CS209265B1 - A process for the production of stable preparations of mutually bound cells of procaid and eukaryotic microorganisms with enzyme activity. - Google Patents

Abstract

The invention protects a general method for the production of stable preparations of bound cells with enzyme activity, usable for various biotransformations; the essence lies in the chemical reaction of individual cells with aldehydes, resulting in individual covalently cross-linked cells, which are further permeabilized by the action of chemical, physicochemical or physical factors. In the next stage, the cross-linked and permeabilized cells are chemically linked to each other by means of dialdehydes and diamines, or under the action of physical factors. No organic or inorganic water-insoluble carrier is required for the chemical binding of cells to each other.

Description

The invention relates to a method for producing stable preparations of mutually bound cells of prokaryotic and eukaryotic microorganisms with enzymatic activity, for example bacteria, yeasts and other imperfect fungi, usable for biotransformations.

Known methods of binding the said cells to form stable preparations; can be characterized as physical, physicochemical or chemical, depending on whether the binding of the cells with the carrier or the enzyme in the cell is mediated, for example mechanically when capturing the cells in a gel, by van der Waals forces, polar interactions or covalent bonding. Various water-insoluble synthetic or natural organic or inorganic carriers are commonly used, which primarily improve the hydrodynamic and sedimentation and thus also the separation properties of the bound cell preparations and contribute to the stabilization of the desired enzyme activity. Various known methods of binding cells, their advantages and disadvantages and their economic limits are described, for example, by Vandamme (Chem. Ind., No. 24, 1070, 1976) and Jack and Zajic (Adv. Biochem. Eng. 5, 126, 1977). The physical method of binding various enzyme-active cells, namely by embedding native cells into a gel of natural origin, is described, for example, in Czechoslovak patent No. 113908, the chemical method, for example, in Czechoslovak author's certificate No. 200802, using spherical particles of synthetic polymers based on methacrylamide, glycidyl methacrylate, hydroxyalkyl methacrylate, etc., obtained by targeted polymerization, for binding cells. These particles must first be chemically modified in advance by attaching distal spatial linkers with terminal amino groups, and only then can cells be bound to the thus modified polymer particles using polyfunctional agents.

Similarly, the process of binding various enzyme-active cells according to DOS 2,513,929 consists in reacting the cells with a reactive monomer and in their subsequent polymerization or copolymerization, or in reaction with a glycidyl methacrylate polymer, optionally in combination with covalent fixation of the enzyme inside the cells with a bifunctional agent.

Carriers of these and similar types are expensive preparations, the price and availability of which limit the industrial use of bound cell preparations. A certain progress in the development of cell binding technology with industrial significance is the method described in the Czechoslovak author's certificate No. 197101, the principle of which consists in binding production cells to ^cell carriers of various types of organisms, which are either intact or physically, chemically or biochemically treated cells or their insoluble fragments, chemically, by covalent binding, using polyfunctional agents. With this method of cell binding, the economic limit of the price of the carrier is eliminated, since the carriers used are waste cells (waste cell mass) after various fermentation productions, the price of which is mostly negligible, or zero. The aforementioned Czechoslovak author's certificate also describes the method of binding production cells to each other, i.e. without the use of a carrier. However, the cells must be largely naturally or artificially autolyzed in order for their mutual binding to occur, not to mention that the bound cells prepared in this way have poorer mechanical and sedimentation properties and, as a result of the previous partial autolysis, lower specific activity. This disadvantage is solved by the method of covalent crosslinking of individual production cells using bifunctional aldehydes (Czech Patent No. 195 063). However, in the case of repeated use, the cells must be isolated using centrifuges, since the dimensions of the individual crosslinked cells are also very small (approximately 1 pm), which also results in their poor sedimentation properties, which do not significantly differ from these properties of native cells. Another advantageous method of chemical binding of production cells to each other, without the use of a carrier, is described in Czech Patent No.

203,607. This method, like the previous one, is limited to the binding of cells with penicillin acylase activity for the production of 6-aminopenicillanic acid by enzymatic hydrolysis of penicillin.

It has now been found that the basic principle described for the method of producing bound cells with penicillin acylase activity according to Czechoslovak Patent No. 203 607 is, after further modifications and improvements, also applicable for binding cells of various production strains, or cells with different enzyme activities, regardless of whether it is a bacterial producer, yeast or other microscopic fungus and regardless of whether the desired enzyme activity (desired enzyme) is localized in the periplasmic or intracellular space (in the cytoplasm). Likewise, within a sufficiently wide range, which is documented by the examples of implementation, the quality of the enzyme, or the type of the desired enzyme-catalyzed biochemical reaction, is not fundamentally decisive. For a given type of prokaryotic or eukaryotic cell, it is in connection with the localization of the desired enzyme in the cell and the quality of the enzyme, or Depending on the type of enzyme-catalyzed biochemical reaction and the known or assumed chemical composition of the cell surfaces, it is necessary to specifically select the reaction conditions for the covalent crosslinking of the intracellular content of individual cells and, in particular, the technical and reaction conditions for their permeabilization after crosslinking, as well as individual technical, methodological procedures and reaction conditions for the subsequent mutual binding of crosslinked and permeabilized cells with the aim of obtaining bound cell preparations with sufficiently high specific activities, with a good yield of enzyme activity and other advantageous properties, in particular in relation to the sedimentation rate and the size distribution of bound cell particles, to their hydrodynamic properties and to their mechanical and enzyme stability.

The invention therefore relates to a method for producing stable preparations of mutually bound cells of prokaryotic and eukaryotic microorganisms with enzymatic activity, for example bacteria, yeasts and other imperfect fungi, usable for biotransformations. Its essence lies in the fact that first the content of the individual cells of the starting organism is crosslinked by reaction with dialdehyde, preferably glutardialdehyde, after this reaction the crosslinked cells are permeabilized by the action of physical or physicochemical or chemical factors, then they are allowed to react in an aqueous medium with a soluble compound containing at least two primary and/or two secondary amine groups, preferably with polyethyleneimine or hexamethylenediamine, and with dialdehyde, preferably with glutardialdehyde, the resulting mutually bound cells are washed with water after separation and optionally mechanically strengthened by temporary partial dehydration by the action of organic water-miscible solvents, preferably acetone or its mixture with water, at a temperature of 10 to -30 °C.

The aforementioned permeabilization of cross-linked cells can be carried out, for example, by stirring the cell suspension in an aqueous medium at temperatures from 0 to 70 °C, at a pH value of 4.0 to 9.0, in the presence of a surfactant or an organic water-miscible or immiscible solvent, for example butyl acetate, chloroform or dimethyl sulfoxide, optionally with simultaneous or subsequent changes in the ionic strength of the suspension.

The reaction of cross-linked and permeabilized cells with a water-soluble compound containing at least two primary and/or two secondary amino groups is conveniently carried out at temperatures below the melting point of the reaction mixture, optionally at a temperature of 0 to 30 °C, at a relative centrifugal force of 1 to 50,000 G or at a filtration pressure of 0 to 50,650 kPa.

