DE10118554A1 - New system of two or more different enzymes, useful e.g. for degradation of alkylpolyglycoside detergents, and as biosensors, bioreactors and chromatography materials - Google Patents

Abstract

New enzymatic system (A) comprises an enzyme degradation chain of at least two different enzymes (I). An Independent claim is also included for a biosensor, bioreactor and chromatography material containing (A).

Description

The present invention relates to enzymatic systems with enzyme degradation chains of different types of enzymes, preferably immobilized Condition, and their use, especially for use in biore actuators, biosensors and chromatographic systems.

It is in applied microbiology, especially in biotechnology known, enzymes, enzyme-producing microorganisms or cells to fix certain carriers, for example if they are used as biocatalysts be used. This process is commonly called immobilization designated.

As native enzymes during storage or with a one-time batch approach through biological, chemical or physical effects in their actions due to the high manufacturing costs of Enzymes have a need to stabilize these enzymes. Through immobilization They are reusable. After use, the En zyme can be easily separated. This way they can be ho local concentration and in continuous flow. The Substrate specificity and the specificity of the reaction as well as the reactivity of the Enzyme must not be lost through immobilization.

There are generally three reasons for the immobilization of enzymes different methods, namely firstly by immobilization Cross-linking, secondly the immobilization by binding to a carrier thirdly, immobilization by inclusion.

When immobilized by cross-linking, cross-linked enes are obtained enzymes that are fixed to one another without affecting their activity. The However, enzymes are no longer soluble. The cross-linking takes place at for example with glutardialdehyde.

If the immobilization is carried out by connection to a carrier, the Binding takes place by adsorption, ion binding or covalent binding.  

The bond to the carrier can also be within the original micro biological cell take place. The enzyme is not in its fixation Activity affects, it can be carrier-bound multiple or continuous be used.

When immobilized by inclusion, the enzyme is usually between se mipermeable membranes and / or gels, microcapsules or fibers closed. The encapsulated enzymes are e.g. B. by a semipermeable Separated membrane from the surrounding substrate and product solution. It cells can also be encapsulated. The enzyme is due to the fixation in the Space is not affected in its activity.

Immobilized enzymes, enzyme-producing microorganisms or cells are used in particular in biotechnological processes. The first Technical processes with immobilized cells have been empirically optimized and are still used today. B. the wastewater treatment in the drip body. Another older method is the vinegar production with the gene ratorverfahren. In the food sector, the use of cells with glu cose isomerase for the production of fructose syrup the most important Method. Glucose amylase for glucose production in the starch process is also used immobilized. The splitting of lactose with the help of mobilized β-galactosidase from yeast to glucose and galactose is flat if a common practice. Further technical processes with immobilized Systems exist in the production of amino acids, in the cleavage of penis cillin G to 6-aminopenicillinic acid and in the production of ethanol with growing immobilized cells from Saccharomyces sp.

Immobilized enzyme and cell systems are not only used in biotechnology ongoing production processes, but also used in analytics, so z. B. in so-called biosensors. The principle of analytics with the help of immobilized systems is based on the fact that a substrate to be determined is implemented by an immobilized system, changing the Product, substrate or cosubstrate concentration can be tracked, for example with several coupled methods (e.g. enzyme electro the).  

Often, however, molecules are not broken down or converted through a single enzyme but along a so-called enzyme breakdown chain of several enzymes, d. H. the reaction is enzymkata in several stages lyses with different enzymes. Sometimes the parallel Be mood of several substances by several different enzymes or due to enzyme (degradation) chains required. In conventional biosensors and Prior art bioreactors can use such a multiple enzyme table-catalyzed reaction sequence but often only rarely carried out who that because the bioreactors and bio sensors preferably comprise a type of enzyme.

The problem underlying the present invention is a To provide a system with which such processes can also be enzymatically catalyzed Allow lysing that takes place in a multi-stage enzymatic manner, d. H. where the Mon leküle, in particular biomolecules or technical molecules, in several hours enzymatically catalyzed or converted into end products the, the implementation within only a single enzymatic Sy stems, but with different enzymes.

Furthermore, the present invention addresses the problem of a Provide system with which the parallel implementation of several substances by several different enzymes or by enzyme (degradation) chains is possible.

In particular, such a system for use in bioreactors, bio sensors and chromatographic systems.

It has now surprisingly been found here that the previously ge mentioned problem can be solved by combining at least two different enzymes, i. H. at least two different types of enzymes, which are preferably each in immobilized form can.

The present invention thus relates to an enzymatic system which comprises an enzyme degradation chain, which in turn has at least two different ones  or different enzymes, d. H. Enzymes different Type.

The at least two enzymes of the enzyme (degradation) chain are based on this coordinated with one another so that they, in particular, selectively or essentially lective, at least one specific molecule - also as a "target molecule" draws - dismantle.

According to a first embodiment of the present invention, the Enzymes in the enzyme (degradation) chain must be coordinated with one another in such a way that in particular selective or essentially selective, at least one not na naturally occurring molecule, in particular a technical molecule to build. The enzymes of the enzyme (degradation) chain can, for example be coordinated so that they do not occur naturally Molecule, especially technical molecule, according to a not natural reduce upcoming metabolism, d. H. a non-natural one Form metabolism chain. Alternatively, the enzymes of En zym (degradation) chain can also be coordinated so that they do not naturally occurring molecule, especially technical molecule, according to break down a naturally occurring metabolism, d. H. one of course Form occurring metabolism chain, which occurs naturally regulates the breakdown of other, namely naturally occurring, molecules.

According to a second embodiment of the present invention the enzymes of the enzyme (degradation) chain must be coordinated with one another in such a way that they, in particular selectively or essentially selectively, at least one na naturally occurring molecule according to a non-naturally occurring one Reduce metabolism. The naturally occurring molecule can for example, be a natural product.

