JPH0325159B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to cellular catalysts for bioconversion based on immobilized microbial prokaryotic and eukaryotic cells and plant cells with various enzymatic activities. The cellular biocatalysts of the invention can be used in industrial bioconversions, allowing the production in batchwise or continuous mode of compounds of importance in the pharmaceutical industry, food industry and agriculture. Generally, known methods for immobilizing cells include physical methods, physicochemical methods, and chemical methods.
The binding of the cells to the carrier is carried out mechanically by embedding the cells in a gel, by van der Walls forces, or by polar action or covalent bonding. Has improved hydraulic properties, sedimentation and separation of immobilized cells;
and a variety of water-insoluble synthetic or natural organic or inorganic carriers that promote the stability of the required enzymatic activity are generally used. Other cell immobilization methods, their advantages and disadvantages, and their economic limitations are described, for example, by Vandamme.
(Chem.Ind., No. 24, 1072 1976), Abott (In: Advances Appl. Microbiol., D.
Perlman, 20, 203, Acad.Press, New York, San Francisco, London, 1976), Jack and Zajic.
(Advances Biochem.Eng.5, 126, 1977) Durand and Navarro (Prc.
Biochem. 13, No. 9, 14, 1978) and Vojtisek et al. (Biologicke listy 44, 192,
(1979). Physical immobilization methods for cells include, for example, Thiecoslovakia Patent No. 113908 (agar), Italian Patent No. 836462 (triacetate cellulose fiber),
It is described in US Pat. No. 3,791,926 and USSR Pat. No. 6,59611 (method for embedding cells in radical polymers of acrylamide and methylene-bis-acrylamide). Currently, 10 patents are known for chemical immobilization of cells. In these methods, for example, various synthetic or natural polymers are used for covalent bonding after preliminary chemical modification and activation with various agents. Many synthetic organic or natural materials, inert or chemically active polymers such as methacrylaldehyde, glycidyl methacrylate, hydroxyalkyl methacrylates, natural polymers such as cellulose and its derivatives, collagen, gelatin, inorganic materials such as glass , titanium, zirconium and vanadium compounds are used as supports. This type of carrier offers low specific activity when bound to cells, and the resulting particles have poor mechanical resistance to abrasion, are expensive, and their price, availability, and often Various chemical denaturation and activation techniques using aggressive and toxic agents limit the production and industrial use of immobilized cells. Czechoslovak Inventor's Certificate No. 197101 describes the development of cell conjugation techniques and their industrial importance. This method consists of combining living cells with penicillin acylase activity with cellular carriers of various organisms. untreated and/or
Alternatively, physically, chemically or biochemically treated cells, or insoluble fragments thereof, are used as carriers. Chemical methods include prior physicochemical flocculation with a flocculant having two or more primary and/or secondary amino groups, followed by covalent bonding using a polyfunctional agent, especially glutaraldehyde. This method includes: In this immobilization method, waste cells obtained after various fermentations, their crushed mixtures or waste cell mass can be used, and their cost is usually negligible or zero, so it is economical. limits are avoided. An alternative, even more advantageous chemical cell binding method that does not use a carrier is Ciekoslovakia National Inventor Certificate No. 203607.
The general method for mutually immobilizing microbial prokaryotic and eukaryotic cells with various enzymatic activities without the use of carriers is described in Czechoslovak National Inventor Certificate No. 209265. has been done. The methods cited in this specification produce very stable products of covalently cross-linked cells of various microorganisms with the desired enzymatic activity, as integrally formed particle units with good sedimentation properties. Obtainable. In principle, this method covalently cross-links the intracellular components of individual cells by chemically reacting untreated cells with a bifunctional cross-linking agent, in particular glutaraldehyde, and then It consists of creating resistance to natural or artificial autolysis.
The cross-linked cells are then subjected to physical, physicochemical or chemical treatments, either alone or in combination, to render them permeable and to form part of the cell surface structure that does not react with glutaraldehyde. The release of macromolecular substances, especially lipids, causes the loss of the diffusion barrier of cross-linked cells. As a final step, under conditions favorable for the cells to come into contact with each other during the reaction, such as freezing, centrifugal force, overpressure, etc.
In a mixture of coreacting water-soluble compounds containing two or more primary and/or secondary amino groups, namely polyethyleneimine and hexamethylene diamine,
Cross-linking in the presence of a free cross-linking agent, particularly glutaraldehyde, causes the permeabilized cells to covalently bond to each other. The immobilized cell production method cited above has already made it possible to produce cell biocatalysts in a sufficiently economical manner. However, small aggressive cross-linking agent molecules, such as glutaraldehyde, easily enter cells even at very low concentrations;
Some enzymes present in cells are rapidly and irreversibly inactivated, which greatly reduces the activity of some sensitive enzymes, and the methods cited above cannot be universally used. Such inactivation is likely to occur through the formation of covalent cross-links and subsequent irreversible deformation of the active site of the enzyme. This has been shown experimentally, especially in cells with racemase and lyase activities. The above-mentioned disadvantages may be caused by the presence of untreated or possibly previously permeabilized cells with enzymatic activity, or fragments of these cells and intracellular granules of these cells; and/or untreated cells. Mixtures of cells and cell fragments, intracellular granules, and cell elution components; and/or individual cells that have been cross-linked and permeabilized are treated with two or more primary and/or secondary amino groups. chemically immobilized by water-soluble reactive polymers produced by reacting water-soluble compounds such as polyethyleneimine, hexamethylene diamine, lysine and polylysine with difunctional aldehydes, such as glutaraldehyde, and in some cases Furthermore, this can be avoided by using the cell catalyst of the present invention obtained by permeabilizing and/or mechanically curing the immobilized product. The catalyst includes, as cellular components, prokaryotic cells or eukaryotic cells, i.e. Gram-positive bacteria, Gram-negative bacteria and Gram-labile bacteria;
Other microscopic organisms such as mycobacteria, actinobacteria, yeasts, and filamentous fungi, as well as higher or parasitic organisms, and plant cells, or even fragments and intracellular granules of these cells, containing enzymes. Enzymes and/or biochemical pathways with activities, i.e. hydrolases, isomerases, lyases, decarboxylases, oxido-reductases and other activities, in particular degradative metabolism, glycolysis, conversion of hormones, and alkaloids, solan alkaloids ( solane alkaloids),
It may contain all or part of the enzymatic activity of the enzyme system that catalyzes the production of sitosterols (phtosterols) and especially ergot alkaloids. It is an object of the present invention to provide a method for producing the above-mentioned catalyst, which method comprises the steps of:
A water-soluble compound containing one or more primary and/or secondary amino groups, such as polyethyleneimine, and a difunctional aldehyde, such as glutaraldehyde, are mixed in water at 0°C to 50°C, pH 4.0 to 12.0. and a water-soluble and chemically active polymer obtained by reacting the cell components in an aqueous environment.
