (5) open at the bottom on its inner wall. Patent attorneys Gram + Lins
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