DE3416142A1 - Process for the production of immobilised biological material in bead form - Google Patents

Abstract

The invention relates to a process for the production of immobilised biological material by entrapment in polymerised compounds in bead form, to the immobilised biological material obtainable by this process, and the use thereof for biotransformations. The production process comprises dispersing an aqueous phase which contains both the biological material and high molecular weight polymerisable compounds in a second, non-aqueous inert phase to give beads, and polymerising this aqueous phase in the form of beads.

Description

Process for the production of immobilized biological

Chemical material in pearl form The invention relates to methods for Manufacture of immobilized biological material by inclusion in polymerized Compounds in bead form, the immobilized obtained by this method biological material and its use for biotransformation.

The immobilization of biocatalysts usually leads to process engineering and economic benefits. It can be chemical and physical properties, sometimes the selectivity and specificity of the biocatalyst as a result of the immobilization modify.

The methods of immobilization can be broadly divided into the following Allocate groups - adsorption on prefabricated carriers - covalent bonding to carriers - Cross-linking - inclusion in a polymer matrix.

The containment method relies on the biomaterial in terms of its range of motion during the polymerization in a network more or less uniform mesh and pore size is included. This polymer network should be impassable for the biocatalyst, while the network is the substrate and the product of a reaction carried out on this catalyst practically none Should offer resistance.

Conventional processes use low molecular weight, hydrophilic monomers to build up the network, such as hydroxyethyl acrylate, hydroxyethyl methacrylate, Hydroxypropyl acrylate, hydroxypropyl methacrylate, acrylamide, and others, those with the aqueous Solution or dispersion of the biomaterial mixed and then polymerized will.

For example, in US Pat. No. 3,788,950, monofunctional acrylic acid derivatives are disclosed such as acrylamide with a small amount of crosslinking agent such as N, N-methylenebisacrylamide used.

For example, US Pat. No. 3,859,169 discloses monoesters of acrylic acid with glycols used in conjunction with a crosslinker and a soluble polymer. With all However, these processes can only be used by external shaping of materials such as films, Films, castings, etc. are obtained. There is often a need to use these materials Subsequent processing is necessary, which, however, is the case for catalytic converters favorable spherical shape can hardly be achieved.

U.S. Patent 3,860,490 mentions the possibility of making pearls by suspension polymerization in an inert medium, however, the process is disadvantageous due to the use of low molecular weight hydroxy-alkyl acrylates and methacrylates, because - as with all of the methods mentioned above - difficulties in the control and reproducibility of the permeabilities for the biological material as well as for whose substrates and products occur. For suitability as an immobilization material However, these sizes are essential. In addition, the use of low molecular weight Acrylic acid derivatives disadvantageous because of their toxicity. It can be used during polymerization damage to the biocatalyst or those in the polymer Residual monomers limit the use of the immobilized biocatalyst for production of products for the food industry or for the pharmaceutical industry strong one.

There has now been a method of making immobilized biological Material found by inclusion in polymerized compounds, which is characterized is that an aqueous solution or dispersion of a biological material with a aqueous solution of highly water-soluble, high molecular weight polymerizable compounds, the two or more polymerizable functional groups per molecule and a molecular weight of over 400, is mixed and this mixture in an inert liquid Medium is divided into pearls and polymerized. As an inert phase, in particular Substances that cannot be mixed with water such as

aliphatic, cycloaliphatic and aromatic hydrocarbons how e.g. hexane, cyclohexane, toluene and also halogenated derivatives thereof, furthermore Alcohol, ethers, esters or other non-water-miscible substances as well as silicone oils and paraffin oils are used, or

also mixtures of these with one another.

By using the higher molecular weight, two or more unsaturated Group-containing, curable water-soluble compounds can be better Control and reproducibility of the permeabilities achieve, as these properties essentially result from the higher molecular weight water-soluble compounds.

It also consists of the use of higher molecular weight polymerizable Compounds no toxicity problem. Low molecular weight toxic impurities can also be removed prior to mixing with the biological material.

The readily water-soluble compounds according to the invention can initially dissolved in a certain amount of water, buffer solution, saline solution and the like be, making the 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. Particularly sensitive biocatalysts suffer thereby no abrupt change in the surrounding medium and it can in this way Loss of activity can be avoided.

