CS209265B1 - Manufacturing process of stable preparates of interbonded cells of procaryonte and eucaryonte microorganisms with enzyme activity - Google Patents

Abstract

The invention protects a general stable manufacturing method enzyme-bound cell preparations activity useful for a variety of biotransformations; the essence lies in the chemical reaction of the individual cells with aldehydes, resulting in a single covalently cross-linked cells that are further permeabilize by chemical, physicochemical or physical factors. In another step with crosslinked and permeabilized cells with each other chemically bond through dialdehydes and diamines, optionally under physical action factors. There is no mutual chemical binding of cells for example, no organic or inorganic matter water-insoluble carrier.

Description

The invention relates to a process for the production of stable preparations of inter-linked cells of prokaryotic and eukaryotic microorganisms with enzymatic activity, for example bacteria, yeast and other imperfect fungi, useful for biotransformation.

Known methods for binding said cells to form stable preparations; may be characterized as physical, physicochemical, or chemical, depending on whether the binding of the cells to the carrier or enzyme in the cell is mediated, for example, by mechanically capturing the cells in the gel, van der Waals, polar interactions or covalent binding. Various water-insoluble synthetic or natural organic or inorganic carriers are commonly used, which primarily improve the hydrodynamic and sedimentation and thus separation properties of the bound cell preparations and contribute to the stabilization of the desired enzyme activity. Various known cell binding methods, their advantages and disadvantages, and their economic limitations 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 by incorporating native cells into a gel of natural origin is described, for example, in U.S. Pat. No. 113908, a chemical method, e.g. No. 200802 using synthetic methacrylamide, glycidyl methacrylate, hydroxyalkyl methacrylate and the like, spherical particles obtained by targeted polymerization. These particles must first be chemically modified by attaching distal spatial junctions with terminal amino groups and only then can the cells be bound to the modified polymer particles by means of polyfunctional agents.

Similarly, the process of binding various enzyme-active cells according to DOS 2,513,929 involves reacting the cells with the reactive monomer and subsequently polymerizing or copolymerizing them, or reacting with a glycidyl methacrylate polymer, optionally in combination with a covalent fixation of the enzyme inside the cells with a bifunctional agent.

Carriers of these and similar types are expensive preparations whose cost and availability limit the industrial use of bound cell preparations. Some progress in the development of bonding technology; cells of industrial importance is the method described in MS. No. 197101, the principle of which is based on the binding of production cells to ¹ cell carriers of different kinds of organisms, which are either whole intact or physically, chemically or biochemically treated cells or their insoluble fragments, by chemical, covalent binding, polyfunctional agents. In this method of cell binding, there is no economic limit to the cost of the carrier, since the carriers used are waste cells (waste cell mass) after various fermentation processes, the cost of which is largely negligible, respectively. zero. In the above MS. The method of binding the production cells to each other, i.e. without the use of a carrier, is also described. For this, however, the cells must to a large extent be autolysed naturally or artificially in order for them to bind to each other, despite the fact that the thus prepared bound cells have inferior mechanical and sedimentation properties and consequently lower specific activity as a result of previous partial autolysis. This disadvantage is solved by the method of covalent cross-linking of individual production cells with bifunctional aldehydes (cf. No. 195 063). However, in the case of re-use, cells must be isolated by centrifugation, since the dimensions of individual cross-linked cells are very small (approx. Another advantageous method of chemical binding of the production cells to each other, without the use of a carrier, is described in the Czech Patent Certificate No. 1 (pm), which results in their poor sedimentation properties which do not differ significantly from these properties in native cells.

203 607. This method, as before, is limited to the binding of cells with penicillin acylase activity to produce 6-aminopenicillanic acid by enzymatic hydrolysis of penicillin.

It has now been found that the basic principle that has been described for the method of producing bound cells with penicillin acylase activity according to U.S. Pat. No. 203 607, after further modifications and improvements, it is also applicable to the binding of cells of various production strains, respectively. cells with different enzymatic activities, regardless of whether they are a bacterial producer, yeast or other microscopic fungus, and regardless of whether the desired enzyme activity (the desired enzyme) is located in the periplasmic or intracellular space (in the cytoplasm). Similarly, the quality of the enzyme and the quality of the enzyme, respectively, is not critical in a sufficiently wide range, as illustrated by the examples. the type of enzyme-catalyzed biochemical reaction desired. For a given type of prokaryotic or eukaryotic cell, it depends on the localization of the desired enzyme in the cell and the quality of the enzyme, respectively. on the type of enzyme - catalyzed biochemical reaction and the known or predicted chemical composition of cell surfaces, the reaction conditions for the covalent cross - linking of the intracellular contents of individual cells and in particular the technical and reaction conditions for their permeabilization after crosslinking as well as individual technically must be chosen. methodologies and reaction conditions followed by cross-linking of cross-linked and permeable cells to obtain bound cell preparations with sufficiently high specific activities, good enzyme activity yield and other advantageous properties, particularly in relation to sedimentation rates and bound size distributions of cell particles, their hydrodynamic properties and their mechanical and enzymatic stability.

The invention therefore relates to a process for the production of stable preparations of inter-linked cells of prokaryotic and eukaryotic microorganisms with enzymatic activity, for example bacteria, yeast and other imperfect fungi, useful for biotransformation. It is based on the fact that the cell content of the parent organism is first crosslinked by reaction with a dialdehyde, preferably glutardialdehyde, after which the crosslinked cells are permeabilized by physical or physicochemical or chemical factors, then reacted in an aqueous medium with a soluble compound containing at least two primary or two secondary amine groups, preferably with polyethyleneimine or hexamethylenediamine, and with dialdehyde, preferably glutardialdehyde, the resulting bound cells are washed after separation with water and optionally mechanically solidified by transient partial dehydration by treatment with organic water-miscible solvents, preferably acetone or mixtures thereof with water, at a temperature of 10 to -30 ° C.

