CS231656B1 - Enzyme catalysts for biotransformation and process for their production - Google Patents

Abstract

The invention relates to enzyme catalysts for biotransformations based on immobilized enzymes of microbial, animal and plant origin and a method for their production. The enzyme catalysts obtained by the process according to the invention consist of various enzymes of varying purity, preferably technical or crude enzyme preparations, carrying in particular hydrolase, isomerase, lyase or racemase activity, which are immobilized by a chemically reactive water-soluble polymer formed by the reaction of a water-soluble substance containing at least two primary and/or secondary amino groups, such as polyethyleneimine, hexamethylenediamine, lysine and polylysine with bifunctional aldehydes of dicarboxylic acids, such as glutaraldehyde. The resulting water-insoluble particles of immobilized enzymes are further solidified in various ways with the aim of obtaining well-sedimenting particles with good mechanical and hydrodynamic properties and high specific activity of the desired enzyme. The method of producing immobilized enzymes does not require any organic synthetic, organic or natural water-insoluble carriers, which significantly simplifies the production of these catalysts. The immobilized catalysts are useful for molecular biotransformations and allow for discontinuous, repetitive or continuous biotechnologies for the production of pharmaceutical, food or agriculturally important products or intermediates.

Description

The invention relates to enzyme catalysts for biotransformation based on immobilized enzymes of microbial, animal and plant origin and to a process for their preparation. The enzyme catalysts obtained by the process according to the invention consist of various enzymes of different purity, preferably technical or crude enzyme preparations, carrying, in particular, hydrolase, isomerase, lyase or racemase activity, which are immobilized by a water-soluble chemically reactive polymer. containing at least two primary and / or secondary amino groups such as polyethyleneimine, hexamethylenediamine, lysine and polylysine with bifunctional dicarboxylic acid aldehydes such as glutaraldehyde. The resulting water-insoluble particles of the immobilized enzymes are further strengthened in various ways to obtain well-settling particles with good mechanical and hydrodynamic properties and high specific activity of the desired enzyme. The process for the production of immobilized enzymes does not require any organic synthetic, inorganic or natural water-insoluble carriers, thereby substantially economizing the production of these catalysts. Enzyme catalysts are useful for industrial biotransformation and allow the development of discontinuous repeated or continuous biotechnologies for the production of pharmaceutically, food or agriculturally important products or intermediates.

231 656

The present invention relates to enzyme catalysts for biotransformation based on mobilized enzymes of microbial, animal or plant origin and a process for their production.

The enzyme catalysts of the invention are useful for industrial biotransformation, respectively. they allow the development of discontinuous or continuous biotechnologies for the production of pharmaceutically, food or agriculturally important products or intermediates.

Known methods of enzyme immobilization can generally be characterized as physical eg sorption and inclusion, physico-chemical eg polar bond and chemical eg covalent bond. For this purpose, various water-insoluble synthetic or natural organic or inorganic materials are used as carriers of the desired enzyme activity. In summary, these enzyme binding procedures are detailed in, for example, the Immobilized Enzymes / edit monographs. 0. Zaborsky, CRC Press, A Division of the Chemical Rubber Co., USA Cleveland, Ohio, 1974 /, Immobilized Enzymes for Industrial Reactors / edit. R.A. Messing, Acad.Press New York,

San Francisco, London, 1975 / Insolubilized Enzymes / edit. Salmon M., Saronia C., Garattini S., Raven Press, New York, 1974. and Industrial Enzymes, Recent Advances (J.C.). Johnson, Noyes Data Corporation, Park Ridge, New Jersey, Cemical Technology Review 85, USA, 1977]. A variety of synthetic organic inert or chemically active polymers obtained by targeted polymerization, for example based on methacrylaldehyde, glycidyl methacrylate, hydroxyalkyl methacrylate, natural polymers such as cellulose and its derivatives, inorganic materials such as glass etc. are used as carriers for enzyme immobilization.

Carriers of these and similar types are generally expensive preparations whose cost and availability and their possible prior modification and activation by various agents limit the preparation and use of the immobilized enzymes. For these reasons, especially in the industrial use of immobilized enzymes, there has been a tendency in the past few years in both the scientific and patent literature to move from more or less defined carriers based on synthetic polymers or porous glass to less expensive materials. From an economically acceptable point of view, a variety of natural materials, especially chitin, collagen and, above all, waste biomass after various fermentation processes seem to be attractive, whose price is largely zero. These are mainly waste yeast after beer production, waste mycelium after penicillin production, etc., ie waste cell3.

231 856 various microorganisms which can be used as insoluble carriers for predominantly covalent or coordinate covalent binding of enzymes to the surface of individual cells or cellular aggregates.

Even in the Czechoslovak literature and especially in patent literature, the shift of interest forced by economization of production has been observed in the last few years, for example by elaborating the procedure of immobilization of penicillin acylase enzyme for production of 6-aminopenicillanic acid to differently treated or native microbial cells or their fragments using bifunctional aldehydes, forming a mutually covalently crosslinked network of the mobilized enzyme together with cells having the character of an insoluble carrier, according to the author's certificate no. 2088931. A similar method, but unlike the previous method using the flocculation of the enzyme with the carrier-like cells, the flocculants entering into reaction with the bifunctional aldehydes, is described for immobilization. Amyloglucosidase enzyme in MS. No. UStbH, the use of this catalyst for the production of glucose and other enzymatic conversion products from starch is disclosed in U.S. Pat. Certificate No.