The enzymatic system according to the invention is particularly suitable for consecutive and / or multi-stage degradation of molecules. It runs the degradation is preferably selective or substantially selective with respect to the molecule or molecules to be broken down.  

Furthermore, the enzymatic system according to the invention can in particular used for catalysis of multi-stage enzymatically catalyzed reactions become.

The enzymatic system according to the invention is equally suitable for parallel implementation of different molecules, each with a difference enzymes.

According to a particular embodiment of the present invention in the enzymatic system according to the invention at least one of the enes zyme the enzyme (degradation) chain in immobilized form. Preferably can in this embodiment, all enzymes of the enzyme (degradation) chain in immobilized form.

According to the invention, the immobilization of the enzymes can be carried out according to the usual The previously described methods of the prior art take place, the Reactivity of the enzymes immobilized in this way at least in the week substantial should be preserved.

Thus, the immobilization of at least one of the enzymes of the invention according to the enzymatic system, for example by cross-linking it consequences.

There is also the possibility of at least one of the enzymes of the inventin Enzymatic system according to the invention by inclusion or encapsulation to immobilize, in particular by inclusion or encapsulation in a semipermeable membrane or between several semipermeable membranes, but also between one and / or more semipermeable membranes and finally an ion permeable but gas permeable membrane, e.g. B. from PTFE or silicone rubber.

The immobilization of at least one of the enzymes of the invention enzymatic system can also by binding to a suitable Trä ger done. Here, the binding due to adsorption, ion bin tion and / or covalent bond. For example immobilization by connection to a suitable, preferably  chemically inert carrier must have been carried out, the carrier previously by plasma chemical surface modification may have been activated.

Finally, there is also the possibility of inventing the enzyme or enzymes to immobilize the inventive enzymatic system by a method is as described in the Henkel KGaA outgoing German patent application with the official file number DE 101.. , (internal file number: 00.495.6.do) from the same filing date as the present application, the entire contents of which hereby express Lich is included by reference.

This process comprises the following process steps:

• a) Activation of the chemically inert carrier surface by modification the carrier surface using plasma chemical methods, in particular wherein the activation of the chemically inert carrier surface Functionalization of the carrier surface includes and preferably there by that at least under plasma chemical conditions a suitable one, towards the enzyme or enzymes to be bound active functional group directly on the chemically inert support surface surface is attached, in particular being the functional group preferably a carboxyl, amino, hydroxy and / or thio group, optionally in activated, especially protonated or deproto form, includes; subsequently
• b) binding of the enzyme or enzymes to be immobilized, if appropriate after transfer to an activated, connectable Zu stood on the carrier surface activated in step (a); and close Lich
• c) optionally crosslinking the in process step (b) bound enzyme,

in particular with the process steps (b) and (c) possibly increasing summarized, in particular can be carried out simultaneously and / or the immobilization, if necessary in layers, in particular with Glutardialdehyde, can be carried out.  

The present invention therefore relates to a special embodiment form of an enzymatic system in which at least one of the enzymes, preferably all enzymes in the enzyme (degradation) chain by fixation and / or binding to a chemically inert, plasma-chemically activated and / or functionalized carrier surface according to that previously described Procedures are immobilized.

Activation of the chemically inert carrier surface using plasmachemi Scher methods in step (a) of the method described above is before preferably done selectively only on the surface, so that the bulk property properties of the chemically inert carrier surface are otherwise retained, wherein the activation of the chemically inert carrier surface in step (a) of the previously described method in reactive plasma, in particular high frequency plasma, has occurred. The chemically inert carrier surface can in particular special precious metals such as platinum and their alloys, precious steel or polyhalogenated polymers, especially polyhalogenated poly mere hydrocarbons such as in particular polytetrafluoroethylene or Po lyvinyl chloride, or cellulose acetate or combinations of these Ma include materials. For example, PTFE (Teflon®) are particularly suitable and cellulose acetate membranes.

The connection or coupling of the enzyme or enzymes to be immobilized to the plasma-chemically modified carrier surface according to step (b) of the The method described above can in particular by connection or Coupling of the enzyme or enzymes via the reak attached in step (a) tive functional groups have taken place, the enzyme or enzymes un indirectly to the reactive function applied to the carrier surface nelle groups can be connected or indirectly via a ge suitable linker.

According to a particular embodiment, the present invention relates an enzymatic system in which at least one of the enzymes is preferred all enzymes in the enzyme (degradation) chain by fixation and / or Binding to a chemically inert, plasma-chemically activated and / or radio tionalized carrier surface are immobilized. You can do this Enzymes directly or indirectly on a chemically inert carrier surface  fixed, in particular tied or coupled. In particular, can the attachment or coupling of the enzyme or enzymes to the chemically Suitable, reactive functional group attached to inert support surface pen.

The enzymes of the enzymatic system according to the invention can in particular especially selected from the group of oxidoreductases, transferases, Hydrolases (e.g. esterases such as lipases), lyases, isomerases and ligases (Synthetases) and their mixtures or combinations with one another.

In the enzymatic system according to the invention there are the mind at least two enzymes either within a single reaction zone or but in separate, consecutive, sequentially switched react tion zones, which then together form the enzymatic system. With at whose words can in the enzymatic system according to the invention Enzymes of the enzyme (degradation) chain within a unit, preferably in within a reaction zone, a compartment or a cell, in particular special measuring cell, be arranged or in separate, in particular their successive units, preferably in separate, behind successive reaction zones, compartments or cells, in particular whose measuring cells, which together form the enzymatic system, are arranged his. However, a parallel connection of Measuring cells take place.

According to a particular embodiment of the present invention ent holds the inventive enzymatic system amyloglucosidase and Glu coseoxidase, optionally in combination with mutarotase and / or α-Glu cosidase (maltase). Such a system is particularly useful for breaking down nonionic surfactants, especially of alkyl polyglucosides.