Said permeabilization of the 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 of 4.0 to 9.0, in the presence of a surfactant or an organic water-miscible or immiscible solvent such as butyl acetate, chloroform or dimethylsulfoxide, optionally with simultaneous or subsequent changes in the ionic strength of the suspension.

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

The process according to the invention yields stable preparations of interconnected cells with very good sedimentation and hydrodynamic properties and good stability of the desired enzyme activity, the size of the bound cell particles being controlled to a certain extent by the cell concentration, reaction conditions and technical method. The material obtained is also characterized by good mechanical abrasion stability due to agitation and friction of the particles. In principle, it can be used for various industrial biotransformations, implemented both by batch and semi-continuous and continuous technology, depending on the type of reactor. The choice of technology using bound enzymatically active cells prepared in the form of stable preparations according to the method of the invention should be based on the type of enzyme-catalyzed biochemical reaction and reaction conditions. The most preferred use of the preparations of the invention is in fixed bed reactive cell reactors. Another possibility is batch repeated conversion in the desired direction in the stirred reactor. The particles formed from interdependent cells are solid, in most cases sandy in nature, in a microscopic image visible as sharply bounded or unbounded platelets or irregular morphologies. Macroscopically, this material resembles a light brown, dark brown, reddish or black resin. The shape, size and color of the particles vary depending on the type of starting production cells used and the permeabilization and bonding technique used. The yield of immobilization is in the range of 20 to 80% and is also dependent on the aforementioned factors.

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

1. Chemical reaction of production cells in a mixed aqueous suspension with bifunctional cross-linking agents, thereby causing covalent cross-linking - crossing the intracellular content of individual cells and thereby fixing and stabilizing the desired activity of the key enzyme (s) contained in the periplasmic or intracellular space of individual cells. increasing the resistance of these cells to autolysis. Key factors that influence the success of this process step are cell concentration, crosslinker concentration, reaction time, suspension pH, reaction mixture temperature, and agitation intensity. These factors must be selected individually.

2. Dilution of cell suspension after reaction with water, 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 physicochemical or chemical factors, thereby leaching unreacted surface and intracellular surfaces components of a predominantly lipid nature will improve the diffusion properties of the cross-linked cells, particularly in the enzymes located in the intracellular space, and reveal other responsive groups capable of their surface and within the cellular space. The basic factors influencing the sufficient permeabilization of the cross-linked cells are the concentration of the cells in the aqueous suspension, the mixing intensity ^: temperature, presence of a suitable surfactant and its concentration, presence or absence of a suitable organic solvent and its concentration, pH and ionic strength of the suspension individual factors, etc. These factors must be selected individually for each cell type, alone or in combination.

3. Dilution of a cross-linked and washed cell suspension after permeabilization with water, centrifugation, washing with water and subsequent chemical reaction in the presence of a water-soluble compound containing at least two primary or secondary amine groups together with bifunctional cross-linking agents, conveniently performed in most cases conditions in which individual cells are in close contact with each other and in minimal movement. In many cases, it is preferred to bind the enzyme-active cross-linked and permeabilized cells to each other when the reaction mixture is cooled below the melting point or at a relative centrifugal force of greater than 100 G or at a filtration pressure of greater than 101 kPa. Sometimes, especially with some enzymatically active eukaryotic organisms, it is preferable to stir or leave the reaction mixture gently. Under these conditions, covalently cross-linked cellular particles are formed over time. The main factors influencing the properties of bound cells, particularly particle size, the desired entrapped enzyme activity, sedimentation, mechanical and hydrodynamic properties and their stability, as well as the immobilization winners, are cell concentration, concentration and quality of crosslinker, concentration and quality of water-soluble compounds containing amine groups, temperature and pH of the reaction mixture, reaction time and, last but not least, the technique used to conduct the reaction. These factors must also be selected individually.

After binding the cells, the product obtained is decanted several times with water, in the case of using the technique of binding at temperatures below the melting point of the reaction mixture, the product is allowed to thaw spontaneously and also decanted several times with water and suctioned off. The well-vacuumed wet mass of bound cells is either dried at about 60% air humidity at room temperature, in some cases it is advantageous to incorporate the aspirated material into chilled acetone with stirring, or ^- into its mixture with water, thereby mechanically solidifying the particles by their partial transient dehydration. The product is then swelled in an aqueous medium, sucked off after swelling, and optionally dried at about 60% humidity to constant weight at room temperature. The bound cells may be stored in well sealed glass or polyethylene containers at temperatures ranging from 0 to 10 ° C. The water content of the bound cell preparations varies between 40 and 65% by weight. Most preparations can be stored for several months without loss of original activity under the conditions described.

By the method of the invention, for example, stable preparations can be obtained in which the bound cells contain enzymes with, for example, hydrolase, isomerase, lyase, decarboxylase, oxidoreductase or other activity, either with a single activity or with any combination of enzyme activities, depending on the purpose. Thus, the bound cells may comprise, 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-aspartic acidase decarboxylase, and decarboxylase.

In the process for producing stable cell-to-cell preparations according to the invention, production cells containing various enzymes may be used, including different species and strains of bacteria, actinomycetes, yeast and other imperfect fungi. Obviously, it is advantageous to employ mitotic organisms obtained by breeding or targeted genetic manipulation which contain a high amount of the desired enzyme or enzymes, since the obtained bound cells then have a high specific enzyme activity.

