CS252560B1 - Heterogeneous biocatalysts for biotransformation and their preparation - Google Patents

Abstract

Heterogeneous biocatalysts for biotransformations consist of an enzyme-active biological material embedded in a gen- -carrageenan gel. The method of their preparation consists in transferring the enzyme-active biological material, after suspension in a sol of 0.5 to 8% by weight of gen-carrageenan, preferably 1 to 3% by weight at a temperature of 20 to 70 °C, into a cold curing solution, cooled to 4 to 15 °C, containing potassium, calcium, aluminum, ammonium ions, low-molecular substances containing amino groups or a mixture thereof, or the gen-carrageenan sol with the suspended enzyme-active biological material is covered with a curing solution after cooling. Heterogeneous biocatalysts are useful for industrial biotransformations in the food, pharmaceutical and chemical industries in continuous and discontinuous biotechnologies.

Description

The invention relates to heterogeneous biocatalysts based on mobilized enzymes and cells with different enzymatic activity and a process for their preparation. The heterogeneous biocatalysts of the invention are useful for industrial biotransformations in the food, pharmaceutical and chemical industries in continuous and discontinuous biotechnologies.

A number of processes have been developed for the preparation of biocatalysts based on immobilized enzymes and cells, which can basically be divided into immobilizations into different types of carriers (sorption, chemisorption, covalent bonding and combinations thereof), aggregate formation using bifunctional reagents and embedding procedures enzymes or cells from various gels, both natural and synthetic.

The issue of enzyme immobilization is described in detail in many monographs and review articles such as Wingard et al .: Immobilized Enzyme Principles, Academic Press, New York (1976), Messing: Immobilized Enzymes for Industrial Reactors, Academic Press, New York 1975), Chibata : Immobilized Enzymes, Research and Development, Kodansha, Tokyo (1978), Marek and Kas: Chem. industry 34, 149 and 377 (1984); Immobilized cells are devoted to eg review articles by Vojtíšek and Jirků: Folia Microbiol. 28, 309 (1983), Marek and Kas: Chem. Industry 34, 546 (1984), Vandamme: Chem. Indian. 24, 1072 (1976), Jack and Hare: Adv. Biochem. Eng. 5, 126 (1977).

Immobilization of whole cells has its merit especially in those cases where the applied enzyme is intracellular, unstable in isolated form, possibly even in the form of immobilized preparation and where the substrate and the product of the catalyzed reaction are low molecular weight substances. Furthermore, cell immobilization is particularly advantageous for multi-step transformations involving the sequential treatment of many different enzymes, possibly with appropriate regeneration of the necessary cofactors. The use of immobilized cells also has economic advantages over immobilized enzymes, since the costly isolation of the enzyme is often omitted, which results in a lower cost of the prepared biocatalyst (Demnerová et al., Chem. Sheets 78, 199 (1984)).

While immobilized enzymes are most commonly used for binding to a suitable carrier, it is most common for immobilized cells to be incorporated into a matrix of different gels, whether of synthetic or natural origin.

From the point of view of technological applications, the method of cell immobilization into polyacrylamide was most often implemented (Chibata et al .: Appl. Microbiol. 27, 878 (1974). Biocatalysts prepared by incorporating various microorganisms into a synthetic gel formed by radical polymerization of acrylamide with N, N'-methylene-bis -acrylamide (U.S. Pat. No. 3,791,926) have been used for many bioconversions (L-aspartic acid, L-citrulline, L-lactic acid, etc.) The half-life of the prepared biocatalysts is up to 120 days.

However, this process for preparing biocatalysts has a number of disadvantages. It is not known to carry out a polymerization process according to which gel particles of a spherical shape and at least approximately of the same size can be prepared which guarantee optimal hydrodynamic properties of the biocatalyst.

The greatest drawback of this process, however, is that the short-term contact of the cell material with acrylamide monomers and N, N ' -methylene-bis-acrylamide often results in a decrease in the respective enzyme activity. Loss of enzyme activity of cellular material also occurs under the influence of heat released during polymerization. Disadvantages of the immobilization of cells into polyacrylamide include the danger that substances produced using the biocatalyst prepared in this way may contain toxic residues of unpolymerized monomers, which is unacceptable for many products from a hyphenic and medical standpoint.

These disadvantages of polyacrylamide-based biocatalysts have led to the development of a novel method of cell immobilization into a polysaccharide matrix, of which calcium alginate has been more prominent (Kierstan and Bucke: Biotechnol. Bioeng. 19, 387 (1977)) and kappa-carrageenan. Since the presence of calcium ions (insolubility of substrates or products of biotransformation etc.) is not possible in many technologies, the use of kappa-carrageenan has found wider technological application. Kappa-carrageenan is a polysaccharide that is obtained by extracting seaweed Chondrus crispus with hot water and then separating it from lambda-carrageenan by fractional precipitation with potassium ions (Rees: J. Chem. Soc., Part II, 1963, 1821).