The method according to the invention provides stable preparations of mutually bound cells with very good sedimentation and hydrodynamic properties and good stability of the required enzyme activity, while the size of bound cell particles can be regulated to a certain extent by the cell concentration, reaction conditions and technical-methodological procedure. The obtained material is also characterized by good stability during mechanical abrasion due to mixing and friction of particles. It can be used in principle for various industrial biotransformations, implemented by both batch, semi-continuous and continuous technology, depending on the type of reactor. When choosing a technology using bound enzyme-active cells prepared in the form of stable preparations by the method according to the invention, it is necessary to proceed from the type of enzyme-catalyzed biochemical reaction and the reaction conditions. The most advantageous use of the preparations according to the invention is in reactors with a fixed bed of bound cells. Another option is batch repeated conversion in the desired direction in a stirred reactor. The particles formed from the interconnected cells are solid, in most cases of a sandy nature, visible in the microscopic image as sharply demarcated or ill-defined plates, or irregular morphology. Macroscopically, this material resembles a resin of light brown, dark brown, reddish or black color. The shape, size and color of the particles vary depending on the type of initial production cells used and the use of the permeabilization and mutual binding technique. The immobilization yield ranges from 20 to 80% and is also dependent on the previously mentioned factors.

The production of bound enzyme-active cells according to the invention includes the following operations:

1. Chemical reaction of production cells in a stirred aqueous suspension with bifunctional crosslinking agents, which results in covalent crosslinking — crosslinking of the intracellular contents of individual cells, thereby fixing and stabilizing the required activity of the key enzyme(s) contained in the periplasmic or intracellular space of individual cells and increasing the resistance of these cells to autolysis. The key factors that influence the success of this technological step are cell concentration, crosslinking agent concentration, reaction time, pH of the suspension, temperature of the reaction mixture and intensity of mixing. These factors must be selected individually.

2. Dilution of the cell suspension after the reaction with water, their separation by centrifugation, washing with water and subsequent permeabilization of individual cross-linked cells in a stirred aqueous suspension under the influence of physical or physico-chemical or chemical factors, which washes out unreacted surface and intracellular components of predominantly lipid character, improves the diffusion properties of cross-linked cells, especially for enzymes localized in the intracellular space and reveals other groups capable of reactions on their surface and inside the cellular space. The basic factors that influence sufficient permeabilization of cross-linked cells are the concentration of cells in the aqueous suspension, the intensity of stirring, temperature, the presence of a suitable surfactant and its concentration, the presence or absence of a suitable organic solvent and its concentration, the pH value and ionic strength of the suspension, the duration of the action of individual factors ^, etc. The aforementioned factors must be selected individually for each type of cell, either separately or in combination.

3. Dilution of the suspension of crosslinked and washed cells after their permeabilization with water, separation by centrifugation, washing with water and subsequent chemical reaction in the presence of a water-soluble compound containing at least two primary and/or secondary amine groups together with bifunctional crosslinking agents, carried out expediently in most cases under conditions where the individual cells are in the closest possible mutual contact and with minimal movement. In many cases it is advantageous to carry out the mutual binding of the enzymatically active crosslinked and permeabilized cells by cooling the reaction mixture below the melting point or at a relative centrifugal force higher than 100 G or at a filtration pressure higher than 101 kPa. Sometimes, especially for some enzymatically active eukaryotic organisms, it is more advantageous to stir the reaction mixture slightly or to leave it at rest. Under the aforementioned conditions, after a certain time, mutually bound, covalently crosslinked cell particles are formed. The basic factors that influence the properties of the bound cells, especially the particle size, the desired captured enzyme activity, sedimentation, mechanical and hydrodynamic properties and their stability, as well as the immobilization yield, are the cell concentration, the concentration and quality of the crosslinker, the concentration and quality of the water-soluble compound containing amine groups, the temperature and pH of the reaction mixture, the reaction time and, last but not least, the reaction technique used, as already indicated. These factors must also be selected individually.

After binding the cells, the obtained product is decanted several times with water; in the case of using the binding technique at temperatures below the melting point of the reaction mixture, the product is allowed to thaw spontaneously and is also decanted several times with water and suctioned off. The well-vacuum-suctioned wet mass of bound cells is either dried at about 60% air humidity at room temperature; in some cases, it is advantageous to introduce the suctioned material with stirring into cooled acetone ^or into its mixture with water, which results in mechanical strengthening of the particles by their partial temporary dehydration. The product is then allowed to swell in an aqueous environment; after swelling, it is suctioned off and, if necessary, dried at about 60% air humidity to a constant weight at room temperature. Bound cells can be stored in well-sealed glass or polyethylene containers at temperatures between 0 and 10 °C. The water content in bound cell preparations varies between 40 and 65% by mass. Most preparations under the described conditions can be stored for several months without losing their original activity.

The method according to the invention can, for example, obtain such stable preparations in which the bound cells contain enzymes, for example, with hydrolase, isomerase, lyase, decarboxylase, oxidoreductase or other activity, namely preparations with either a single activity or with any combination of enzyme activities, depending on the purpose of use. The bound cells can therefore contain, for example, the enzyme penicillin acylase, β-galactosidase, L-α-amino-ε-caprolactam hydrolase, L-asparagine amidohydrolase, L-aspartate ammonia lyase, glucose isomerase, L-aspartic acid decarboxylase, L-lysine decarboxylase, glucose oxidase, etc.

In the method of producing stable preparations of mutually bound cells according to the invention, production cells containing various enzymes can be used, namely, various species and strains of bacteria, actinomycetes, yeasts and other imperfect fungi. It is of course advantageous to use for binding cells of mitant organisms, obtained by breeding or targeted genetic manipulation, which contain a high amount of the desired enzyme or enzymes, because the obtained bound cells then have a high specific enzyme activity.

The size of the bound cells in the stable preparations according to the invention was assessed microscopically (with an optical microscope) using a calibrated measuring eyepiece. Some preparations were assessed with a scanning electron microscope. The sedimentation properties were assessed by suspending 2.5 g of the wet mass of the preparation in a total volume of 25 ml of demineralized water in a calibrated graduated cylinder and the time required for quantitative sedimentation of the particles was monitored. Metal sieves were also used for sieve analysis to analyze the particle size and the bound cells were divided according to size using three sets of these sieves, the sieves having openings ranging from 0.03 to 2.0 mm.

In some cases, the mechanical stability of bound cell preparations to abrasion and the stability of key enzyme activity were also evaluated by long-term mixing of the bound cell suspension, for example, 5 to 10 g of wet mass of bound cells in 100 ml of demineralized water was shaken at 30 °C in 500 ml boiling flasks on a rotary shaker with a speed of 260/min. Samples of the suspension were taken at selected time intervals and the desired enzyme activity in the sediment and supernatant was evaluated.

For example, the activity of penicillin acylase was assessed spectrophotometrically by the color reaction of p-dimethylaminobenzaldehyde with 6-aminopenicillanic acid, using the potassium salt of penicillin as a substrate, the activity of β-galactosidase chromatographically with densitometric evaluation of the increments in the concentration of glucose and galactose using lactose as a substrate. L-α-amino-ε-caprolactam hydrolase activity was assessed by measuring the decrease in the concentration of L-α-amino-ε-caprolactam as a substrate and the increase in the product, L-lysine, also chromatographically with densitometric evaluation. The activity of L-asparagine amidohydrolase was measured by the increase in the concentration of ammonium ions by Nesslerization colorimetrically, using L-asparagine as a substrate. L-aspartate ammonia lyase activity was assessed chromatographically with densitometric evaluation of the increase in the concentration of L-aspartic acid using the ammonium salt of fumaric acid as a substrate. The activities of L-aspartic acid and L-lysine decarboxylase were measured using a Warburg apparatus by determining the rate of carbon dioxide formation using the respective amino acids as substrates. The activity of glucose oxidase was determined colorimetrically after oxidation of o-dianisidine with hydrogen peroxide by peroxidase, and the activity of glucose isomerase was also determined colorimetrically by determining fructose after reaction with cysteine and carbazole. In both cases, glucose was used as the substrate.

