DE3427889A1 - Immobilisation of Protaminobacter rubrum and use of the immobilised product for the conversion of sucrose into isomaltulose - Google Patents

Abstract

The invention relates to a process for the immobilisation of Protaminobacter rubrum by entrapment in polymerised compounds, to the product obtainable by this process, and to the use thereof for the preparation of isomaltulose from sucrose. The process comprises mixing aqueous solutions or dispersions of the biological material with an aqueous solution of high molecular weight polymerisable compounds which are readily soluble in water and have two or more polymerisable functional groups per molecule and a molecular weight above 400, and subsequently polymerising.

Description

Immobilization of Protaminobacter rubrum and ver

application of the immobilized preparation for the conversion of sucrose to isomaltulose The invention relates to methods for immobilizing Protaminobacter rubrum by inclusion in polymerized compounds obtained by this process available preparation and its use for the production of isomaltulose from Sucrose.

From EP-sen 49 742 and 49 801 it is known that isomaltulose consists of Sucrose using immobilized cells from Protaminobater rubrum or using purified free or immohilized sucrose mutase produced from Protaminobacter rubrum can be. Thereby a) the Protaminobacter cells are flocculated or immobilized by inclusion in calcium alginate or in te-carrageenan, b) solutions the purified enzyme sucrose mutase for immobilization in hollow fibers, bound covalently to solid or soluble carriers or adsorptively bound to solid carriers.

The economics of a manufacturing process for isomaltulose is among other things also from the costs of the biocatalyst used for this purpose and determines its long-term stability.

To avoid a loss of activity, a gentle immobilization process is required necessary for Protaminobacter rubrum, which has high activity yields and high reproducibility offers. It is also advantageous to use carrier materials that are as cheap as possible for immobilization to use.

Inclusion processes in 3t-carrageenan or in Ca-alginate stand out Although the conditions are gentle, the customary ones are often available Prices for these substances oppose their use on a technical scale.

In addition, the matrices made of carrageenan or calcium alginate are very good susceptible to changes in various parameters of the aqueous reaction solution are, unless by additional cross-linking of the carrier matrix, e.g. by means of Glutaraldehyde stabilization took place.

Crosslinkers such as Glutaraldehyde, however, are often more biological for degradation Activities responsible.

The production of immobilized cells by flocculation is used usually inexpensive flocculants, but processing is for storable, granular catalyst is often very labor-intensive and time-consuming and associated with high technical effort Separation, drying, Grinding and classifying devices.

This multitude of mechanical operations also leads to mitigation the total yield of catalyst material of up to 30%.

There has now been a method of immobilizing Protaminobacter rubrum found by inclusion in polymerized compounds, which is characterized is that Protaminobacter rubrum in an aqueous solution or dispersion with a aqueous solution of readily water-soluble, high molecular weight polymerizable compounds with two or more functional groups per molecule and a molecular weight of over 400 is mixed and this mixture, if necessary after this previously has been broken up into beads in an inert liquid medium, is polymerized.

The cells immobilized as a result of this process can be used to produce of isomaltulose from sucrose can be used.

The fermentation broth of Protaminobacter rubrum is used, if necessary without purification steps, by microfiltration or by continuous centrifugation concentrated, particularly advantageous by a factor of 10. In the cell sludge obtained in this way the water-soluble, higher molecular weight, pre-dissolved with a small amount of buffer polymerizable compounds introduced and stirred homogeneously.

By pre-dissolving the higher molecular weight, polymerizable compounds in a small volume of aqueous buffer, the similar pH, viscosity, conductivity etc. as the cell preparation exhibits, abrupt changes may occur during entrapment avoided and so the biological activities of the cells are largely preserved. The mixed with readily water-soluble polymerizable high molecular weight compounds Cell sludge from Protaminobacter rubrum is either used as a film, possibly also under Incorporation of a support fabric, e.g. made of polyamide (Monodur PA 250 N, Verseidag industrial textiles GmbH), or polymerized in another geometric shape. To those for catalysts To achieve a particularly favorable bead shape, it is necessary to use the polymerizable Compounds mechanically turned cell sludge into pearls in an inert phase divide e.g. by stirring, whereby the bead diameter depends on the stirrer speed can be controlled.

The polymerization can be chemical or photochemical by irradiation take place with the help of radical initiators or photosensitizers.

