CS234059B1 - Method of microbial cells immobilization with plant celis - Google Patents

Description

The invention relates to a method of immobilizing microbial cells, plant cells and enzymes to activated organic and inorganic polymers to form immobilized cellular or enzyme biocatalysts.

The cellular or enzyme biocatalysts of the present invention are useful for industrial biotransformation, respectively. make it possible to develop discontinuous or continuous biotechnologies for the production of pharmaceutically, food or agriculturally important products or intermediates

Known methods of cell and enzyme immobilization can generally be characterized as physical e.g. sorption and inclusion, physico-chemical e.g. polar binding and chemical e.g. covalent bond. These procedures are mentioned in more detail with respect to cell immobilization, for example, in review articles by Vandamme / Chem. Ind., 1070 (1976), Jack and Zajic (Adv, Biochem. Eng., 5, 126 '(1977); Durand and Navarro (Proc. Biochem., 13, 14, 1978], Vojtisek et al. / Biol. leaves, 44, 192, 1979] and Chibata and Elk / Ann. Roar. Biophys. Bioeng., 10, 197, 1981] as regards the immobilization of enzymes, e.g. in the monographs Immobilized Enzymes / edit. 0. Zaborsky, CRC Press, A Division of Chemical Rubber Co., USA Cleveland, Ohio, 1974 / Insolubilized Enzymes / edit. Salmon M., Saronia C., Gerattini S., Raven Press, New York, 1974. and Industrial Enzymes, Recent Advances. Johnson Johnson, Noyes Data Corporation, Park Ridge, New Jersey, Chemical Technologist (Review No 85, USA, 1977).

A number of synthetic organic inert or chemically active polymers obtained by targeted polymerization, eg based on methacrylaldehyde, glycidyl methacrylate, hydroxyalkyl methacrylate, natural

234 059 organic polymers such as cellulose and its derivatives, chitin, polysaccharides, inorganic natural or artificial materials such as bricks, sand, porous clay, clay, ceramic materials, glass, inorganic zirconium and titanium gels etc.

From a number of different, more or less chemically defined materials that can be used as insoluble carriers for cell and enzyme immobilization, these materials will differ primarily in price and availability and their use for immobilization will be limited primarily by territorial conditions, especially synthetic, chemically defined organic and inorganic polymers. For example, a method of immobilizing bacterial cells to the surface of modified macroporous copolymers of MS. provenance, in particular glycidyl methacrylate with ethylene dimethacrylate and copolymers of methacrylaldehyde with divinylbenzene after their conversion to amino derivatives by treatment with organic amines and cross-linking with glutaraldehyde in MS. No. 200 802, the method of immobilization of yeast to organic polymer particles. provenance based on hydroxyalkyl methacrylate, after previous chemical activation of carrier particles with epichlorohydrin, modification with hexamethylenediamine and subsequent covalent bonding of the cells with glutaraldehyde, in the Czech Republic. No. 201 786. Although preparations of immobilized cells with good biochemical and other properties have been obtained, the cost and availability and possible methods of chemical activation and modification of said types of organic polymers do not permit a realistic presumption of industrial use of these materials as carriers of desired biological activities for industrial use. biotransformation. From an economically attractive point of view, organic and inorganic artificial or abrasive materials of various sorbent type seem to be the most useful, the cost and availability of which do not significantly affect their industrial use.

Various synthetic organic and inorganic polymeric sorbents have been developed, and some of them are industrially produced, in Czechoslovakia. It is a polymeric microporous organic sorbent based on polyethylene terephthalate (SOHSILEN), the method of manufacture of which is given in Czech. No. 171 917 and polymer microporous organic sorbents based on poly (-2,6-dimethyl) phenyloxide

- 4,234 OSB (SOSFIX), poly-6-caprolactam (APA and SILQNA) differing in degree of polymerization), based on poly-6-caprolacttaw copolymer with ethylene oxide (AMEDAP) polymers and based on vinyl chloride-vinyl acetate block copolymer, whose production methods are listed in MS. Certificate No. 202 215. Of other organic sorbents MS. provenance may be mentioned polycondensates of melamine and formaldehyde (W) and urea and formaldehyde (MOR), the method of manufacture of which is the subject of MS. 205 586. Finally, inorganic microporous sorbent based on polymeric silica under the commercial name SILOXID is industrially manufactured in Czechoslovakia. All of the above-mentioned types of polymeric organic and inorganic sorbents can in principle be prepared in the desired quality, in particular with the desired particle size distribution, specific surface area, porosity, swelling and the like, allowing the use of these materials for both whole cell and enzyme immobilization.

The limiting factor of the universal use of these polymeric sorbents, MS. However, as a carrier for the immobilization of biologically active materials, it is a universal method of immobilizing whole enzymatically active cells and enzymes, with the aim of achieving a high specific activity of the obtained immobilized catalysts and a high immobilization yield while maintaining high stability of biological resp. enzyme functions, irrespective of the chemical composition of these polymeric sorbents, on the one hand, organic and inorganic sorbents, the basic chemical composition of the monomer, the type of chemical bonds and the degree of polymerization, and the type and quality of the starting biologically resp. enzyme-active material that is immobilized, microbial cells, plant cells, enzymes of microbial, animal or plant origin, on the other side.

