WO1990010696A1

WO1990010696A1 - Biocatalytic process and magnetic glass or ceramic carrier particle and device for implementing the process

Info

Publication number: WO1990010696A1
Application number: PCT/EP1990/000425
Authority: WO
Inventors: Ralf Kindervater; Rolf Dieter Schmid
Original assignee: GESELLSCHAFT FüR BIOTECHNOLOGISCHE FORSCHUNG MBH (GBF)
Priority date: 1989-03-15
Filing date: 1990-03-15
Publication date: 1990-09-20
Also published as: JPH03505163A; EP0417227A1

Abstract

The invention relates to a catalytic process taking place in a fluid, especially in an aqueous, medium and carrier particles and a device for implementing the process. The biocatalyst is borne on glass or ceramic particles, especially porous glass, and the carrier particles are magnetic. The surface of the carrier particles is coated with a separating layer of an organic polymer, for example glutaraldehyde or polyurethane.

Description

Biocatalytic process and carrier particles consisting of magnetic glass or ceramic particles and device for carrying them out.

It. It is known to immobilize biocatalysts, such as proteins, DNA structures or microorganisms, on surfaces in order to bring them into contact with a chemical or biological substance that is to be measured. The chemical or biological substance can also be measured indirectly by removing the substance or the reaction products from the reaction site and then allowing a further reaction to take place at the reaction site with which the course of the first reaction can be measured. This second reaction can thus be carried out without any interference from starting materials or products of the first reaction.

It is also already known to immobilize biocatalysts on magnetic supports. Examples can be found in the following table.

Catalyst carrier source

Enzyme polyacrylamide / LKB Ltd. Agarose gel Bromma / Sweden pearls with embedded iron oxide

Enzymes magn. Particles Koch-Light (iron oxide) Laboratories Antibodies magn. Particle scan. J. Immunol. , (Rare earths) 22 (1985) 207;

Tissue Antigens, 28 (1986) 46

On the other hand, it is known to immobilize biocatalysts on ceramic material or glass, for example porous glass (CPG). CPG offers the advantage that beads of practically any size can be provided. CPG can be used to immobilize the biocatalysts used. Chen modification have been subjected, for example with the help of aminopropylsilane. Enzymes can be bound, for example, to such surface-modified CPG using the known Carbodii id, thiourea or azo method. Those skilled in the art are familiar with these techniques; see. for example Filbert, Controlled-Pore Glasses for Enzyme Immobilization. In: Messing, I mobilized Enzymes for Industrial Reactors, 39-62, NY, 1975.

So far, however, it has not been satisfactory that the processes carried out with immobilized biocatalysts have to be interrupted if the activity of the biocatalysts drops, since then the catalyst supports with the used biocatalyst have to be removed and replaced by fresh biocatalyst.

According to the invention, a catalytic process which runs in liquid, in particular aqueous media, is now proposed, in which a biocatalyst is used which is fixed on glass or ceramic particles, in particular on porous glass (CPG) as a support, the process thereby resulting in is characterized in that the carrier particles are magnetic or magnetizable.

Such particles can be held in the liquid medium with the help of a magnetic field. If the process is carried out in a reactor through which the liquid medium flows, the carrier particles can easily be removed by switching off the magnetic field.

As in the prior art, biological macromolecules, such as antibodies, lectins, enzymes or DNA structures, organisms, such as microorganisms, or parts thereof, such as animal or plant tissue, are suitable as biocatalysts. The process according to the invention can be carried out with carrier particles which may be surface-modified ceramic material or glass, in particular porous glass (CPG), on which a fur / Felll mixed oxide / mixed hydroxide has been deposited.

Also suitable for carrying out the method according to the invention is a device which is characterized by a reactor for carrying out the catalytic process, a permanent magnet and an electromagnet, the reactor being arranged in the field of the permanent magnet and the permanent magnet using the Electromagnets can be switched off.

According to a special embodiment, the device according to the invention can be used

- A pot or gugelhupf-like permanent magnet and

a cover covering the opening of the permanent magnet and made of a magnetically neutral material, such as brass, is marked,

- The cover being a magnetizable element for the force connection between the poles of the permanent magnet and

- As a reactor carries a line loop lying in the force field of the permanent magnet.

The invention is explained in more detail below by means of figures and examples.

Figure 1 shows a schematic of a flow injection analysis (FIA); FIG. 2 shows a device according to the invention for carrying out an FIA;

FIGS. 3 and 4 show further embodiments of the device according to the invention.