The enzymatic system according to the invention can be used in many ways, in particular especially in biosensors, bioreactors or chromatographic systems (e.g. chromatographic columns). Subject of the present invention are therefore also biosensors, bioreactors and chromatographic systems, which comprise the enzymatic system according to the invention.  

As described above, the enzymatic Sy systems in biosensors. There are at least two ver different types of immobilized enzymes combined in one another an enzyme chain or enzyme degradation chain can be used. You can the different enzymes either in a single reaction system (e.g. in a measuring cell) or sequentially one after the other be switched (e.g. in successive measuring cells), which then together form the enzymatic system according to the invention. This is how it works for example the parallel determination of several substances by several En zyme (or enzyme chains) in a measuring cell or the sequential degradation nes starting material (analyte) in several series-connected measurements cells with simultaneous electrical analysis in one and / or several measuring cells len.

According to the invention, “biosensors” are understood to mean sensors with a bio active component, based on the coupling of biomolecules as Recognize receptors in the broadest sense specifically analytes, with physiko chemical transducers that produce a biologically generated signal (e.g. Sauer substance concentration, pH value, dye etc.) into electrical measurement signals men.

Fig. 1 shows schematically the typical structure of a biosensor for specific detection of an analyte 1 , the biosensor comprising a receptor 2 and a transducer 3 which converts the biological signal generated by the receptor 2 into an electronic signal 4 which is sent to an elec tronik 5 is forwarded.

Different biomolecules can be used for specific recognition especially enzymes. Can be used as transducers potentiometric sensors, amperometric electrodes, piezoelectric Sensors, thermistors or optoelectronic sensors. Due to the Re action or interaction of the analyte with the receptor there are two basic types of biosensors, namely bio affinity sensors, the change in complex formation occurring Exploit electron density, and on the other hand, metabolism sensors based on the specific detection and implementation of substrates.  

Biosensors are found in the ge - especially in the form of enzyme electrodes healthcare, to control biotechnological processes in life medium industry or environmental protection use. With biosensors different systems are analyzed z. B. glucose, galactose, lac tose, ethanol, lactic acid or uric acid.

When using the enzymatic system according to the invention in bio sensors can, for example, the various enzyme molecules for Im mobilization either in polymer matrices (such as PVC, gels, graphite or zeolites) or between foils (e.g. cellulose acetate) the. Furthermore, sensors, for example on the enzyma based generation or consumption of oxygen and a have chemically inert membrane (e.g. a Teflon® membrane), this ge can be used to bind the various enzymes. The production the biosensors according to the invention can be carried out, for example, as is the case is described in the already cited, also for Henkel KGaA declining German patent application with the same filing date and the official file number DE 101.. , (Internal file number: 00.495.6.do), the entire content of which is included by reference.

The biosensors according to the invention enable the manufacturers, for example Microelectrodes (arrays) for small volumes and high samples throughput (e.g. for combinatorial application).

Application examples for the biosensors according to the invention are ana lytic determination of surfactants, especially ionic surfactants such as nonionic surfactants (e.g. alkyl polyglucosides), of polyaspartic acid and fatty alcohol derivatives.

According to a particular embodiment, the present invention relates a biosensor, in particular for qualitative and / or quantitative loading mood of nonionic surfactants, preferably of alkyl polyglucosides, the biosensor as an enzyme (degradation) chain around an enzymatic system summarizes which are given as enzymes amyloglucosidase and glucose oxidase if necessary in combination with mutarotase and / or α-glucosidase (maltase),  contains. The above-mentioned enzymes are in particular immobi form, preferably by binding to a chemically inert surface surface and / or membrane, preferably to a polytetrafluoroethylene or Cellulose acetate.

As described above, the enzymatic system according to the invention can can also be used in bioreactors.

According to the invention, “bioreactors” are understood to mean the physical loading ratio, in which biological material conversions, especially with enzymes be performed.

The immobilized enzymes of the enzymati according to the invention systems, for example on the wall surfaces of the bioreactor be attached or - especially in the case of fixed bed reactors the stationary carrier material or bulk material must be attached. To single ten can be referred to the one already cited, also to the handles KGaA declining German patent application from the same filing date with the official file number DE 101.. , (Internal file number: 00.495.6.do), the entire contents of which are hereby included by reference is. This way, a more efficient generation of reactors be realized in which no separation of the enzymes from the Re action solution is required. Reactor surface suitable according to the invention Chen can be made of metal (e.g. precious metal such as platinum) or with all of them common polymers that are used for reactor manufacture, be be layered (e.g. Teflon®).

Fig. 2 shows by way of example and schematically different types of Bioreakto ren of the prior art:

Fig. 2A shows a stirred tank reactor in which the energy input is accomplished by mechanically moving units. Here G denotes the gas flow and M the mechanical drive device (e.g. motor). Because of their versatility, stirred tank reactors are used most frequently.

Fig. 2B shows a bubble column reactor, wherein the mixing is carried out by supplying air or other gas. Here G denotes the gas flow.

Fig. 2C shows a so-called airlift fermenter with inner passage, generally by the introduction of air or another gas, a liquid circulation and thorough mixing is generated. Here G denotes the gas flow.

Fig. 2D shows a so-called airlift fermenter with outer flow, generally by the introduction of air or another gas, a liquid circulation and thorough mixing is generated. Here G denotes the gas flow.

In the prior art bioreactor types described above the enzymatic system according to the invention are used, for example wise by modifying the wall surface (e.g. by coupling the enzymes on the chemically inert reactor walls) or in the case of fixed bed bioreactors by linking the enzymes to the stationary one Carrier material or bulk material.

When using the enzymatic system according to the invention in bio reactors, in particular to modify the wall surface or at the binding of the enzymes to the stationary carrier material or bulk material, there is a possibility of using at least two different types of en to combine zymen in such a way, d. H. the enzyme chains or En use zymab chain in such a way that either the different En zymes are present in a single reaction zone or sequentially are connected in series (e.g. in successive reaction zones). In this way, multi-stage, enzymatically catalyzed Syntheses and processes enable or also the simultaneous, parallel Um different enzymes.