The immobilized cells of Protaminobacter rubrum thus obtained are characterized by high specific sucrose mutase activities and good long-term stability and can be used directly as biocatalysts without further processing or shaping can be used to convert sucrose to isomaltulose.

The storage of the catalyst preparations can also be in aqueous non-aqueous, e.g. organic materials. In addition, additives such as Glycol, polyether diols such as polyethylene glycols can be used.

The use of pearl-shaped immobilized is particularly advantageous Biocatalyst. The pearl shape is particularly useful for small pearl diameters of e.g. 0.2mm -2.0mm, an ideal catalyst shape for use in continuous Column reactors are. Other bead diameters are from 0.5 mm to 1.5 mm, in general however, the pearl diameter can also be 0.05 mm to 5 mm. It is also possible, to dry the immobilized Protaminobacter cells, store dry, and after Sources in the sucrose-containing reaction solution can be reused directly.

The following are the polymerizable ones used in the present invention water-soluble high molecular weight compounds that can entrap Protaminobacter rubrum, described in more detail.

By using the higher molecular weight used according to the invention, curable, water-soluble, preferably containing two or more unsaturated groups Connections can be used for better control and reproducibility of permeabilities because these properties are essentially due to the higher molecular weight, result in water-soluble compounds. In addition, through the use of the Compounds according to the invention do not pose a toxicity problem. Low molecular weight toxic Impurities can also be removed prior to mixing with the biological material will.

Furthermore, it has been found that the compounds are readily soluble in water initially in a certain amount of water, buffer solution, saline solution and the like can be dissolved, making this polymer solution of the aqueous solution or dispersion of the biological material with regard to certain properties such as pH value, salt concentration, Viscosity, among other things, can be adjusted. Then both solutions or the aqueous polymer solution and the cell dispersion mixed and polymerized. This Process has particular advantages for sensitive biocatalysts, which thereby are not exposed to abrupt changes in the surrounding medium, which results in a Loss of activity in the preparation of the polymerizable mixture largely is avoided.

Another advantage of the present invention is that of being good Water solubility associated with the large absorption capacity of the polymer for aqueous Solutions and suspensions. They are weight ratios of aqueous biological Material to solid polymer between 1: 1 and 30: 1 possible, are particularly suitable Weight ratios between 4: 1 and 20: 1.

Due to the possible large water content of the immobilized biological Materials before and after the polymerization, the biomaterials are in one particularly the appropriate, gentle environment, which increases the activity can be kept stable and thereby also the substrate and product flow are favored. By varying the structure and the molecular weight of the polymerizable, Highly water-soluble, higher molecular weight compounds are the properties of the polymer Netzwerkkes can be varied to a large extent. This allows the network density, optimize the permeability, the swelling behavior and other properties in order to achieve the To meet the requirements of the biological material.

Water-soluble, higher molecular weight, polymerizable ones were used for immobilization Compounds that have two or more polymerizable functional groups per molecule and have a molecular weight in excess of 400, particularly with molecular weights from 1000 to 20,000, especially compounds with ethylenically unsaturated groups, used.

According to their structure, they can be described as connections that from the reaction of higher molecular weight, hydrophilic compounds with compounds, the polymerizable ethylenically unsaturated groups are formed. Both Reaction partners must contain functional groups that allow them to be linked chemically with or with one another or with the help of di- or polyfunctional ones To link reagents with or with one another.

For example, such high molecular weight, hydrophilic compounds Polyethylene glycols, ethylene oxide-propylene oxide blocks and copolymers, alkoxylated, especially ethoxylated di- or polyhydric alcohols as well as readily water-soluble Polymers, e.g. B. wholly or partially saponified polyvinyl acetates, readily water-soluble Polycondensates such as B. polyester made from the above higher molecular weight, hydrophilic Connections with Di or. Polycarboxylic acids and highly water-soluble polyaddition compounds such as B. polyether polyurethanes made from the above higher molecular weight, hydrophilic Compounds with di- or polyisocyanates.

Furthermore, those in the above-mentioned higher molecular weight, hydrophilic Compounds containing hydroxyl groups in whole or in part by conventional methods, such as B. Reaction with ammonia, be converted into amino groups.

As compounds with polymerizable, ethylenically unsaturated Groups are e.g. B. suitable: unsaturated carboxylic acids, acid chlorides, dicarboxylic acids, Acid anhydrides or functional derivatives thereof.