Similarly, covalent cross-linking of different enzymes by different bi- and bi-resp. polyfunctional agents, in particular glutaraldehyde or toluene diisocyanate to form a water insoluble covalently crosslinked network of enzymatically active proteins / Jansen E.F. and Olson, A.C; Sheet. Biochem. Biophys. 9, 221, 1969]. These procedures do not require water-insoluble carriers at all. In contrast to the previous processes (binding to cell surfaces), the latter procedure provides lower yields of the immobilized enzyme and usually these preparations are characterized by lower stability of the immobilized enzyme activity. Cross-linking of enzymes by the highly reactive crosslinking agents often leads to considerable or complete inactivation of enzyme activity. For suppressive strong inactivation of enzyme activity in cross-linking of enzymes, eg glutaraldehyde, a competitive reaction with an additional protein such as albumin is used during the cross-linking reaction of the desired enzyme, eventually cross-linking in the presence of

diamines such as hexamethylenediamine (Avrameas, S., Immunochemistry 6, 43, 1969).

A special case is the use of water-soluble chemically inert or chemically reactive polymers, in the first case, for example, by sorption of the dissolved enzyme to a water-soluble polymer, thereby forming a stabilized water-soluble polymer.

In the latter case, the water-soluble polymer is chemically activated and, when activated, is mixed with the enzyme, thereby covalently binding the enzyme to the activated polymer to form a chemically stabilized but water-soluble enzyme. An example of the preparation of stabilized ducally soluble enzymes is dextran-sorbed glycerol kinase (U.S. Pat. No. 3,962,037) or covalently bonded Warbler and cyanobromide-activated water-soluble dextran (NSR DOS 2,312 824) or the same enzyme covalently bound to male-soluble copolymers. with vinylmethyl ether, ethylene, styrene or vinyl acetate. Also described is a method of chemically activating proteins such as albumin with glutaraldehyde to produce water-soluble chemically active proteins [Attalla and Hultin, J.Food.Sci., 42,

7, 19777]. Using the latter methods, immobilized but water-soluble enzymes result, while immobilized enzymes by remaining water-soluble cannot be used for industrial biotransformation by conventional reactor types whose use is limited to special types of ultrafiltration reactors, which is associated with considerable demands. for equipment. This method of immobilization is mostly used to stabilize enzymes for analytical and cine-diagnostic practice.

Recently, we have found that cell catalysts useful for biotransformation based on immobilized prokaryotic or eukaryotic cells of various types of microorganisms and plant cells containing various enzymes can be prepared using reactive water-soluble polymers according to U.S. Pat. The resulting particles of mobilized cells are characterized by very good sedimentation properties, a high specific activity and a high immobilization yield. This procedure has been applied in various modifications in order to verify in principle the feasibility of preparing immobilized water-insoluble enzyme catalysts, in other words, different degrees of purity of purified enzymes isolated from microbial, plant and animal sources have been used instead of enzyme active cells of different species.

Arises _| the question of the expediency of using the present process for obtaining immobilized enzymes if the process can in principle and β good yields be utilized using enzymatically active cells, a process not requiring prior purification and isolation of the desired enzymes from producer cells of various microorganisms.

231 BM

The reasons for the use of the procedure in question arise from the following.

1. Some enzymes are extracellular, ie they are not retained in the production cells of microorganisms, which excludes the use of immobilized cells.

2. Many of the enzymes that remain in cells and have high molecular substrates (proteins, fats, polysaccharides) would be unused because cell wall permeability in microorganisms is limited to low molecular weight compounds, which again excludes the use of immobilized cells in these cases.

3. A number of industrially produced enzymes are of non-microbial origin / e.g. trypsin, chymotrypsin, papain etc./.

4. The cost of a number of immobilized enzyme preparations is known to be significantly influenced by the cost of the carrier; the present process does not require any organic, inorganic or natural water-insoluble carriers.

The present invention provides biotransformation enzyme catalysts consisting of enzymes of microbial, plant or animal origin, immobilized with a chemically reactive, water-soluble, water-soluble substance containing at least two primary and / or secondary amino groups such as polyethyleneimine, hexamethylenediamine, lysine and polylysine with bifunctional aldehydes of dicarboxylic acids, such as gl utaraldehyde, optionally further mechanically strengthened.

As the enzyme material, these catalysts may contain microbial, plant or animal enzymes for various purity and s'v HODO technical or crude enzyme preparations carrying particular hydrolase, isomerase, lyase _t racemázovou and other activity.

The present invention further provides a process for the preparation of said catalysts, characterized in that the above-mentioned aqueous solutions of enzymes having a protein concentration of 5.0-500.0 mg / ml are immobilized by contacting a water-soluble, chemically reactive polymer formed by the reaction. water-soluble substances containing at least two primary and / or secondary amino groups, preferably with polyethyleneimine and 8 bifunctional dicarboxylic acid aldehydes, preferably 8 with glutaraldehyde, at a temperature ranging from 0 to 60 ° C in aqueous medium, at a pH ranging from 4 0 to 12.0, wherein the reaction of said enzyme material with said polymer is carried out at a temperature in the range of -30 to 50 ° C in an aqueous medium and

231 U6 pH in the range of 5.0 to 9.0, the insoluble particles formed after washing from the reaction mixture are washed with water or buffer and then mechanically solidified.