Stable preparations of chemically bound cells obtained by the method according to the invention represent a significant technical and economic advance in both industrially feasible biotransformations.

Further details are given in the examples which illustrate the method according to the invention in more detail, but do not limit it in any way. The examples also show the enzyme activity of native and bound cells.

Example 1

From a wet paste of fresh native Escherichia coli cells containing the enzyme penicillin acylase with a specific activity of 8 pmol 6-APK/hour/mg wet cell mass (42 °C) pH 7.6, 500 ml of a homogeneous aqueous suspension with a cell concentration of 0.25 g w/ml was prepared; the pH of the suspension was adjusted to 7.0 with a 20% NaOH solution. A 25% aqueous solution of glutaraldehyde was added to the stirred aqueous suspension so that its current concentration in the reaction mixture was 1% (w/v). The reaction was carried out for 3 hours with continuous stirring at a temperature of 20 °C. After this time, the suspension was diluted with drinking water to a total volume of 5 liters, mixed and the cells were centrifuged (5000 G/30 minutes). 110 g w/v was obtained. individually covalently cross-linked, washed cells, whose specific activity was 5.1 pmol 6-APK/hour/mg wt.

An aqueous suspension with a concentration of 0.25 g wt/ml was prepared from the wet paste of individually cross-linked cells thus obtained. Butyl acetate and Slovafol 909 were added to the homogeneous stirred suspension, the pH of which was adjusted to 7.0 with a 25% NaOH solution, so that their current concentrations in the suspension were 2.5 vol. % in the first case and 0.5 vol. % in the second case. The suspension was heated in a water bath to 45 °C and, with continuous stirring with a steel propeller stirrer (500 rpm), the cross-linked cells were permeabilized for 3 hours. After this time, the suspension was cooled to 25 °C, diluted with drinking water to a total volume of 5 liters, mixed and the cells were centrifuged (5000 G/30 minutes). 78.3 g wt. individually cross-linked and permeabilized cells, whose specific activity was 6.8 μmol/hour/mg wt.

From a portion of the wet paste of cells obtained in the described manner, 150 ml of a homogeneous aqueous suspension with a pH of 7.0 and a cell concentration of 0.3 g in. wt/ml was prepared. The stirred suspension was cooled to 10 °C and Sedipur KA (polyethylenimine) was added so that its concentration in the suspension was 0.5 vol. %. Then, a 25% aqueous solution of glutaraldehyde was added to the suspension with stirring so that its concentration in the suspension was 1 % (weight/volume) and the mixed reaction mixture was spread into 10 ml polyethylene cuvettes and, depending on the relative centrifugal force used, the formation of mutually bound flocs with penicillinase activity was carried out. The reaction took place at 0 °C in cooled Janetzki K 26 centrifuges for 3 hours. After the reaction, the preparations of bound cells were decanted twice with water, vacuum-drained through a terylene (miller's) cloth and treated with a mixture of acetone:water (1:1) cooled to -20°C, vacuum-drained again and left to swell in water at 10°C for 16 hours. Then vacuum-drained again in the described manner, weighed and their sedimentation rate, particle size, volume after sedimentation and specific activity were evaluated in the manner described in the description of the invention. The results are given in the following table.. ' .

Relative centrifugal force (G) 1100 4500 « » JO 200 . . 28,595. -y—.----_____ - í* ’ Particle size (gm) 25—150 75—150 ?5—300 75—450 1—2 Sedimentary particle velocity (min at 1 G) 7 5 1 1 quantum, don't sit. Particle volume after sedimentation (ml) 6.5 5 . 3 3 — Spec. activity (μ,πιοί 6-APK/hour/mg v. wt. bound cells) 6.2 5.9 5.1 4.65 6.8 Microscopic image unbounded formations with irregular edges unites. cells Macroscopic appearance brown amorphous particles cell suspension

* Standing the reaction mixture at 0 °C for 3 hours at J G, then centrifuging, washing and suspending the sediment in water.

Example 2

The same fresh native paste of Escherichia coli cells with penicillin acylase activity was cross-linked as described in Example 1.

Permeabilization of an aqueous suspension of individually covalently crosslinked cells was performed in the same way, with the only difference that instead of butyl acetate, NaCl at a concentration of 5.8 g/liter (1 M NaCl concentration in the suspension) was used for permeabilization; the concentration of Slovafol 909, the reaction time and the conditions were the same as in Example 1; the pH of the suspension was adjusted to the same value only after the addition and dissolution of NaCl.

From a wet paste of cross-linked, permeabilized and washed cells with a specific activity of 6.1 μπιοί 6-APK/hour/mg v. wt. a homogeneous aqueous suspension with a cell concentration of 0.3 g v. wt./ml was prepared; the pH of the suspension was 6.9. The suspension was heated to 40 °C and continuously stirred at this temperature with a steel propeller stirrer (300 rpm) for 72 hours. After the specified heat treatment time, the suspension was cooled to °C and made up to the original volume with drinking water.

To 100 ml of this suspension, Sedipur KA and glutaraldehyde were added with stirring as described in Example 1 and the mixed suspension was poured into a bag made of linen filter press cloth. The bag was closed and placed horizontally between two metal plates and its contents were subjected to a filtration pressure of 1.25.times.104 ^kPa using a hydraulic press at room temperature for 30 minutes.

The described method produced a brown-black solid mass with a total weight of 25 g in the shape of an irregular plate. The obtained mass was cut into irregular thinner parts and homogenized in 200 ml of drinking water intermittently for about 15 minutes at laboratory temperature using a kitchen mixer. After homogenization, the material was decanted twice with 1 liter of water, vacuum-dried well through a terylene sheet and weighed.

21 g of wet mass of bound cells were obtained *with an irregular particle size distribution of 50 to 5000 gm; the specific activity of the bound cell homogenate was 3.3 gmol 6-AKP/hour/mg wt. and their quantitative sedimentation was achieved after 2 minutes of standing. '

Example 3

From a moist cross-linked, permeabilized and washed paste of Escherichia coli cells with penicillin acylase activity with a specific activity of 4.64 gmol 6-APK/hour/mg wt., where the cross-linking of individual cells was carried out according to Example 1 and their permeabilization and further heat treatment for 72 hours according to Example 2, 500 ml of an aqueous suspension with a cell concentration of 0.25 g wt./ml was prepared.