Acrylic acid, methacrylic acid, crotonic acid, acrylic acid chloride are preferred, Methacrylic acid chloride, itaconic acid, fumaric acid, maleic acid, maleic anhydride, Itaconic anhydride, glycidyl acrylate, glycidyl methacrylate, hydroxyethyl acrylate, Hydroxypropyl acrylate, hydroxyethyl methacrylate, hydroxypropyl methacrylate, isocyanatoethyl acrylate, Isocyanatoethyl methacrylate, 4-isocyanato-3-methyl-but-2-ylacrylate.

Linking the higher molecular weight, hydrophilic compounds with the polymerizable, ethylenically unsaturated compounds are therefore very different Nature, for example, results in ester, ether, urethane, amide, amine and urea bonds. The linkage can be established by processes known from the literature, d. H. for example by reacting the hydroxyl or amino groups with acids, Acid halides or acid anhydrides to the corresponding esters or amides, with epoxides to the corresponding ethers or amines or with isocyanates to the corresponding urethanes or ureas.

Furthermore, in addition to the direct linkage of the higher molecular weight, hydrophilic Compounds with the compounds containing the ethylenically unsaturated groups, the linkage can also be carried out by means of bifunctional or multifunctional reagents.

Examples of such bi- or polyfunctional reagents are di- or multifunctional isocyanates such as isophorone diisocyanate, tolylene diisocyanate, hexamethylene diisocyanate, Polyisocyanates containing biuret groups (e.g. Desmodur N, reaction product of hexamethylene diisocyanate with water), polyisocyanates resulting from the reaction of diisocyanates with polyvalent Alcohols are formed (e.g.

B. Desmodur L, reaction product of toluene diisocyanate with trimethylolpropane) as well as di- or polyepoxides such. B. bisphenol A digylcidyl ether or hexahydrophthalic acid diglycidyl ester. The linking of these reagents takes place, for. B. with the hydroxyl groups the higher molecular weight, hydrophilic compounds and compounds containing hydroxyl groups, which contain polymerizable, ethylenically unsaturated groups, among urethane or ether formation.

For the preparation of the compounds according to the invention, the various Linkage principles can also be combined with one another.

Type and proportion of the hydrophilic, higher molecular weight compounds, the Compounds which carry the ethylenically unsaturated groups and, if appropriate However, the di- or polyfunctional reagents must be chosen so that the higher molecular weight polymerizable compounds according to the invention produced therefrom are so hydrophilic that good water solubility and large absorption capacity for aqueous solutions or dispersions of biological material is given.

As preferred water-soluble, higher molecular weight, polymerizable compounds may be mentioned: - Compounds made from polyether polyols, some of which are hydroxyl groups Esterified with unsaturated carboxylic acids and partly with isocyanate groups Derivatives of unsaturated carboxylic acids are implemented, are produced.

Particularly preferred are: - Compounds derived from polyethylene glycols with a molecular weight greater than 400, their hydroxyl groups partially Esterified with acrylic acid or methacrylic acid and partly with isocyanatoethyl methacrylate or 4-isocyanato-3-methyl-but-2-ylacrylate are reacted.

The hydroxyl groups of the polyether polyols initially partially with the isocyanate group-containing derivatives of unsaturated carboxylic acids are reacted and then to the other part with the unsaturated carboxylic acids are esterified, but it is preferred first to partially react with the unsaturated Carboxylic acids and then the conversion of the other part of the hydroxyl groups with the isocyanate group-containing derivatives of unsaturated carboxylic acids.

Also preferred are water-soluble, higher molecular weight, polymerizable Compounds called: - Compounds made from polyether polyols, their hydroxyl groups partly esterified with unsaturated carboxylic acids and partly with di- or polyfunctional isocyanates are reacted, are produced.

Particularly preferred are: - Compounds from Polyethylene glycols with a molecular weight greater than 400, their hydroxyl groups partly esterified with acrylic acid or methacrylic acid and partly with Isophorone diisocyanate, tolylene diisocyanate, polyisocyanates containing biuret groups or polyisocyanates resulting from the reaction of diisocyanates with polyhydric alcohols arise, are produced.

The hydroxyl groups of the polyether polyols are initially partially reacted with the di- or polyfunctional isocyanates and then esterified to the other part with unsaturated carboxylic acids.

However, it is more advantageous to first carry out the partial esterification and then carry out the reaction with the di- or polyfunctional isocyanates.