The reaction is carried out at 30°C to 50°C, in the pH range of 5.0 to 9.0, and optionally in the presence of a flocculant, and the cell immobilizations or aggregates thus formed are separated from the reaction mixture and water Alternatively, it is based on the fact that the cellular components can be immobilized by washing with buffers and optionally permeabilizing and/or mechanical hardening. Water-soluble polyethyleneimines in the molecular weight range of 100,000 to 800,000 are useful for the preparation of water-soluble polymers and pre-aggregation of cellular materials. The reaction between the cellular material and the polymer is carried out at 0 to 45°C under a relative centrifugal force of 1 to 50,000 g or at 0 to 45°C.
It is preferable to carry out the reaction under an overpressure of 50,000 KPa while the reaction mixture is left standing and mixed slowly or intermittently. Permeability treatment for untreated cells before fixation or cell aggregates after fixation is carried out in an aqueous environment at 0 to
At 70°C and pH 4.0 to 9.0, surfactants and/or water-miscible or immiscible organic solvents such as acetone, ether, isopropanol, petroleum ether, butyl acetate, chloroform, dioxane,
This is carried out by mixing the cell suspension in the presence of dimethylformamide or dimethyl sulfoxide and optionally by adding salts of strong acids and/or bases at once or successively varying the ionic strength. The immobilized cell particles are partially dehydrated by treatment with an organic solvent, preferably acetone, or optionally with an adhesive solution in an organic solvent or a mixture of organic solvent and water at -20°C to 25°C. Further curing can be effected by further curing, and the immobilized product thus obtained can be further removed and partially dried in a dry or humid environment and, if appropriate, further shaped. The advantage of this invention is that the chemically reactive water-soluble polymer (hereinafter abbreviated as RSP) has high specific activity,
Immobilized cellular catalysts with good sedimentation properties can be produced with high immobilization yields, and unlike methods using small molecule crosslinkers, RSP prepared according to the present invention can be produced intracellularly. Since the polymer is so high that it cannot penetrate the cell surface, a covalent bond (immobilization) occurs only between the cell surface and the reactive aldehyde group of activated RSP.
The advantage is that it can be applied to highly sensitive enzymes to which the methods of Nos. 197101, 203607, and 209265 cannot be applied. The mechanism of formation of a chemically active water-soluble polymer, for example, between polyethyleneimine and a difunctional crosslinking agent, glutaraldehyde, is that the terminal aldehyde group of glutaraldehyde and the primary or secondary amino group of polyethyleneimine An internal cross-linking reaction of the polyethyleneimine moieties that preferentially generates colored Schiff bases (C=N bonds) between the branches and possibly also some groups at the ends of the polyethyleneimine chains. It can be thought of as a reaction in which the molecular weight of the polyethyleneimine moiety increases with simultaneous chemical activation of the primary or secondary amino groups. The crosslinked, chemically active polymer thus produced is still soluble in aqueous solution;
RSP solution is a clear, yellow-brown to reddish-brown liquid. Generally, the method of the invention consists of two technical operations. In the first step, in an aqueous environment, preferably while mixing the reaction mixture, a commercially available flocculant used in wastewater treatment containing polyethyleneimine, such as Sedipur KA or Sedipur CL, is used. −930,
A chemically active water-soluble polymer (RSP) is prepared by use with a difunctional crosslinker, especially glutaraldehyde. The rate of polymerization can be tracked, for example, by the change in absorbance in the range of the visible spectrum (e.g. 550 nm), which reflects the concentration of the colored Schiff base, which primarily reflects the concentration of both reactants, i.e. the crosslinker and the co-reacting compound. and on the reaction time and temperature. The reaction proceeds according to a first-order reaction. In a second step, untreated fresh microbial or plant cells with the desired enzymatic activity, previously separated from the medium, are added to the partially or completely reacted RSP solution. If the sensitivity of the enzyme in question allows the use of a free solution of crosslinker, crosslink and permeabilize cells are added. Additionally, a mixture of untreated cells and a suspension of cell debris can be added. the immobilization technique used;
i.e. freezing, standing, gentle agitation, overpressure effects, centrifugal forces, squeezing of suspended or formed particles by suitable equipment and subsequent drying or partial drying of the particles in a moist environment. The size, shape, properties, porosity, and specific activity of the cell immobilized material can be controlled. Particularly when using untreated cells as starting material, and also when treating the immobilized particles to impart mechanical stability and resistance to the particles, in particular with acetone or other organic solvents, or preferably partially When particles are partially dehydrated using a solution such as a commercially available adhesive in an organic solvent that forms a protective layer of a water-insoluble film on the surface of the particles after drying, during the manufacturing process of the immobilized cells of this invention, Includes permeabilization of cells or permeabilization of cell aggregates after fixation. In order to produce the fixed cells of the present invention, it is generally necessary to determine where in the protoplasm or intracellular region (in the cytoplasm of the cell) the desired enzyme activity (of one type of enzyme or multiple enzymes) is present. Regardless, prokaryotic and eukaryotic microbial cells of various species or strains can be used, and even plant cells can be used. In general, the nature of the enzyme and the type of biochemical reaction catalyzed by the enzyme are not limiting factors. Depending on the given type of prokaryotic or eukaryotic cells, specific chemical techniques, such as freezing, standing, agitation, etc., and in particular techniques for permeabilizing the cells before and/or after fixation are used. You need to choose. The choice of technique depends on the location of the enzyme in the cell, the amount of the enzyme or the type of reaction catalyzed by the enzyme, and the known or expected composition of the cell surface [peptidoglycan,
Lipids, proteins, polysaccharide lipids, chitin, chitosan]. Correct choice of technique is particularly important when fixing untreated living cells. The technique of the immobilization method of this invention described above further includes:
The choice must be made according to the number and availability of reactive groups, which are cell surface components, determined by the type and physical state of the immobilized cells. When using cells with a low number and availability of reactive groups on the cell surface,