The size of the bound cells in the stable preparations of the invention was assessed microscopically (by optical microscope) using a calibrated eyepiece. Some preparations were scanned by scanning electron microscope. The sedimentation properties were evaluated 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 measuring cylinder and monitoring the time required for quantitative sedimentation of the particles. Metal sieves for screen analysis were also used for particle size analysis and bound cells were sized by using three sets of sieves, with sieves having mesh sizes ranging from 0.03 to 2.0 mm.

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

For example, penidlinacylase activity was evaluated spectrophotometrically by color reaction of p-dimethylaminobenzaldehyde with 6-aminopenicillanic acid using penicillin potassium salt as a substrate, β-galactosidase activity chromatographically with densitometric evaluation of glucose and galactose increments using lactose as substrate. L-.alpha.-amino-.epsilon.-caprolactam hydrolase activity was evaluated by measuring the loss of L-.alpha.-amino-.epsilon.-caprolactam as a substrate and the product addition, L-lysine, also chromatographically with densitometric evaluation. L-asparagine amidohydrolase activity was measured by increasing the concentration of ammonium ions by nesslerization colorimetrically, using L-asparagine as a substrate. L-aspartate ammonia lyase activity was evaluated chromatographically with a densitometric evaluation of the L-aspartic acid concentration increase using ammonium as fumaric acid as 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 appropriate amino acids as substrates. Glucose oxidase activity was determined colorimetrically after oxidation of o-dianisidine with hydrogen peroxide by peroxidase, and glucose isomerase activity was also colorimetrically determined by fructose after reaction with cysteine and carbazole. In both cases glucose was used as substrate.

The stable preparations of chemically coupled cells obtained by the process of the invention represent significant technical and economic progress in both industrially feasible biotransformations.

Further details will be apparent from the examples which illustrate the method according to the invention but do not limit it in any way. The enzymatic activity of native and bound cells is also shown in the examples.

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 / hr / mg wet cell mass (42 ° C) pH 7.6, 500 ml of homogeneous aqueous suspension with a cell concentration of 0.25 g in .wt./ml; The pH of the suspension was adjusted to 7.0 with 20% NaOH. A 25% aqueous glutardialdehyde solution was added to the stirred aqueous suspension so that its actual concentration in the reaction mixture was 1% (w / v). The reaction was allowed to stir for 3 hours at 20 ° C. After this time, the suspension was diluted with potable water to a total volume of 5 liters, mixed and centrifuged (5000 G / 30 minutes). 110 g / wt. individually covalently cross-linked, washed cells whose specific activity was 5.1 pmol 6-APK / hr / mg v / w.

An aqueous suspension was prepared from the thus obtained wet paste of individually crosslinked cells at a concentration of 0.25 g v / w. To a homogeneous stirred suspension whose pH was adjusted to 7.0 with 25% NaOH was added butyl acetate and Slovafol 909 so that their actual concentrations in the suspension were 2.5 vol% for the first and 0.5 vol% in the latter case. The suspension was allowed to warm to 45 ° C in a water bath 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 centrifuged (5000 G / 30 minutes). 78.3 g / wt. individually cross-linked and permeabilized cells with a specific activity of 6.8 μπιο! 6-APK / hr / mg w / w

150 µm of a homogeneous aqueous suspension having a pH of 7.0 and a cell concentration of 0.3 g / wt / ml was prepared from a portion of the wet paste as described above. The stirred suspension was cooled to 10 ° C and Sedipur KA (polyethyleneimine) was added so that its concentration in the suspension was 0.5 vol%. A 25% aqueous solution of glutardialdehyde was then added to the slurry with stirring until the slurry concentration was 1% (w / v) and the agitated reaction mixture was expanded into 10 ml polyethylene cuvettes and depending on the relative centrifugal sphere used. interconnected wells with penicillin activity. The reaction was carried out at 0 ° C in refrigerated Janetzki K 26 centrifuges for 3 hours. After the reaction, the bound cell preparations were decanted twice with water, vacuum aspirated through a terylene cloth and treated with acetone: water (1: 1) cooled to -20 ° C, aspirated again and allowed to swell in water at 10 ° C for 16 hours. They are then aspirated, vacuum-weighed, weighed and evaluated for their sedimentation rate, particle size, sedimentation volume and specific activity in the manner described in the description. lezu. The results are shown in the following table.

Rel. centrifugal force (G) 1100 4500 « »JO 200. . 28 595. -y —. ---- _____  - í * ’ Particle size (gm) 25—150 75—150 ? 5—300 75—450 1—2 Speed sed. particles (min at 1 G) 7 5 1 1 Quantum, not sitting. Particle volume per sed. (ml) 6.5 5 . 3 3 - Spec. activity (μ, πιοί 6-APK / hour / mg in weight of binding cells) 6.2 5.9 5.1 4.65 6.8 Microscopic image borderless formations with irregularities. edge unity. cells Macroscopic appearance brown amorphous particles cellular suspension

* Standing the reaction mixture at 0 ° C for 3 hours at 10 ° C, then centrifuging, washing and suspending the sediment in water.

Example 2

The same fresh Escherichia coli cell paste with penicillin acylase activity was crosslinked as described in Example 1.

The permeabilization of the aqueous suspension of individually covalently crosslinked cells was performed in the same manner except that for permeabilization, NaCl was used at a concentration of 5.8 g / liter (1 M NaCl concentration in suspension); the concentration of Slovafol 909, reaction time and conditions were the same as in Example 1; The pH of the suspension was adjusted to the same value only after 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 / h / mg w / w. a homogeneous aqueous suspension having a cell concentration of 0.3 g / wt / ml was prepared; The pH of the suspension was 6.9. The suspension was allowed to warm to 40 ° C and was continuously stirred at this temperature with a steel propeller stirrer (300 rpm) for 72 hours. After this heat treatment period, the suspension was cooled to ° C and brought to its original volume with potable water.