The polymerization can be carried out under an inert gas and in the presence common free radical initiators such as azo-bis (isobutyronitrile), t-butyl peroctoate, benzoyl peroxide, Dicyclohexyl peroxydicarbonate, methyl ethyl ketone peroxide, cumene hydroperoxide, acetyl cyclohexanesulfonyl peroxide, Dicumyl peroxide, potassium peroxodisulfate or ammonium peroxodisulfate as well as redox systems such as potassium peroxodisulfate-riboflavin, potassium peroxodisulfate-sodium bisulfite, hydrogen peroxide compounds of divalent iron. Numerous accelerators can also be used Compounds serve, e.g., N, N, N ', N'-tetramethylethylenediamine or 13-dimethylaminopropionitrile.

Another possibility for polymerization is irradiation the actinic light mixture.

For example, high-pressure mercury lamps, low-pressure mercury lamps, Fluorescent lamps, xenon lamps, carbon arcs and solar radiation. An irradiation with electron beams or gamma rays is also possible, but must be with some damage to the biological material expected will. Furthermore, the photopolymerization can be accelerated by photosensitizers will.

Known photosensitizers such as OC carbonyl alcohols, e.g. benzoin or acetoin, acyloin ethers such as benzoin methyl ether, benzoin ethyl ether, Benzoin isopropyl ether, OG-substituted acyloins such as ob -methylbenzoin and Oc-methoxybenzoin i.a. as well as ionic groups such as carboxylic acid, sulfonic acid or amino groups modified and thereby water-solubilized derivatives are used.

Furthermore, polycyclic aromatic compounds such as naphthol and hydroxyanthracene, azoamides such as 2-cyano-2-butylazoformamide as well Metal salts such as uranyl nitrate and iron chloride, as well as mercaptans, disulfides, halides or dyes can be used.

The immobilized biomaterial can through during the polymerization Shape can take on almost any shape. There are foils, films, molded parts, etc. representable. Furthermore, the mechanical properties can be improved by incorporating supporting fabrics be modified.

Likewise are coatings of electrodes, membranes, or others Materials feasible.

Furthermore, the polymerization in the form of a dispersion of the not yet polymerized biological material in an inert liquid medium such as aliphatic, cycloaliphatic and aromatic hydrocarbons, as well as halogenated derivatives of the same, des- further in ethers, Esters or other non-water-miscible substances as well as in silicone oils and Paraffin oils, or mixtures of these with one another, are carried out, depending on the distribution, which is achieved by stirring, introducing inert gas, e.g. nitrogen, or other metering in of the aqueous phase, e.g. by pumping in or spraying into the inert phase can be achieved, the size of the pearls very much is variable. Furthermore, through additives to the water or inert phase, their viscosity, Density, surface tension, etc. can be changed, which also changes the pearl diameter influenced.

Due to the high capacity for water - particularly suitable weight ratios of aqueous solution or suspension of the biological material to solid polymer between 4: 1 and 20: 1 - which the immobilized material before and after the polymerization, there are also advantages in terms of economy of the procedure. As a result, on the one hand, large amounts of aqueous solutions or Dispersions of the biological material in a relatively small amount of the polymerizable Compounds are included, on the other hand it can fermenter broths immobilized directly or after concentration, e.g. by microfiltration or centrifugation without further components of the fermentation medium having to be removed.

Furthermore, before and after the immobilization, it is also possible to use smaller amounts of the aqueous solutions, as well as part of the proportion to replace water with other liquids such as alcohol.

It is also possible to use the immobilized Protaminobacter rubrum cells in other than aqueous solutions, e.g. in aliphatic or aromatic hydrocarbons, to use.

The Protaminobacter rubrum immobilized by the above process Cells can be used as biocatalysts for biotransformations.

Examples Fermentation of Protaminobacter rubrum For the production of Sucrose Mutase, the Protaminobacter rubrum strain (CBS 574.77) was used.

The nutrient solution consists of 5% syrup, 2% corn steep water and 0.05 % (NH4) 2 HPO4, the pH value is 7.1. With 1 ml of a Protaminobacter rubrum wash-off 200 ml of nutrient solution were inoculated in a 1 1 Erlenmeyer flask. The fermentation runs for 15 hours at 310C on the rotary shaker.

This preculture was used to create a 300 l fermenter with 200 l of the above nutrient solution inoculated and fermented for 16 h.