It is preferred that an aqueous solution of polyethyleneimine with a molecular weight distribution of 100,000 to 800,000 is used to prepare the water-soluble polymer.

Preferably, the reaction of said enzymes with said polymer is carried out at a temperature of 0 to 45 ° C, at a relative centrifugal force of 1 to 50,000 g, at a filtration pressure of 0 to 50,000 KPa, while standing or stirring or intermittently stirring and standing.

The particles of immobilized enzymes are optionally further mechanically consolidated by partial dehydration by treatment with organic solvents, preferably acetone or adhesive solutions in organic solvents or mixtures thereof with water, at temperatures of +30 to -25 ° C, optionally before or after mechanical consolidation. they are appropriately postformed and partially dried.

The advantages of the process according to the invention are that immobilized water-insoluble enzymes with good specific activity and very good sedimentation properties and good immobilization yield, which of course fluctuates according to the type, are obtained by means of chemically reactive water-soluble polymers (hereinafter RRP). and in the case of highly sensitive enzyme activities compared to the use of free highly reactive crosslinking agents, in particular glutaraldehyde. The mechanism of RRP formation is outlined in the description of the invention. AO 231 458 does not require any water-insoluble organic, inorganic or natural carrier for immobilization.

The manufacturing process of the present invention consists essentially of two technological operations. In a first stage, a chemically reactive water-soluble polymer (RRP) is prepared, preferably by reacting commercially available flocculants used to purify wastewater containing a polyethyleneimine fraction, e.g. Sedipur KA or Sedipur CL-930, with a bifunctional crosslinking agent, e.g. glutaraldehyde, in aqueous preferably by stirring the reaction mixture.

'· * »

For example, the polymerization rate can be monitored by varying the absorbance of the color Schiff base increments in the visible spectrum (e.g. at 550 nm), the concentration of which is mainly dependent on the ratio of the two reactants, i.e. crosslinker: reacting compound, reaction time and temperature. The reaction can be

-7231 SM to describe first-order kinetic. In the second phase, an aqueous solution of the desired enzyme in the range of actual protein concentrations in the reaction mixture of 5.0 to 5.0 is mixed into the partially or quantitatively reacted RRP solution (depending on the sensitivity of the immobilized enzyme to the partially unreacted glutaraldehyde), usually after 1 to 24 hours of reaction. 500.0 mg / ml, and depending on the immobilization technique used, in particular mixing, standing, freezing, filtration pressure, centrifugal field, extrusion of the resulting particles by suitable equipment and subsequent partial dry or wet drying, the size, shape, character, porosity and specific activity of the particles of immobilized enzymes.

The process for obtaining the immobilized enzymes according to the invention also comprises processes for the subsequent treatment of the immobilized particles to increase their mechanical stability and strength, in particular partial dehydration of the particles by treatment with acetone or other organic solvents, optionally and preferably solutions of such commercial adhesives in organic solvents which form a protective coating. micro-layer of the water-insoluble film on the particle surface, under individual treatment conditions, provided that the enzymatic reaction rate of diffusion is not very significant from a technological point of view.

In principle, various microbial, plant and animal enzymes, in particular extracellular microbial and plant and animal proteases, for example differently pure preparations of alkaline, neutral and acidic proteases from different microbial strains, for example of the genus Bacillus, plant proteases for example papain and animal, can be used for the production of the immobilized enzymes. for example trypsin and chymotrypsin and other hydrobases of microbial origin isolated to varying degrees of purity from culture liquid or microorganism cells, such as penicillin.1 inamidohydrolase and beta-galactosidase from E. coli cells, yeast invertase, glucose isomerase from Streptomycetes and the like. The use of so-called technical, not very pure enzyme preparations is preferred, where the content of the enzyme in the preparation is 1.0-10.0% of the total amount of protein and is expected to be used in larger production volumes for industrial biotransformation. In the preparation of such immobilized enzymes, it does not matter if the solutions of the technical enzymes contain a certain proportion of cell detritus from the individual microbial cells or plant or animal tissues resulting from the isolation process prior to the immobilization, i.e. if the above crude enzyme preparations are used. 231 858

The particle size of the immobilized enzymes obtained by the process of the invention was assessed microscopically (optical microscope) using a calibrated eyepiece. Some preparations were scanned by scanning electron microscope. For screening of particle sizes, metal screens were used for screen analysis and bound enzymes were sized according to size using three sets of screens, the screens having openings ranging from 0.1 to 2.0 mm. The sedimentation properties were evaluated by suspending 2.5 wet masses of the preparation in a total volume of 25 ml of demineralized water in a calibrated graduated cylinder and monitoring the time required to quantitatively settle the particles.

The activities of the mobilized enzymes were evaluated for. mixing and tempering a defined amount of particles in the substrate solution under measurement conditions in the zero order kinetics region of the enzyme reaction and enzyme activity respectively. specific activity expressed as the amount of product umoles produced, respectively. % product / mg or gram wet mass of the particles, or as a percentage of conversion of the converted substrate to product, unless otherwise indicated in the examples.