To 100 ml of this suspension was added Sedipur KA and glutardialdehyde as described in Example 1 with stirring, and the mixed suspension was poured into a bag of linen filter cloth. The bag was sealed and placed in a horizontal position between two metal plates and its contents were subjected to a filter pressure of 1.25 x ^{10 4} kPa using a hydraulic press at room temperature for 30 minutes.

A brown-black solid mass with a total weight of 25 g in the form of an irregular plate was formed as described. The obtained mass was cut into irregular then-pieces and homogenized in 200 ml of drinking water intermittently for about 15 minutes at room temperature using a kitchen mixer. After homogenization, the material was decanted twice with 1 liter of water, vacuum sucked well through a terylene sheet and weighed.

2 g of wet bound cell mass * was obtained with an irregular particle size distribution of 50-5000 gm; the specific activity of the bound cell homogenate was 3.3 gmol of 6-AKP / hr / mg in wt. and their quantitative sedimentation was caused by standing for 2 minutes. '

Example 3

From wet crosslinked, permeabilized and washed Escherichia coli cell paste with penicillin acylase activity with a specific activity of 4.64 gmol 6-APK / hr / mg w / w, wherein the cross-linking of individual cells was performed 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 in weight / ml were prepared.

A 10% w / v aqueous solution of hexamethylenediamine at pH 7.0 was added to the homogeneous slurry at 15 ° C and pH 7.0 with stirring so that its concentration in the slurry was 0.5% (w / v). , and after 15 minutes of standing, a 25% aqueous glutardialdehyde solution was added with stirring to a concentration of 2% (w / v) in the reaction mixture. The reaction mixture was centrifuged (10 minutes / 5000G) and a circular wet cake of 5 cm diameter and 1 cm height was made on a pad of linen cloth. Moist> cake was inserted into a bag of the same material and the bag is placed between two metal plates in a horizontal position and subjected to filtration pressure 1,25.10 ⁴ psi using a hydraulic press at room temperature for 24 hours. A portion of the dark brown solid (10 g) was mixed in water and aspirated as described in Example 2; Bound cells with penicillin acylase activity were obtained with a particle size distribution of 100 to 1000 gm whose specific activity was 2.75 gmol 6-APK / h / mg v / w.

and their quantitative sedimentation was caused by standing for 3 minutes.

AND!

Example 4

To 80 ml of an aqueous suspension of cross-linked, permeable, heat-treated, Escherichia i coli cells with penicillin acylase activity (specific activity was 5.16 gmol 6-APK / hr / mg w / w, cell concentration in suspension 0.25 g in A 10% strength aqueous solution of hexamethylenediamine at pH 7.0 was added to give a suspension concentration of 0.5% (w / v). The mixture was stirred briefly and then allowed to stand at 11 ° C for 20 hours. A 25% aqueous glutardialdehyde solution was then added to the aqueous suspension with stirring to a concentration of 2% (w / v) in the suspension. The reaction mixture was then poured onto a 14 cm diameter round dish made of aluminum foil and placed in a horizontal freezer _; boxes; the layer thickness on the dish was about 0.5 cm, the reaction was run at -23 ° C for 48 hours. After this time, the dish was removed from the roasting box and poured about 100 ml of drinking water and the material allowed to thaw at room temperature. After thawing, the bound cells were decanted three times with 100 ml of drinking water, vacuum aspirated through a terylene sheet and weighed.

14.3 g / wt. aspirated bound cells. This amount was suspended in 107 ml of acetone p.a., pre-cooled to -23 ° C. The material was stirred in chilled acetone for 3 minutes and then vacuumed immediately and sharply as described. The bound cells were swelled overnight in 0.05 M phosphate buffer pH 7.6 at 8 ° C and then vacuum aspirated as described and aspirated on the cloth for about 20 minutes.

14.2 g / wt. brown, scaled-bound cells that sediment quantitatively at 10 minutes on standing; the particle size distribution ranged from 75 to 300, gm and their specific activity was 4.25 gmol 6-APK / hr / mg in wt. The material was subjected to stability testing of its enzyme activity and mechanical stability of the particles under the conditions described in the disclosure. Po 528; hours (22 days) of continuous shaking of the suspension of bound cells, their specific activity was 85% of the activity of the original while maintaining 70% of the total mass of the material relative to the weight of the original.

Example 5 g wt. (paste) fresh native Alcaligenes faecalis cells with penicillin acylase activity were suspended in 80 ml of drinking water; The pH of the suspension was adjusted to 7.0 with 20% NaOH solution. The suspension contained 0.25 g w / w. of native cells / ml with a specific activity of 0.25 gmol and 6-APK / hr / mg in wt. (42 ° C) pH 7.6. Single cell cross-linking was performed according to Example 1. Permeabilization of the cross-linked cells: Example 2. Heat treatment of the cross-linked and permeabilized suspension also according to Example 2, except that the stirred suspension temperature was 37 ° C under otherwise identical treatment conditions.

To 100 ml of an aqueous suspension of cross-linked, permeabilized and heat-treated cells. And, after cooling to 10 ° C with stirring, Šedipur KA (polyethyleneimine) was added to a suspension concentration of 0.5% (w / v). The suspension was then allowed to stand for 30 minutes, and a 25% aqueous solution of glutardialdehyde was added with stirring to a concentration of 1% (w / v) in the suspension, mixed briefly and the procedure was continued as above. Example 4, except that the reaction time during freezing was 5 hours.