The Czechoslovak patent literature has already described certain methods of immobilization, especially enzymes, to some of the types of polymeric microporous sorbents of Czechoslovakia. provenience. These are mainly MS. No. 214,096, which discloses the use of microporous particles of polymers based on polyethylene terephthalate (SORSIIEN), polydimethylphenyloxide (SORFIX) and polycaprolactam (APA) for the production of immobilized enzymes by their sorption and 5,254,059 sequentially covalent bonds using sorbed crosslinking agents in particular glutaraldehyde, tertiary diisocyanate, divinylsulfone and the like, or by sorption of enzyme solutions to particles of these organic polymeric sorbents and subsequent covalent crosslinking of the sorbed enzymes by these multifunctional crosslinking agents. The cited author's certificate includes only the use of the three types of microporous organic polymeric sorbents and is limited to the method of producing the mobilized enzymes in the manner described. In MS. author's certificate no. 209743 _W is described again only a method of immobilizing enzymes on a limited microporous organic polymeric sorbents MS. provenance based on poly-6-caprolactam, differing in degree of polymerization and polycondensates of melamine with formaldehyde and urea with formaldehyde, ie exclusively organic polymeric sorbents containing primary or secondary amino groups in the polymer molecule, the actual immobilization of enzymes is based on previous chemical reaction of particles of these synthetic aminopolymers. with free glutaraldehyde, while simultaneously chemically activating these polymer particles, eluting excess glutaraldehyde and subsequent covalent enzyme bonds via chemically active aldehyde groups, forming Schiff bases between the enzyme protein and the sorbents activated as described above. No. 217,857 describes a method of preparing mobilized yeast cells limited to the use of a single type of organic polymeric sorbent of MS. a polydimethylphenyloxide (SOBFIX) -based provenance consisting in the treatment of these sorbents with glutaraldehyde, subsequent washing of glutaraldehyde and anchoring of yeast cells by a combination of sorption and covalent bonding. The cited author's certificate is thus limited to only one type of utilization of the organic polymeric sorbents, to a single type of microbial cell - yeast and the only method of immobilization with sorbed glutaraldehyde. In the same way immobilized plant cells, to the same type of microporous organic polymeric sorbents, have been described in the literature (Biotechnol). Lett. 8447, 1981].

We have recently found that various microbial and plant cells, their subcellular particles and cell fragments and various enzymes of microbial, plant or

6 234 059 of animal origin, by means of reactive water-soluble polymers containing at least two primary and / or secondary amino groups per molecule, in particular based on polyethyleneimine, polylysine and hexamethylenediamine and bifunctional crosslinking agents, in particular glutaraldehyde. However, these largely universal and economically acceptable methods of immobilizing cells and enzymes, in some cases, provide soft preparations of immobilized spongy-like biocatalysts with inferior hydrodynamic properties, and in particular using higher organism cells as the starting immobilized biological material, especially higher cells. fungi and plant cells, or enzymes attacking high-molecular substrates / proteases - proteins, amyloglucosidase - starch, etc.), provide preparations with a lower biological / enzymatic. activity and shorter operating half-lives in their batch reuse or continuous utilization in the desired biotransformation direction.

Therefore, we have verified experimentally the possibility of using these reactive soluble polymers to activate all microporous polymeric organic and inorganic sorbents of MS. and elaborated methods of immobilization biologically resp. enzymatically active microbial and plant cells and enzymes of microbial, plant and animal origin, in order to obtain immobilized biocatalysts with improved hydrodynamic and other advantageous properties, enabling their industrial use in the desired biotransformation direction in the pharmaceutical, food and agriculture industries.

The method for the immobilization of microbial and plant cells and enzymes according to the invention is characterized in that the particles of water-insoluble organic polymers, in particular based on polyethylene terephthalate, poly-2,6-dimethylphenyloxide, poly-6-caprolactam, poly-6-caprolactam block copolymer with polyethylene oxide , vinyl chloride-vinyl acetate copolymer, melamine-formaldehyde polycondensates and formaldehyde-ureas and inorganic polymers, in particular based on polymeric silica, are activated with chemically reactive water-soluble polymers prepared by reaction of water-soluble substances containing at least 2 primary and / or

7,234,059 secondary amino groups, in particular branched polyethylene oxide of molecular weight 50,000 to 1,000,000, or polylysine of molecular weight 1,000 to 50,000, lysine or hexamethylenediamine and bifunctional aldehydes of dicarboxylic acids such as glutaraldehyde, whereupon the insoluble polymer particles are activated The aqueous or buffered aqueous suspension of whole microbial or plant enzyme-active cells and / or aqueous or powdered aqueous enzyme solution is added and the resulting immobilized biocatalyser is separated and washed. The immobilization of the cells and enzymes is carried out with water-insoluble particles of organic and inorganic polymers having a size of 0.001 to 5 mm, a specific surface area of from 1 to 700 m @ 2 / g and a particle porosity of from 0.01 to 2.5 m @ 2 / g. The activation of the water-insoluble polymer particles, in particular based on polyethylene terephthalate, poly-2,6-dimethylphenyloxide, vinyl chloride-vinyl acetate block copolymer and polymeric silica, is carried out by mixing them with preformed reactive soluble polymers which are prepared by reacting water-soluble substances containing at least two primary and / or secondary amino groups with bifunctional dicarboxylic acid aldehydes, whereupon? after removal of the excess solution, it is contacted with a cell suspension or enzyme solution. The particles of poly-6-caprolactam-based polymers, poly-6-caprolactam-block copolymer with polyethylene oxide and polycondensates of melamine with formaldehyde and urea with formaldehyde are contacted sequentially with the individual reactive components of the reactive soluble polymers, first exposing the particles of said polymers by contact with a solution of polyethylene oxide, polylysine, lysine or hexamethylenediamine and after sorption and removal of the excess solution, the particles are exposed to a bifunctional dicarboxylic acid aldehyde such as glutaraldehyde, after which the excess solution is removed after formation of the reactive polymer. the particles are exposed to a cell suspension or enzyme solution.