To a homogeneous suspension at a temperature of 15 °C and pH 7.0, a 10% (w/v) aqueous solution of hexamethylenediamine at pH 7.0 was added with stirring so that its concentration in the suspension was 0.5% (w/v), and after 15 minutes of standing, a 25% aqueous solution of glutaraldehyde was added with stirring so that its concentration in the reaction mixture was 2% (w/v). The reaction mixture was centrifuged (10 minutes/5000 G) and a circular wet cake with a diameter of 5 cm and a height of 1 cm was made from the paste thus formed on a filter press linen fabric. The wet cake was inserted into a bag made of the same material and the bag was placed between two metal plates in a horizontal position and subjected to a filtration pressure of 1.25.10 ⁴ kPa using a hydraulic press at room temperature for 24 hours. A portion of the dark brown solid (10 g) was mixed in water and suctioned off as described in Example 2; bound cells with penicillin acylase activity were obtained with a particle size distribution of 100 to 1000 gm, the specific activity of which was 2.75 gmol 6-APK/hour/mg wt.

and their quantitative sedimentation was achieved after 3 minutes of standing.

AND!

'< Example 4

To 80 ml of an aqueous suspension of cross-linked, permeabilized and heat-treated Escherichia coli cells with penicillin acylase activity (specific activity was 5.16 gmol 6-APK/hour/mg wt., cell concentration in suspension 0.25 g wt./ml) at pH 7.0, which was obtained by the procedure described in Example 3, a 10% aqueous solution of hexamethylenediamine at pH 7.0 was added so that its concentration in the suspension was 0.5% (w/v), the mixture was stirred briefly and then left to stand at a temperature of 11 °C for 20 hours. Then, a 25% aqueous solution of glutaraldehyde was added to the aqueous suspension with stirring so that its concentration in the suspension was 2% (w/v). Then the reaction mixture was poured onto a circular dish with a diameter of 14 cm made of aluminum foil and the dish was placed in a horizontal position in a freezer _; the layer thickness on the dish was approximately 0.5 cm, the reaction took place for 48 hours at a temperature of -23 °C. After this time, the dish was removed from the freezing box and approximately 100 ml of drinking water was poured over it and the material was left to thaw at room temperature. After thawing, the bound cells were decanted three times with 100 ml of drinking water, vacuum-dried through a terylene sheet and weighed.

14.3 g of aspirated bound cells were obtained. This amount was suspended in 107 ml of acetone p. a., previously cooled to a temperature of — 23 °C, the material was stirred in the cooled acetone for 3 minutes and then immediately and sharply, as described, aspirated by vacuum. The bound cells were left to swell overnight in 0.05 M phosphate buffer pH 7.6 at 8 °C and then aspirated by vacuum as described and aspirated on a cloth for about 20 minutes.

14.2 g in. wt. of brown scaly bound cells were obtained, which quantitatively sedimented in 10 minutes upon standing; the particle size distribution ranged from 75 to 300, gm and their specific activity was 4.25 gmol 6-APK/hour/mg in, wt. The material was subjected to stability testing of its enzyme activity and mechanical stability of the particles under the conditions specified in the description of the invention. After 528 ; hours (22 days) of continuous shaking of the suspension of ' bound cells, their specific activity was 85% of the original activity while maintaining 70% of the total mass of the material based on the original weight.

Example 5 g of fresh native cells of Alcaligenes faecalis with penicillin acylase activity were suspended in 80 ml of drinking water; the pH of the suspension was adjusted to 7.0 with a 20% NaOH solution. The suspension contained 0.25 g of native cells/ml with a specific activity of 0.25 gmol of 6-APK/hour/mg of native cells (42 °C) pH 7.6. Crosslinking of individual cells was carried out according to example 1. Permeabilization of crosslinked cells: according to example 2. Heat treatment of the crosslinked and permeabilized suspension was also according to example 2, with the difference that the temperature of the stirred suspension was 37 °C under otherwise identical treatment conditions.

To 100 ml of an aqueous suspension of cross-linked, permeabilized and heat-treated cells of A. faecalis, Šedipur KA (polyethylenimine) was added with stirring after cooling to 10 °C so that its concentration in the suspension was 0.5% (mass/volume). Then the suspension was left to stand for 30 minutes and then a 25% aqueous solution of glutaraldehyde was added with stirring so that its concentration in the suspension was 1% (mass/volume), the suspension was briefly mixed and the procedure was continued as described in Example 4, with the difference that the reaction time during freezing was 5 hours.

11.8 g of dark brown bound cells were obtained, which sedimented quantitatively in 4 minutes upon standing; the particle size distribution ranged from 150 to 300 pm and their specific activity was 0.23 pmol 6-APK/hour/mg of mass. The enzymatic and mechanical stability of the particles monitored under the conditions specified in the description of the invention for 10 days (240 hours) was 100% of the original activity during the specified period of continuous shaking while maintaining 94% of the original mass of the material.

Example 6

250 g of fresh native Escherichia coli cell paste containing the enzyme beta-galactosidase was suspended in 750 ml of drinking water; a homogeneous cell suspension of pH 7.0 was prepared, which contained 0.33 g of native cells (ml with a specific activity of 0.035 pmol glucose + galactose) min/mg of native cells (37 °C) pH 7.0.

Covalent crosslinking of the intracellular content of individual cells and their washing was carried out according to Example 1. Permeabilization of cells was carried out by the combined effect of changing the ionic strength, the presence of a surfactant and the temperature in a stirred suspension of crosslinked cells according to Example 2, with the difference that instead of Slovafol 909, Slovafol 910 was used in the same concentration and the temperature during the 3-hour permeabilization in the presence of 1 MNaCl was only 40 °C instead of 45 °C. 190 g of v. wt. (paste) of crosslinked, permeabilized and washed cells was obtained, the specific activity of which was 0.029 pmol glucose + galactose/min/mg v. wt. The paste of these cells was suspended in drinking water so that the cell concentration was 0.3 v. wt./ml and heat-treated with stirring at 40 °C for 72 hours, also as described in Example 2.

After the suspension volume was made up to the original volume with drinking water and the suspension was cooled to 15 °C, the cells were bound together according to Example 5, with the difference that the reaction time during freezing was 4 hours and that the reaction mixture was immediately filled into 375 ml polyethylene bags after brief mixing after the addition of glutaraldehyde, sealed and placed horizontally on aluminum trays. The method of treating the bound cells after the reaction (thawing, decanting, filtration, treatment with acetone, swelling, etc.) was carried out according to Example 4, with the difference that the bound cells were swollen overnight in demineralized water under otherwise identical conditions.

110 g of bound cells with beta-galactosidase activity with a specific activity of 0.021 pmol glucose + galactose/min/mg of weight were obtained, macroscopically resembling a dark brown resin of sandy character, microscopically appearing as unbounded irregular plates with a particle size distribution of 75 to 300 pm. Quantitative sedimentation of the particles occurred in 4 minutes. The moisture content of the preparation was 60%. The material was subjected to mechanical and enzyme stability testing as stated in the description of the invention; after 1632 hours (68 days) of continuous shaking of the material in an aqueous suspension, the specific activity of the bound cells was 110% of the original activity and the balance of the total weight of the material after this time was 75% of the original mass. 25% of the material mass due to abrasion and thus the reduction of the particles after the specified shaking time passed through the cloth during filtration and suction. The cell-bound material remained unchanged in the described characteristics including specific activity during its storage in a closed polyethylene bottle kept at 10°C for 6 months. Example 7;

From the same amount of wet mass of fresh native Escherichia coli cells with beta-galactosidase activity as in Example 6, 80 g of wet mass of bound cells was produced by the procedure also described in Example 6, with the difference that instead of changing the ionic strength (1 M NaCl), the combined effect of the same temperature and the same surfactant in the presence of 2.5 vol. % chloroform and 1 vol. % dimethyl sulfoxide under otherwise identical reaction and handling conditions was used to permeabilize the cross-linked cells.