11.8 g / wt. dark brown bound cells that sedimented quantitatively at 4 minutes on standing; the particle size distribution ranged from 150 to 300 µm and their specific activity was 0.23 pmol 6-APK / h / mg v / w. The enzymatic and mechanical stability of the particles observed under the conditions described in the specification for 10 days (240 hours) was 100% of the original activity at that continuous shaking time while maintaining 94% of the original material mass.

Example 6

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

The covalent cross-linking of the intracellular contents of the individual cells and their washing was carried out according to Example 1. The permeabilization of the cells was accomplished by the combined effect of ionic strength change, surfactant presence and temperature in the stirred suspension of crosslinked cells according to Example 2 except that Slovafol 910 was used the same concentration and temperature during the 3 hour permeabilization in the presence of 1 MNaCl was only 40 ° C instead of 45 ° C. 190 g / wt. (pastes) of cross-linked, permeabilized and washed cells having a specific activity of 0.029 pmoles glucose + galactose / min / mg in wt. The paste of these cells was suspended in drinking water so that the cell concentration was 0.3 w / w and heat treated at 40 ° C for 72 hours with stirring as described in Example 2.

After the volume of the suspension was adjusted to the original volume by drinking water and the suspension was cooled to 15 ° C, the cell binding was carried out as described in Example 5 except that the reaction time during freezing was 4 hours and the reaction mixture was immediately quenched after addition of glutardialdehyde. 375 ml in polyethylene bags, sealed and placed horizontally on aluminum trays. The method of treating the bound cells after the reaction (thawing, decanting, filtration, acetone treatment, swelling, etc.) was performed according to Example 4 except that the bound cells were swelled overnight in demineral. water under otherwise identical conditions.

110 g / wt. Bound cells with beta-galactosidase activity with a specific activity of 0.021 pmoles glucose + galactose / min / mg in wt., macroscopically resembling a dark brown resin of a sandy nature, microscopically appearing as unbound irregular platelets with a particle size distribution of 75-300 µm. 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 described in the disclosure; after 1632 hours (68 days) of continuous shaking of the material in the aqueous suspension, the specific activity of the bound cells was 110% of the original activity, and the remaining mass of the material after this time was 75% of the original mass. 25% of the mass of the material due to abrasion and thus reduction of the particles after said shaking time passed through the sheet during filtration and suction. The bound cell material remained unchanged as described, including specific activity during storage in a sealed polyethylene bottle stored 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 were produced as described in Example 6 except that it was used instead of changing ionic strength to permeabilize the cross-linked cells. 1 M NaCl) combined effect of the same temperature and the same surfactant in the presence of 2.5 vol% chloroform and 1 vol% dimethylsulfoxide under otherwise identical reaction and handling conditions.

The characteristics of the preparation were similar to those in Example 6 except that the specific activity of the material was 0.028 gmols of glucose + galactose / min / mg in wt. bound cells. The stability of the material under the storage conditions of Example 6 did not change for 3 months of follow-up.

Example 8

250 g wt. fresh native Escherichia coli cell paste containing beta-galactosidase as in Example 6 was crosslinked, permeabilized and heat treated in an aqueous suspension as described in Example 6. The cell binding in the presence of hexamethylenediamine and the treatment of bound cells after binding were performed as described in Example 6. 4.

96 g wt. orange-brown, scaled-bound cells that sedimented quantitatively after 12 minutes; the particle size distribution ranged from 75 to 350 µm and their specific activity was 0.019 pmoles glucose + galactose / min / mg wet material.

Example 9

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

The covalent cross-linking of the intracellular content of individual cells including the method of washing and separating the wet cross-linked biomass was carried out according to Example 1. Permeabilization of 100 ml of an aqueous suspension of yeast with a cell concentration in suspension of 0.25 g v / w and pH 7.0 was performed at 40 ° C in the presence of 1 vol% dimethylsulfoxide and 0.5 vol% toluene in the absence of surfactant by vigorously mixing 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, briefly mixed, cells centrifuged (5000 G / 20 minutes), milky turbid decant and cell sediment suspended in the same volume of drinking water and centrifuged again. A total of 20 g wt. individually cross-linked, permeabilized and washed yeasts having a specific activity of 0,022 μιηοΐύ glucose + galactose / h / mg. v. wt.

g in wt of the yeast treated as described above was suspended in 380 ml of drinking water and the pH of the homogeneous suspension of cells adjusted to 7.0 with 20% NaOH with stirring. _x The suspension was then poured into 1 liter erlenmaye- graves ⁱⁿ a flask and added Sedipur KA (polyethyleneimine) so that its concentration in the suspension was 0.5 vol.%. The suspension was stirred briefly and allowed to stand for about 15 minutes. A 25% aqueous glutardialdehyde solution was then added with stirring to a concentration of 1% by volume in the reaction mixture. The flask was placed on a low-speed reciprocating shaker at 40 / min. The reaction was carried out at 29 ° C for 3 hours under the stated stirring conditions. After this time, the reaction mixture was diluted with potable 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 sucked off under vacuum on a terylene cloth and weighed. 9.7 g wt. the aspirated and washed material was suspended with stirring in 100 ml of acetone cooled to -20 ° C, stirred for 3 minutes and then immediately suctioned off sharply in a vacuum-like manner and then sieved for about 10 minutes before being suspended in 100 ml of 0, 05 M phosphate buffer> pH 7.0 and swelled at 10 ° C overnight. 8.1 g in wt. yeast-linked yeast with beta-galactosidase activity.

The specific activity of the material was 0.009 μιηοΐύ glucose + galactose / h / mg v / w. bound cells. The particle size distribution ranged from 100 to 850 μιη and resulted in quantitative sedimentation for 2 minutes. The storage stability of the material under the conditions of Example 6 was constant for 3 months.