The sucrose mutase activity of the fermenter solution was 9.1 U / ml. 1 U = 1 pmol sucrose / min converted.

The 200 l fermenter solution were by continuous centrifugation concentrated by a factor of about 10, and this concentrated Protaminobacter cell suspension used directly for subsequent immobilization experiments on a pilot scale.

Example A (see Fig. 1) 2189 g of polyethylene glycol with a medium Molecular weight of 1550 (Chemische Werke Hüls) were taken with 98.4 g of acrylic acid Addition of 22.8 g of p-toluenesulfonic acid, 2.3 g of di-tert-butylhydroquinone and 2.3 g of p-methoxyphenol esterified in toluene.

1100 g of the ester were added with 74.2 g of isophorone diisocyanate implemented by 0.15 g Desmorapid SO.

100 g of this polymerizable acrylate resin thus obtained was mixed with 1 g Irgacure (Ciba-Geigy) and mixed with 40 g (0.01 M) phosphate buffer pH 7 added so that a solution was formed.

This solution was concentrated with 348 g of the microfiltration Fermenter broth of Protaminobacter rubrum mixed and as 500 pm thick films (format 40 cm x 60 cm) polymerized using four high-pressure mercury lamps.

The films were then cut into small pieces (approx 0.5 cm2) and placed in a 1 1 column that can be thermostatically controlled. The bioreactor was at 300C with a 50% sucrose solution after sterile filter (Plillistak, Millipore) flows through. After setting a flow rate of about 130 ml / h, a sucrose conversion was established achieved by about 70-80%. The product solution was analyzed by HPLC. After 40 days there was still no reduction in activity.

1 shows the continuous conversion of sucrose into Palatinose with enclosed cells of Protaminobacter rubrum: 1 L reactor volume, 50% sucrose, 300C.

Flow rate / ml / h 7% converted sucrose; Palatinose / kg / d / example B (see FIG. 2) The mixture containing Protaminobacter rubrum cells from Example A (175 g) was put into pieces of polyamide fabric (Monodur 250 N, Verseidag industrial textiles GmbH) of the format 40 cm x 60 cm and polymerized by exposure. The reinforced films produced in this way were combined with uncoated polyamide fabric wound so that there is a layer of the uncoated between each 2 layers of the film Tissue. The cylindrically wound material was placed in a 1 liter column pushed in, the column inside diameter was chosen so that the material filled the whole column diameter, and at 300C with a 50% sucrose solution, pH 6.5, flowed through. The sucrose stock solution was filtered through a Milli-Stak sterile filter (Millipore) pumped through the reactor column by peristaltic pump. After setting a flow rate of about 60 ml / h, a constant sucrose conversion of about 75% achieved. The product solution was analyzed by HPLC and contained 68.5% isomaltulose 4.5% 1,1'-disaccharide, 1.2% fructose, 0.8% glucose.

The sucrose mutase activity of the immobilized Protaminobacter cells was stable for 50 days.

In Fig. 2 is the continuous conversion of sucrose to palatinose shown with enclosed Protaminobacter rubrum cells.

1 L reactor volume, 50% sucrose, 300C.

Flow rate / ml / h 7% converted sucrose; Palatinose / kg / d / example C 1000 g polyethylene glycol with an average molecular weight of 1000 (chemical Werke Hüls) was treated with 72 g of acrylic acid with the addition of 9.2 g of p-toluenesulfonic acid and 1.0 g of di-tert-butylhydroquinone and 1.0 g of p-methoxyphenol esterified in toluene.

750 g of the ester thus obtained was mixed with 83.3 g of isophorone diisocyanate with the addition of 0.09 g Desmorapid So implemented.

From 500 g of the polymerizable acrylate resin thus obtained, 5 g of Irgacure (Ciba-Geigy), 100 g phosphate buffer, 0.01 M, pH 7, and 2750 g concentrated fermenter broth small pieces of film were produced analogously to Example A, with which a 5 1 column was filled.

This bio-rector was at 300C after connecting a Millistak sterile filter (Millipore) with 50% sucrose solution, pH 7.0, flowing through. After hiring a With a flow rate of about 500 ml / h, a sucrose conversion between 78.7 and 80.0% measured.