The characteristics of the preparation were similar to those of Example 6, except that the specific activity of the material was 0.028 gmol glucose + galactose/min/mg wt. of bound cells. The stability of the material under the storage conditions specified in Example 6 did not change over a 3-month observation period.

Example 8

250 g of fresh native paste of Escherichia coli cells containing beta-galactosidase as in Example 6 was cross-linked, permeabilized and heat-treated in aqueous suspension by the procedure also described in Example 6. The mutual binding of the cells in the presence of hexamethylenediamine and the treatment of the bound cells after binding were carried out by the procedure described in Example 4.

96 g of orange-brown bound scale-shaped cells were obtained, which sedimented quantitatively in 12 minutes on standing; the particle size distribution ranged from 75 to 350 pm and their specific activity was 0.019 pmol glucose + galactose/min/mg wet mass of the material.

Example'9

From fresh native wet mass (paste) of yeast cells Kluyveromyces fragilis containing the enzyme beta-galactosidase, 100 ml of aqueous suspension was prepared in 0.05 M phosphate buffer pH 7.0; the concentration of native cells in the suspension was 0.35 g wt. (ml and the specific activity of these cells was 0.017 μιηοΐύ glucose + galactose) hour/mg wt. (37 °C) pH 7.0.

Covalent crosslinking of the intracellular content of individual cells including the washing method and separation of the wet crosslinked biomass was carried out according to Example 1. Permeabilization of 100 ml of an aqueous yeast suspension with a cell concentration in suspension of 0.25 g wt/ml and pH 7.0 was carried out at a temperature of 40 °C in the presence of 1 vol. % dimethyl sulfoxide and 0.5 vol. % toluene in the absence of surfactant by sharp stirring of the cell suspension with a steel propeller stirrer (800 rpm) for 3 hours. Then the suspension was cooled to room temperature and diluted with drinking water to a total volume of 1 liter, mixed briefly, the cells were centrifuged (5000 G/20 minutes), the milky turbid supernatant was decanted and the cell sediment was suspended in the same volume of drinking water and centrifuged again in the described manner. A total of 20 g wt. individually cross-linked, permeabilized and washed yeast, whose specific activity was 0.022 μιηοΐύ glucose + galactose/hour/mg. v. wt.

g v. hm of yeast treated in the described method was suspended in 380 ml of drinking water and the pH of the homogeneous cell suspension was adjusted to 7.0 with 20% NaOH solution while stirring. _x Then the suspension was poured into a 1 liter Erlenmeyer ^flask and Sedipur KA (polyethyleneimine) was added so that its concentration in the suspension was 0.5 vol. %. The suspension was briefly mixed and left to stand for about 15 minutes. Then a 25% aqueous solution of glutaraldehyde was added while stirring so that its concentration in the reaction mixture was 1 vol. %. The flask was placed on a slow-speed reciprocal shaker with a number of oscillations of 40/min. The reaction took place for 3 hours at a temperature of 29 °C under the specified mixing conditions. After this time, the reaction mixture was diluted with drinking water to a total volume of 2 liters and centrifuged (5000 G/10 minutes). The sediment of the bound yeast was suspended in 1 liter of drinking water and vacuum-sucked through a terylene sheet and weighed. 9.7 g by weight of the aspirated and washed material was suspended in 100 ml of acetone cooled to -20 °C with stirring, stirred for 3 minutes and then the particles were immediately vacuum-sucked off sharply in the described manner and then sieved for about 10 minutes, after which they were suspended in 100 ml of 0.05 M phosphate buffer > pH 7.0 and left to swell overnight at 10 °C. 8.1 g by weight of the bound yeast with beta-galactosidase activity was obtained.

The specific activity of the material was 0.009 μιηοΐύ glucose + galactose/hour/mg v. wt. of bound cells. The particle size distribution ranged from 100 to 850 μιη and their quantitative sedimentation was achieved after 2 minutes of standing. The stability of the material during storage under the conditions given in Example 6 was constant for 3 months.

Example 10

470 g of fresh native yeast cells of Cryptococcus laurentii with L-alpha-amino-epsilonaminocaprolactam hydrolase activity were suspended in 0.05 M phosphate buffer pH 7.0 to a total volume of 4.7 liters; the pH of the homogeneous suspension was adjusted to the buffer value with 20% KOH solution. A suspension containing 0.1 g of native cells/ml with a specific activity of 6.01 μηιοίύ L-lysine/hour/mg of native cells (37 °C) pH 7.5 was obtained.

Covalent crosslinking of the intracellular content of individual yeasts in suspension was carried out according to Example 1. After the reaction, the suspension was diluted with drinking water to a total volume of 50 liters, briefly mixed and centrifuged on a Sharples separator. 320 g of crosslinked and washed cells were obtained in the form of a wet paste whose specific activity was 3.83 μΜίθΐύ L-lysine/hour/mg of mass. * »

320 g of cross-linked and washed cells were suspended to a total volume of 3.2 liters in drinking water and permeabilized with vigorous stirring (800 rpm) using a steel propeller stirrer in the presence of 1 vol. % dimethyl sulfoxide at a temperature of

42.5 °C for 3 hours. The suspension was then cooled to room temperature and diluted to a total volume of 35 liters with drinking water and centrifuged to form a wet cell paste on a Sharples separator. 283 g of individually cross-linked, permeabilized and washed yeast cells were obtained, the specific activity of which was 4.15 μιηοΐύ L-lysine/hour/mg of mass.

From a part of the wet mass of the cells obtained in the described method, 200 ml of a homogeneous aqueous suspension was prepared, the pH of which was adjusted to 7.0 with a 20% KOH solution; the suspension contained 50 mg of cells/ml. The suspension was divided into two equal volume parts (each 100 ml) designated A and B, and Sedipur KA (polyethyleneimine) was added to each of them with stirring so that its concentration in the suspension was 0.5 vol. %, after which enzyme-active cells bound to each other were prepared from them using various techniques in the following manner:

A. The suspension was cooled to 10 °C with stirring and left to stand for 30 minutes. Then, a 25% aqueous solution of glutaraldehyde was added with stirring so that its concentration in the suspension was 1 vol. %. After brief mixing, the cells were linked together by freezing the reaction mixture under the conditions specified in Example 5; the method of treating the particles after linking was carried out according to Example 4.

3.8 g of bound yeast with L-alpha-amino-epsilon-caprolactam hydrolase activity were obtained; the specific activity of the material was 0.52 μιηοΐύ L-lysine/hour/mg of mass. The particle size distribution ranged from 75 to 1450 gm and their quantitative sedimentation was achieved after standing for 1 minute. Microscopically, the particles formed needle-like, sharply demarcated formations resembling crystals. Macroscopically, the material resembled heterogeneously crystallized potassium dichromate, including a typical brown-red color. The stability of the material during storage under the conditions given in Example 6 was constant for 6 months.

B. Glutardialdehyde was added to the slowly stirred suspension at laboratory temperature in the same concentration as in variant A and the cells were further bound together using the slow stirring technique of the reaction mixture as described in Example 9, including the method of treating the resulting particles after binding.