Example 10

470 g w. fresh native yeast Cryptococcus laurentii cells with L-alpha-amino-epsilonamino-caprolactam 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 a buffer value with 20% KOH. A slurry containing 0.1 g / wt. of native cells / ml with a specific activity of 6.01 μηιοίύ L-lysine / h / mg in wt. (37 ° C) pH 7.5.

The covalent cross-linking of the intracellular content of the individual yeast in the 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, mixed briefly and centrifuged on a Sharples Separator. 320 g / wt. crosslinked and washed cells in the form of a wet paste having a specific activity of 3.83 μπίθΐύ L-lysine / hr / mg v / w. * »

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

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 in wt. individually cross-linked, permeabilized and washed yeast cells having a specific activity of 4.15 μιηοΐύ L-lysine / hr / mg v / w.

From a portion of the wet mass of the cells obtained in this way, 200 ml of a homogeneous aqueous suspension was prepared, the pH of which was adjusted to 7.0 with 20% KOH; the suspension contained 50 mg wt. cells / ml. The suspension was divided into two equal volumes (each 100 ml) labeled A and B, and Sedipur KA (polyethyleneimine) was added to each of them, with stirring, to a concentration of 0.5% by volume, of which the enzyme-linked cells bound to each other by a variety of techniques as follows:

A. The suspension was cooled to 10 ° C with stirring and allowed to stand for 30 minutes. A 25% aqueous glutardialdehyde solution was then added with stirring to a 1% v / v suspension concentration. After brief mixing, cell binding was performed by freezing the reaction mixture under the conditions of Example 5; the method of treating the particles after binding was carried out according to Example 4.

3.8 g in wt. bonded yeast with L-alpha-amino-epsilon-caprolactam hydrolase activity; the specific activity of the material was 0.52 μιηοΐύ L-lysine / hr / mg in wt. The particle size distribution ranged from 75 to 1450 gm and resulted in a quantitative sedimentation of 1 minute standing. Microscopically assessed, the particles consisted of acicular sharply bounded crystal-like formations. Macroscopically, the material resembled heterogeneously crystallized potassium dichromate, including a typical brown-red coloration. The storage stability of the material under the conditions of Example 6 was constant for 6 months.

B. To the slowly stirred suspension at room temperature, glutardialdehyde was added at the same concentration as in variant A and the cells were coupled to each other by the slow agitation technique of the reaction mixture as described in Example 9, including a method of treating the resulting particles after binding.

2.75 g (w / w) were obtained. bonded yeast with L-alpha-amino-epsilon-caprolactam hydrolase activity; the specific activity of the material was 1.8 μηιοίύ L-lysine / h / mg in wt. The particle size distribution ranged from 15 to 900 µm and was quantitatively sedimented by standing for 5 minutes. Microscopically assessed particles formed irregular shapes with irregular edges. Macroscopically, the material formed amorphous cotton-like structures. The storage stability of the material under the conditions of Example 6 was constant for 6 months.

Example 11

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

Covalent cross-linking of the intracellular contents of individual cells was performed according to the example

1. Permeabilization of the cross-linked cells was performed according to Example 2 except that the temperature of the stirred suspension during permeabilization was 40 ° C. The heat treatment of the cells was also performed according to Example 2. Cell binding was performed by the freezing technique as in Example 6; a method of treating the bound cells after binding is also exemplified

6.

198 g of wet mass of bound cells with L-aspartate ammonia lyase activity were obtained; the specific activity of the material was 0.5 μηιοί L-aspartic acid / min / mg in wt. The particle size distribution ranged from 75 to 300 μιη and was quantitatively sedimented by standing for 3 minutes. Microscopically assessed material consisted of boundless formations with irregular edges. Macroscopically the material consisted of light brown scales of mica-like character. The glass stability of the glassware under the conditions of Example 6 was constant for 1 month.

Example 12

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

Covalent cross-linking of the intracellular content of individual cells was performed according to Example 1; their permeabilization was performed as described in Example 2, except that Slovafol 910 was used in the same concentration under otherwise identical conditions instead of Slovafol 909. The heat treatment of the cells was also performed according to Example 2. Cell binding was performed by freezing the reaction mixture as described in Example 6, including the method of treating the post-binding material.

312 g / wt. interconnected cells with L-aspartate ammonia lyase activity; the specific activity of the material was 0.63 μηιοί L-aspartic acid / min / mg in wt. The particle size distribution ranged from 50 to 250 μιη and was quantitatively sedimented by standing for 10 minutes. Microscopically assessed material consisted of boundless formations with irregular edges. Macroscopically, the material formed beige scales. The storage stability of the material under the conditions of Example 6 was constant for 3 months.

Example 13

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

The covalent cross-linking of the intracellular contents was carried out according to Example 1, including the method of permeabilizing them. The heat treatment of the cells after permeabilization was carried out according to Example 2. 150 ml of a homogeneous aqueous suspension containing 0.3 g v / w of the treated cells were used to prepare the bound cells by centrifugation as described in Example 1, including the method of their treatment. detention. The results are shown in the following table.

Rel. centrifugal force (G) i 100 4500 10,200 28,595 1 * Particle size (μιη) 25—150 50—320 75—400 75—425 1—2 Speed sed. particles (min at 1 G) 12 6.5 1 1 Quantum, not sitting.

Particle volume per sed. (ml) 10 7.5 4 4 - Spec. activity (/ L-aspartic acid / min / mg v / w binding b. 0.2 0.13 0.09 0.09 0.28 Microscopic image neohr. formations with irregularities. edge unity. cells Macroscopic appearance amorphous light brown particles cellular 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 the wet paste of fresh native Mycobacterium sp. containing L-aspartic acid decarboxylase enzyme with a specific activity of 7,1 units / gram in weight (1 unit of activity is the amount of enzyme which releases 100 μΐ CO ₂ from L-aspartic acid in 5 minutes at 30 ° C and pH 5.5) 100 ml of a homogeneous aqueous suspension with a native cell concentration of 0.3 g in .wt./ml; The pH of the suspension was adjusted to 7.0 with 20% NaOH with stirring.