After decanting and vacuum filtration of the particles of mobilized enzymes their average moisture / water content / 90 Z.

In the following examples, commercial flocculants of the Sedipur type, namely Sedipur KA or Sedipur CL-930, containing polyethyleneimine having a molecular weight of 200,000 to 300,000 at a concentration of approximately 30 weight percent are used as substances containing at least two primary and / or secondary amino groups. or polylysine hydrochloride having a molecular weight of about 40,000 to 50,000.

In the following, the invention is illustrated in more detail by the examples without limiting them.

-9Examples

Example 1 231 6M

50 ml of a 2 Z (w / v) aqueous solution of Sedipur CL-S30 (BASF, A.G.) in demineralized water was prepared. To this solution, whose pH was 6.1, was added a 25 Z aqueous solution of glutar aldehyde (MERCK) so that its concentration in the reaction mixture was 1.0 Z (v / v). The reaction mixture was placed on a laboratory shaker and the polymerization rate was monitored spectrophotometrically at 550 nm (Unicam, SP-500, 10 x 10 mm square glass cells) at selected time intervals. The reaction was carried out with continuous stirring at room temperature. Absorbance values from individual samples taken over time from the reaction mixture were measured against demineralized water. The reaction was allowed to proceed for 24 h.

0.75 g of freeze-dried technical preparation of beta-galactosidase isolated from Escherichia coli cells with specific activity 30 / µmol o-nitrophenol / min / mg protein / 37 ° C, pH 7.0, o-nitrophenyl beta-D-galactopyranoside substrate / was dissolved in 0.05 M phosphate buffer pH 7.0 to a total solution volume of 25 ml; a technical enzyme solution was produced with a total protein concentration of 30 mg / ml

To 25 ml of the above-described technical beta-galactosidase solution was added 25 ml of the above-described RRP, the reaction mixture was stirred briefly, and the resulting precipitate containing 15 mg protein / ml was poured onto a 20 cm aluminum foil dish and reaction mixture dish. stored in a freezer at -20 ° C for 3 hours. After this time, the dish was removed from the hox, poured the frozen contents on the dish with approximately 50 ml of the same buffer in which the enzyme was dissolved and allowed to thaw slowly at room temperature for 30 min.

After this time, the formed voluminous particles were poured into 200 ml of the same buffer and decanted several times with this buffer each time after quantitative sedimentation of the particles by standing. The particles were then sucked off well on a frit through a filter paper and weighed (0.92 g).

0.92 g of wet mass of the particles of mobilized enzyme, after previous mechanical homogenization of the voluminous mass, were suspended in 20 ml of acetone previously cooled to -20 ° C and stirred vigorously for 2 minutes using a propeller stirrer. After this time, the particles were sharply sucked off on the frit several times

-10231 651 washed with the same buffer and well aspirated and washed material weighed (0.45 g).

The specific activity of the material was 8.6 µmol o-nitrophenol / min / mg wet mass of immobilized enzyme. The material consisted of beige-colored flakes of beige in size of 0.5 to 2 mm, which sedimented quantitatively on standing within one minute. A microscopic image and a scanning electron microscope image showed some porosity of particles resembling polyurethane foam by their nature; the particles formed amorphous formations with irregular edges.

Example 2

50 ml 27 w / v / v aqueous solution of Sedipur CL-930 in demineralized water was prepared. The flocculant solution was vacuum filtered through a S-4 porcelain frit to completely remove the insoluble fraction of hydrophilic groats.

To a 50 ml perfectly soluble clear filtrate was added a 25 Z aqueous solution of glutaraldehyde to a concentration of 1.0 X (v / v) in the reaction mixture, and proceeded as described in Example 1 except that the reaction proceeded 3. hours under otherwise identical reaction conditions and polymerization rate monitoring conditions.

2.5 g of the lyophilized technical preparation of beta-galactosidase as in Example 1 was dissolved in 25 ml of phosphate buffer as described in Example 1. A technical enzyme solution with a total protein concentration of 100 mg / ml was formed.

To 25 ml of the technical beta-galactosidase solution was added 25 ml of the RRP prepared as described and mixed. The reaction mixture containing 50 mg / ml protein was placed on a rotary shaker at 160 rpm and stirred continuously for one hour and then allowed to stand for 1 hour at room temperature. The resulting insoluble particles were suctioned off through the textile material and thoroughly washed on a frit with two liters of 0.05 M phosphate buffer pH 7.0, sucked again thoroughly under vacuum and weighed; 3.75 g of wet mass of immobilized enzyme were obtained.

The wet particles of the immobilized beta-galactosidase were suspended in acetone under the conditions and method described in Example 1 and the well aspirated and washed material weighed in the same way (1.12 g).

The specific activity of the material was 15.1

231 658 µmol o-nitrophenol / min / mg wet mass of irradiated particles. The material consisted of irregular beige plates with a particle size distribution of 0.3 mm and 2.8 mm, which sedimented quantitatively on standing for a few tens of seconds.