The method of the invention can be modified by pre-agglomerating the untreated cell suspension with a flocculant having two or more primary and/or secondary amino groups. These physicochemically formed aggregates can be aspirated or added directly to the RSP solution, thus promoting mutual contact of the cells. Furthermore, the cell surface is enriched with reactive groups. The immobilized cells of this invention can be prepared using cells containing various enzymes, namely various species and strains of bacteria, actinomycetes, yeasts, filamentous fungi such as deuteromycetes,
Highly parasitic filamentous fungi as well as plant cells can be used. It is advantageous to use cells of mutant organisms, which have been obtained by genetic improvement, selection, enrichment methods and genetic treatments, so that the immobilizate has a high specific activity. The size of the bound cells (particles) obtained by the method of this invention was measured microscopically (light microscopy) using a graduated eyepiece. Several specimens were observed with a scanning electron microscope. Sedimentability was tested by the following method. Briefly, 2.5 g of the wet preparation is suspended in demineralized (deionized) water in a graduated cylinder to a total volume of 25 ml, and the time required for quantitative settling of the particles is determined. A metal sieve was also used to measure particle size. 0.03mm～
Bound cells were separated using three types of sieves with a mesh size of 2.00 mm. The enzymatic activity of the immobilizates was measured using a fixed amount of bound cell particles in the substrate solution with stirring at constant temperature. The activity was determined within the scope of a zero-order enzymatic reaction and, unless otherwise stated, is expressed as specific activity, ie micromoles μmol of product per mg or g of wet weight of particles. The enzymatic activity of the whole chain of immobilized cells was determined using a manometer by the total amount of CO ₂ generated from sucrose during the measurement. In particular, the production of alkaloids, solan alkaloids and sitosterol
Measured by HPLC. The resulting immobilized cell particles are solidified, optionally with an organic solvent, preferably acetone, or with a commercially available adhesive solution, filtered with suction and partially dried, and further deformed and their size increased. The size, shape, properties, porosity, specific activity and sedimentation properties can be adjusted. In the following examples, as compounds having two or more primary and/or secondary amino groups, the molecular weight
A commercial flocculant of the Cedaipur type (i.e. Cedaipur CL-930 or Cedaipur-KA) containing about 30% w/v polyethyleneimine of 200,000 to 300,000 was used. Next, examples will be described to explain the invention in more detail, but the scope of the invention is not limited thereby. Example 1 100 ml of a 1.0 wt/vol% solution of Cedaipur CL-930 (manufactured by BASF) in deionized water and 2.0 wt/vol% solution
A 100 ml volume % solution was prepared. The solution was transferred to a 500 ml round bottom flask and attached to a rotary shaker (260 times/min). A 25% aqueous solution of glutaraldehyde (manufactured by Merck & Co.) was added to this solution with a pH of 6.1 so that the concentrations in the reaction mixture were 0.5% by volume and 1.0% by volume, respectively. The flask containing the reaction mixture was covered with aluminum foil, stirring was started, and the polymerization rate was measured at 550 nm using a spectrophotometer (Unicam, SP-
500, a 10 x 10 mm glass cuvette]. The reaction was carried out at 20° C. with continued stirring. The absorbance at predetermined time intervals was measured compared to deionized water. The measurement results showing the increase in the concentration of Schiff's base in both reaction mixtures are shown in Table 1.

[Table] The values marked with a (+) mark in the table and the values listed below are the values calculated after diluting the sample and measuring it. After 24 hours of reaction, a reddish-brown colored solution with pH 4.9 was obtained. Alcaligenes metalligenes (Alcaligenes) with aspartate ammonium lyase activity obtained by centrifugation
25g of unwashed wet bacterial cell paste (metmlcaligenes)
was added to the reacted RSP mixture while stirring vigorously. The specific activity of the cells was 550 μmol L-aspartic acid/min/g wet weight (37°C, PH8.5, substrate
1.35M ammonium fumarate). After mixing the cells into each reaction mixture, the homogeneous suspension was transferred to two aluminum plates with a diameter of 20 cm and placed in a -20°C freezer for 4 hours. The depth of the suspended layer was approximately 1.5 cm. After this time had elapsed, the plates were removed, the frozen material was covered with approximately 100 ml of deionized water, and the plates were left at room temperature for 1 hour. After this thawing time, the formed aggregates are transferred to a glass container containing 1000 ml of deionized water, gently stirred, allowed to settle quantitatively, the wash water is discarded, and the aggregates are removed. Decanted three times with 1000 ml of drinking water. The particles were then thoroughly suctioned through the fabric onto a fritted glass and weighed. 16.0 g of wet-fixed cells were obtained from the first reaction mixture and 18.2 g of wet-fixed cells were obtained from the other. Both types of immobilized products were biochemically analyzed and further characterized. The results are shown in Table 2 below.

[Table] Example 2 Prepare the RSP solution in the same manner as in Example 1 (the starting concentrations of both reaction components are Cedaipur CL-930 at 2.0%,
glutaraldehyde (1.0%) was prepared. 25 ml of bacterial cells were mixed vigorously with 100 ml of RSP solution as described in Example 1.
17.2 g of well-aspirated aggregates with a specific activity of 512 μmol L-aspartic acid/min/g wet weight were obtained. This was suspended in 50 ml of acetone containing 5% Kanagom, previously cooled to -20°C. This was mixed at room temperature for 3 minutes, then quickly vacuumed and vacuumed for 15 minutes until the adhesive on the individual particle surfaces was partially dry. After this, the weight was measured (10.5g), and the pH was 8.5.
Suspended in 100ml of 1.35M ammonium fumarate solution,
Stored overnight at 8°C. Suction this again and
It was washed with 1000 ml of water on a fritted glass, filtered with suction and weighed (13.0 g). The specific activity of this product is 180 μmol L-aspartic acid/min/g wet weight, and the average particle size is 400 μmol L-aspartic acid/min/g wet weight.
It consisted of coarse, distinct plate-like solids of ~600 μm. After standing for 2 minutes, quantitative precipitation occurred. Example 3 An RSP solution was prepared as in Examples 1 and 2.
However, the reaction time was 3 hours, and the other reaction conditions were the same. Similarly to examples 1 and 2, 100
25 g of bacterial cells were added to ml of RSP solution, and the subsequent procedure was the same as in Example 2. However, before treating the aggregates with the adhesive solution in acetone, 100 ml of a 1.0 M aqueous ammonium sulfate solution at pH 8.5 containing 0.1% surfactant (Slovafol 909) was added.