A product distribution of, for example, 21.25 was obtained by HPLC % Sucrose, 69.91 isomaltulose, 5.81% 1,1'-disaccharide, 1.84% fructose, 1.19% Glucose. There was no reduction in the activity of the biocatalyst over 40 days established.

Example D 1658 g of the ester prepared in Example A were with 182 g of isocyanatoethyl methacrylate (DOW Chemicals) with the addition of 0.36 g of Desmorapid SO implemented.

700 g of this polymerizable acrylate resin thus obtained was with 210 g phosphate buffer, 0.01 M, pH 7.0 and 35 g 1,2-diphenyl-2-hydroxy-3- / Ñ (N-methyl) -pyrrolidinium-7-propan-1-one-methylsulfate mixed. 4.9 kg of that concentrated by microfiltration were added to this solution Fermenter broth, which contains Protaminobacter robrum cells, is added and this mixture in 32.2 kg of a paraffin oil-silicone oil mixture with a spec.

³ density of 0.9 g / cm dripped. After adding 5 g of emulsifier (Span 80) was broken up by vigorous stirring and by introducing nitrogen the above cell-retaining mixture in pearls, which is achieved by means of 4 high-pressure mercury lamps and polymerized for 1 1/2 hours of irradiation.

The obtained beads (mean diameter 1 mm) were in a 10 1 column filled and at 300C with a 50% sucrose solution after sterile filtration flows through Millistak filters (Millipore). The pH was between 6.4 and 6.6 are set. After a short time, the flow rate was relatively constant of about 2.8 l / h is achieved and the product solution is analyzed by HPLC.

A typical range of products looked like this: 2.53% fructose, 1.86% glucose, 15.11% sucrose, 74.42% isomaltulose, 6.08% 1,1'-disaccharide. No tri-, tetra- or other oligosaccharides were detectable.

Example E 1200 g of the ester from Example A were mixed with 63.3 g of Desmodur N and 38.3 g of isophorone diisocyanate reacted with the addition of 0.2 g of Desmorapid SO.

From 800 g of this polymerizable acrylate resin obtained in this way, 40 g Irgacure (Ciba-Geigy), 320 g phosphate buffer, 0.01 M, pH 7.0, and 4.0 kg Protaminobacter Cell suspension, concentrated by a factor of 10, and 30 kg of silicone oil with one density of 0.95 g / cm3, beads (mean diameter 1.5 mm) were produced analogously to Example D and filled into a 10 1 column.

The 10 l bioreactor filled with immobilized Protaminobacter rubrum was perfused by a sterile-filtered 50% sucrose solution at 300C and after Setting a flow rate of about 3.05 l / h operated continuously. The pH was regulated to 6.4-6.6.

The reactor was operated for 2 months without any significant loss of activity operated. For a typical analysis example, which was determined by HPLC: 31.1 % Sucrose, 60.7% isomaltulose, 1.7% fructose, 1.4% glucose, 5.1% 1,1'-disaccharide, no oligosaccharides.

The flow rate of the bioreactor was now reduced to about 1.75 l / h and the following product spectrum determined: 4.3% fructose, 3.9% glucose, 0.9% sucrose, 81.7% isomaltulose, 9.2% 1,1'-disaccharide, no oligosaccharides.

Example F 55.5 g of the polymerizable used in Example A Resin were mixed with 12 g of phosphate buffer (0.01 M, pH 7.0), so that a solution originated.

This solution was mixed with 500 g of the cross-flow microfiltration concentrated fermenter broth of Protaminobacter rubrum mixed and 1.1 g ammonium peroxodisulfate dissolved. After adding 1.1 g of N, N, N ', N', - tetramethylethylenediamine the mixture was stirred vigorously and poured into a bowl so that the layer thickness was about 3 mm. The polymerization started within a few minutes. That polymerized product was cut into small pieces of 5 x 5 x 3 mm in size and packed in a 1 L column.

In continuous operation at 300C, 50% sucrose solution, one turned up A flow rate of 225 ml / h results in a conversion of 58% of the sucrose offered. After 8 days there was still no decrease in productivity.