One g wet mass of the immobilized beta-galactosidase particles was suspended in 25 ml of a 5 Z / v / v lactose solution dissolved in 0.1 M phosphate buffer pH 7.0, placed in a thermostat at 37 ° C and at a time the rate of enzymatic hydrolysis of lactose to glucose and galactose during continuous stirring was monitored.

Quantitative conversion of (100 Z) lactose to the two monosaccharides occurred under the reaction conditions in 3 hours. The enzymatic hydrolysis with the same catalyst feed was repeated a total of five times so that after completion of the reaction, the reaction mixture was poured through a thick polyamide mesh, the collected particles were washed with 100 ml of said buffer and resuspended in fresh lactose feed under otherwise identical reaction conditions at the same reaction time. . The average degree of conversion / five repeated cycles of using the same batch of immobilized galactosidase was 98.2 Z.

Example 3

50 ml RRP was prepared as described in Example 1 except that the polymerization reaction time was one hour. Next, 50 ml of a technical acacia solution were prepared by dissolving 6 g of the lyophilized enzyme in 0.05 M phosphate buffer pH 7.6 and making up to the indicated volume. This resulted in a technical enzyme solution containing 120 mg of protein / ml with specific activity of penicinaselase 260 yumol of 6-aminopenic acid (/ h / mg of protein) pH 7.6, temperature 42 ° C, K-salt-benzylpenicillin substrate. The enzyme was isolated from high-production mutant E cells. coli.

The enzyme solution was quantitatively poured into a 200 ml glass vial into which a vertical steel propeller stirrer was introduced and stirring was performed at 120 rpm. The RRP solution was then mixed with the enzyme solution in a 1: 1 volume ratio to a total volume of 100 ml; the actual protein concentration in the reaction mixture was 60 mg / ml. The reaction mixture was continuously stirred for one hour at room temperature, then the stirrer was switched off and the resulting suspension was allowed to stand for 30 minutes before being poured over

-12231 Ml gently mixed onto a 0.1 mm steel sieve and the sieve washed by demineralized water float. The voluminous particles were suspended in 100 ml of 0.05 M phosphate buffer pH

7.6 and sucked off well under vacuum and suspended with stirring in 50 ml of acetone previously cooled to -20 ° C. The material was vigorously stirred for three minutes and then drained vigorously, sucked off on the frit for about 5 minutes and then washed with one liter of said buffer, aspirated and weighed (4.3 g).

The specific activity of the material was 12.6 µmol of 6-APK / h / mg wet mass of the imohilized particles. The material consisted of light brown particles of approximately spherical shape with an average size distribution of 0.45 - 2.0 mm, and its quantitative sedimentation was caused by standing for several tens of seconds.

g of the wet mass of the immobilized warbler acylase was suspended in 50 ml of 6 ml of a solution of benzylpenicillin K-salt dissolved in demineralized water and continuously stirred and tempered at 37 ° C with continuous pH adjustment. to a value of 7.8 with dilute ammonia water (15 Z v / v), the reaction rate of converting the benzylic foam 1in to 6-APK was monitored. Quantitative conversion of the sulfate to said product occurred in the reaction conditions in 90 minutes. The enzymatic hydrolysis with the same catalyst feed was repeated a total of ten times so that after the reaction was complete, the reaction mixture was poured through a thick polyamide mesh, the collected particles were washed with 100 ml of water and resuspended in fresh feed of benzyl foaming solution under otherwise identical reaction conditions at the same reaction time. and the enzyme biotransformation process. The average degree of conversion / 10 repeated cycles of using the same batch of immobilized foamy acylase was 97.3 Z, the residual penicillin concentration was zero and the average amount of penicillin and 6-APK degradation products was 2.5 Z.

Example 4

50 ml RRP pos was prepared as described in Example 1 except that it was used in place of Sedipur CL-930. Sedípur KA. Further, 50 ml of a technical activity warbler iinacylase with specific activity was prepared as described in Example 3 except that the enzyme solution in the buffer contained 60 mg / ml protein.

ml of RRP solution was mixed with 50 ml of enzyme solution; a reaction mixture with a current protein concentration of 30 mg / ml was formed.

-13231 S5B

Further, the enzyme immobilization was carried out as in Example 1 (freezing the reaction mixture for three hours). The formed particles were then overflowed with buffer and decanted several times with approximately 100 ml of 0.05 M phosphate buffer pH 7.6 after quantitative sedimentation of the particles by standing, then vacuum-sucked well through the textile material and weighed off (2.3 g).

Said amount of immobilized penicillin acylase was treated with precooled acetone as described in Example 1 and well aspirated and phosphate buffer washed particles weighed (1.46 g) and homogenized into smaller pieces with a sharp spear. The immobilized and treated material was then swelled in 50 ml of said buffer overnight at +8 ° C.

The specific activity of the material was 4.5 µmol of 6-APK / h / mg wet mass of the immobilized particles. The swelling material consisted of beige bounded solid irregularly shaped plates with an average size distribution of 0.8 «4.0 mm and sedimented quantitatively by standing for a few seconds.