Another advantage is that it is readily soluble in water large absorption capacity of the polymer for aqueous solutions and Suspensions. There are weight ratios of aqueous biological material too solid polymer between 1: 1 and 30: 1 possible and advantageous, particularly suitable are ratios between 4: 1 and 20: 1.

The pearl shape obtained directly by this process has a special one Advantages of using the immobilized biomaterial. On the one hand, it is suitable The spherical shape is particularly suitable for filling column reactors, with good flow is guaranteed, blockages can be avoided and at the same time through the large surface a good reaction speed for the conversion on the biocatalyst results.

Even in flowing systems, the spherical shape is due to the hydrodynamic Properties particularly suitable.

Furthermore, the present method represents a simplification and improvement of the previous procedures.

The spherical shape is obtained directly during the polymerization and is easily controllable by the process conditions.

For example, the polymerization can be 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, functional derivatives of the same or also silicone oils, paraffin oils be, depending on the distribution, by stirring, introducing Inert gas, e.g. nitrogen, or other Adding the aqueous Phase, e.g. by pumping or spraying into the inert phase can, the size of the pearls is very variable. Furthermore, additives to the water or inert phase whose viscosity, density, surface tension etc. are changed, which also affects the pearl diameter.

In this way, the mean bead diameters are from 0.05 mm to 5 mm possible, with pearls having a larger diameter, e.g. larger than 2 mm, the desired Oppose the reaction with a high diffusion resistance. Are particularly advantageous Pearls with an average diameter of 0.2 mm to 2 mm.

In addition to the simplicity of production of the pearls, there are advantages for the immobilization in terms of handling under sterile conditions of the Biomaterials as the process from mixing to the finished immobilized material can be carried out in a closed system. The immobilized biomaterial can then easily be recovered by filtration. However, it is also possible to cool the entire mixture under OOC to allow the material to last longer to be able to store. The cooling can also take place above the freezing point of the external medium can also be carried out, the freezing point of which is also due to additives or the addition of other substances can be reduced.

Water-soluble, higher molecular weight, polymerizable ones were used for immobilization Connections that are two or more polymerizable functional Groups per molecule and a molecular weight of over 400, especially between 1,000 and 10,000, especially compounds with ethylenically unsaturated Groups, used.

Preferred are those modified with unsaturated carboxylic acids Reaction products of polyether polyols, particularly preferably polyethylene glycols, with di- or polyfunctional isocyanates, particularly preferably isophorone diisocyanate, Tolylene diisocyanate, Desmodur N or Desmodur L.

- Reaction products of polyether polyols, preferably polyethylene glycols, with unsaturated carboxylic acids, preferably acrylic acid, methacrylic acid, itaconic acid, and / or with derivatives containing isocyanate groups, preferably isocyanatoethyl methacrylate, Isocyanatoethyl acrylate and reaction products of urethane-modified, unsaturated Carboxylic acids with polyether diols.

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 or dicumyl peroxide as well as redox systems such as potassium peroxodisulfate riboflavin, Potassium peroxodisulfate-sodium bisulfite, hydrogen peroxide compounds of the bivalent Made of iron. Numerous compounds can also serve as accelerators, e.g. N, N, N ', N' -tetramethylethylenediamine.

Another possibility for polymerization is irradiation the actinic light mixture. For example, high-pressure mercury lamps are suitable, Low pressure mercury lamps, fluorescent lamps, xenon lamps, charcoal arcs as well Exposure to sunlight.

Irradiation with electron beams or gamma rays is also possible possible, but a certain damage to the biological material must be expected will. Furthermore, the photopolymerization can be accelerated by photosensitizers will. Known photosensitizers such as carbonyl alcohols, e.g. Benzoin or acetoin, acyloin ethers such as benzoin methyl ether, benzoin ethyl ether, benzoin isopropyl ether, o -substituted acyloins such as -methylbenzoin and i -methoxybenzoin and others as well as through modified ionic groups such as carboxylic acid, sulfonic acid or amino groups 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 and dyes can be used.