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Claims (18)

Claims 1. A method for immobilizing Protaminobacter rubrum by inclusion in polymerized compounds, characterized in that aqueous solutions or dispersions of the biological material with an aqueous one Solution of highly water-soluble, higher molecular weight polymerizable compounds that two or more polymerizable functional groups per molecule and one molecular weight own over 400, mixed and then polymerized. 2. The method according to claim 1, characterized in that a radical Polymerization initiator and / or a radical polymerization initiator system and / or an accelerator is added. 3. The method according to claim 2, characterized in that a water-soluble radical polymerization initiator, in particular ammonium peroxodisulfate and a water-soluble accelerator such as N, N, N ', N'-tetramethylethylenediamine was added will. 4. The method according to claim 1, characterized in that a photosensitizer is added and the polymerization takes place by irradiation. 5. The method according to any one of the preceding claims, characterized in, that the weight ratio of the aqueous solution or dispersion of Protaminobacter rubrum to the water-soluble poly- merizable higher molecular weight Compound or their aqueous solutions between 1: 1 to 30: 1, in particular between 4: 1 to 20: 1. 6. The method according to any one of the preceding claims, characterized in that that the readily water-soluble, higher molecular weight, polymerizable compounds Polyether polyols, the hydroxyl groups of which are partially with unsaturated carboxylic acids esterified and partly unsaturated with derivatives containing isocyanate groups Carboxylic acids are implemented, are produced. 7. The method according to claim 6, characterized in that a) the polyether polyols Are polyethylene glycols with a MW of 400 and greater, b) the unsaturated carboxylic acids Acrylic acid and / or methacrylic acid are, c) the derivatives containing isocyanate groups unsaturated carboxylic acids isocyanatoethyl acrylate, isocyanatoethyl methacrylate and / or Are 4-isocyanato-3-methyl-but-2-ylacrylate. 8. The method according to claim 1 to 5, characterized in that the highly water-soluble, higher molecular weight, polymerizable compounds made from polyether polyols, whose hydroxyl groups are partially esterified with unsaturated carboxylic acids and for other part are reacted with di- or polyfunctional isocyanates, produced are. 9. The method according to claim 8, characterized in that a) the polyether polyols Are polyethylene glycols with a MW of 400 and greater, b) the unsaturated carboxylic acids Acrylic acid and / or methacrylic acid are, c) the di- or polyfunctional isocyanates Isophorone diisocyanate, tolylene diisocyanate, hexamethylene diisocyanate, biuret group-containing Polyisocyanates and / or polyisocyanates resulting from the reaction of diisocyanates are formed with polyhydric alcohols. 10. The method according to the preceding claims, characterized in that that for immobilization directly fermentation broths, optionally after the preceding one Concentration, to be used without removing any constituents of the fermentation medium. 11. The method according to any one of the preceding claims, characterized in, that cells or enzymes in the presence of disinfectants, bactericides or Fungicides are immobilized, thus chemically sterilized and transferred to the bioreactor and the sterilization agents are removed from the closed, sterile reactor system are washed out. 12. The method according to any one of the preceding claims, characterized in, that a cell suspension of Protaminobacter rubrum, preferably those with a Solids content from 5% to 25%, with an aqueous solution of the highly water-soluble, higher molecular weight, polymerizable compound is mixed and polymerized. 13. The method according to claim 12, characterized in that the weight ratio from aqueous cell suspension to solid polymer from 1: 1 to 30: 1, preferably from 4: 1 up to 20: 1. 14. The method according to any one of the preceding claims, characterized in, that the Protaminobacter rubrum containing aqueous solution of the readily water-soluble, higher molecular weight, polymerizable compound in a second, non-aqueous inert phase dispersed into pearls and this pearl-shaped water phase is polymerized. 15. The method according to any one of the preceding claims, characterized in that that - the weight ratio of the water phase to the inert phase is between 1: 1 and 1:20, - The division of the aqueous phase into pearls by stirring and / or passing through of inert gas, or by spraying or pumping the aqueous phase into the inert phase takes place, pearls with an average diameter of 0.05 mm to 5 mm, preferably 0.2 mm to 2 mm, especially preferably 0.5 mm to 1.5 mm accordingly produced the inert phase is a substance that is difficult to mix or immiscible with water, such as aliphatic, cycloaliphatic or aromatic hydrocarbons, their derivatives as well as silicone oils and paraffin oils or mixtures of these with one another. 16. Immobilized Protaminobacter rubrum cells obtainable after a of the preceding claims. 17. Use of the immobilized Protaminobacter rubrum cells after one of the preceding claims on biotransformations. 18. Use of the prepared according to the preceding claims immobilized material containing Protaminobacter rubrum cells for conversion from sucrose to isomaltulose.