Mix for 3 hours (30℃, rotary mixer,
260 times/min), the aggregate was then filtered by suction as described above, washed with 1000 ml of water, filtered again by suction, weighed (16.0 g), and Further processing was performed as described in Example 2. 10 g of immobilized wet bacterial cells having an activity of 250 μmol L-aspartic acid/min/g wet weight were obtained. This was a coarse, well-defined plate-like solid with an average particle size of 300-550 μm, which settled quantitatively after standing for 3 minutes. Example 4 8.0μmol6-APA/hour/mg wet cell (42℃, PH
7.6, using potassium salt of benzylpenicillin as substrate) 300 g of fresh paste of untreated cells of Escherichia coli with penicillin acylase activity (using potassium salt of benzylpenicillin as substrate) were mixed with 2% Cedaipur KA and 1% by the method described in Example 1. % of glutaraldehyde at 30° C. for 24 hours, the suspension was suspended in 1000 ml of RSP solution with vigorous stirring using a steel propeller to obtain a homogeneous suspension. Next, divide the suspension into approximately 4 equal parts (approximately 320
ml), each separately in a polyethylene bag,
The edges of the bag were folded and bent several times and secured with metal clips, and the bag was placed horizontally in a freezer at -20°C to -25°C for approximately 5 hours until the layer of horizontally placed reaction mixture was 0.5 ~2.0cm. After this, remove the bag from the freezer, mechanically crush the frozen contents, cut the bag open, and pour the contents into 25°C deionized water in a glass container.
I put it in 10000ml. The contents were thawed with occasional mixing for 1-2 hours, the resulting particles were allowed to settle quantitatively, the wash water was discarded, and 5000 ml of
Decanted three times with drinking water. The aggregates were then suctioned off with a cloth, washed again with 5000 ml of potable water, and then suctioned off again to obtain a wet cake. 215g of well-sucked wet cake, 500ml
of a pre-chilled acetone-water mixture (1:
1) was suspended with mixing at -15°C and mixed vigorously for 3 minutes in diluted acetone at room temperature. After this, the product is quickly sucked off and
For another 5 to 10 minutes, use a vacuum filter to aspirate the
Washed with 5000 ml of potable water, aspirated well again and suspended in 1000 ml of 0.05M phosphate buffer, pH 7.6 and allowed to swell overnight at 8°C. A total of 180 g of wet-fixed cells with a penicillin acylase specific activity of 6.5 μmol 6 -APA/hr/mg wet weight were obtained. This one has an average particle size of 150
Distinct platelets of ~450 μm, which settled quantitatively after standing for 5 minutes. Example 5 Penicillin acylase activity (specific activity:
7.0 μmol6-APA/hour/mg wet weight), cross-linked and permeabilized E. coli obtained by the method described in Ciekoslovakia Inventor's Certificate No. 203607 Wet weight of 230g
cells were prepared by the method described in Example 1.
Mixed with 1000ml of RSP. However, the reaction time for RSP preparation was 5 hours. Immobilized cells were prepared according to the method of Example 4, which included treatment with an acetone-water mixture after the cells were mixed in RSP solution until a homogeneous suspension was obtained. Total amount with penicillin acylase activity: 166g
Wet-fixed cells were obtained. This substance has a specific activity of
6.0μmol6−APA/hour/mg wet weight, 180~
Distinct reddish-brown platelets with an average particle size of 400 μm and settled quantitatively after standing for 3 minutes. Example 6 40 g of fresh, untreated wet cells of E. coli with penicillin acylase activity similar to that in Example 4.
were homogeneously suspended in 150 ml of the same RSP solution as in Example 4, and the mixture was placed in a polyethylene cuvette and a given relative centrifugal force was applied to the immobilized cell particles. The reaction was carried out at 20°C for 3 hours. After the reaction, the product was decanted twice with water, vacuumed through a cloth, treated with an acetone-water mixture as in Example 4, and allowed to swell overnight as in Example 4. Then, it was further suctioned, the weight was measured, and the properties were further investigated. The results are shown in Table 3 below.

[Table] Note: Immobilized products were evaluated as described in this specification.
Example 7 Fresh, untreated E. coli cell paste containing β-galactosidase enzyme was added to 100 ml of RSP solution prepared by the 24-hour reaction method described in Example 1.
suspended in. However, the starting concentrations of both reaction components were 5% by weight/volume for Sedipur CL-930 and 2.0% by volume for glutaraldehyde. The specific activity of clean, untreated cells was 0.42 μmol glucose + galactose/min/g wet weight (37° C., PH 7.0, using lactose solution as substrate). The homogeneous cell suspension was allowed to stand at room temperature for 2 hours to react. After this, the spongy precipitate of cell aggregates is suctioned off by the method described above, and the resulting wet cake-like product is transferred to a vacuum cloth bag.
The edges of the bag were bent and secured with metal clips, the bag was placed horizontally between two metal plates, and an overpressure of 1.25 x 104 KPa (hydraulic) was applied for 2 hours at room temperature. After this, the disk-shaped solid was crushed into small pieces and transferred to 100 ml of water in a food mixer.
The mixture was crushed into small particles by rotating the mixer at low speed while switching on and off the mixer. After crushing and homogenization, the product was decanted three times with 500 ml of water, filtered thoroughly with suction, weighed (16.2 g) and treated with 0.2% surfactant (Surovafurol 910) and 1% dimethyl. 100ml of 1M NaCl solution containing sulfoxide
The suspension was equilibrated at 40° C. and mixed continuously for 3 hours. After permeabilization, the particles were decanted three times with 1000 ml of potable water, filtered thoroughly as described above, transferred to 0.1 M phosphate buffer, pH 7.0, and allowed to swell overnight. After straining and washing the fixed cells, a small, irregular, dark, amorphous wet mass with a total weight of 13.2 g was obtained which settled very rapidly. The specific activity of this product is 0.12 μmol glucose + galactose/min/g
wet weight, and its average particle size is 0.5~2.6mm
It was hot. Example 8 30 g of untreated cells of E. coli having the same β-galactosidase activity as in Example 7 were mixed with 0.5
% surfactant (Slobafur 910), this suspension was thoroughly homogenized by intensive mixing, and 500 ml
Transfer the flask to a round-bottomed flask, cover the flask with aluminum foil, and shake the rotary shaker (260 times per minute).