2.75 g of bound yeast with L-alpha-amino-epsilon-caprolactam hydrolase activity were obtained; the specific activity of the material was 1.8 μηιοίύ L-lysine/hour/mg of mass. The particle size distribution ranged from 15 to 900 μm and their quantitative sedimentation was achieved after standing for 5 minutes. Microscopically, the particles formed irregular shapes with irregular edges. Macroscopically, the material formed amorphous cotton-like shapes. The stability of the material during storage under the conditions given in Example 6 was constant for 6 months.

Example 11

500 g of wet mass (paste) of fresh native cells of Alcaligenes metalcaligenes containing the enzyme L-aspartate ammonia lyase was suspended in 0.05 M phosphate buffer to a total volume of 2 liters; a homogeneous suspension of pH 7.1 was formed containing 0.25 g of native cells/ml. The specific activity of the native cells was 0.75 μηιοίύ ’ * L-aspartic acid/min/mg of mass (37 °C) pH: j 8.5. : ‘

Covalent crosslinking of the intracellular contents of individual cells was performed according to the example

1. Permeabilization of cross-linked cells was carried out according to Example 2, with the difference that the temperature of the stirred suspension during permeabilization was 40 °C. Heat treatment of the cells was also carried out according to Example 2. Cell binding was carried out by freezing technique as described in Example 6; the method of treating the bound cells after binding is also described in Example

6.

198 g of wet mass of interconnected cells with L-aspartate ammonia lyase activity was obtained; the specific activity of the material was 0.5 μιοί L-aspartic acid/min/mg v. wt. The particle size distribution ranged from 75 to 300 μιη and their quantitative sedimentation was achieved after 3 minutes of standing. Microscopically, the material formed unbounded formations with irregular edges. Macroscopically, the material formed light brown scales of a micaceous character. The stability of the material during storage under the conditions given in Example 6 was constant for 1 month.

Example 12

750 g of wet mass (paste) of fresh native Corynebaeterium glutamicum cells containing the enzyme aspartate ammonia lyase was suspended in 0.05 M phosphate buffer to a total volume of 3 liters; the homogeneous suspension contained 0.25 g w. w. native cells/ml and their specific activity was 1 μηιοί aspartic acid/min/mg w. w. (37 °C) pH 8.5.

Covalent crosslinking of the intracellular content of individual cells was carried out according to Example 1; their permeabilization was carried out in the same way as in Example 2, with the only difference that instead of Slovafol 909, Slovafol 910 was used in the permeabilization in the same concentration under otherwise identical conditions. Heat treatment of the cells was also carried out according to Example 2. Mutual binding of the cells was carried out by the technique of freezing the reaction mixture in the manner described in Example 6, including the method of treating the material after binding.

312 g of bound cells with L-aspartate ammonia lyase activity were obtained; the specific activity of the material was 0.63 μιοί L-aspartic acid/min/mg of mass. The particle size distribution ranged from 50 to 250 μιη and their quantitative sedimentation was achieved after 10 minutes of standing. Microscopically, the material formed unbounded formations with irregular edges. Macroscopically, the material formed beige scales. The stability of the material during storage under the conditions given in Example 6 was constant for 3 months.

Example 13

From a wet paste of fresh native Escherichia coli cells containing the enzyme aspartate ammonia lyase with a specific activity of 0.35 pmol L-aspartic acid/min/mg wt. of native cells (37 °C) pH 8.5, 500 ml of a homogeneous aqueous suspension with a cell concentration of 0.25 g wt./ml was prepared; the pH of the suspension was adjusted to 7.0 with stirring with a 20% NaOH solution.

Covalent crosslinking of intracellular contents was carried out according to Example 1 including the method of their permeabilization. Heat treatment of cells after permeabilization was carried out according to Example 2. 150 ml of homogeneous aqueous suspension containing 0.3 g w/w/ml of cells treated in the described method was used to prepare bound cells by centrifugation technique in the method described in Example 1 including the method of their treatment after binding. The results are shown in the following table.

Relative centrifugal force (G) and 100 4500 10,200 28,595 1* Particle size (μιη) 25—150 50—320 75—400 75—425 1—2 Sedimentary particle velocity (min at 1 G) 12 6.5 1 1 quantum, don't sit.

Particle volume after sedimentation (ml) 10 7.5 4 4 — spec. activity (/imole acid. L-asp./ min/mg in. wt. weight. b. 0.2 0.13 0.09 0.09 0.28 Microscopic image unplayable formations with irregularities. edges unites. cells Macroscopic appearance amorphous light brown particles cell suspension

* Standing the reaction mixture at 0 °C for 3 hours at 1 G, then centrifuging, washing and suspending the sediment in water.

Example 14

From a wet paste of fresh native Mycobacterium sp. cells containing the enzyme L-aspartic acid decarboxylase with a specific activity of 7.1 units/gram of mass (1 unit of activity is the amount of enzyme that releases 100 μΐ CO ₂ from L-aspartic acid in 5 minutes at a temperature of 30 °C and pH 5.5) 100 ml of a homogeneous aqueous suspension with a native cell concentration of 0.3 g mass/ml was prepared; the pH of the suspension was adjusted to 7.0 with stirring with a 20% NaOH solution.

Covalent crosslinking of the intracellular contents of individual cells was performed according to the example

1. Permeabilization of cross-linked cells was carried out according to Example 2 with the following differences: the concentration of cross-linked cells in the stirred suspension was 0.1 g wt/ml, the pH of the suspension was 6.1, Slovafol 910 was used in the same concentration instead of Slovafol 909 and chloroform was used in the actual concentration of 5 vol. % instead of 1 M NaCl, the temperature of the suspension was 35 °C and the duration of permeabilization was 1 hour. No further heat treatment was performed after permeabilization of cross-linked cells. The washed, cross-linked and permeabilized cells were bound to each other by freezing using the procedure given in Example 5 with the difference that the concentration of cells in the reaction mixture was 0.1 g wt/ml. Treatment of bound cells after their mutual reaction was carried out according to Example 4.

12.1 g of bound cells with decarboxylase activity of L-aspartic acid with a specific activity of 0.2 u/g of material were obtained. The particle size distribution ranged from 150 to 1000 pm; the particles did not sediment spontaneously, but could be filtered and suctioned off well. Macroscopically, the particles formed amorphous gray-yellow flakes with a poorly wettable surface. The stability of the material during storage under the conditions given in Example 6 was constant for 1 month.

Example 15

From a wet paste of fresh native Bacterium cadaveris cells containing the enzyme L-lysine decarboxylase with a specific activity of 16.4 units/g wt. (1 unit of activity is the amount of enzyme that releases 100 μΐ CO ₂ from L-lysine at a temperature of 30 °C and pH 5.5) 100 ml of a homogeneous aqueous suspension with a pH of 7.0 and a concentration of native cells of 0.25 g wt./ml was prepared.

Covalent crosslinking of the intracellular contents of individual cells was performed according to the example

1. Permeabilization of cross-linked cells according to example 2 with the differences that the concentration of cells in the permeabilized suspension was 0.1 g wt/ml, the pH of the suspension was 6.3 and instead of Slovafol 909, Slovafol 910 of the same concentration was used, while the temperature during permeabilization was 35 ° C. The procedure was then carried out according to the reference given in the previous example (example 14).