One gram of the wet mass of the immobilized foamed cell was suspended in 50 ml of a 6 Z / w / v solution of desacetoxycephalosporin-G K salt dissolved in demineralized. The reaction rate of the conversion of desacetoxycephalosporin G to 7-degradation of tox.ycef alosporanic acid (7-) was monitored during continuous stirring and tempering at 37 ° C with continuous pH adjustment to 7.6 with dilute NaOH solution (0.1 M). ADCK /. The substrate was quantitatively converted to the product under 150 minutes. Enzyme hydrolysis with the same catalyst feed was repeated three times as described in Example 3. The average conversion rate / 3 cycles of the same immobilized penicillin acylase feed was 93.6 Z, the residual desacetoxyceph alosporin concentration was 1 Z, the average amount of desacetoxycephalosporin degradation products and 7- ÁDCK was 4.3 Z.

Example 5

100 ml RRP solution was prepared as described in Example 1. A protease solution of animal origin was prepared as follows. 3.0 g dry mass of lyophilized chymotrypsin / n.p. Drugs - SPOFA / with a specific activity of 3.0 U / mg / casein substrate, temperature 35 ° C, pH 8.0 /, was dissolved in 40 ml of 0.05 M phosphate buffer pH 7.6 and with continuous stirring the pH was enzyme solution

-14231 8M adjusted to 7.2 with 10N NaOH solution and supplemented with said buffer to a total volume of 50 ml. A protease solution of pH 7.2 and a protein concentration of 60 mg / ml was formed. The solution was divided into two equal volumes (25 25 ml) and proceeded as follows.

To 25 ml of this solution was added 25 ml of RRP, mixed briefly and rapidly, and the precipitate was poured onto an aluminum foil dish and allowed to freeze at -20 ° C in a freezer for three hours; the actual protein concentration in the reaction mixture was 30 mg / ml. After this time, the dish was removed, poured over with the same phosphate buffer and allowed to thaw at room temperature, then decanted several times with the same buffer with gentle stirring with a stick, then pour the liquid and filter through a filter press in vacuum. The wet, well aspirated material, after filtration with a sharp spear, was homogenized into approximately equal particles and divided into two equal parts by weight.

One portion of the material was overflowed with 50 mL of the same phosphate buffer and allowed to swell at +8 ° C until the next day (sample labeled α). The second part was treated with 25 ml of acetone pre-cooled to -20 ° C with stirring for 2 minutes, then the material was immediately sucked sharply under vacuum and decanted several times with the same phosphate buffer, sucked again, homogenized with a eighth spear, overflowed with about 50 ml of the same buffer. allowed to swell at +8 ° C the next day / sample labeled and? 1.

To 25 ml of the same enzyme solution (second volume) was added 25 ml of the same RRP solution to give a solution of the actual protein concentration in the reaction mixture of 30 mg / ml and stirred for 30 minutes on a laboratory shaker at room temperature. The precipitate was allowed to stand at this temperature for two hours. After this time, the reaction mixture was centrifuged at 5000 g for 5 minutes, the clear supernatant was decanted and the sediment was stirred in 100 ml of the same buffer, decanted several times with the same buffer, vacuumed after decanting the particle through the textile material, washed with buffer and homogenized well as described.

Next, the immobilized material was treated with acetone, washed, homogenized and swelled as described for the sample and this example. The sample was labeled b ^.

The enzymatic activities of the bound chymotrypsin by these immobilization methods are summarized in the following table.

-15231 3M

Sample identification ^and i ^and 2 ^b 1 obtained wet mass of material / g / 2.3 1.6 1.5 specific activity / j / mg wet mass / 7,3.10 ^-3 4,4.10 ^-3 0.015 immobilization yield / 2 / 0.75 0.3 1.0

+ / based on the starting soluble chymotrypsin.

All obtained immobilized chymotrypsin samples sedented quantitatively in a few tens of seconds, the freeze-dried immobilized protease particles showed lower particle porosity in the scanning electron microscope images compared to the samples obtained by the agitation / standing centrifugation technique, in accordance with the observed specific activity of immobilized materials.

Example 6

50 ml RRP was prepared as described in Example 1 except that the polymerization reaction time was 16 hours. A protease solution of plant origin was prepared as follows. 6.0 g of Papain dry matter (Fluka, A.G., Switzerland) with a specific activity of 1.0 J / mg was dissolved in 50 ml of 0.05 M phosphate buffer pH 7.6. A papain solution having a protein concentration of 120 mg / ml was formed.

To a 50 ml solution of this enzyme was added 50 ml RRP and the reaction mixture was continuously stirred using a vertically placed 60 rpm steel stirrer at room temperature for 30 min and then allowed to stand for 30 minutes. The resulting suspension was vacuum filtered through a filter press, washed with one liter of the same buffer and vacuum vacuumed again, and the insoluble material weighed (10.2 g). After homogenization, the immobilized material was divided into two equal parts by weight (approximately g5 g), the first part being suspended in 50 ml of precooled acetone as described in Example 5, including washing and swelling, the second part being introduced into a syringe and extruded into cylindrical formations by a syringe without needle on aluminum foil. After extrusion, the material was air dried at room temperature for three hours. After partial drying, the material was homogenized with a knife into approximately the same rolls, poured over with 50 ml of the same buffer and allowed to swell overnight at +8 ° C.