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 Procedure. On the one hand, this allows 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 will.

Furthermore, before and after the immobilization, it is also possible to use smaller amounts of water or aqueous solutions, as well as a part to replace the proportion of water with other liquids such as alcohols. It is also possible to use the immobilized biological materials in other than aqueous solutions, e.g. in aliphatic or aromatic hydrocarbons, to use.

The biological materials immobilized according to the above procedure, in particular cells or enzymes can be used as biocatalysts for biotransformations insert.

As interesting microorganisms for the immobilization according to the invention The following are possible: Aspergillus niger, Gluconobacter suboxydans, Gluconobacter oxydans, Escherichia coli, Saccharomyces cerevisiae, Protaminobacter rubrum Serratia plymuthica, Pseudomonas putida, Cunninghamella elegans, Olostridia such as Clostridium thermoaceticum, Clostridium kluyveri, Clostridium butyricum, Clostridium sporogenes, Bacillus licheniformis, Streptomyces olivaceus.

Examples Example A Immobilization of alcohol dehydrogenase 48.7 g polyethylene glycol with an average molecular weight of 800 (manufactured by the chemical works Hüls) was with 4.7 g of acrylic acid with the addition of 0.54 g of p-toluenesulfonic acid and 50 mg di-tert. -butylhydroquinone and 50 mg p-methoxyphenol esterified in toluene.

25.0 g of this ester were prepared with 3.9 g of isocyanatoethyl methacrylate ( from Dow Chemicals) with the addition of 4 mg Desmorapid S0 (Rheinchemie Rheinau GmbH) implemented.

1 g of the polymerizable resin thus obtained was 50 mg of 1 , 2-Diphenyl-2-hydroxy-3N (N-methyl) pyrollidinium7-propan-1-one-methylsulfate mixed and 15 g of phosphate buffer (0.01 M, pH 7) are added. In this solution was 200 mg alcohol dehydrogenase (from yeast, Boehringer, Mannheim, containing 120 mg enzyme protein, 400 U / mg) and the solution in 100 g of a mixture of silicone oil / paraffin oil with a spec. Density of 0.9 g / cm3 dripped, with vigorous stirring and bubbling with nitrogen formed beads, which were formed by irradiation for 15 minutes polymerized using a high pressure mercury lamp.

1.0 g of the beads thus obtained were in 50 ml Erlenmeyer flasks with 10 ml 0.1 M potassium phosphate pH 8.5, 10 mM NADH, 1% acetaldehyde on the rotary shaker, 230 rpm, incubated at 300C.

After 6 h, about 50% of the NADH used, 50 FMol, was oxidized to NAD, the initial speed was 0.23 mol / min.

Example B Immobilization of L-lactate dehydrogenase 25.0 g of the prepared Esters from Example A were made with 2.3 g of Desmodur N (BAYER AG Leverkusen) and 1.4 g isophorone diisocyanate with the addition of 7.5 mg desmorapid (Rheinchemie Rheinau GmbH) implemented.

Analogously to Example A, 0.6 mg L-lactate dehydrogenase (from pig muscle, Boehringer, Mannheim, containing 2 mg enzyme protein, approx. 550 U / mg) instead of the Alcohol dehydrogenase immobilized in pearl form.

The pearls obtained were in a 100 ml Erlenmeyer flask with 20 ml of 0.1 M potassium phosphate buffer, pH 8.0, 10 mM NADH, 0.05 M pyruvate sodium salt 300C, 200 rpm, incubated.

After dilution, taken samples were photometrically for the NADH decrease tested against a blank sample.

After 3 hours, the entire amount of NADH was oxidized to NAD.

The initial speed was 1.4 mol / min.

Example C Immobilization of Baker's Yeast 100.0 g of polyethylene glycol with an average molecular weight of 1000 (Chemische Werke Hüls) was 7.2 g of acrylic acid with the addition of 0.92 g of p-toluenesulfonic acid and 0.1 g of di-tert.-butylhydroquinone and 0.1 g of p-methoxyphenol esterified in toluene.

75.0 g of the ester thus obtained was mixed with 8.3 g of isophorone diisocyanate implemented with the addition of 9 mg Desmorapid S0.