9.35 g of bound cells with L-lysine decarboxylase activity with a specific activity of 0.67 u/g of material were obtained. The particle size distribution ranged from 75 to 300 μπι and their quantitative sedimentation was achieved after 4 minutes of standing. Microscopically, the particles were formed by scaly plates with irregular edges. Macroscopically, the particles resembled a fine resin of light brown color. The stability of the material during storage under the conditions given in Example 6 was constant for 1 month of monitoring at one-week intervals.

Example 16

300 g of wet mass (paste) of fresh native Escherichia coli cells containing the enzyme L-asparagine amidohydrolase was suspended in drinking water to a total volume of 1 liter. The homogeneous suspension contained 0.3 g of native cells/ml and their specific activity was 0.09 μμιοί L-aspartic acid/min/mg of mass (37 °C) pH 5.0.

Covalent crosslinking of the intracellular content of individual cells was carried out according to Example 1 and 394 g of wet mass of crosslinked, washed cells with a specific activity of 0.015 pmol L-aspartic acid/min/mg wt. were obtained. Permeabilization of a suspension containing 0.25 g wt. of crosslinked cells/ml was carried out in the presence of only 1 M NaCl, the pH of the suspension was 6.0 and the suspension was shaken in 500 ml boiling flasks with three seals (stops) on a rotary shaker with a speed of 260/min at a temperature of 30 °C for 3 hours. After permeabilization, the cells were washed by diluting with drinking water to ten times the volume of the suspension | and centrifuged (5000 G/30 minutes). From 250 g wt.

181.5 g of cross-linked cells were obtained.

: cross-linked -permeabilized and washed cells Q specific activity 0.029 pmol L-aspartic acid/min/mg wt. Heat treatment of cells after permeabilization was carried out according to example 2, mutual binding of cells by freezing technique according to example 6 and treatment of cells after mutual binding according to example 4.

1Ó2 g in. wt. of interconnected cells with L-asparagine amidohydrolase activity were obtained; the specific activity of the material was 0.039 μπιοί. ' L-aspartic acid/min/mg. The particle size distribution ranged from 75 to 750 pm and their quantitative sedimentation was achieved after standing for 8 minutes. Microscopically, the material formed unbounded formations with irregular edges. Macroscopically, the material formed light brown scales. The stability of the material during storage under the conditions given in Example 6 was constant for 3 months.

Example 17

150 g of wet mass (paste) of fresh native Erwinia aeroidea cells containing the enzyme L-asparagine amidohydrolase was suspended in drinking water to a total volume of 500 ml; the homogeneous suspension contained 0.3 g of native cells/ml with a specific activity of 0.15 pmol of L-aspartic acid/min/mg of mass (37 °C) pH 5.0. The pH of the suspension was adjusted to 7.0 with 20% NaOH solution.

The procedure was then carried out according to Example 16; 100 g of cross-linked, permeabilized and heat-treated cells were obtained, the specific activity of which was 0.12 gmol L-aspartic acid/min/mg of mass. When binding the cells together by the filtration pressure technique, the procedure was then carried out according to Example 2, with the difference that the reaction time was 2 hours.

The described method produced a solid brown mass with a total weight of 75 g in the shape of an irregular plate. The obtained mass was cut into irregular smaller pieces and ground using a meat grinder; a fragile granular material was formed, which was suspended in 250 mg of acetone cooled to -20 °C and stirred intensively for 3 minutes. The material was then immediately sucked off with a sharp vacuum through a terylene sheet and sucked off on the sheet for about 20 minutes. It was then suspended in 250 ml of demineralized water and left to swell overnight at 10 °C. The material was then sucked off well and weighed.

63 g of bound cells with L-asparagine amidohydrolase activity with a specific activity of 0.05 μμιοί L-aspartic acid/min/mg of mass were obtained. The particle size distribution ranged from 550 to 1650 pm and their quantitative sedimentation was achieved after standing for 1 minute. Macroscopically, the particles formed brown amorphous granules. The stability of the material during storage under the conditions given in Example 6 was constant for 6 months.

Example 18

150 g of wet mass of fresh native Aspergillus niger cells containing the enzyme glucose oxidase with a specific activity of 0.105 pmol H ₂ O ₂ /min/ mg wt. of native cells was suspended in 1 liter of drinking water; the suspension contained 0.15 g wt./ml., the suspension was adjusted with stirring to a pH of 7.0 with 20% NaOH solution.

Covalent crosslinking of the intracellular content of individual cells was carried out according to Example 1 and 125 g of crosslinked cells with a specific activity of 0.08 μπιοί H ₂ O ₂ /min/mg of mass were obtained. Permeabilization of crosslinked and washed cells was carried out in an aqueous suspension containing 0.05 g of crosslinked cells/ml in three-well flasks according to the procedure described in Example 16. 107 g of crosslinked and permeabilized cells with a specific activity of 0.1 μπιοί H ₂ O ₂ /min/mg of mass were obtained. Heat treatment of the cells after permeabilization was not carried out. Mutual binding of the cells was carried out by the freezing technique according to Example 6 with the difference that the concentration of crosslinked and permeabilized cells in the reaction mixture was 0.08 g of crosslinked and permeabilized cells in the reaction mixture was 0.08 g of mass/ml. The treatment of the material after bonding was also carried out according to example 6.

62 g of wet mass of interconnected cells with glucose oxidase activity was obtained; the specific activity of the materials was 0.095 μπιοί H ₂ O ₂ / _; min/mg v. wt. The particle size distribution ranged from 100 to 1000 gm and their quantitative sedimentation was achieved after 12 minutes of standing. Macroscopically, the material formed dark gray amorphous pellets. The stability of the material during storage under the conditions specified in Example 6 was constant for 3 months. The material was subjected to mechanical and enzymatic stability testing according to the procedure specified in Example 6 under the conditions specified in the description of the invention; after 288 hours (12 days) of continuous shaking of the aqueous suspension of the bound cells, the specific activity was 70% of the original activity.

Example 19

150 g of wet mass (paste) of fresh native cells of Streptomyces phaeochromogenes with glucose isomerase activity was suspended in 1 liter of 0.05 M phosphate buffer pH 7.0.

The concentration of native cells in a homogeneous suspension was 0.15 g wt/ml and their specific activity was 0.021 μmol fructose/min/mg wt.

(60°C) pH 6.85.

Covalent crosslinking of the intracellular content of individual cells, including the method of their separation and washing, was carried out according to Example 1. 119 g of crosslinked and washed cells with a specific activity of 0.012 gmol fructose/min/mg of crosslinked cells were obtained. Permeabilization of crosslinked cells was carried out according to Example 6, with the difference that the concentration of crosslinked cells in the suspension was 0.08 g of crosslinked cells/ml and the temperature of the suspension during the 3-hour permeabilization was 60 °C. Further heat treatment of the crosslinked cells after their permeabilization was not carried out. Mutual binding of the cells was carried out by the freezing technique according to the procedure given in Example 5, with the difference that the concentration of cells in the reaction mixture was 0.12 g of crosslinked cells/ml. Treatment of the material after binding was also carried out according to Example 5.