Whole intact enzyme-active cells of micro-organisms shall be used as the starting biological material for immobilization,

234 0S9 in particular Gram-positive and Gram-negative bacteria, actinomycetes, acid-resistant bacteria, yeasts and other imperfect fungi and enzyme-active plant cells and raw technical or to various degrees of pure enzymes of microbial, plant or animal origin.

In the following examples, commercially available polyethyleneimine-based flocculants having a molecular weight of 200,000 to 300,000 at a concentration of about 30 weight percent in the flocculant, or polylysine having a molecular weight of from 25,000 to 50,000 are used as substances containing at least two primary and / or secondary amino groups. 000 or oligomers of polylysines formed by the reaction of technical lysine with glutaraldehyde.

The enzymatic activities of the mobilized biological materials were evaluated by stirring and tempering a defined amount of particles in the substrate solution under conditions of zero order kinetics of the enzyme reaction and enzyme activity respectively. specific activity expressed as the amount of umoles produced, respectively. products / mg or g wet or dry particulate matter, unless otherwise indicated in the examples.

Production cells containing various enzymes can be used in the method of immobilizing the cells of the invention, the use for immobilizing cells of mutant organisms obtained by breeding, selection and accumulation methods and genetic manipulation is preferred because the obtained mobilized preparations have a high specific activity.

The immobilized microbial and plant cells may carry various enzyme activities, in particular hydrolase, lyase, isomerase, decarboxylase, oxidoreductase and other activities and / or part or all of the sequence of enzymes of the logical sequence of the biochemical pathway, especially degradative metabolism, glycolysis, hormone biotransformation enzymes, alkaloids, phytosterol, tosteral, campesterol, stigmasterol and other substances.

- 9,234,059

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

Example 1

100 ml of a 2% (w / v) aqueous solution of ethyleneimine poles (Sedipur CL-930) was prepared and the solution was vacuum filtered through a S4 porcelain frit to remove a slight insoluble portion of this commercial flocculant. A 25% (v / v / v) aqueous solution of glutaraldehyde was added to 100 ml of a perfectly dissolved clear Sedipur solution to a final concentration of 1% (v / v). The reaction mixture, pH 6.8, was poured into a 500 ml glass flask whose neck was covered with aluminum foil and the flask was placed on a rotary shaker at 250 rpm and the mixture continuously; stirred for 24 h at 30 ° C. A reactive polymer solution was prepared by this procedure.

g of dry mass of the microporous SOSFIX poly- (2,6-dimethyl) phenyloxide carrier having an average particle size distribution of 0.2-0.5 mm, a porosity of 0.459 cm @ 2 / g and a specific surface area of 400 m @ 2 / g was suspended in 100 ml. ethanol: water (volume ratio, 1: 3) and allowed to swell in the mixture overnight. The following day, the suspension of carrier particles was vacuum filtered and the material washed thoroughly with 1 liter of water and aspirated.

g of the wet mass of the carrier was suspended in 100 ml of a reactive water-soluble solution of P155nne.ru (hereinafter referred to as RRP) and after homogenization of the particles, the mixture was continuously stirred for 1 hour on a reciprocal shaker and allowed to stand at room temperature for 2 hours, vacuum filtered and well aspirated. the activated carrier was divided into 5 equal parts by weight and 2 g wet mass into 5 50 ml glass beakers. In addition, 5 aqueous suspensions of whole native cells of yeast Saccharomyces cerevisiae containing 100, 200, 250, 300 and 400 mg wet, well centrifuged cell mass / ml were prepared. Thereafter, 4 ml of homogeneous aqueous cell suspensions of different concentrations were added to 2 g of the wet mass of the activated carrier, mixed, the beakers were covered with foil and placed on a reciprocal shaker and mixed continuously for 7 hours. After this time, the samples were decanted successively several times

10,234,059 with water until turbidity of the upper liquid layer after quantitative sedimentation of the particles has occurred. Then the samples were gradually transferred to a Buchner funnel whose bottom. It was covered with a textile cloth Aalolis cloth / and sucked off well under vacuum and washed thoroughly with water on the funnel, dried after suction to constant weight (105 ° C) and the yield of cell immobilization was monitored according to their concentration at the same weight. , according to the increment of bound nitrogen. The results are summarized in the following table.

Cell concentration (mg / ml) 100 ALIGN! 200 250 300 400 Nitrogen addition /% / 1,25 2.03 2.68 3.02 3.16

From the values given in the table, the logic of selecting a cell concentration of 300 mg / ml per the amount of the same activated carrier is used in the following examples where microbial cells are used as bound material.

Example 2

Since the contact time of the cells with the activated carrier as described in Example 1 was empirically selected (7 hours), the time dependence of the cell contact with the same type and the same activated carrier was further monitored.

100 ml 1-t? was prepared as described in Example 1, as well as a method of treating the same type of carrier prior to activation including activation alone. To 5 equal amounts of well aspirated, moist, activated carrier were added 4 ml of an aqueous cell suspension each containing 300 mg of wet mass of native yeast Saccharomyces cerevisiae and immobilization was performed by stirring as described in Example 1. At various time intervals, the reaction mixtures were successively decanted, vacuum, filtered, washed and dried as described in Example 1 and the optimal contact time of the cells with the activated carrier was calculated from the amount of captured biological nitrogen. The results are summarized in the following table.

- 11 234 059

Contact time / h / 1 2 3 4 24 Nitrogen addition /% / 3.72 3.73 3.73 3.68 3.55

From the values shown in the table, the experimentally found contact time of the cells with the activated carrier of 3 hours was used, which is used in the following examples, as well as the experimentally found cell concentration given in Example 1.