Figs. 3 shows by way of example and schematically some embodiments of bioreactors according to the present invention:

Fig. 3A shows a bioreactor, the chemically inert walls are modified by coupling an immobilized type A enzyme and an immobilized type B enzyme, which are arranged in different, successive reaction zones. Here G denotes the gas flow.

Fig. 3B shows a bioreactor, the chemically inert walls are modified by coupling an immobilized enzyme of type A and a type B enzyme immobilisier th, which are arranged within a single re action zone.

Fig. 3C shows a bioreactor in the form of a fixed bed reactor, at whose Trä germaterial or bulk are coupled immobilized enzymes of the type A and type B, which are arranged within a reaction zone.

Numerous other variants are possible, which the person skilled in the art Reading the present description will readily consider without departing from the scope of the present invention.

There is also the possibility of the enzymatic according to the invention System in chromatographic systems, especially in chromatographi columns. This can be done for synthetic purposes (e.g. carrying out enzymatically catalyzed reactions on a chroma graphical column) or for analytical purposes (e.g. for the analytical column chromatography). The immobilized Enzymes of the enzymatic system, for example on the stationary carrier material or bulk material, especially the stationary column material, the chromatographic system are bound. For more details, can Reference is made to the one already cited, to the applicant of the present German patent application from the same declining patent application Chen filing date with the official file number DE 101.. , (Firm-internal File number 00.495.6.do), the entire contents of which are hereby incorporated by reference name is included.

The use of the enzymatic system according to the invention has the advantage that with the system according to the invention such processes are also enzymatic  catalyze the multi-stage enzymatic process, d. H. at them the molecules, in particular biomolecules or technical molecules, in meh rere stages and enzymatically catalyzed with various enzymes builds or be converted into end products.

Furthermore, the use of the enzymatic according to the invention enables Systems also the parallel implementation of several substances by different Enzymes or by enzyme (degradation) chains and thereby, for example, the parallel analytical determination of several substances side by side with one single measuring cell, which an enzyme degradation chain from different Enzy contains or the sequential degradation of a starting material (analyte) in several measuring cells connected in series with simultaneous electrical analysis in one and / or several measuring cells.

Finally, the use of the enzymatic system according to the invention also the advantage that on the one hand the enzymes due to the immobilization can be reused and on the other hand after their use easy separation is possible (e.g. after synthesis in the bio reactor, for example by draining the reaction mixture). To this In this way, the enzymes can be efficiently and inexpensively used in high local areas Use concentration and in continuous flow. The substrate Specificity and the specificity of the reaction as well as the reactivity of the enzymes however, they are not lost.

The concept of the present invention is therefore different Combine enzymes in such a way that molecules (e.g. technical molecules or biomolecules) can be broken down. This can be used, for example, for analytical purposes to degraded molecules via certain parameters as previously described (e.g. change in pH value, oxygen consumption / generation etc.) sen. With regard to bioreactors, this means that the possibility of simultaneous or sequential implementation of one or more substances stands.

According to the concept of the invention, the production of a substance and the reaction tracking with sensors that measure oxygen or H ₂ O ₂ , for example, are identical and thus also enable efficient reaction control.

Further configurations and variations of the present invention are readily recognizable to the person skilled in the art when reading the patent specification and realizable without ver ver the scope of the present invention leaves.

The present invention is based on the following exemplary embodiments illustrates which, however, in no way be the present invention should restrict.  

embodiments Enzymatic degradation chains for alkyl polyglucosides (APG®) in invent biosensors according to the invention

For APG® (alkyl polyglucoside, a group of non-ionic surfactants, distributed by the company Henkel) an enzymatic degradation chain was ent wraps, consisting of amyloglucosidase and glucose oxidase as well as gege also mutarotase and / or α-glucosidase (maltase).

To produce the biosensor according to the invention, the previously ge called immobilized initially.

The immobilization was carried out by the aforementioned enzymes on a Cellulose acetate membrane or was applied to a Teflon® membrane as described in the Henkel KGaA outgoing German patent application with the official file number DE 101.. , (internal file number: 00.495.6.do) from the same Filing date as the present patent application. The mem bran modified or known according to known plasma chemical methods activated, d. H. in reactive high-frequency plasma among those known per se Functionalized conditions, thereby suitable, enzyme-reactive functional groups bound to the chemically inert membrane surface are, in particular amino and / or carboxyl groups. Here were then the enzymes are bound using methods known per se.

Alternatively, the immobilization can also be carried out from the state of the art Techniques known in the art can be carried out, for example by introduction between foils (e.g. from cellulose acetate) or in polymeric matrices (e.g. PVC, gels, graphites, zeolites etc.).

APG® could then be detected using this enzymatic degradation chain or can be determined electroanalytically.

1. Alkyl polyglycosides

For about 10 years, there has been a significant increase in the production of Alkyl polyglycosides can be observed. They are called phosphate-free Neutraltenside surface-active cosmetic preparations or detergents  added. One to seven glucose units, in the frequency of mono- drastically decreasing to heptaglycosides are glycosidic with one mostly linked long-chain alcohol such as dodecanol or tetradecanol.

Because all of the biochemical information on the basis of enzymes Detection of the analyte can be incorporated into a membrane system bioelectrochemical membrane electrodes are particularly suitable ders attractive measuring principle for the alkyl polyglycosides.

Of the various molecular selective APG® Membranes with immobilized enzymes are here eleven Mem Bransystemen reported the principles of the invention.

The water-soluble alkyl polyglycoside APG® 220 was used for analysis Henkel KGaA in Düsseldorf is available. All measurement solutions were the phosphate buffered to a pH of 5.0.