71g of interconnected cells with glucose isomerase activity were obtained; the specific activity of the material was 0.01 μιηοΐ fructose/min/njg of wt. The particle size distribution ranged from 200 to 1300 pm and their quantitative sedimentation was achieved after standing for 4 minutes. Microscopically, the particles formed irregular plates with defined edges. Macroscopically, the material formed light brown scales. The stability of the material during storage under the conditions given in Example 6 was constant for 6 months.

F

Example 20

Native E. coli cells with a specific penicillin acylase activity of 7.0 pmol 6-APK/hr/mg wet mass were cross-linked and permeabilized as described in Example 1, except that the cross-linked cells had an activity of 5.8 pmol 6-APK/hr/mg wet mass and that permeabilization was performed with a suspension of cross-linked cells of 0.5 g wet mass/ml at concentrations of butyl acetate 5% v/v and Slovafol 909 1% v/v during 4 hours of continuous stirring at 25°C (500 rpm), so that the specific activity of the permeabilized cells was 8.0 pmol 6-APK/hr/mg wet mass.

An aqueous suspension of 50 g/100 ml was prepared from the permeabilized cells in the described manner, the pH of which was adjusted to 7.6. Then 5 ml of a 10% aqueous solution of Sedipur CL-930 was added to the suspension and, after 5 minutes of standing, 4 ml of a 25% aqueous solution of glutaraldehyde. The mixture was mixed intensively, filled into a syringe equipped with a needle with an internal diameter of 1.6 mm and extruded onto an aluminum tray in the form of cylindrical long shapes. After 2 hours of air drying at room temperature, about 2/3 of the material was decanted 3 times with water and then swelled in water for another 30 minutes to wash out the unreacted components, and about half of this amount was left to dry in air at room temperature for 24 hours after suction, after which the material was swollen in water for 24 hours and then suctioned (variant a). The second half of the material after 30 minutes of swelling and soaking was intensively mixed for 5 minutes with cooled acetone, the temperature of which was -27 °C, then sucked off, swollen for 24 hours in water and sucked off again by vacuum (variant b). The remaining 1/3 of the material after the initial 2-hour drying was further dried for a total of 24 hours, i.e. in the presence of unreacted reaction components (glutaraldehyde, Sedipur). Then the preparation was decanted 5 times with water, swollen for 24 hours in water and sucked off (variant c).

The dark brown aspirated materials were mechanically processed into the final form of short cylinders with a diameter of 1 mm. The specific penicillin acylase activities of the individual preparations showed the following values in pmol 6-APK/hour/mg of wet mass:

(a) 0.80 (drying without reagents) / (b) 0.75 (drying with acetone) / (c) 0.60 (drying with reagents).

Example 21

A wet paste of cross-linked and permeabilized cells with a penicillin acylase activity of 8.0 pmol 6-APK/hour/mg wet mass obtained according to Example 20 was used. A 10 g/20 ml suspension was prepared from this cell paste using a 51.4% aqueous solution of Sedipur CL-930 with vigorous stirring. A thin paste was prepared from this suspension by centrifugation (10,000 G/20 minutes), which was extruded into a 10% glutaraldehyde solution using a needleless syringe with an internal diameter of 2 mm. The process was accompanied by the formation of drops that remained on the surface of the aqueous glutaraldehyde solution for a while and then fell onto a sieve in the form of granules. The granules were left to pickle in the glutaraldehyde solution at laboratory temperature for 30 minutes and were then washed on the sieve for 1 hour with a slow flow of water. The aspirated granules, which were still soft, were dried for 24 hours in the open air at room temperature. After 48 hours of swelling in water, the dark brown granules were aspirated into the final form of the preparation, the penicillin acylase activity of which was 1.25 μμιοί 6-APK/hour/mg of wet matter.

Example 22

600 g of wet cell mass of Alcaligenes metalcaligenes containing aspartate ammonia lyase with a specific activity of 0.45 μμιοί L-aspartic acid/min./mg wet mass (pH 8.5, 37 °C) was suspended in drinking water to a total volume of 2 liters. The resulting cell suspension at pH 7.5 was sieved by stirring in the presence of 0.5% glutaraldehyde (w/v) for 1 hour at 25 °C. The reaction mixture was then diluted 10 times with water and the cells were separated by centrifugation. The cross-linked cell paste was suspended in a 1.5 M aqueous solution of ammonium fumarate containing 1 mg/ml MgSO ₄ .7H ₂ O at pH 8.5 to a concentration of 300 g wet cell mass/l and after adding Slovafol 909 so that its concentration in the suspension was 0.1%, the suspension was stirred for 48 hours at 30 °C. Then the suspension was centrifuged (6,000 G/30 min.) and after washing with 2x31 water, 416 g of wet cell mass with a specific activity of 0.35 μμιοί L-aspartic acid/mg wet mass was obtained.

g of wet paste of cross-linked, permeabilized and activated cells with aspartase activity was suspended in a 3% (w/v) solution of Sedipur CL-930 to a total concentration of 0.2 g cells/ml. At pH 7.0, the suspension was left to stand for 30 minutes and then centrifuged (6000 g/30 minutes). The obtained paste was extruded through a 1.6 mm diameter orifice into a 0.5% aqueous solution of glutaraldehyde pH 7.0, where it was left for 15 minutes at room temperature. The obtained material was then washed with 2 x 50 ml of water and air-dried at room temperature for 18 hours. The dried material was mechanically crushed into cylindrical particles with a length of 1-3 mm. After 10 hours of swelling in a 1.5 M aqueous solution of ammonium fumarate containing magnesium ions at 29°C, 3.8 g of solid, rapidly sedimenting particles with good hydrodynamic properties and an activity of 0.05 gmol L-aspartic acid/min./mg of wet mass of the materials were obtained.

Claims (4)

SUBJECT OF THE INVENTION Method for the production of stable preparations of inter-linked cells of prokaryotic and eukaryotic microorganisms with enzymatic activity, in particular bacteria, yeasts and other imperfect fungi, useful for biotransformation, characterized in that the content of individual cells of the parent organism is first crosslinked with dialdehyde, preferably glutaraldehyde. this reaction crosslinked permeabilized cells (by physical or physico-chentických or cherfiic- _(sneeze factors néépájí then reacted in vodj 'cool environment with soluble compound containing · ^. or least two primary or two secondary, amine groups, preferably with polyethyleneimine or hexamethylenediamine, and with dialdehyde, preferably glutardialdehyde, the resulting bound cells are, after separation, penetrated by water and optionally mechanically solidified by temporary partial dehydration by the action of organic salts. water-miscible solvents, preferably acetone or mixtures thereof with water, at a temperature of 10 to -30 ° C. * 2. A process according to claim 1, wherein the permeabilization of the cross-linked cells is carried out by stirring the cell suspension in an aqueous medium at temperatures of from 0 to 70 [deg.] C. at pH 4.0 to 9.0. a miscible or non-quenching solvent, in particular butyl acetate, chloroform or dimethylsulfoxide, optionally with simultaneous or subsequent changes in the ionic strength of the suspension, 3. The method of initiating is the reaction of the crosslinked cells with a water-soluble compound containing at least two primers. or two secondary amino groups with dialdehyde is carried out at temperatures below the melting point of the reaction mixture. 4. The process according to claim 3, wherein the reaction is carried out at a temperature of 0 to 30 [deg.] C., at a relative centrifugal force of 1 to 50,000 G or at a filtration pressure of 0 to 50,650 kPa.