(minutes). Suspension of untreated cells at 30℃
Cells were permeabilized by continuous mixing for 20 minutes. Next, the cells were centrifuged (20,000 g/10 minutes), the supernatant was discarded, and the weight of the pasty cell precipitate was measured. 26 g of wet cells treated as described above were suspended in 100 ml of RSP solution as described in Example 2, the suspension was homogenized, and the cells were isolated by freezing the reaction mixture according to the method described in Example 2. The fixed cells were fixed and treated according to the method described in Example 2, and 17 g of the wet particles thus obtained were pre-treated with -
It was suspended in 50 ml of pure acetone cooled to 20°C. The acetone treatment was completed in 3 minutes, and the treated material was quickly suctioned onto a fritted glass, washed with water, and swelled overnight at 8° C. in 0.1M phosphate buffer, pH 7.0. In this way, a total weight of 12.6 g of immobilized wet cells with a specific β-galactosidase activity of 0.38 μmol glucose+galatose/min/g wet weight was obtained. The average particle size of this material is 85-400μm,
platelet-shaped with irregular edges, and 5
After standing for 1 minute, quantitative precipitation occurred. Example 9 38 azocasein units/g wet weight (37℃, PH7.5,
Fresh untreated wet cells of Bacillus megaterium harvested before the end of the exponential growth phase with azocasein as substrate
2.5 Kg were suspended in 10000 ml of demineralized water, the suspension was homogenized by vigorous mixing and cooled to 5°C. This suspension was crushed three times at 250 atmospheres in a micropore mechanical crusher. Continuous cooling was performed between each crushing process to ensure that the temperature of the crushing mixture did not exceed 35°C. Adjust the pH of a total of 1000 ml of the disrupted cell suspension to 7.5 with a 20% NaOH solution while continuously mixing, and add a flocculant (Cedeipur KA) so that the final concentration in the disrupted suspension is 0.25% by volume. Ta. This suspension was thoroughly mixed and allowed to stand at room temperature for about 1 hour, during which time the cell pieces were completely aggregated. After this, 2000 ml of RSP solution prepared by a 24 hour reaction as described in Example 1 were added to the 1000 ml suspension. However, unlike Example 1, the starting concentrations of both reaction products in the preparation of RSP were 4% by weight/volume for Sedipur KA and 2% by volume for glutaraldehyde. Both components were mixed gently using a stainless steel stirring bar while avoiding breaking the preformed agglomerated particles. After standing for 2 hours, the viscous partially reacted suspension was gently mixed and transferred in approximately 0.3 portions to 10 polyethylene bags. Further processing was carried out in the same manner as in Example 4. However, the immobilized particles were treated with acetone previously cooled to -20°C for 3 minutes while stirring vigorously. The process was then quickly aspirated, decanted several times with water, transferred to 1000 ml of 0.1M phosphate buffer, pH 7.5, and allowed to swell overnight at 8°C. The thus obtained suspension was slowly processed while being washed on a sieve to obtain 1.28 kg of wet immobilized particles. The specific activity of this immobilized product is 3.62 azocasein units/g wet weight, and the average particle size is 0.4
~2.0 mm, and the particles settled quantitatively upon standing for 2 minutes. Example 10 50 g of fresh, untreated wet proteolytic Bacillus megaterium cells were suspended in 100 ml of 0.05% NaCl aqueous solution and the pH was adjusted to 7.0 with 20% NaOH as described in Example 9. After homogenizing the suspension, the pH was adjusted to 7.0 again, freeze-dried egg lysozyme was added to a final concentration of 100 μg/ml, and after 30 minutes, the pH of the suspension was carefully adjusted to 8.6 with NaOH. and the suspension was heated to 50° C. for several minutes until the viscosity of the suspension changed rapidly and the cell walls were enzymatically degraded. RSP solution containing the reaction components described in Example 9
100 ml was added to the suspension without prior agglomeration. This suspension was mixed vigorously and further immobilized as in Example 8 (freezing of the reaction mixture).
The particles were treated with acetone. 30 g of immobilized wet particles with an activity of 2.4 azacasein units/g wet weight were obtained. The average particle size is
The diameter was 0.2 to 1.0 mm, and it settled quantitatively after standing for 3 minutes. Example 11 0.017 μmol glucose + galactose/min/mg
Wet weight of β-galactosidase (37℃, PH6.0,
The yeast Kluyveromyces fragilis (using lactose as a substrate)
fragilis) untreated wet cell paste at 0.1%
of surfactant (Slobafol 910) and 1% dimethyl sulfoxide in 100 ml of 0.1 phosphate buffer, pH 7.0, and this suspension was
℃ and stirred continuously for 30 minutes. After this, the permeabilized cells were centrifuged (5000 g, 10 min), the supernatant was discarded, and the weight of the precipitated cell paste was determined. 25 g of permeabilized wet cells were immobilized according to the method of Example 8. However, the particles after immobilization were treated with a 1% solution of canagon in acetone. Other conditions for processing and swelling were as described in Example 8. 0.02 μmol glucose + galactose/min/mg
15 g of immobilized wet cells with specific activity of wet weight were obtained. The particle size distribution ranged from 0.2 to 1.5 mm, and the particles settled quantitatively after standing for 2 minutes. The aggregates were linear and the precipitates were relatively large. Example 12 Specific activity 5.9μmolL-lysine/hour/mg wet weight (37
The yeast Cryptococcus laurentii having L-α-amino-ε-aminocaprolactam hydrolase activity (L-α-amino-ε-caprolactam aqueous solution)
laurentii) were suspended in 100 ml of RSP solution prepared according to the method of Example 2.
After gentle stirring for 1 hour, the highly viscous linear suspension was immobilized using the freezing technique of Example 2 and the aggregates after immobilization were treated with an adhesive solution in acetone according to the method of Example 2. . The aggregates obtained by suction were allowed to swell overnight in a phosphate buffer of pH 7.0 at 8°C. 15 g of immobilized wet cells with a specific activity of 3.2 μmol L-lysine/h/mg wet weight were obtained. The average particle size ranged from 0.125 to 1.5 mm, and quantitative settling occurred after 2 minutes of standing. Macroscopically, this immobilized material had an amorphous linear structure, and microscopically, it had an irregular structure with irregular edges. Example 13 7.1 units/g wet weight (1 unit is 100μ of L-aspartic acid in 5 minutes at 30℃ and PH5.5)
In Example ₂ , 30 g of a wet paste of fresh, untreated cells of Mycobacterium sp. containing L-aspartate decarboxylase with a specific activity of The suspension was suspended in 100 ml of the same RSP solution as above to form a homogeneous suspension. The reaction mixture was then frozen, decanted, washed and aspirated as in Example 2, yielding 19.6 g of immobilized wet cells. The above amount of particles was added to a 1M NaCl solution at PH 6.5 containing 0.5% surfactant (Slobafol 910).