Covalent cross-linking of the intracellular contents of individual cells was performed according to the example

1. Permeabilization of the cross-linked cells was performed according to Example 2 except that the concentration of cross-linked cells in the stirred suspension was 0.1 g v / w, the pH of the suspension was 6.1, instead of Slovafol 909 Slovafol 910 was used at the same concentration. and instead of 1 M NaCl, chloroform at a current concentration of 5 vol% was used, the suspension temperature was 35 ° C and the permeabilization duration was 1 hour. Further heat treatment after permeabilization of the cross-linked cells was not performed. The washed, crosslinked and permeabilized cells were bound to each other by the freezing technique as described in Example 5, except that the concentration of cells in the reaction mixture was 0.1 g v / w. Treatment of the bound cells after their mutual reaction was performed according to Example 4.

It was. 12.1 g. cells bound to each other with L-aspartic acid decarboxylase activity having a specific activity of 0.2 J / g w / w. material. The particle size distribution ranged from 150 to 1000 µm; the particles sedimented spontaneously but could be well filtered and sucked off. Macroscopically, the particles consisted of amorphous gray-yellow flakes with poorly wet surface. The storage stability of the material under the conditions of Example 6 was constant for 1 month.

Example 15

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

Covalent cross-linking of the intracellular contents of individual cells was performed according to the example

1. Permeabilization of the cross-linked cells according to Example 2 with the difference that the concentration of the cells in the permeabilized suspension was 0.1 g v / w, the pH of the suspension was 6.3 and Slovafol 910 of the same concentration was used instead of Slovafol 909. it was 35 ° C during permeabilization. Next, the reference given in the previous example (Example 14) was followed.

9.35 g in wt. cells bound to each other with L-lysine decarboxylase activity with a specific activity of 0.67 J / g w / w. material. The particle size distribution ranged from 75 to 300 μπι, and their quantitative sedimentation was caused by standing for 4 minutes. Microscopically, the particles consisted of flaky plates with irregular edges. Macroscopically, the particles resembled a fine resin of a light brown color. The storage stability of the material under the conditions of Example 6 was constant for 1 month of follow-up at weekly intervals.

Example 16

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

Covalent cross-linking of the intracellular content of individual cells was performed according to Example 1 and 394 g of wet mass of cross-linked, washed cells with a specific activity of 0.015 pmol L-aspartic acid / min / mg w / w were obtained. Permeabilization of a suspension containing 0.25 g / wt. cross-linked cells / ml were performed in the presence of only 1 M NaCl, the pH of the suspension was 6.0, and the suspension was shaken in 500 ml three-liter flasks (stoppers) on a rotary shaker at 260 rpm at 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.

of cross-linked cells, 181.5 g / wt.

crosslinked -permeabilized and washed Q cells with a specific activity of 0.029 pmol L-aspartic acid / min / mg in wt. The heat treatment of the cells after permeabilization was carried out according to example 2, the cell binding by freezing technique according to example 6 and the treatment of cells after mutual binding according to example 4.

1 g g / wt. interconnected cells with L-asparagine amidohydrolase activity; the specific activity of the material was 0.039 μπιοί. L-aspartic acid / min / mg. The particle size distribution ranged from 75 to 750 µm and was allowed to quantitate sedimentation for 8 minutes. Microscopically assessed material consisted of boundless formations with irregular edges. Macroscopically, it formed a scales of light brown color. The storage stability of the material under the conditions of Example 6 was constant for 3 months.

Example 17

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

Example 16 was followed; 100 g in wt. cross-linked, permeabilized, and heat-treated cells whose specific activity was 0.12 gmol L-aspartic acid / min / mg in wt. The cells were coupled to each other by filtration pressure, as described in Example 2, except that the reaction time was 2 hours.

In this way, a solid brown mass with a total weight of 75 g was formed in the form of an irregular plate. The mass obtained was cut into irregular smaller pieces and ground by means of a meat grinder; a brittle granular material was formed, which was suspended in 250 mg acetone cooled to -20 ° C and stirred vigorously for 3 minutes. Subsequently, the material was immediately vacuum-sucked sharply through a terylene sheet and sucked on the sheet for about 20 minutes. It was then suspended in 250 ml of demineralized water and allowed to swell at 10 ° C overnight. The material was then well aspirated and weighed.

63 g. cells bound to each other with L-asparagine amidohydrolase activity with a specific activity of 0.05 μπιοί L-aspartic acid / min / mg in wt. The particle size distribution ranged from 550 to 1650 µm and resulted in quantitative sedimentation for 1 minute. Macroscopically, the particles formed brown amorphous granules. The storage stability of the material under the conditions of Example 6 was constant for 6 months.

Example 18

150 g wet mass of fresh native Aspergillus niger cells containing glucose oxidase enzyme with specific activity 0,105 pmol H ₂ O ₂ / min / mg w / w native cells were suspended in 1 liter of drinking water; the suspension contained 0.15 g w / w, the pH of the suspension was adjusted to 7.0 with 20% NaOH with stirring.