1, a water sample is to be examined for impurities, for example inhibitors of cholinesterase. The water sample is included With the help of one of the pumps of the pump block 4 to 6 via the line 3 and the valve 12, it is fed to a reactor 17 in which the impurity sought is subjected to a reaction with the aid of a biocatalyst. The reactor can be a reaction or line loop in a permanent magnet system which can be switched off and in which magnetizable CPGs are held by means of the magnetic field, on which, for example, cholinesterase has been immobilized. In the selected example, the immobilized choline esterase is gradually poisoned by the impurity sought. The outflow takes place via the valve 13 and the line 20. After a certain time interval, the valve 12 is actuated so that the inflow of the water sample via line 3 is interrupted and the reactor via line 1 with the help of one of the pumps of pump block 4 to 6 a substrate is fed which is subjected to a reaction by the not yet inactivated biocatalyst. In the selected example, the substrate is choline ester. The substrate supplied to the reactor 17 via the line 1 and the valves 11 and 12 can be diluted with a liquid carrier which meets the substrate in the valve 11. The liquid carrier can be a buffer solution. The stream leaving the reactor 17 is fed via the valve 13 to a secondary reactor 21, in which the reaction product of the biocatalytic reaction is subjected to a further reaction, the result of which can be determined in the downstream detector 22. In the selected example, the subsequent reaction is the oxidation of the choline released in the reactor 17 with the aid of choline oxidase, which produces hydrogen peroxide, which can be measured in the detector 22.

Carrier with unused biocatalyst is kept ready in the storage vessel 14 and can be stirred there with the aid of a stirrer 15 slurried. If the biocatalyst in the reactor 17 is used up, the permanent magnet (not shown) can be switched off, so that the used biocatalyst can be discharged via the valve 13 and the line 20 with the aid of the carrier stream 2. Unused biocatalyst can be fed to the reactor 17 from the storage vessel 14 via the line 16 and the valve 12.

The reactor 17 (FIG. 1) is shown in more detail in FIG. 2. The permanent magnet 100 is formed like a gugelhupf, so that its two poles 101, 102 are formed by the edge of the ring wall 101 and the central pin 102. This permanent magnet 100 is additionally provided with an electromagnet, of which only the connections 103, 104 can be seen. The permanent magnet 100 can be switched off by the electromagnet, not shown. The permanent magnet 100 carries a cover 105 made of a magnetically neutral material, for example brass. This cover 105 is provided on its side facing the permanent magnet 100 with a steel disk 106 which has a smaller diameter than the cover 105 and which produces the force fit between the poles 101 and 102. A feed line 118 is brought to the periphery of the steel disk 106 and, as the actual reactor 117, runs around the steel disk 106 and then leads away as a line 119. The actual reactor is thus placed as an approximately omega-shaped conductor loop 117 around the steel disk 106 and can lie on the periphery of the steel disk 106 in a groove in the cover 105. 3 shows a further embodiment of a device according to the invention using a module.

The module consists of a base plate on which the coil of an electromagnet with a circular core is mounted so that a hose is guided between a gap in the core with tapered ends, which transports the magnetic particles. A block made of non-magnetic material and fitted with a device for receiving a hose serves as the hose holder. By switching on a current, the magnetic field is activated and magnetic particles are held in the core gap. If the electrical current is switched off, the particles can continue to flow with the flow.

4 shows a further embodiment in the form of a device according to the invention on the basis of a further module.

The module consists of a base plate on which a permanent magnet can be moved back and forth in the longitudinal direction by an actuator. A block made of non-magnetic material which is firmly attached to the base plate and is equipped with a device for receiving a hose serves as the rear stop. At both ends of the block there are devices for holding the hose tight in the guide.

To fix the magnetic particles, the servomotor is controlled so that the permanent magnet is moved against the stop block. So the tube through which the magnetic particles can flow comes under the direct influence of the magnetic field forces, which are concentrated in the slot of the magnet, between the two poles. After performing the appropriate analysis steps that the mag. Require fixation, the magnet can be moved backwards by actuating the servomotor, so that the magnetic field acting on the hose is eliminated. She likes. Particles in the hose are no longer held and transported away in the direction of flow.

Production Example 1.

2 g of aminopropyl CPG were introduced into 20 ml of a solution of 8.0 g / 1 iron (II) chloride and 21.6 g / 1 iron (III) chloride in distilled water. By applying vacuum (50 bar) the capillary spaces of the CPG were freed of air and filled with the solution mentioned. The shaken-up solution was then stirred into 50 ml of a solution of 100 g / 1 sodium hydroxide heated to 70 ° C. in distilled water. The black precipitate formed was stirred together with the CPG for a further 1 hour. The CPG particles provided with a magnetic incrustation were cleaned in several washing processes with subsequent decantation of the colloidal supernatant. The CPG particles were dried in a porcelain bowl in a desiccator.