2. Construction of molecular selective APG® sensors 2.1. APG® sensors

The sensors of the invention are inte in a flow measuring system griert, whereby the enzyme membranes for the APG® substrate conversion Form the roof of a trough-shaped, flattened flow chamber radial inflow and outflow channels. The tangential flow of the Enzyme membrane is made by sucking in the APG®-containing solutions with a roller pump downstream of the measuring cell, measuring and Calibration solutions can be fed in separately from air bubbles.

2.1.1. H ₂ O ₂ -sensitive-enzymatic APG® sensors 2.1.1.1. Modular measuring system

These are amperometric APG® biosensors with H ₂ O ₂ detectors in disk-electrode design without internal electrolyte made of Pt measuring anode and Ag reference cathode, each with a diameter of 2 mm, in two separate, serially arranged and connected through flow chambers , Measuring anode and reference cathode are made in "spark plug-shaped" design and are screwed sealingly against the edges of the flow chambers. Only the end faces of the precious metal electrodes are freely accessible and are electro-analytically active. The membrane systems with a diameter of 5 mm are protected against cavities and are in direct contact with the Pt measuring anode.

Special designs of the enzyme membranes see the purpose Surface enlargement against the cylindrical lining of the cavity if necessary with a coating of the Pt surface (SBC-1412-APG®). Kon vex electrode surface design in connection with thin enes zymmembranen promotes the speed of adjustment.

The installation described here was installed for a special form of application benen measuring anodes and reference cathodes in the lid and bottom of Plastic vessels (SBC-1420-APG®) for stationary measurement e.g. B. for a customer service measurement kit. In another embodiment these measuring system components were made into the side walls of an acrylic glass tube res integrated, the top and bottom end with inlet and outlet taps was provided.

2.1.1.2. Measuring system with anodic window

The Pt measuring anode with cavity-protected enzyme membrane from 5 mm has direct access through an anodic window to the measuring solution, while the rod-shaped Ag reference cathode (Diameter 1 mm) is located in a side space of the measuring cell and above an internal electrolyte is connected to the Pt measuring anode.

2.1.2. O ₂ -sensitive-enzymatic APG® sensors

These APG® sensors consist of a Pt cathode with a membrane system (see 2.1.2.1. And 2.1.2.2.) Based on PTFE and immobilized enzymes. The Ag / AgCl reference anode is in turn analogous to 2.1.1.2. in a side area of the measuring cell and is connected via a buffered internal electrolyte to the Pt measuring electrode in the cathodic window, which is sealed by the PTFE membrane, but is gas-permeable, so that O ₂ acts as a transducer between the immobilized enzymes and the Pt cathode can act.

2.1.2.1. Membrane system with chemically unmodified PTFE membrane bran

The membrane system does not have a chemical one on the electrode side modified PTFE membrane, two dialysis membranes towards the measuring solution of cellulose acetate, between which - before bacterial Ab protected by construction - the enzymes on a strip of cellulose fibers Adsorption and / or ionic binding immobilized and / or covalent networked.

2.1.2.2. Membrane system with chemically modified PTFE membrane

Coupled to the PTFE membranes aminated in high-frequency plasma covalently the bifunctional reagent glutardialdehyde with simultaneous Cross-linking enzymes that convert APG®.

3. APG®-selective enzyme membranes 3.1. Bienzyme membranes

The enzymatic attack on the α-1,6-glucosidic bonds of APG® 220 by the amyloglucosidase provides glucose,

whose β-form is further converted by glucose oxidase (GOD):

Due to the reaction, the amperometric measurement at the end of the enzyme degradation chain can take place via the consumption of O ₂ by oxygen electrodes or the formation of H ₂ O ₂ with water peroxide detectors.

Fig. 1 shows a U / I diagram for bienzyme membranes for alkyl polyglycoside biosensors with hydrogen peroxide detectors (t = 20.0 ° C).

For the APG® sensors with type 2.1.1.1 hydrogen peroxide detectors. with the membranes SBC-1408, SBC-1417 and SBC-1419, Fig. 1 graphically shows the relationship between the _total enzyme concentration in U / membrane, the current measured at 7500 ppm APG® 220 (marking points) and the response time.

The bienzyme membranes contain the two enzymes amyloglucosidase and glucose oxidase in the same concentration based on units, the total concentration of which is shown in the graph in Fig. 1. The enzymes are present in the membrane systems in cross-linked form due to glutardialdehyde.

SBC-1419-APG® has the highest analytical resolution capacity among the three measuring systems with bienzyme membranes shown in Fig. 1.

Increasing the enzyme concentration (SBC-1408-APG®) extends the intramembranous diffusion pathways. The membrane thickening increases the response time. At the same time this is accompanied by a decrease in the resolving power. It seems worthy of discussion that this could be caused, among other things, by a larger decay of H ₂ O ₂ on the longer diffusion path to the Pt measuring anode, with additional loss due to diffusion across the membrane surface.

As an expression of lower enzyme concentration (SBC-1417-APG®) decreases the substrate turnover and accordingly the measuring current. The faster ones Response times are a consequence of the shorter intramembranous diffusi onswege (membrane thinning).

In principle, the bienzyme membrane SBC-1419-APG® is also suitable for use on the H ₂ O ₂ sensitive enzymatic measuring system with an anodic window (2.1.1.2.), As is the trienzyme membrane described below.

3.2. Trienzym membranes

To further increase the sensitivity of the APG® sensor, through additional covalent bonding or cross-linking of mutarotase with glutaraldehyde the trienzyme membrane SBC-1425- APG® designed (Tab. 1).

Tab. 1

Bi- and trienzyme membranes for alkyl polyglycoside biosensors with hydrogen peroxide detectors

As is known, GOD reacts very selectively with β-D-glucose, while the α-form is not implemented by this enzyme.