Example 5

400 ml RRP was prepared as described in Example 1. SORFIX (10 g) was treated with ethanol / water as before as described in Example 1 »Other microporous SILOXID carriers (with different particle size distributions) and SORSILEN in amounts and 10 g of dry matter were wetted with approximately 1 liter of water, decanted several times and well vacuum filtered. Further, 100 ml of an aqueous suspension of Saccharomyces cerevisiae with invertase activity was prepared at 300 mg wet cell mass / ml. The invertase activity in this suspension was 13.8 U / mg dry cell mass.

In each case, 10 g of wet mass of well aspirated microporous carriers of said commercial names were suspended in 100 ml RRP and the particle suspension of these materials was activated as described in Example 1. After activation and separate aspiration of RRP from activated carriers, these were separately suspended in 20 ml of aqueous cell suspension. and after homogenization and suspension, cell immobilization was performed by stirring for 1 hour and then standing for 2 hours at room temperature. Thereafter, the suspensions were decanted separately several times with water until the turbidity of the upper liquid layer did not occur after quantitative sedimentation of the particles. Subsequently, the materials were sequentially separated by filtration through a textile material on the filter, yet thoroughly washed with water, and well aspirated wet materials weighed and further analyzed. The results are summarized in the following table.

- 12 234 059

Carrier type Poly- / 2,6-di- Polymeric methyl / phenyl-silica polymer Polyethylene- terephthalate / SORSILEN / oxide / SORFIX / I / SILOXIDE / II Avg. distribution particle size 0.2 to 0.5 and 0.2 to 0.5 0.02 to 0.03 / mm / 1 0.01 to §80 Porosity of particles 0,048 / cm ⁵ / g 0.459 unknown 0,800 Specific surface 400.0 62.0 66.0 / nr / g / 100.0 / + / Spec. activity u immobil.quasine <k 563.0 110.0 145.0 on carriers / j / g 263.0 dry matter Nitrogen increment /% / 0.09 0.43 0.11 0.26

A / Invertase activity with sucrose as substrate was determined according to J.B. Lloyd and W.J. Whelana / Analyt. Biochem., 30,

No. 3, 467 (1969)].

Example 4

Immobilization of whole bacterial cells of Escherichia coli with benzylpenicillin amide hydrolase activity to solid particles of microporous carriers of SORFIX, SILOXID and SORSILEN type activated by RRP. Preparation of RRP, carrier wetting and activation, concentration of starting cell material with specific activity of 14.5 J / mg dry cell mass, immobilization technique, decanting, washing and filtering of immobilized cell materials were performed as in Example 3. The results are summarized in the following table. .

Carrier type Poly- (2,6-di- Polymeric methyl / phenyl-silica) / SORFIX / (SILOXID) Polyethylene terephthalate / SORSILEN / Avg.Distribution particle size (mm) 0.2 to 0.5 0.2-0.5 0.01-0.8 0.02-0.03 Porosity of particles / cm? / G / 0.459 0.048 unknown 0.80

Specific surface area / m ² / g / 400.0

62.0

100.0

66.0

- 13 234 059

/ + / Spec. activity enzyme u immobil. bacteria on carriers / j / g dry matter / 2760.0 8830.0 7560,0 9530,0 Nitrogen addition /% / 5.10 5.44 4.82 4.49

(+) The activity of benzyl foamsilinamidohydrolysis with benzylpenicillin as a substrate was measured according to Balasingham et al., Biochem. Biophys. Acta, 276, 250 (1972).

Example 5

In contrast to the procedures described in the previous examples, 2 types of poly-L-lysines of different molecular weights were used for the preparation of RRP, (a) polylysine hydrochloride having a molecular weight of from 40,000 to 50,000 and / b) Both types of polylysines were prepared at the Institute of Organic Chemistry and Biochemistry of the Czechoslovak Academy of Sciences. The reactive polymer was obtained in (a) for 24 hours by reacting an aqueous solution of the reaction mixture in a concentration ratio of 0.1% polylysine. HCl (mass / volume) and 0.3% glutaraldehyde (volume / volume) in a total volume of 20 ml with continuous agitation on a shaker at 30 ° C and in case (b) under the same reaction conditions as (a) except that 20 ml of the aqueous solution of the reaction mixture contained 1% lysine (w / v) and 0.5% glutaraldehyde (v / v).

To activate the microporous SORFIX-type carrier whose physicochemical and physical characteristics are shown in Example 1, 10 g of wet mass of carrier particles were used, which were mixed with 20 ml of each of the RRPs prepared as described above; b /. Carrier activation, method of immobilization of aqueous suspension of cells of yeast Saccharomyces cerevisiae with invertase activity, including cell concentration and decantation, washing and filtration of the obtained material was performed as described in Example 3. The obtained material was determined for biological nitrogen growth . The results are summarized in the following table.

Carrier type Poly - / '2,6-dimethyl / phenyloxide / SORFIX /

- 14 234 059

The type of SRP used to activate the carrier basalt /and/ / b / / + / Invertase-specific activity in immobilized yeast carrier / j / g dry matter / 9 492.3 1 901.3 Nitrogen increment /% / 3.39 1.57

/ + / shown in example 3. ·

Example 6

A series of spectrophotometric measurements were performed to monitor the kinetic of the L-lysine reaction. HG1 with glutaraldehyde to form colored Schiff bases. Based on the results obtained, RRP was used to subsequently activate the described microporous carriers, which was obtained 24 hours by reacting the mixture of reactants at the current concentration of 5% (w / v) L-lysine. HCl and 4% (v / v) glutaraldehyde in 60 ml of 0.1 M phosphate buffer pH 8.0, with stirring on a shaker at 30 ° C.

10 g of wet mass of microporous carriers SOSFIX, SILOXID and SORSILEN each were mixed separately with 20 ml of this RRP. Carrier activation, immobilization technique, including the weight ratios of the aqueous suspension of cells to the mass of activated carriers, decantation, washing and separation was performed as in Example 3. Yeast cells with invertase activity were used as well as shown in Example 3. The results are listed in the following table.