Example 1.

Cholinesterase was immobilized on the CPG particles obtained in Production Example 1 as follows. First with 5 percent. Glutaraldehyde activated in 0.1 M potassium phosphate buffer of pH 7.5. 50 mg glass and 2 ml aldehyde solution were slowly agitated in a sealed vessel for 30 minutes. The glass took on a strong pink color over the course of this time. The glass thus activated was washed six times with buffer, after which it was decanted and the supernatant was discarded. After the last washing, a solution of 10 mg / ml enzyme preparation in buffer could be added, after which the mixture was slowly rotated for 1 hour. The supernatant of the mixture was used for the Bradford protein determination. The slides were washed three times with buffer and then stored in buffer at 4C.

Example 2.

Glucose oxidase (GOD) was immobilized on CPG analogously to Example 1 with an enzyme concentration of 10 mg / ml buffer.

A flow injection analysis was carried out in a device according to FIGS. 1 and 2, with the reactor every 40th - 3 -

sec was loaded with a substrate injection. The composition of the substrate was as follows: 0.1 M potassium phosphate buffer of pH 7.5; dissolved therein per ml 21, uM glucose, 2 ^ uM ABTS and 10 units POD. 0.1 M potassium phosphate buffer of pH 7.5 was used as the carrier stream. The hydrogen peroxide formed by oxidation of the dye ABTS was detected photometrically at a wavelength of 420 nm.

Example 3.

Alcohol oxidase (AOD) was immobilized on CPG at an enzyme concentration of 10 mg / ml buffer according to Example 1.

With a device shown in FIGS. 1 and 2, a flow injection analysis is carried out, pulsing every 40 seconds with a substrate solution of the following composition: 0.1 M potassium phosphate buffer of pH 7.5; dissolved per ml: 300.UM ethanol, 2.uM ABTS and 10 units POD. The detection in the photometer was carried out at 420 πm.

Claims

- 3 "claims:

1. In a liquid, in particular aqueous medium, the catalytic process which uses a biocatalyst which is fixed to glass or ceramic particles, in particular porous glass (CPG) as the support, characterized in that the support particles are magnetic or magnetizable.

2. The method according to claim 1, characterized; that the surface of the carrier particles is provided with a separating layer made of an organic polymer, for example glutaraldehyde or polyurethane.

3. The method according to claim 1 or 2, characterized in that the carrier particles are surface-modified for fixing the biocatalyst.

4. The method according to any one of the preceding claims, characterized in that the carrier particles are held in the liquid medium with the aid of a magnetic field.

5. The method according to claim 4, characterized in that one carries out the method in a reactor which is flowed through by the liquid medium.

6. The method according to any one of the preceding claims, characterized in that biological macro-molecules, such as antibodies, enzymes, DNA structures or lectins, or organisms, such as microorganisms, or parts thereof, such as animal or plant tissue, are used as biocatalysts.

7. carrier particles in particular for performing the method according to any one of the preceding claims, characterized gekennzeich¬ net that it is ceramic material or glass, in particular porous glass (CPG), on which a fur / Felll mixed oxide / mixed hydroxide has been deposited and which is optionally provided on its surface with a separating layer made of an organic polymer, for example with glutaraldehyde or polyurethane and optionally surface-modified.

8. carrier particles according to claim 7, producible in that one

- brings ceramic or glass particles together with an aqueous solution containing fur and fur ions,

- Fills the particle cavities with the solution by means of negative pressure and

- a fur / Felll mixed oxide / mixed hydroxide falls alkaline on the particles,

optionally coating the carrier particles with an organic polymer, for example with glutaraldehyde or polyurethane and

- If necessary, carries out a surface modification.

9. Device for carrying out the method according to any one of claims 1 to 6, characterized in that it comprises one or more mechanically movable permanent magnets, electromagnets that can be switched on and off, or permanent magnets that can be switched on and off electromagnetically, with the aid of which the bio ¬ catalyst can be kept temporarily in the liquid medium flowing through the device.

10. The device according to claim 9, characterized in that the device is provided for carrying out a chemical or biological analysis, for example a Fliessinjek¬ tion analysis. - II-

11. The device according to claim 9 or 10, characterized by a reactor for carrying out the catalytic process, a permanent magnet and an electromagnet, wherein the reactor is arranged in the field of the permanent magnet and the permanent magnet can be switched off with the aid of the electromagnet.

12. The device in particular according to claim 11, characterized by

- A pot-like permanent magnet and

a cover which covers the opening of the permanent magnet and is made of a magnetically neutral material, such as brass,