Von Rybinski, W. and Hill, K. describe in their work on "Alkyl polyglycosides - properties and applications of a new surfactant class" in Angew. Chem. 110 (1998), pages 1395 to 1412 that in the industrial process the α-anomers and β-anomers are in a ratio of approximately 2: 1. However, since, almost inversely after adjustment of the mutarotation equilibrium, α-D-glucose with 36% and β-D-glucose with 64% are present after approximately 2 hours from freshly prepared α-D-glucose, it was expected that with the help of the additionally incorporated Mu tarotase

a faster intramembrane sub in accelerated reaction strate supply for glucose oxidase can be enforced if one assumes that in the product APG® 220 a similar ratio of α- and β-anomers as given in the technical process.

It was possible to prove on modular measuring systems (cf. 2.1.1.1.) With H ₂ O ₂ detectors that the bee enzyme membrane SBC-1419-APG® can be distinguished by the additional presence of the mutarotase in the trienzyme membrane SBC -1425-APG® had the sensor sensitivity increased by approx. 30%, although the two other enzymes were present in the trienzyme membrane system even in slightly lower enzymatic concentrations (see Table 1).

3.3. Tetra enzyme membranes

The first molecular selective membrane electrodes for the determination of APG® were realized on the basis of four enzymes and contained in addition to amyloglucosidase, glucose oxidase, mutarotase additionally α-Glu cosidase, the latter imagining possibly the fatty alcohol to be released from its glycosidic bond (Tab. 2).

Tab. 2

Tetraenzyme membranes for O ₂ sensitive APG® multienzyme electrodes

A turnover of α-1,6-glucosidic bonds through that as well as times Unfortunately, this enzyme is slow. No attack takes place on β- glucosidic bonds instead. In contrast, α-1,4-glucosidic bin the preferred target.

The four enzymes were immobilized by adsorption and / or ionic binding on a strip of cellulose fibers between two dialysis membranes made of cellulose acetate. The enzyme membrane was covered with a chemically unmodified PTFE membrane and stretched on an acrylic glass ring by means of an O-ring and finally positioned with the help of an inner electrolyte film in front of the Pt cathode of the O ₂ sensor, which was covered by an Ag / AgCl reference cathode The common internal electrolyte for the measuring cell was completed: O ₂ sensitive APG® multi-enzyme membrane electrodes.

3.4. APG® biosensors with enzymes on aminized PTFE mem membranes

On chemically modified PTFE membranes in aminized form (2.1.2.2.) for oxygen electrodes (2.1.2.) were determined by the bifunctional Rea genz glutardialdehyde amyloglucosidase and glucose oxidase covalently bound. In further layers, these enzymes were cross-linked and coupled to the previous layer. Among those listed in Tab. 3 Enzyme membranes draw the APG® sensor with 10.) SBC-1322-APG®- HDKS5 no. 1 in addition to high measurement stability, response times of only 3 to 6 minutes. This is the result of a particularly aerodynamic Geometry with tangential flow of the entire membrane system and the lack of enzyme-encapsulating dialysis membranes.

Tab. 3

Aminated PTFE membranes with covalently bound enzymes for O ₂ detectors of molecularly selective APG® sensors

4. Results 4.1. Response time

In addition to the changing number of individual enzymes and the streamlined positioning of the membrane systems, the concentrations of immobilized enzymes influence the response time of the APG® sensor membranes via the related length of the diffusion paths. The ratio of mass (mg) to substrate turnover (unit) of the enzymes immobilized in the APG® sensor membranes leads to the fact that the rate-determining step in the intramembrane diffusion process starts from the amyloglucosidase in Fig. 2. A comparison of Fig. 3 with Fig. 4 confirms this relationship (see also Fig. 1). This explains the creeping setting behavior of SBC-1402. In contrast to this, other APG®-selective membrane electrodes with O ₂ detectors and immobilized enzymes on aminated PTFE membranes also show short response times of a few minutes with a tangential flow and without dialysis membranes: cf. 10.) SBC-1322-APG®-HDKSS-No. 1 under 3.4 and Tab. 3, while APG® sensors with hydrogen peroxide detectors offer superior sensitivity or greater resolution (see Fig. 1).

Fig. 2 shows the relationship of mass (mg) to substrate turnover (U) of the enzymes used in the APG® sensor membranes (1 unit = 1 µmol substrate turnover / min)

Fig. 3 shows the enzyme concentrations in the U / APG® selective biosensor membrane.

In the Fig. 3 and 4, the columns for mutarotase and maltase (α-glucosidase) with the "Bi". (Abbreviation for bienzyme membrane) marked membranes set to zero for SBC-1425-Tri. (Tri. = Trien zymmembran) this only affects the α-glucosidase (maltase).

Fig. 4 shows the enzyme concentrations in mg / APG®-selective biosensor membrane

By increasing the temperature of the measuring medium, a shortening of the response times of APG® sensors can be expected. In order to reliably rule out thermal denaturation of the enzymes, a temperature rise above 40 ° C was avoided. For the trienzyme membrane SBC-1425-APG®, the H ₂ O ₂ detector according to 2.1.1.1. built-up modular flow measuring system determines the factor 1.3 when the temperature rises by 10 ° C, which also indicates that the physical process of diffusion as a rate-determining step is decisive for the response times of this APG® sensor; because, as is well known, according to the RGT rule, the speed of a chemical reaction increases two to four times when the temperature increases by 10 ° C. Obviously, this chemical process is superimposed by the intramembrane diffusion processes, in which case a factor of only about 1.2 can be expected.

4.2. longevity

Since the previous focus in the development of membrane chemical Principles, the continuous long-term perfusions of the APG® Sensors with a phosphate buffer with a pH value of 5.0 and intermediate Measurements in APG®-containing solutions stopped after a few weeks chen.