The covalent cross-linking of the intracellular content of the individual cells was carried out according to Example 1 to obtain 125 g / wt. cross-linked cells with a specific activity of 0.08 μπιοί H ₂ O ₂ / min / mg in wt. Re-destabilization of the cross-linked and washed cells was carried out in an aqueous suspension containing 0.05 g / wt. of cross-linked cells / ml in three-well flasks as described in Example 16. 107 g / wt. cross-linked and permeabilized cells with a specific activity of 0.1 μπιοί H ₂ O ₂ / min / mg in wt. Thermal treatment of the cells after permeabilization was not performed. Cell binding was performed by the freezing technique of Example 6, except that the concentration of cross-linked and permeabilized cells in the reaction mixture was 0.08 g v / w. Treatment of the post-bonded material was also carried out according to Example 6.

62 g of wet mass of mutually bound cells with giucose oxidase activity were obtained; the specific activity of the materials was 0,095 μπιοί H ₂ O ₂ / _; min / mg w / w The particle size distribution ranged from 100 to 1000 gm and resulted in quantitative sedimentation for 12 minutes. Macroscopically, the material was a dark gray amorphous pellet. The storage stability of the material under the conditions of Example 6 was constant for 3 months. The material was subjected to mechanical and enzyme stability testing as described in Example 6 under the conditions described in the disclosure; after 288 hours (12 days) of continuous shaking of the aqueous suspension of bound cells, the specific activity was 70% of the original activity.

Example 19

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

Concentration of native cells in homogeneous sus! the pension was 0.15 g in wt / ml and their specific! the activity was 0.021 μπιοί fructose / min / mg in wt.

(60 ° C) pH 6.85.

The covalent cross-linking of the intracellular contents of individual cells, including the method of separation and washing, was carried out according to Example 1. 119 g / wt. cross-linked and washed cells with a specific activity of 0.012 gmol fructose / min / mg in wt. Permeabilization of the cross-linked cells was performed according to Example 6 except that the concentration of cross-linked cells in the suspension was 0.08 g v / w and the temperature of the suspension during the 3-hour permeabilization was 60 ° C. Further heat treatment of the cross-linked cells after their permeabilization was not performed. Cell binding was performed by freezing as described in Example 5, except that the concentration of cells in the reaction mixture was 0.12 g v / w. Treatment of the post-bonded material was also performed according to Example 5.

71g of bound cells with glucose isomerase activity were obtained; the specific activity of the material was 0.01 μιηοΐ fructose / min / njg v. wt. The particle size distribution ranged from 200 to 1300 µm and was quantitatively sedimented by standing for 4 minutes. Microscopically assessed were irregular plate particles with bounded edges. Macroscopically, the material was a light brown scale. The storage stability of the material under the conditions of Example 6 was constant for 6 months.

F

Example 20

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

An aqueous suspension of 50 g / 100 ml was prepared from the permeahilized cells described above, the pH of which was adjusted to 7.6. 5 ml of a 10% aqueous solution of Sedipura CL-930 was then added to the suspension and after standing for 5 minutes 4 ml of a 25% aqueous solution of glutardialdehyde. The mixture was vigorously mixed, filled into a syringe equipped with a 1.6 mm ID needle and extruded onto an aluminum tray to form cylindrical long formations. After air drying at room temperature for 2 hours, about 2/3 of the material was decanted 3 times with water and then swelled in water for 30 minutes to wash out unreacted components, and about half of this amount was allowed to air dry at room temperature after aspiration. After 24 hours, the material was swelled in water for 24 hours and then aspirated (variant a). The other half of the material, after swelling and occupying for 30 minutes, was vigorously stirred for 5 minutes with chilled acetone at -27 ° C, then aspirated, swelled in water for 24 hours and vacuum evacuated again (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 reactants (glutaraldehyde, Sedipur). The preparation was then decanted 5 times with water, swelled 24 hours in water and aspirated, (variant c).

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

(a) 0.80 (reagent-free drying) / (b) 0.75 (acetone-drying) / (c) 0.60 (reagent-drying).

Example 21

A wet paste of cross-linked and permeabilized cells with a penicillin acylase activity of 8.0 pmol 6-APK / h / mg wet mass obtained according to Example 20 was used. A 10 g / 20 ml suspension was prepared from this cell paste with 51.4% aqueous solution. Sedipuru CL-930 with vigorous stirring. A thin paste was prepared from this suspension by centrifugation (10,000 G / 20 minutes) and extruded into a 10% glutardialdehyde solution using a syringe without a 2 mm ID needle. The process was accompanied by the formation of drops which remained on the surface of an aqueous solution of glutardialdehyde for a while and then fell onto the underlying sieve in the form of granules. The granules were allowed to pickle in a glutardialdehyde solution at room temperature for 30 minutes and then washed on the sieve for 1 hour by slow water flow. The aspirated granules, which were still soft, were air-dried at room temperature for 24 hours. After 48 hours of swelling in water, the dark brown granules were aspirated to a final formulation whose penicillin-lysis activity was 1.25 μπιοί 6-APK / hour / mg wet mass.

Example 22

600 g of wet mass of Alcaligenes metalcaligenes containing ammonia aspartate aspartate with a specific activity of 0,45 μπιοί 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 crosslinked by stirring in the presence of 0.5% glutardialdehyde (w / v) for 1 hour at 25 ° C. The reaction mixture was then diluted 10x with water and the cells were collected by centrifugation. The cross-linked cell paste was suspended in a 1.5 M aqueous ammonium fumarate solution containing 1 mg / ml MgSO ₄ .7H ₂ O at pH 8.5 to a concentration of 300 g wet cell mass / lt and after adding Slovafol 909 such that the suspension was 0.1% and the suspension was stirred at 30 ° C for 48 hours. Thereafter, the suspension was centrifuged (6,000 G / 30 min) and after washing with 2 x 31 water, 416 g of wet mass of cells with a specific activity of 0.35 μπιοί L-aspartic acid / mg wet mass were obtained.

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

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.