3.2 g of immobilized papain having a specific activity of 0.017 j / mg wet matter for the first (acetone treated material) and 3.6 g of the same immobilized enzyme having a specific activity of 0.0095 j / mg wet matter in the second case were obtained (postformed). partially dried material. Both materials sedimented quantitatively within a few tens of seconds, the average particle size distribution of the first (treated) material was 0.45 d df * 1.75 mm, in the second sample (postformed material), the particles formed a cylindrical formation of approximately 1.0 1.5 mm thick and 1.5 * 4

4,0 mm long.

Example 7

25 ml of a 0.1 Z (w / v) aqueous solution of polylysine.HCL having a molecular weight of 40,000 to 50,000 were prepared in distilled water. To this solution was added a 25 Z aqueous glutaraldehyde solution so that its concentration in the reaction mixture was 0.15 percent (v / v). The reaction mixture was placed on a laboratory shaker and the polymerization rate was monitored spectrophotometrically at 415 nm at selected time intervals.

The reaction was carried out with continuous stirring at room temperature for 4 hours.

ml of crude preparation of invertase obtained by cell breakdown and partial centrifugation of cellular detritus of brewing waste yeast Saccharomyces car1 collectgensis at protein concentration

11.6 mg / ml and a specific activity of 2.8 U / mg (sucrose substrate, pH 4.9, temperature 30 ° C) were mixed with 25 ml RRP, the preparation of which was mentioned in the previous paragraph. The reaction mixture was vigorously stirred and poured onto an aluminum foil dish and stored in a freezer at -25 ° C for 4 hours. After this time, the dish was removed from the freezer and poured over 0.05 M acetate buffer pH 4.9 and allowed to thaw spontaneously at room temperature. The resulting sponge precipitate was vacuum filtered and introduced into a syringe and extruded into cylindrical form by syringe without needle. on aluminum foil. After extrusion, the material was dried for 5 hours

-1 7231 BSB air at room temperature. After partial drying, the material was homogenized with a knife into approximately the same rollers, which were poured over. . Ml of the same acetate buffer and swelled overnight at.

0 ° C.

1.04 g of cylindrical immobilized invertase postformed particles were obtained. The indicated amount of immobilized enzyme was suspended in 50 ml of 10% sucrose solution dissolved in said acetate buffer pH 4.9 and was stirred at 30 ° C with continuous stirring. an enzyme inversion of sucrose to a mixture of glucose and fructose was performed. Enzyme biotransformation was quantitative under the indicated reaction conditions at 6 hours. _Λ This procedure was repeated three times with the same catalyst bed, wherein each of the cycles, enzymatic inversion of terminated, the catalyst was "separated via polyamide-strainer, washed with said buffer and reused. The average degree of enzyme inversion / 3 cycles of reuse of the same catalyst feed was 86.2 Z.

EXAMPLE 8 liters of a concentrated aqueous suspension of Streptomyces phaeochromogenes cells (40 vol.% Hiomase) containing glucose isomerase enzyme were cooled to + 5 ° C. Then, the suspension was disintegrated by passing twice through a slotted mechanical disintegrator at a pressure of 250 atm. The suspension between the individual disintegrations was continuously cooled so that the maximum temperature of the disintegrated mixture did not exceed 45 ° C. Then the suspension was centrifuged (Janetzki S-70, 5,000 g, 30 minutes) and the cloudy, strongly opalescent supernatant was decanted. 2.8 liters of crude enzyme preparation with a concentration of 105.6 mg / ml protein and a specific glucose isomerase activity of 0.8 yumol fructose / min / mg protein (temperature 60 ° C, pH 9.3, substrate glucose) were obtained.

To 100 ml of the crude glucose isomerase obtained above was added 100 ml RRP, the preparation of which was given in Example 1, except that the reaction time * of polymerization was 1 hour and the concentration of the flocculant was 4 Z * and the concentration The actual protein concentration in the reaction mixture was 52.8 mg / ml. The reaction mixture was continuously stirred for one hour using a propeller steel stirrer at 60 rpm at room temperature. After this time, the resulting spongy precipitate was centrifuged (5,000 g / 15 min), super-1,831 an natant slit and the resulting mass was introduced in the form of a wet cake into a filter press bag, the bag opening was folded and secured with metal clamps. metal plates in a horizontal position and briefly subjected to a hydraulic pressure of 1.25.10 kPa using a hydraulic press at room temperature for 10 minutes.

After this time, the mass in the form of a plate was mechanically disrupted by breaking into smaller pieces and introduced into a meat grinder with a special steel insert having a pore size of 1 mm and extruded through this insert into amorphous granules. The material was suspended in 100 ml 2 of a solution of glue (Kanagom) in acetone previously cooled to -20 ° C and stirred for 2 minutes, then sucked vigorously through a filter press and sucked off for 15 minutes on the suction. The material was then transferred to 100 ml Britton-Robinson buffer pH 8.0 and allowed to swell at +8 ° C overnight in the refrigerator.

A total of 8.4 g of wet mass of granulated solid immobilized glucose isomerase material with specific activity was obtained

0.12 yumol fructose / min / mg wet mass. The material settled quantitatively within seconds. 5 g of this material was suspended in 50 ml of 1 M D-glucose solution, pH 9.0. The glucose solution was pre-warmed to 60 ° C. The reaction mixture was stirred continuously. After 4 hours of reaction under the above conditions, glucose isomerized to a mixture of fructose + glucose in the ratio of the monosaccharides in the order of 40:60%. The procedure was repeated with the same catalyst feed five more times under the same reaction conditions with the same result. After repeated use, the catalyst was separated from the reaction mixture and washed with water as described above.