4.3. molecular selectivity

In addition to the substrate selectivity of the enzymes, detector-related cross-sensitivities have to be taken into account with APG® sensors. With O ₂ detectors, these cause surface-active or gaseous substances that permeate the PTFE membrane and can be converted cathodically, such as the halogenated hydrocarbons chloroform (trichloromethane) or the inhalation anesthetic halothane (1,1,1-trifluoro-2-bromo-2-chloroethane) ).

Hydrogen peroxide detectors act as measuring anodes also other anodically oxidizable substances. The one with a Polarisati on voltage of +950 mV measuring against reference electrode on freshly po gelled platinum anodes observed glucose interference current in 5.55 mmol / l phosphate buffer solutions containing glucose with a pH value of 7.04 showed an e-functional drasti within two weeks waste that after this period is almost negligible has been.

At a concentration of 7500 mg / l APG® 220 in to a pH value of 5.0 phosphate buffered solutions did not provide freshly polished Pt Measuring anodes in a modular measuring system according to 2.1.1.1. unmarried Lich 1.33% of the measuring current compared to one with the bienzyme membrane SBC-1419 coated. This minor due to the APG® molecules Additional current that can be triggered directly at the detector depending on the concentration Strength cannot be considered an interference signal because it is synergi acts on the measuring current triggered by the enzyme membrane and can be calibrated.  

5. Electronic components and computer-aided measurement data evaluation

The nanoamperemeter transducer supplies the amperometric sensor with a polarization voltage that is adapted to the plateau and has a transducer constant of 1 mV / nA. The polarization voltage for O ₂ sensitive enzymatic measuring systems is -750 mV measuring against reference electrode and for H ₂ O ₂ sensitive enzymatic measuring systems with anodic oxidation +950 mV measuring against reference electrode.

The 21-bit AD converter replaces that of the nano-amperemeter Current / voltage conversion supplied electrical DC to corresponding digital signals via a serial Interface (RS 232) transmitted to an IBM compatible computer be a computer-based measurement of the measured currents perform data analysis. The curve shape with a measurement value within about 2 seconds Track continuous display.

Other applications in the field of biosensors are besides Alkyl polyglucosides and other surfactants, for example, also the determinations tion of polyaspartic acid and fatty alcohol derivatives.

Substances in the area of the reactors can e.g. B. substances that are on site at Customers are needed, e.g. B. peroxides or surfactants, lubricants or high-quality specialty chemicals.

Claims (42)

1. Enzymatic system comprising an enzyme (degradation) chain that its partly comprises at least two different enzymes. 2. Enzymatic system according to claim 1, wherein the enzymes of the En zym (degradation) chain are coordinated so that they, esp special selective or essentially selective, at least one particular Degrade t molecule (target molecule). 3. Enzymatic system according to claim 1 or 2, wherein the enzymes Enzyme (degradation) chain are coordinated so that they, esp special selective or essentially selective, at least one not na naturally occurring molecule, especially a technical molecule, dismantle. 4. Enzymatic system according to claim 3, wherein the enzymes of the En zym (degradation) chain are coordinated so that they do not naturally occurring molecule, in particular technical molecule, break down according to a non-naturally occurring metabolism. 5. Enzymatic system according to claim 3, wherein the enzymes of the En zym (degradation) chain are coordinated so that they do not naturally occurring molecule, in particular technical molecule, break down according to a naturally occurring metabolism. 6. Enzymatic system according to claim 1 or 2, wherein the enzymes Enzyme (degradation) chain are coordinated so that they, esp special selective or essentially selective, at least one natural occurring molecule according to a non-naturally occurring one Reduce metabolism. 7. Enzymatic system according to claim 6, which occurs naturally molecule is a natural product.   8. Enzymatic system according to one of the preceding claims consecutive and / or multi-stage degradation of molecules, in particular the latter being selective or substantially selective with respect to degradation on the molecule or molecules to be broken down. 9. Enzymatic system according to one of the preceding claims Catalysis of multi-stage enzymatically catalyzed reactions. 10. Enzymatic system according to one of the preceding claims parallel implementation of different molecules with differ enzymes. 11. Enzymatic system according to one of the preceding claims, where at least one of the enzymes of the enzyme (degradation) chain in immo bilized form is available. 12. Enzymatic system according to one of the preceding claims, since characterized in that all enzymes in the enzyme (degradation) chain in mobilized form. 13. Enzymatic system according to one of the preceding claims, where at least one of the enzymes passes through the enzyme (degradation) chain Crosslinking, preferably with glutardialdehyde, is immobilized. 14. Enzymatic system according to one of the preceding claims, where at least one of the enzymes of the enzyme (degradation) chain immobili is based on inclusion or encapsulation, especially in a semi permeable membrane or between several semipermeable membranes or between one and / or more semipermeable memes branches and finally an ion-impermeable but gas permeate blen membrane, especially made of PTFE and / or silicone rubber. 15. Enzymatic system according to one of the preceding claims, where at least one of the enzymes passes through the enzyme (degradation) chain Connection to a suitable, preferably chemically inert carrier  is immobilized, in particular where the carrier is previously plasma chemical surface modification has been activated. 16. An enzymatic system according to claim 15, wherein the binding is due to physical and / or chemical bond, especially due to of adsorption, ion binding and / or covalent binding. 17. Enzymatic system according to one of the preceding claims, wherein at least one of the enzymes, preferably all of the enzymes in the enzyme (degradation) chain, are immobilized by a method by fixing and / or binding to a chemically inert, plasma-chemically activated and / or functionalized carrier surface, which comprises the following process steps:

• a) Activation of the chemically inert carrier surface by modifying the carrier surface using plasma chemical methods, in particular wherein the activation of the chemically inert carrier surface comprises functionalizing the carrier surface and preferably taking place in that at least one suitable, reactive towards the enzyme or enzymes to be bound under plasma chemical conditions functional group is attached directly to the chemically inert support surface, in particular the functional group preferably comprising a carboxyl, amino, hydroxyl and / or thio group, optionally in activated, in particular protonated or deprotonated, foam; subsequently
• b) binding of the enzyme or enzymes to be immobilized, if appropriate after their conversion into an activated, bindable state, to the surface of the carrier activated in step (a); and finally
• c) if appropriate crosslinking of the enzyme bound to process step (b),

in particular where process steps (b) and (c) can be combined, in particular carried out simultaneously and / or the immobilization can be carried out in layers, in particular with glutardialdehyde. 18. Enzymatic system according to claim 17, characterized in that activation of the chemically inert carrier surface by means of plasma chemical methods in step (a) selectively only on the surface follows and the bulk properties of the chemically inert carrier surface the rest of the area is retained, especially with the activation the chemically inert carrier surface in step (a) in the reactive plasma, in particular high-frequency plasma. 19. Enzymatic system according to claim 17 or 18, characterized records that the chemically inert support surface precious metals such as ins special platinum and its alloys, stainless steel or polyhaloge nated polymers, especially polyhalogenated polymeric carbon substances such as in particular polytetrafluoroethylene or polyvinyl chloride, or else cellulose acetate or combinations of these materials summarizes. 20. Enzymatic system according to one of claims 17 to 19, characterized characterized in that in step (b) the connection or coupling of the or the enzymes to be immobilized on the plasma chemical modes Defined carrier surface by connecting or coupling the or of the enzymes via the reactive radio attached in step (a) tional groups has taken place, in particular wherein in step (b) the or the enzymes directly on the applied to the carrier surface, reactive functional groups are linked or indirectly via a suitable linker. 21. Enzymatic system according to one of the preceding claims, since characterized in that at least one of the enzymes, preferably all enzymes in the enzyme (degradation) chain by fixation and / or Binding to a chemically inert, plasma-chemically activated and / or functionalized carrier surface are immobilized, the or which Enzymes directly or indirectly to a chemically inert carrier surface fixed, in particular connected or coupled, in particular  the connection or coupling of the enzyme or enzymes via suitable, attached to the chemically inert support surface reactive functional groups has occurred. 22. Enzymatic system according to one of the preceding claims, since characterized in that the enzymes from the enzyme (degradation) chain are selected from the group of oxidoreductases, transferases, Hy drolases such as in particular esterases such as lipases, lyases, isomerases and ligases (synthetases) and their mixtures or combinations with each other. 23. Enzymatic system according to one of the preceding claims, the enzymes of the enzyme (degradation) chain within a unit, preferably within a reaction zone, a compartment or a cell, in particular a measuring cell. 24. Enzymatic system according to one of the preceding claims, the enzymes of the enzyme (degradation) chain in separate, in particular successive units, preferably in separate, behind successive reaction zones, compartments or cells, ins special measuring cells, which together form the enzymatic system, are arranged and / or wherein, in particular for the purpose of difference measurement, also a parallel connection of reaction zones, compartments elements or cells, in particular measuring cells, can take place. 25. Enzymatic system according to one of the preceding claims, containing amyloglucosidase and glucose oxidase, optionally in Combination with mutarotase and / or α-glucosidase (maltase). 26. Enzymatic system according to claim 25 for the degradation of non-ioni tensides, in particular of alkyl polyglucosides. 27. Use of an enzymatic system according to one of claims 1 to 26 in bioreactors.   28. Use according to claim 27, wherein the enzyme or enzymes of the enzy immatic system and especially on the walls surfaces of the bioreactors are fixed or bound. 29. Use according to claim 27, wherein the enzyme or enzymes of the enzy immobilized system and in particular to the stationary Carrier material or bulk material of the bioreactors fixed or bound are, especially in the case of fixed bed reactors. 30. Use of an enzymatic system according to one of claims 1 to 26 in chromatographic systems, especially chromatogra phical pillars. 31. Use according to claim 30 for analytical or preparative syn theoretical purposes. 32. Use according to claim 30 or 31, wherein the enzyme or enzymes immobilized of the enzymatic system and in particular to the sta tional carrier material or bulk material of the chromatographic sy stems, in particular the stationary column material, fixed or bound are. 33. Use of an enzymatic system according to one of claims 1 to 26 in biosensors. 34. Use according to claim 33, wherein the enzyme or enzymes of the enzy immatic system and in particular immobilized on a membrane of the Biosensors are fixed or bound. 35. Biosensors, bioreactors or chromatographic systems, incl tend an enzymatic system according to one of claims 1 to 26. 36. Biosensor, in particular for qualitative and / or quantitative loading mood of nonionic surfactants, preferably of alkyl polyglucosi the, with the biosensor as an enzyme (degradation) chain an enzymatic System which comprises amyloglucosidase and glucose oxidase as enzymes,  optionally in combination with mutarotase and / or α- Contains glucosidase (maltase), especially where the enzymes in mobilized form are present, preferably by binding to a che mixed inert surface and / or membrane, preferably a polyte trafluoroethylene or cellulose acetate membrane. 37. Biosensor according to claim 35 or 36, characterized in that it is an H ₂ O ₂ -sensitive-enzymatic or O ₂ -sensitive-enzymatic sensor for alkyl polyglucosides. 38. Biosensor according to one of claims 35 to 37, characterized in net that the membrane system or the biosensor at least one include chemically modified polytetrafluoroethylene membrane. 39. Biosensor according to one of claims 35 to 38, characterized in net that the membrane system or membranes of the biosensor tight are protected and / or in direct contact with the measuring electrode, in particular another of a platinum anode. 40. Biosensor according to one of claims 35 to 39, characterized net that the biosensor at least one multi-enzyme membrane, in particular re comprises a bi-, tri- and / or tetraenzyme membrane. 41. Biosensor according to one of claims 35 to 40, characterized net that it is an amperometric biosensor. 42. Biosensor according to one of claims 35 to 41 for cleaving 1,4- and / or 1,6-glycosidically linked bonds, in particular of α-1,4- and / or α-1,6-glucosidically linked bonds.