Suspended in 100ml. Chloroform (5% by volume) was added to the suspension and while mixing the suspension was brought to a temperature of 35°C and mixed vigorously for 1 hour at this temperature. Dilute this with 1000ml of water,
The permeabilized particles were decanted three times with 0.5 ml of water and aspirated as described above.
Suspended in 50 ml of acetone cooled to 0.degree. C. and mixed vigorously for 3 minutes at room temperature. Aspirate the particles again, wash them thoroughly with water on a fritted glass, and reduce the pH to 6.0.
The suspension was suspended in 50 ml of 0.05M acetate buffer and allowed to swell at 8°C overnight. 14.2 g of immobilized wet cells with a specific activity of 4.0 units/g wet weight were obtained. Particle size distribution is 75-800μm
It was within the range of Macroscopically, the particles had an amorphous linear flake shape with little wetted surface.
Fixed cells hardly spontaneously sedimented. That is, the particles remained in the upper layer of the liquid. However, hypersensitivity and aspiration hyperactivity were good. Example 14 Specific activity 16.4 units/g wet sample (1 unit is 30
100 μ CO ₂ from L-lysine at ℃, PH 5.5
fresh, untreated wet cells of Bacterium cadaveris containing L-lysine decarboxylase
30 g were suspended in 100 ml of RSP solution as in Example 2. Further processing was carried out as in Example 2, including freezing, decanting, washing, and aspiration. 24g of immobilized wet cells were obtained. The above amount will aspirate the particles at a pH of 6.5 containing 0.1% surfactant (Slobafol 909).
Suspend in 100 ml of 1M NaCl solution and keep this suspension at 37℃.
The mixture was mixed continuously for 1 hour. The fixed and permeabilized cells were further processed according to the method described in Example 13. 19.1 g of immobilized wet cells with a specific activity of 11.8 units/g wet weight were obtained. This immobilized product has an average particle size of 180 to 600 μm, microscopically the particles are clearly platelet-like, macroscopically they are amorphous brown particles, and quantitatively determined after standing for 3 minutes. It settled. Example 15 20 g of fresh, untreated wet cells of the yeast Saccharomyces cerevisiae containing invertase with a specific activity of 148 units/g wet weight (30°C, PH 4.75, using sucrose as substrate) Same RSP solution as for 100
ml to obtain a homogeneous suspension. Furthermore, the fixation process, including freezing and all other manipulations, was carried out according to the method described in Example 2. Cells were treated with acetone as in Example 8. 12.1 g of immobilized wet cells with a specific activity of 90 units/g wet weight were obtained. The average distribution of particle size is from 0.32 to
2.0 mm, the particles microscopically have an irregularly shaped, indistinct structure with cracks at the edges, and macroscopically are light brown to beige colored amorphous particles;
After standing for 2 minutes, quantitative precipitation occurred. Example 16 Centrifugation of medium 1 after liquid culture of yeast Saccharomyces coreanus [Janetzki]
S-70, 3000g, 10 minutes], discard the supernatant liquid,
Then, 28 g of fresh, untreated, wet bacterial cells were obtained. The amount of pasty fresh yeast cells thus obtained was suspended in 100 ml of RSP solution prepared by the method described in Example 2, the suspension was thoroughly mixed by vigorous mixing, and the reaction mixture was It was attached to a reciprocating laboratory shaker and was shaken (50 times/min) at temperature for 1 hour. Mixing was then stopped, and the suspension was allowed to stand at the above temperature for 3 hours. After this, add 500 ml of highly viscous spongy mash of the generated aggregates.
into a polyethylene cuvette and centrifuged as above, the supernatant was discarded, the precipitate was suspended in deionized water, mixed gently and decanted three times with 250 ml of water. After decanting and quantitative precipitation of the aggregates, the immobilization was vacuum filtered, washed again with approximately 100 ml of water and weighed (14 g). This was suspended in 25 ml of acetone pre-cooled to -25°C, mixed vigorously, quickly suctioned through a fritted glass, and then approximately 1000 ml of
Wash with deionized water and aspirate thoroughly again.
Then, the weight was measured (9 g). The obtained immobilized wet bacterial cells were suspended in the above-mentioned centrifugation supernatant (medium after culturing the yeast) and allowed to swell at room temperature for several hours. The immobilized material was thoroughly vacuum-filtered again to remove 100% of wet bacterial cells.
mg into a Warburg container, add 2 ml of centrifuged clear fermentation liquid (PH4.6), and add 0.5 ml of 12.5% seuclose to the side chamber of the Warburg container.
The first target vessel contained untreated immobilized bacterial cells (100 mg of untreated wet paste) and 2 ml of centrifuged fermentation liquid was added. In the second target container, put only the supernatant liquid (no bacterial cells added), put sucrose in the side chamber, attach a manometer, turn on the shaker, and keep it at 30℃ for 15 minutes.
Incubate for minutes. The manometer was evacuated, the sucrose solution was injected from the side chamber, and the measurement of the generated CO ₂ was started. In the first target (untreated unfixed bacterial cells)
CO ₂ was generated at 3064 μ/hour, and no CO ₂ was generated in the second target (only supernatant containing no bacterial cells).
In the third measurement (using the same concentration of immobilized bacteria as in the first subject), 165 μ/h of CO ₂ was generated. This amount corresponds to 5.4% of the total metabolic activity of the initial (non-immobilized) bacterial cells. The yeast immobilized product was observed microscopically as a clear particle with an irregular shape, and macroscopically as a beige-colored amorphous particle. Quantitative sedimentation after standing for 1 minute, particle size distribution is 300 ~
It was 900 μm. Example 17 A fresh sample of Erwinia aroidea containing L-asparagine aminohydrolase enzyme with a specific activity of 100 μmol L-aspartate/min/g wet weight (37°C, PH 5.0, using L-asparagine as substrate). The untreated wet bacterial cell paste was mixed with RSP solution 100% by the method described in Example 7.
suspended in ml. The suspension was homogenized by vigorous mixing, gently stirred for 30 minutes as in Example 16, and for 1 hour at room temperature. After this, the viscous sponge-like material was poured into a 500 ml polyethylene cuvette and centrifuged at 5000 xg for 30 minutes.