Example 9

2.5 kg of Alcalígenes metalcaligenes cell paste containing the enzyme aspartate ammonia lyase were suspended in demineralized water to a total volume of 10 liters. The cellular suspension was cooled to + 5 ° C and then disintegrated at 250 atm three times by passing through a slotted mechanical disintegrator. The suspension between the individual disintegrations was continuously cooled so that the maximum temperature of the disintegrated mixture did not exceed 35 ° C. Thereafter, the disintegrated suspension was centrifuged in portions as described in Example 8. 5.8 liters of crude enzyme solution having a concentration of 110 mg / ml of proteins having a specific activity of 165 / µmol L-aspartic acid / min / mg protein / fumaran substrate was obtained. ammonium, heat 37 ° C, pH 8.5).

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To 100 ml of RRP, the preparation method of which was given in Example 1, was added 100 ml of crude aspartate ammonia lyase prepared as described in the previous paragraph. The protein concentration in the reaction mixture was 55 mg / ml. the reaction mixture was continuously stirred for one hour, then centrifuged under the conditions and handling method of Example 8. Further, the material treatment / filter pressure treatment, grinding / procedure was also carried out as described in Example 8, except that the postformed unwashed granular material was not treated. with acetone or glue in acetone, but air dried for 5 hours. After this time, the material was transferred to a 1.35 M ammonium fumarate solution at pH 8.5 and allowed to swell overnight at +8 ° C.

A total of 7.5 g of granulated solid material immobilized with aspartate ammonia lyase having a specific activity of 750 yusol L-aspartic acid / min / g wet mass was obtained; The immobilized material sedimented quantitatively within a few tens of seconds. 5 g of this material was suspended in 50 ml of a 1.35 M ammonium fumarate solution at pH 8.5. The substrate solution was pre-equilibrated to 37 ° C. The reaction snow was continuously stirred. 'BA. After 2.5 hours of reaction under the above conditions, the substrate was quantitatively converted to the product, L-aparonic acid. The process was repeated with the same catalyst feed a total of five times under the same reaction conditions. After repeated use, the catalyst was separated from the reaction mixture using a polyamide mesh, washed with water on the mesh and reused. The average conversion rate / 5 cycles of reuse was 99.6%.

Claims (7)

1. Enzyme catalysts for biotransformation consist of 4 4 enzymes of microbial, plant or animal origin, mobilized by a chemically reactive water-soluble polymer, formed by the reaction of a water-soluble substance containing at least two primary and / or secondary amino groups such as polyethyleneimine, hexatnethylenediamine , lysine, and polylysine, with bifunctional dicarboxylic acid aldehydes such as glutaraldehyde, optionally further mechanically strengthened. 2. Enzyme catalysts according to claim 1, characterized in that said enzyme material contains enzymes of different purity, preferably technical or crude enzyme preparations, preferably carrying hydrolase, isomerase, lyase or racemase activity. O 3. The enzyme catalyst of claim 1 wherein said enzyme material is immobilized with a chemically reactive water-soluble polymer formed by reaction of polyethylenimine having a molecular weight distribution of 100,000 to 800,000 with glutaraldehyde. 4. A process according to claim 1, wherein the aqueous solutions of microbial, vegetable or animal enzymes having a protein concentration of 5.0-500.0 mg / ml are immobilized by contacting a water-soluble, chemically reactive polymer. , formed by the reaction of a water-soluble substance containing at least two primary and / or secondary amino groups, preferably polyethyleneimine, with bifunctional dicarboxylic acid aldehydes, preferably glutaraldehyde, at a temperature in the range of 0 to 60 ° C in aqueous medium at pH 4 0 to 12.0, wherein the reaction of said enzyme material with said polymer is carried out at a temperature in the range of -30 to +50 ° C in an aqueous medium and at a pU in the range of 5.0 to 9.0, the resulting insoluble particles are separated The reaction mixtures are washed with water or buffer and then mechanically solidified. -and. I .231 SM 5. The method of item. 4, characterized in that the water-soluble polymer is prepared from an aqueous solution of polyethyleneimine having a molecular weight distribution of 100,000 to 800,000 and / or an aqueous solution of polylysine having a molecular weight distribution of 5,000 to 50,000. O 6. The process of claim 4 wherein the reaction of said enzyme materials with said reactive polymers is carried out at a temperature of from 0 ° C to 45 ° C at a relative centrifugal force. 1 to 50,000 g, at a filtration pressure of from 0 to 50,000 KPa, with standing or stirring, or with intermittent stirring and standing of the reaction mixture. 7. Process according to claim 4, characterized in that the particles immobilized in water-insoluble enzymes are optionally further mechanically consolidated by partial dehydration by the action of organic solvents, preferably acetone or adhesive solutions in organic solvents or mixtures thereof with water, at temperatures +30 to -25 ° C, optionally the formed particles are suitably deformed and partially dried before and / or after mechanical consolidation.