Carrier type Poly- (2,6-dimethyl) phenyloxide (SOBFIX) Polymeric silica / SILOXID / Polyethylene- terephthalate / SOSSILSN / Avg. particle size distribution (mm) 0.2 to 0.5 0.01 to 0.80 0.02 to 0.03 Porosity Particles / cm3 / g / 0.459 unknown 0,800 Specific surface particles / ř »^ / g / 400.0 100.0 66.0

/ + / Spec. invertase activity in immobilized, ₂ > ₂₈ θ yeast on carriers ^{, y} / j / g dry matter / '

1831.8

56.2

234 059

Nitrogen addition

1.47

1.51

1.76 (+) In Example 5.

Example 7

In contrast to the previous examples, solid microporous polyamide-based carriers containing primary and secondary amino groups were used to immobilize whole cells. Three types of carriers, APA, AMIDAP and SILONA, were used. The chemical composition of these polymers is shown below. In contrast to the above examples, the process for preparing RSP and activating the particles of these carriers was carried out in direct contact with the carrier particles. Among the many methods of activation of these types of microporous polyamide carriers investigated, the method that provides the highest captured specific activity of immobilized cells was selected by first adsorbing polyethyleneimine to the surface of the carrier particles and secondly treating the carrier particles with glutaraldehyde. at the same time chemical activation of the carrier particles according to the following procedure.

In each case, 10 g of the abovementioned types of polyamide supports, which were washed with water and vacuum-suctioned, were suspended in. 40 ml of a 2% (w / v) vacuum-filtered aqueous solution of Sedipur CL-930 and the suspension was stirred at room temperature for 1 hour. Thereafter, the carrier particles were separately vacuum aspirated and suspended in 40 ml of a 1% (v / v) aqueous glutaraldehyde solution. After a 1 hour reaction during continuous stirring at room temperature, the activated carrier particles were vacuum filtered and used to immobilize the cells. The cell suspension of Saccharomyces cerevisiae, at a concentration of 500 mg of wet cell mass / ml of distilled water, was mixed in 10 ml each with 5 g of wet aspirated, activated carrier and placed on a laboratory shaker. with the exception that the efficiency of cell immobilization was monitored depending on the time of its contact with the activated carrier particles during continuous stirring at room temperature. The degree of immobilization was determined at two time intervals, 1 and 5 hours, after previous quantitative decantation, washing and aspiration.

234 059 reindeer mentioned in previous embodiments. The results are summarized in the following table.

Carrier type Poly-6-caprolactam, polymerization degree 160 (APA) Copolymer of poly6-caprolactam with polyethylene oxide (AMIDAP) Poly-6-caprolactam, polymerization degree 90 / SILONA / Avg. particle size distribution (mm) 0.25 0.2 to 0.5 0.5 Porosity of particles / cm5 / g / 0 * 67 0.393 unknown Spec. particle surface / m ² / g / 6.6 1.6 to 2.0 0.5 Contact time / h / ¹ . 5 1 5 13 / + / Spec. activity invertase immobil. yeast on carriers / j / g / dry matter / 8353.3 1900, 0 105.0 10473.6 438.0 152.0 Nitrogen increment /% / 0.27 0.28 0.58 0.52 0.68 1.17

/ + / As shown in Example 3.

Example 8

An aqueous suspension of fresh native washed cells of an Aspergillus niger imperfect filamentous fungus was prepared at a concentration of 300 mg wet cell mass / ml aqueous suspension. The invertase specific activity of this microorganism was 0.3 J / g dry cell mass. Cells were harvested after 24 hours of submersible one-time cultivation in a fermentation tank under conditions preventing cell growth in pellets in sucrose as a carbon source. Next, 50 ml of an aqueous RRP solution was prepared for 24 hours by reacting the Sedipur CL-930 / glutaraldehyde reagents in their concentration ratio and as described in Example 1.

In each case, to 20 g of wet, well aspirated mass of washed microporous SORFIX and SORSILEN carriers was added 25 ml RRP and activation of the carriers was performed by stirring and standing as described in Example 1, including the described method of separating chemically activated particles of both carrier types.

g wet, aspirated scrubbed microporous SILONA microporous support was activated in direct sequential contact

As previously described in Example 7, this amount was suspended in 80 ml of a filtered 2% (w / v) aqueous solution of SEDIPUR CL-930, 10 min. the mixture was stirred and then the suspension was left for 50 min. The particles were aspirated and 80 ml of a 1% (v / v) aqueous solution of glutaraldehyde was added to the well aspirated particles, followed by activation by stirring and standing at the same time intervals, followed by separation of carrier particles and quantitative aspirations.

To 20 g of wet mass as described by the SORPXX, SORSILEN and SILONA activated carriers as described above, 40 ml of an aqueous Aspergillus niger cell suspension was added and the cells were immobilized for 1 hour with stirring and 2 hours on standing. Then the particles of the individual preparations of immobilized cells were vacuum filtered through a textile material, decanted several times with several liters of distilled water, sucked off again and washed several times thoroughly with water. Furthermore, the preparations were evaluated for specific invertase activity and biological nitrogen content. The results are summarized in the following table.

Carrier type Poly- / 2,6-di- methyl / phenyl- oxide / SORFIX / Polyethylene- terephthalate / SORSILEN / Poly-6-caprolactam; polymerization stage 90 / SILONA / Avg. Size distribution. particles / mm / 0.5 to 1.0 0.5 to 1.2 0.5 Porosity of particles / cm3 / g / 0.459 2.14 0.5 Particle surface area / m2 / _g / 400.0 56.0 0.5 / + / Invertase specific activity in immobil. A. niger fibers on carriers / j / g dry matter / 1.5. 10-5 1,2. 10 ' ⁵ 7.3. 10-4 Nitrogen addition / <$> / 0.06 0.08 1,3.2,

/ + / As shown in Example 3.