The supernatant was discarded and the precipitate was overpressurized as described in Example 7. However, the precipitated grains are squeezed by hydraulic means for about 5 minutes (to squeeze out the RSP liquid part),
The brittle solids are crushed and placed in a meat homogenizer with a special steel insert of 1 mm hole diameter, extruded through this insert into rod-shaped granules, and the granules are air-dried at 25°C for about 3 hours and soaked with water for 3 hours. Decanted twice and allowed to swell overnight in deionized water. A granular solid material having a total weight of 8 g of immobilized bacterial cells having a specific activity of 64 μmol L-aspartic acid/g wet weight and having a rod shape with a length of 1 to 3 mm and an irregular particle size was obtained. This substance precipitated quantitatively in several tens of seconds. Example 18 Specific activity 0.21μmol fructose/min/g wet weight (60℃, PH6.85, using glucose as substrate)
50 g of fresh, untreated wet cell paste of Streptomyces phaeochromogenes containing glucose isomerase enzyme was mixed with 0.1 M NaCl at pH 7.0.
The suspension was heated at 70°C for 10 minutes with continuous stirring. The suspension was then cooled and centrifuged (5000 x g, 15
minutes). The supernatant was discarded and the precipitate was suspended in 150 ml of RSP solution prepared according to the method of Example 7. Further immobilization procedures were performed according to the method described in Example 17, including centrifugation, brief squeezing, crushing, and drying. However, the granules of immobilized bacterial cells after molding are 2%
of adhesive (Kanagon) for 3 minutes at -28°C.
Then, quickly aspirate the particles and collect about 1000ml.
Washed with 0.05M phosphate buffer, aspirated again and suspended in 100ml of the same buffer and stored overnight at 8°C. A total of 20 g of granular wet solid matter of immobilized bacterial cells was obtained. The specific activity was 0.1 μmol fructose/g wet weight, the particles were rod-like, and the particle size was irregular and ranged from 0.5 to 3.0 mm. The immobilized material precipitated quantitatively within several tens of seconds. Example 19 45 g of untreated wet bacterial cell paste of Aspergillus niger containing glucose oxidase enzyme and catalase with a specific activity of 0.1 μmol H ₂ O ₂ /min/mg wet weight was added to the RRP solution prepared by the method of Example 7. Added to 150ml. Furthermore, a fixation treatment was performed by the method described in Example 7. however,
The post-formed granules were dried in 40% humid air (H ₂ SO ₄ ) for 24 hours at room temperature. After this, the cells were transferred to deionized water and allowed to swell at 8°C for 24 hours. A total of 29 g of immobilized wet bacterial cells having a specific activity of 0.045 μmol H ₂ O ₂ /ml wet bacterial cells was obtained. Particle size was irregular and ranged from 0.5 to 3.0 mm. The shape of the particles was rod-like. This immobilized material precipitated quantitatively in several tens of seconds. Example 20 20 g of freshly washed wet cells of the plant Solanum aviclare (single cell culture) which synthesizes solan alkaloids and sitosterol in a hypertonic environment with seuucrose are added to 100 ml of RSP solution prepared according to the method of Example 2. The homogeneous suspension obtained by suspending and vigorously mixing was transferred to an aluminum dish (15 cm diameter) and placed in a -19°C freezer for 4 hours. The frozen reaction mixture was removed from the freezer and overlaid with approximately 50 ml of 0.05M phosphate buffer, pH 5.7, containing 0.75M KCl. After 2 hours at room temperature, the plant cell aggregates were
Transferred to 1000 ml of the above buffer and decanted several times with this buffer, always allowing the particles to settle quantitatively. The fixed cells were then thoroughly vacuum filtered, washed with buffer on a fritted glass, filtered again and weighed (18 g). The particles were then transferred to 100 ml of a sterile 8% weight/volume aqueous sucrose solution and the suspension was stirred with a magnetic stirrer three times at 24 hour intervals at 20°C. After separating the aggregates, the extracellular production of solan alkaloids and sitosterol was determined by Jirku et al. (Biotechnol. Letters, 3, No. 8).
447, 1981),
Followed at 24 hour intervals using HPLC. Production of the substance by the immobilized cells was maintained for 24 hours x 3 periods. When the particles were observed using a scanning electron microscope, it was found that they bonded between plant cells. Example 21 30g of fresh unwashed plant cells as in Example 20 were
Suspended in 100 ml of 0.05M phosphate buffer containing 0.75M KCl. A flocculant (Sedaipur KA) was added (0.5% by volume) to the homogeneous cell suspension while mixing.
After mixing, it was left at room temperature for 1 hour until cell aggregation was visually observed. After this, the suspension was carefully transferred onto a fritted glass (so as not to disintegrate the flakes formed) and passed gently and thoroughly under vacuum through a fabric. The excess liquid was removed by suction, and the resulting solid was added, without mixing, to 100 ml of the RSP solution prepared according to the method of Example 10, and the suspension was carefully homogenized by gentle stirring. It became. After homogenization, the suspension was kept at room temperature for 1 hour and further fixed according to the method of Example 12. However, the freezing temperature was -25°C. After freezing for 4 hours, the frozen material was removed from the freezer and further processed according to the method of Example 20. However, the thawing time at room temperature was 1 hour. 20 g of fixed, washed and well aspirated cells were obtained. The particles were then transferred to the same medium as described in Example 20. However, the biosynthesis of solan alkaloids and sitosterol was performed by recycling the sucrose solution through the biocatalyst bed for 5 x 24 hours. Production of solan alkaloids and sitosterol was followed at 24 hour intervals by the method described in Example 20. The production of the above material and the production cycle were maintained for 24 hours x 5 periods. Example 22 A solution containing 0.1% by weight/volume of polylysine with a molecular weight of 40,000 to 50,000 and a solution containing 1% by weight/volume of polylysine with a molecular weight of 26,200 are each
25ml each was prepared. Polylysine with a molecular weight of 40,000 to 50,000 was dissolved in distilled water, and polylysine with a molecular weight of 26,200 was dissolved in 0.1M Tris buffer (PH8.0). In the first solution (polylysine solution with a molecular weight of 40,000 to 50,000), the actual concentration in the reaction mixture was 0.3.
The second solution (molecular weight 26200
(polylysine solution), a 25% aqueous glutaraldehyde solution was added to both solutions to give a concentration of 0.5% by volume. The container containing the reaction mixture was attached to a rotary shaker, and the reaction mixture was heated at 30℃ for 10 minutes.
Mixed continuously for hours. The rate of polymerization, ie the rate of Schiff base formation, was followed spectrophotometrically according to the method described in Example 1. However, the measurement was performed at 430 nm. 6 g of fresh, untreated cells of the yeast Saccharomyces cerevisiae having invertase activity (specific activity 14 μmol glucose/min/mg dry weight, 30°C, pH 4.8, using sucrose as substrate)
A homogeneous suspension was prepared by suspending in RRP solution.