5.0 g of the polymerizable resin was mixed with 0.1 g of phenylglyoxylic acid and 1.0 g of buffer solution (0.01 M, pH 7) mixed. A mixture became this solution from 13.7 g of baker's yeast (25% solids content) and 13.7 g of pH 7 buffer solution. The mixture thus obtained was then poured into 200 ml of a mixture of paraffin oil and silicone oil with a spec. Density of 0.9 g / cm3 while stirring and passing through divided into pearls by nitrogen and polymerized using a high-pressure mercury lamp.

15 g of these pearls were in a 200 ml Erlenmeyer flask with 50 ml 0.01 M potassium phosphate, pH 8.0, 1 mM NADH (grade II, Boehringer, Mannheim) incubated at 25 "C on a rotary shaker at 230 rpm.

The NADH oxidase activity of the immobilized yeast cells was over the decrease in the NADH content photometrically against a blank sample without yeast cells certainly.

15 g of immobilized preparation had an activity of 0.24 U (1 U = 1 µmole NADH / min. oxidized).

This reaction is used for NAD regeneration in coupled enzyme systems applicable.

Example D Immobilization of Baker's Yeast 218.9 g of polyethylene glycol with an average molecular weight of 1550 (Chemische Werke Hüls) were with 9.8 g of acrylic acid with the addition of 2.3 g of p-toluenesulfonic acid, 0.23 g of di-tert-butylhydroquinone and 0.23 g of p-methoxyphenol esterified in toluene.

165.8 g of the ester thus obtained were mixed with 18.2 g of isocyanatoethyl methacrylate (Dow Chemicals) implemented with the addition of 36 mg Desmorapid SO.

9.0 g of this polymerizable resin was mixed with 90 mg of phenylglyoxylic acid mixed and mixed with 2.70 g of phosphate buffer (0.01 M, pH 7).

This solution thus obtained became a mixture of 20.0 g of baker's yeast and 20.0 g of phosphate buffer (0.01 M, pH 7) are added and pearls are made from them analogously to Example C manufactured.

15 g of moist pearls were placed in a 200 ml Erlenmeyer flask with 50 ml of 0.01 M potassium phosphate pH 8.0, 1 mM NADH (Boehringer, Mannheim) at 250C a rotary shaker, 230 rpm, incubated. Based on the photometric NADH determination 15 g of pearls had an activity of 0.14 U.

Example E Immobilization of Protaminobacter rubrum for production the strain Protaminobacter rubrum (CBS 574.77) is used by sucrose mutase.

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 wash-off are placed in a 1 1 Erlenmeyer flask Inoculated 200 ml of nutrient solution.

The fermentation runs for 15 hours at 310C on the rotary shaker. With this preculture, 20 liters of the above nutrient solution are inoculated in a 30 l fermenter and fermented for 16 h at 310C. The fermentation broth is made by microfiltration concentrated to a solids content of 5%.

10 g of the polymerizable acrylate resin obtained in Example D. were mixed with 3.0 g of phosphate buffer 0.01 M pH 7 and 500 mg of Irgacure (Ciba-Geigy). To this solution, 70 g of a fermenter broth concentrated by microfiltration, which contains Protaminobacter rubrum cells and this mixture in 460 g of one Silicone oils with a spec.

Density of 0.95 g / cm3 dripped. Through intensive stirring and through Nitrogen introduction became a division of the above cell-containing mixture achieved in pearls using 2 high pressure mercury lamps and 1/2 hour irradiation have been polymerized.

6.0 g of the pearls thus obtained were in 200 ml conical flasks with 50 ml 50% sucrose solution, pH 7.0, incubated on the rotary shaker at 230 rpm.

The reaction solution was analyzed by HPLC.

The immobilized Protaminobacter cells put 0.32 g / h of sucrose around.

Example F Immobilization of Protaminobacter rubrum. Cultivation of Protaminobacter rubrum (CBS 574.77) was carried out analogously to Example E.

110 g of the ester from Example D were mixed with 7.4 g of isophorone diisocyanate implemented with the addition of 15 mg Desmorapid S0.