Example 9

From a 24 hour and 48 hour culture of Aspergillus niger cells with invertase activity, homogenous aqueous suspensions of washed fibers were prepared at a cell concentration of 300 mg wet weight / ml.

- 18 2M0ST suspension. The cell invertase specific activity of 24 hr culture was 0.3 J / g dry mass, the specific activity of the same enzyme in cells of 48 hr culture was 0.138 J / g dry cell mass.

In addition, a polylysine / glutaraldehyde RRP was prepared, principally as described in Example 5, by first preparing 25 ml of a 1% (w / v) solution of lysine poles of 26,200 molecular weight, which was dissolved in 0.1 M Tris buffer pH 8.0 and a 25% aqueous glutaraldehyde solution was added to the solution so that its initial concentration in the reaction mixture was 0.5% (v / v), with continuous stirring for 24 hours at 30 ° G.

g of the wet mass of the polymeric carrier type, SORFIX, was mixed with 25 ml of the above-prepared RRP, and the carrier particles were activated for 1 hour by stirring and 2 hours at room temperature, then vacuum dried and divided into 2 equal parts by weight. 10 g of wet mass / and to one part 20 ml of a 24 hour cell suspension culture was added, to the other part 20 ml of a 48 hour cell suspension culture of the same microorganism. The cell immobilization proceeded in the same way as the activation of the carriers, i.e. 1 hour stirring and 2 hours standing at room temperature. Then, the two materials were separated, decanted, and washed with water as described in the previous examples. The results are summarized in the following table.

Carrier type Poly- (2,6-dimethyl) phenyloxide / SORFIX /

Particle size distribution

/ mm / 0.5 to 1.0 Porosity of particles (cm5 / g). ...................... .. 0.459 Specific surface area / m ^ / g / 400.0 Age of cell culture of A. niger / h / 24 48 A / Default spec. activity invertase in native A. niger fibers / j / g dry matter / 3. 10 “! 1.38. 10-1 / + / Spec. invertase activity in immobilized fibers A. niger on . 10 “5 1.5 2.8. 10 ^ carriers / j / g dry matter / Nitrogen increment /% / 0.10 0.13

/ + / As shown in Example 3.

- 19 Example 234 059

50 ml of polyethyleneimine / glutaraldehyde-based RRP was prepared for 24 hours by reacting the two reactants as described in Example 1.

g of the wet mass of the SORFIX-type polymer (Poly-2,6-dimethylphenyloxide) having a particle size of 0.25-0.5 mm, a specific surface area of 600 m ^ / g and a porosity of 0.5 cm ^ / g was after wetting, swelling, washing and aspirated as in Example 1, mixed with 50 ml RRP and the carrier was activated for 1 hour by stirring and 2 hours standing as in Example 1, including separation of activated carrier particles.

A single cell culture suspension of Solanum aviculare, obtained by aseptic culture under constant aeration without phofoperiod, was filtered, the individual cells were washed with 0.05 M phosphate buffer containing 0.75 M KG1 pH 5.7 after separation of the medium and the suspension contained in this buffer contained 150 mg wet mass of washed plant cells / ml. To 3 g wet aspirated mass of activated carrier 12 ml of plant cell suspension was added and immobilization was followed by stirring for 1 hour and standing for 2 hours under the conditions and procedure described in Example 2. The mobilized material was decanted several times with the buffer, filtered, aspirate on the filter and wash again with this buffer.

The obtained mobilized material was packed into a glass column equipped with a tempering jacket (Pharmacia Fine Chemicals K 26/40) and the fixed bed of this biocatalyst was pumped from top to bottom by recirculating 100 ml of 8% (w / v) sterile aqueous sucrose at a rate of 1.5 ml / min at 26 ° C.

. At 24-hour time intervals of continuous recirculation, extracellular production of cytosterol, campesterol, stigmasterol and other, chemically unidentified sterol components were analytically monitored by HPLC chromatography described by Jirka et al., Biotechnol. Lett. 8, 447 (1981)]. It has been found that during a seven-day continuous fixed bed biocatalyst recirculation, the extracellular production of these components occurs at concentrations ranging from 10 to 20 µg / 24 h / 100 µg of wet mass of mobilized plant cells at unchanged intensity and in preserved biorhythms.

They exemplified 234,059 g of the wet washed mass of an inorganic carrier type. SILOXIDE (polymeric silica) having an average particle size of 0.2-0.5 mm, a specific surface area of 62 m ² / g and a porosity of 0.048 cm 2 / g was activated with 50 ml of polyethyleneimine / glutaraldehyde based RRP as in the previous example. Immobilization of S. aviculare plant cells was also carried out as in Example 10, including separation, decanting and washing with said buffer.

The extracellular biochemical activity of the same steroid compounds was monitored in a fixed bed biocatalyst recirculation reactor under conditions and as described in Example 10.

The concentration of these compounds ranged from 8 to 16 µg / 24 h / 100 µg of the wet mass of the immobilized plant cells, and extracellular production was maintained at unchanged intensity and maintained biorhythms throughout the 7 day follow-up.

Example 12

In each case 0.5 g of dry mass of polymer carriers of type ΔΡΔ, AKIDAP, SORj / IX ,. SORSILEN and PVG-PVA 37 (chemical composition, physical and physiological / chemical characteristics of the carriers are given below) were suspended in 10 ml of 96% ethanol and allowed to stand for 30 min. with occasional mixing. Subsequently, the carriers were suspended in water, filtered, washed thoroughly with water on the filter and suctioned off under vacuum.