The suspension was mixed for 1 hour on a reciprocating laboratory shaker and allowed to stand for 2 hours, then the particles were centrifuged (10 minutes, 5000 g) and the precipitate was taken into a syringe without a needle. , the pasty solid was extruded into rods onto aluminum foil. The unwashed post-formed particles were dried at room temperature for 6 hours, homogenized to particles of approximately equal size, and swollen in water at 5° C. overnight. The swollen particles were vacuum filtered, washed several times with water, filtered thoroughly and weighed. In the first case and the second case, respectively
Total wet weight of particles of 5.6 g with a specific activity of 276 μmol glucose/min/g wet weight and 3.6 wet weight with a specific activity of 387.2 μmol glucose/min/g wet weight.
g particles were obtained. In each case, the particles were rod-shaped solids with a diameter of 1.2 mm after swelling and an average length of 1.45 to 4 mm. This immobilized material precipitated quantitatively within a few seconds. Immobilized bacterial cells with a wet weight of 5 g having a specific activity of 387.2 μmol glucose/min/g wet weight were incubated at a pH of 4.9.
Sucrose was dissolved in 0.05M acetate buffer
Conversion of sucrose to a mixture of glucose and fructose, suspended in 50 ml of 0.5 M solution, was carried out at 30 °C.
This was carried out under continuous mixing (an anchor type stirrer rotating at a low speed of 100 times/min). Under the conditions used, the enzymatic conversion was completed in 3 hours. This methodology was repeated 10 times using the same batch of biocatalyst. After each repeated enzymatic conversion, the particles were sieved through a polyamide mesh, washed with the buffer described above, and reused as described above. When the same immobilized bacterial cells were used repeatedly 10 times, the average enzyme conversion rate was 92.6%. Example 23 50 ml of a 10% solution of technical grade L-lysine (active ingredient content 89%) in 0.1 M Tris buffer at pH 8.2 was prepared.
An aqueous glutaraldehyde solution (25%) was added to the above solution so that the concentration of glutaraldehyde in the reaction mixture was 5% by volume. This reaction mixture is
Continuous mixing was carried out under the conditions described in Example 22. However, the reaction time was 24 hours. After this, a wet weight of 12 g of fresh cells of the same microorganism with the same enzymatic activity as described in Example 22 was added.
Suspended in 50 ml of a dark brown solution of RRP. The suspension was homogenized with a low-speed vibrating stirrer, transferred to an aluminum foil dish, and placed in a -20°C freezer for 24 hours. Next, the dish was removed and the contents were allowed to thaw naturally at room temperature. The highly viscous spongy mixture was centrifuged (10,000 g for 15 minutes), the supernatant was discarded, and the precipitate was taken into a syringe. Further operations were carried out as described in Example 22. however,
Air drying of the post-formed particles was completed in 10 hours. After filtration, washing and swelling according to the method described in Example 22, a specific activity of 180 μmol glucose/min/
A wet weight of 2.8 g of particles with an invertase activity of g wet weight and an average particle size of 1.2 to 3.7 mm was obtained.
After swelling, the immobilized material was a sponge-like, rather soft particle that settled quantitatively in a few seconds.

Claims (1)

[Scope of Claims] 1. A water-soluble reactive polymer produced by the reaction of polyethyleneimine, hexamethylenediamine, lysine or polylysine with a difunctional aldehyde, which has enzymatic activity, in untreated cells or in some cases Permeabilized cells or fragments of these cells and intracellular granules; and/or mixtures of untreated cells with cell fragments, intracellular granules and cell eluted components; and/or crosslinking of individual cells. Biological cells are prepared by chemically fixing treated and permeabilized cells, optionally further permeabilizing the immobilized product, and/or mechanically hardening the immobilized product. Cellular catalyst for transformation. 2 hydrolases, isomerases, lyases, decarboxylases, oxidoreductases, or other enzymes, and/or enzyme groups forming part of biochemical metabolic pathways;
or Gram-positive, Gram-negative or Gram-labile bacteria, mycobacteria, actinomycetes, yeasts, filamentous fungi, which are prokaryotic or eukaryotic cells that have enzymatic activity that catalyzes the bioconversion of hormones or the production of alkaloids; Or plant cells,
or a cell fragment and an intracellular granule as described above. 3. The cellular material is chemically immobilized using a water-soluble and chemically active polymer produced by the reaction of polyethyleneimine with a molecular weight of 100,000 to 800,000 and glutaraldehyde. Cell catalyst according to item 2. 4. Untreated cells or, where appropriate, permeabilized cells, or fragments and intracellular granules of these cells; and/or combinations of untreated cells with cell fragments, intracellular granules, and cell eluted components. blend;
and/or cells subjected to cross-linking and permeabilization treatment in an aqueous medium at -30°C to
Polyethyleneimine, hexamethylenediamine, lysine or polylysine and a difunctional aldehyde are mixed at 0°C in an aqueous medium at 50°C, pH in the range 5.0-9.0 and optionally in the presence of a flocculant.
Immobilization by contacting with a water-soluble and chemically active polymer obtained by reaction at ~60°C and pH 4.0 ~ 12.0, separating the immobilized product from the reaction mixture, washing with water or a buffer solution, A method for producing a cell catalyst for biological conversion, which comprises performing a permeability treatment and/or a mechanical hardening treatment depending on the case. 5. The method according to claim 4, characterized in that polyethyleneimine having a molecular weight of 100,000 to 800,000 is used for producing a water-soluble polymer and, in some cases, for pre-coagulating cells. 6. The reaction between the above cells and the above polymer was carried out at 0°C.
It is characterized in that it is carried out at ~45°C and under a relative centrifugal force of 1 to 50,000 g or under an overpressure of 0 to 50,000 Kpa, while standing still, or with gentle mixing, or while standing still and mixing is repeated. A method according to claim 4 or 5. 7 Permeabilization treatment of cells before fixation or cell aggregates after fixation is carried out in an aqueous environment at 0°C to 70°C, PH
4.0 to 9.0 in the presence of surfactants and/or water-miscible and/or water-immiscible organic solvents, optionally by adding salts of strong acids and/or bases. 7. The method according to claim 4, wherein the method is carried out by mixing the cell suspension while changing the ionic strength therein all at once or sequentially. 8. Optionally further mechanical hardening of the immobilized cells by partial dehydration with organic solvents or with adhesive solutions in organic solvents or mixtures of organic solvents and water at temperatures in the range -25°C to 20°C. , and the immobilized product is filtered by suction, and partially dried under dry or humid conditions.
6. The method as claimed in claim 4, further comprising a suitable postforming, as the case may be.