10 g of the polymerizable acrylate resin thus obtained were with 4 g phosphate buffer 0.01 M, pH 7.0 and 500 mg 1,2-diphenyl-2-hydroxy-3 / Ñ (N-methyl) pyrrolidinium-7-propan-1-one-methylsulfate and 35 g of concentrated fermenter broth mixed with 300 g of a silicone oil-paraffin oil mixture with a spec. Given a density of 0.9 g / cm3 and produced beads analogously to Example E.

6.0 g of the beads thus obtained were analogous to Example A with 50% sucrose solution tested. The immobilized Protaminobacter cells convert 0.69 g / h of sucrose.

Claims (18)

Claims 1. Process for the production of immobilized biological Material by inclusion in polymerized compounds, characterized in that that an aqueous phase, which contains both the biological material and higher molecular weight, contains polymerizable compounds in a second, non-aqueous inert phase dispersed into pearls and this pearl-shaped water phase is polymerized. 2. Process for the production of immobilized biological material by inclusion in polymerized compounds according to claim 1, characterized in that that an aqueous solution or dispersion of a biological material with an aqueous Solution of highly water-soluble, higher molecular weight, polymerizable compounds, the two or more polymerizable functional groups per molecule and a molecular weight of over 400, is mixed and this mixture in a liquid inert Medium is divided into pearls and polymerized. 3. The method according to claim 1 and 2, characterized in that the The weight ratio of the water phase to the inert phase is between 1: 1 and 1:20. 4. Process according to claims 1, 2 and 3, characterized in that that the division of the aqueous phase into pearls by stirring and / or by Pass through takes place by inert gas or by spraying the aqueous phase into the inert phase. 5. The method according to claims 1, 2, 3 and 4, characterized in that that as an inert phase, especially water-immiscible substances such as aliphatic, cycloaliphatic and aromatic hydrocarbons and derivatives thereof, silicone oils, Paraffin oils or mixtures of the same with one another can be used. 6. The method according to any one of the preceding claims, characterized in, that pearls with an average diameter of 0.05 mm to 5 mm are produced. 7. The method according to any one of the preceding claims, characterized in, that the polymerization by irradiation with actinic light with the addition of takes place poorly soluble or insoluble photosensitizers in the inert phase. 8. The method according to any one of the above claims, characterized in, that the weight ratio of the readily water-soluble, higher molecular weight, polymerizable Connections to the biological material between 1: 1 and 30: 1, especially between 4: 1 and 20: 1. 9. The method according to any one of the preceding claims, characterized in, that the water-soluble Chen compounds reaction products of Polyether polyols with di- or polyfunctional isocyanates, modified with unsaturated Are carboxylic acids. 10. The method according to claim 9, characterized in that a) the Polyether polyols Polyethylene glycols with a MW of 400 and greater, b) the isocyanates Isophorone diisocyanate, tolylene diisocyanate, Desmodur N and / or Desmodur L and c) the unsaturated carboxylic acids are acrylic acid, methacrylic acid and / or itaconic acid. 11. The method according to claims 1 to 9, characterized in that that the polymerizable compounds are reaction products of polyether polyols with unsaturated carboxylic acids and / or isocyanate-containing derivatives thereof. 12. The method according to claim 9, characterized in that a) polyether polyols Are polyethylene glycols with a molecular weight of 400 and greater, b) the unsaturated ones Carboxylic acids are acrylic acid, methacrylic acid and / or itaconic acid, c) the isocyanate-containing Derivatives are isocyanatoethyl methacrylate and / or isocyanatoethyl acrylate. 13. The method according to any one of the preceding claims, characterized in that that for immobilization directly fermentation broths, optionally after the preceding one Concentration, can be used. 14. 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 and thus chemically sterilized in the bioreactor be transferred and the sterilization agents are removed from the closed, sterile reactor system are washed out. 15. The method according to any one of the preceding claims, characterized in that that Protaminobacter rubrum is included as biological material. 16. Immobilized biological material obtainable according to one of the preceding claims 1 to 15. 17. Use of the immobilized biological material according to claim 16 on biotransformations. 18. Use of the biological material produced according to claim 15 to convert sucrose into isomaltulose.