Further, 50 ml of polyethyleneimine / glutaraldehyde based RRP was prepared in principle as described in Example 1, with the difference that the actual reagent concentration ratio was 4% (limo / volume) of Sedipur CL-930 and 2% (v / v). The reaction time was 16 hours and the reaction temperature was 28 ° C. The washed wet particles of the individual carriers were separately mixed with 10 ml of the RRP obtained as described above, and the materials were activated with constant agitation at 25 ° C for 16 hours. After this time, the individual carriers were decanted 3 times with 50 ml of water and vacuum vacuumed. 1.2 to 1.4 g of wet mass of the individual activated materials were obtained.

To each of 0.5 g of wet mass of washed activated carriers was added 20 mL of chymotrypsin solution dissolved in 0.05 M phosphate buffer pH 7.0. The solution contained 10 mg / ml protein specific activity of 4.6 J / mg. Immobilization of this protease to activated 21 234 059. The carriers were allowed to stir for 18 hours at 28 ° C during continuous stirring. The individual materials were then separately vacuum filtered, washed with 2 times 50 ml of water, 2 times with 50 ml of 1M NaCl solution, 2 times with 50 ml of water and sucked off well. The results are summarized in the following table.

Carrier type Poly-6-ca-Prolactam copolymer; poly-6polymerC- caprolactive degree tamu with poly- Poly-2,6-Polyethydimethyl-lenterephenyloxide phthalate Vinyl chloride vinyl acetate copolymer containing 20% vinyl acetate units 160 lyethylene- oxide Designation / APA / / AMIDAP / / SORFIX / / SORSXLEN / PVG-PVA 57 Avg. particle size distribution (mm) 0.2 to 0.5 0.2 to 0.5 0.2 to 0, 5 0,5 0.25 to 1.2 Porosita of particles / onč / g / 0.588 0.420 0.459 2,454 unknown Specific 6.0 particle surface area / m ² / g / 7.4 575 '100 7.8 / + / Spec. activity immobil. chymo trypsin on carriers (j / g wet mass) 26.0 27.0 55.0 52.0 25.0

(+) The proteolytic activity of soluble and immobilized chymotrypsin with casein as a substrate was measured according to Methods of Enzymatic Analysis, Bergmayer, H. U., Ed., P. 1006, Verlag Chemie, 1974.

Example 15 The HPP was prepared as described in Example 12. The same amount and the same types of carriers whose chemical, physical and physicochemical characteristics are given in Example 12 were also activated as in Example 12, including separation and washing thereof. Immobilization of a neutral protease solution from Bacillus sp _o in 0.05 M phosphate buffer pH 7.0 and activity 1.15

22,234,059 J / mg of protein was also performed in the same manner and under the conditions of Example 12, including washing with water and NaCl solution and separating the immobilized enzyme. The results are summarized in the following table.

/ + / Label carrier_APA / + / Spec. activity immobil. neutralAMIDAP SOBFIX

SORSILEN PVC-PVA 37 proteases on carriers / j / g wet mass /

2.6,0

18.0 33.0

35.0 31.0 (+) In Example 12.

Example 14

The SORPIX, SORSILEN and PVC-PVA 37 carriers, whose chemical and physical characteristics are given in Example 12, were activated by the method and under the conditions set forth in Example 12, method (s), and by mixing with 4% (v / v / v) aqueous. solution of glutaraldehyde for 18 hours at a temperature of 28 ° 0, principally in the manner given in the art. No. 214 096, method / b /. Washed particles activated by both methods / a; (b) were suspended separately in 20 ml of 0.05 M phosphate buffer at pH 7.0 containing benzylpeniline amine hydrolase having a protein content of 100 mg / ml and a specific enzyme activity of 300 U / mg protein. After 20 hours of continuous stirring at 25 ° C, the supports were washed and aspirated as in Example 12. The results are summarized in the following table.

/ + / Label carrier SORPIX SORSILEN PVC-PVA 37 kluva akti- vace / a / / b / / a / / b / / a / / b / Spec. activity

immobilized benzylpenicillinamidone 0.25 0.24 3.48 1.85 0.22 0.22 hydrolase on supports (j / mg wet mass), (or -).

(+) Exemplified in Example 12 / a / The present method of activation of carriers by means of RRP / b / Control method of activation of carriers based on MS. AO 214,096

234 059 (++) The activity of the soluble and immobilized benzylpenicillin amidohydro lesion with benzylpenicillin as a substrate was measured according to Balasingham et al. Biochim. Biophys. Acta, 276, 250 (1972).

Example 15

The SORFIX and SORSILEN type carriers whose chemical and physical characteristics were given in Example 12 were activated by RRP with the composition and method also shown in Example 12, including separation and method of washing the activated particles with water, NaCl solution and again with water.

0.5 g of wet, washed washed microporous carrier each were suspended separately in 20 ml of amyloglucosidase enzyme solution in 0.05 M phosphate buffer pH 7.2, obtained by precipitation with ammonium sulfate and desalting by ultrafiltration from a commercial preparation AL1G 150 L from NOVO Terapeutisk / Denmark/. The solution contained 9 mg / ml protein, the soluble amyloglucosidase activity was 12 J / ml. Immobilization was carried out with continuous stirring for 18 hours at 12 ° C. After this time, the immobilized materials were washed twice with 50 ml of water, twice with 50 ml of 1 M NaCl solution and then again with water and well aspirated. The results are shown in the following table.

Carrier type Poly- (2,6-dimethyl) - Polyethyleneere / + / Phenyloxide phthalate / SORFIX / / SORSILEN / / ++ / Spec. activity immobil. amyloglucosidase on carriers (j / g wet mass) / (+) Example 12 (++) The activity of soluble and immobilized amyloglucosidase with soluble starch as substrate was measured according to GLUCOSE SIRUPE, NOVO Enzyme in der Starke. Industrie, Novo Ind., Gopenhagen, 1976.

Example 16

The organic microporous carriers of the SORFIX and SORSILEN type and the inorganic microporous carrier of the SILOXID type were activated as described in Example 5, including the composition and method of preparing RRP,

24 234 059 method (s). The same carriers were activated for 15 hours by stirring with a 4% (v / v) aqueous glutaraldehyde solution at temperature. 30 ° C, principally in the manner specified in MS, author's certificate No. 214 096, method (b). All 3 types of carriers activated by the two methods described above were separately aspirated and each of the 6 materials was divided into two equal parts by weight (and 1 g), in total in 12 equal parts by weight. Six portions of both activated particle modes were washed with 250 ml of distilled water, 1 M NaCl solution and again with water and well aspirated and mixed with 10 ml of a yeast invertase aqueous solution with a protein concentration of 11.5 mg / ml and an activity of 86 J / ml and immobilization of the enzyme. The reaction was allowed to stir for 1 hour and to stand at room temperature for 2 hours. The remaining 6 parts by weight of well aspirated but not washed, both activated particle method (s); b) 10 ml of a solution of said enzyme of the same activity was suspended. and protein concentration, the immobilization being carried out again;

1 hour by stirring and 2 hours standing as described.

After that time. all 12 samples of the mobilized materials were well vacuum-sucked through the textile material and washed in each case with 200 ml of water, 200 ml of 1 M NaCl solution and again with water and the materials well vacuumed. The specific activity of the mobilized invertase was evaluated in the mobilized enzyme materials obtained. The results are summarized in the following table.

Type. Poly- (2,6-dimethyl) - Polyethylene- Polymer Oxide Carriers phenyloxid / SORFIX / terephthalate / SORSILEN / Silica / SILOSIDE / Avg. particle size (mm) 0.5 to 1.0 0.5 to 1.2 0.2 to 0.5 Particle porosity / cm ^ / g / 0.459 2.14 0,048 Spec. particle surface / m ^ / g / 400.0 56.0 62.0 Method of carrier activation /and/ / b / / a / / b / / a./ / b / Washing of particles after activation + - + - + - -h - + / ++ / Specific activity immobilized. invertases on carriers 16.2 19.8 3.6 2.8 14.1 22.0 8.2 19.6 2.4 3.9 1 / / j / mg wet mass /

- 25 234 039 / a / The subject method of activation of carriers using K2P / b / Control method of activation of carriers based on MS. AO 214 0 $ 6 / + / Washing aspirated carrier particles after activation / - / Failure to wash aspirated carrier particles after activation / ++ / Listed in Example 5

Claims (4)

Method for the immobilization of microbial cells, plant cells and enzymes, characterized in that the particles of water-insoluble organic polymers, in particular based on polyethylene terephthalate, poly (2,6-dimethyl) phenyloxide, poly-6-caprolactam, a poly- block copolymer 6-caprolactam with polyethylene oxide, a copolymer of vinyl chloride with vinyl acetate, polycondensates of melamine with formaldehyde and ureas with formaldehyde and inorganic polymers, in particular based on polymeric silica, are activated with chemically reactive water-soluble polymers which are prepared by reaction between at least water-soluble substances 2 primary and / or secondary amino groups, in particular branched polyethyleneimine having a molecular weight of 50,000 to 1,000,000 or a polylyl alcohol having a molecular weight of 1,000 to 50,000, lysine or hexamethylenediamine and bifunctional aldehydes of dicarboxylic acids such as glutaraldehyde; to the activated particles of insoluble polymeric material is added an aqueous or buffered aqueous suspension of whole microbial or plant enzymatically active cells and / or an aqueous or buffered aqueous solution of the enzyme, and the resultant immobilized biocatalyst is separated and washed. 2. A method according to claim 1, characterized in that water-insoluble particles of organic and inorganic polymers of 0.001 to 5 mm in size, a specific surface area of from 1 to 700 m @ 2 / g and a particle porosity of 0.01 are used to immobilize the cells and enzymes. up to 2.5 cm @ 2 / g. 5. A method according to claim 1 or 2, characterized in that activation of the water insoluble polymer particles in particular ^Z ba zi polyethylene terephthalate, polyethylene / 2,6-dimethyl / fenyloxidu, block copolymer of vinyl chloride with vinyl acetate and a polymer of silicon dioxide is carried out by mixing with pre-prepared reactive soluble polymers which are prepared by reacting water-soluble substances containing at least two primary and / or secondary amino groups with bifunctional dicarboxylic acid aldehydes and then contacting the cell suspension after removal of the excess reactive polymer solution; enzyme solution. 4. A process according to any one of claims 1 to 2 wherein the particles of poly-6-caprolactam-based polymers, poly-6-caprolactam-block copolymer with polyethylene oxide and polycondensates of melamine with formaldehyde and urea with formaldehyde are contacted with the individual reactants. reactive soluble polymers sequentially, wherein in the first stage the particles of said polymers are contacted with a solution of polyethyleneimine, polylysine, lysine or hexamethylenediamine, and after sorption and removal of excess solution, the particles are exposed to a bifunctional aldehyde of dicarboxylic acids such as glutaraldehyde; after the formation of the reactive polymer on the surface of the particles, the excess solution is again removed and said activated polymer particles are exposed to a cell suspension or enzyme solution. , 5. The method according to claims 1 to 4, characterized in that whole, intact, enzymatically active cells of microorganisms, in particular gram-negative and gram-positive bacteria, acid-resistant bacteria, actinomycetes, yeast and other imperfect fungi, enzyme active plant cells and microbial, plant enzymes are immobilized. or animal origin.