Kappa-carrageenan contains D-galactose and 3,6-anhydro-D-galactose in a molar ratio of 1.1 to 1.5: 1 with a content of one SO 4 H group per 2 to 2.5 monosaccharide units. Lambda-carrageenan is mainly composed of 1,3-D-galactose-2-sulfate and 1,4-D-galactose-2,6-disulfate. Potassium, ammonium and other ions or substances (Tosa et al., Biotechnol. Bioeng. 21, 1697 (1979)) can be used to form rigid cured particles of different physical properties and shapes from kappa-carrageenan, while lambda-carrageenan in the presence of these substances. they do not create these structures.

By incorporating cells or other enzyme-active material into the kappa-carrageenan gel, a biocatalyst is prepared which, in addition to a high proportion of retained enzyme activity and very good physical and hydrodynamic properties, also has a very high stability. In the literature (Nishoda et al., Enzyme Microb. Technol. 6, 85 1984), an operating half-life of up to 680 days is reported for biocatalysts prepared by incorporating microbial cells into kappa-carrageenan.

Said excellent properties of these biocatalysts led to the gradual replacement of previously realized immobilized cell-based biotechnologies in polyacrylamide by procedures associated with biocatalysis using cells immobilized in kappa-carrageenan. The disadvantage of these biocatalysts is only the relatively demanding isolation of kappa-carrageenan and the associated rather high cost of this material.

This deficiency can be overcome by the use of the heterogeneous biocatalysts of the invention, which consists of an enzyme-active biological material incorporated into the gene of carrageenan. As enzymatically active biological material, these heterogeneous biocatalysts may comprise prokaryotic or eukaryotic cells of organisms, in particular gram-positive, gram-negative and gram-labile bacteria, acid-resistant bacteria, yeast, actinomycetes and other microscopic fungi, or fragments thereof and cellular particles carrying enzymatic activities, , lyases, oxidoreductases and other activities and / or particles of the entire sequence of enzymes of the logical sequence of the biochemical pathway, in particular degradative metabolism, glycolysis, enzymes catalyzing hormone biotransformation and the production of antibiotics and alkaloids, or mixtures of whole native cells with fragments, cellular particles and cells or whole cell conjugates or fragments thereof and added enzymes or enzymes alone, enzyme conjugates or synthetic glycoproteins.

The invention further provides a process for the preparation of said heterogeneous biocatalysts by suspending said enzyme-active biological material in a gene of carrageenan, transforming the suspension into a gel-like form exhibiting sufficient strength of the biocatalyst while maintaining a high proportion of enzyme activity using potassium, ammonium and other ions gel-like particles of various shapes, which have, besides sufficient strength, excellent hydrodynamic properties. By incorporating the enzyme-active biological material into the gel-carrageenan, according to the invention, a heterogeneous biocatalyst is prepared which has, in addition to high enzyme activity, also considerable stability.

According to the process of the invention, the enzyme-active biological material suspended in a sol of 0.5 to 8% gene-carrageenan at 20 to 70 ° C is transferred to a curing solution cooled to 4 to 15 ° C or cooled first in a thin layer and then overlayed with cold. with a curing solution and the cured gel is then converted to a suitable particle size by cutting or mixing. A solution containing potassium, ammonium, calcium, aluminum or other low molecular weight amine-containing substances such as 1,6-diaminohexane, ethylenediamine, histamine, hydrazide or D, L-histidine may advantageously be used to cure the gel of carrageenan.

The process according to the invention advantageously produces heterogeneous biocatalysts with spherical gel particles formed by dripping warm soles of gene-carrageenan with suspended enzyme-active biological material through a layer of liquid of lower specific gravity than the curing solution into this cooled solution, for example through a layer of paraffin oil into potassium chloride solution.

For some biotransformations, it is advantageous, especially in those cases where gas evolution occurs during the reaction, to further increase the rigidity of the gel particles formed. By the method of the invention, the strength of the gel-formed particles is increased by crosslinking its surface portion by addition of 1,6-diaminohexane or polyethyleneimine or other amino-containing substances and subsequent cross-linking with glutaraldehyde. The gel particle strength can also be increased by polymerizing N, N'-methylene-bis-acrylamide cross-linked acrylamide in the surface or even subsurface portion of the gel formed.

The cost of the heterogeneous biocatalyst according to the invention is considerably lower than that of immobilization to kappa-carrageenan, since the cost of the gene-carrageenan is approximately 40 times lower with respect to simpler isolation compared to kappa-carrageenan. Another great advantage over other methods of immobilization is the possibility of preparing a biocatalyst without the use of any toxic, potentially concerogenic or the like substances that could contaminate the biotransformation product.

This possibility is particularly important for the preparation of catalysts with a focus on use in the food or pharmaceutical industry. (Genu-carrageenan is commonly produced for the food industry.)

The biocatalysts intended for these areas of application can also crosslink the gel of the formed particles in its entire volume by the addition of non-toxic substances having a high hydroxyl group content, for example tannin. In this way, the stability of the formed biocatalysts is further increased.

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

Example 1

The wet cell paste of the production strain Escherichia alcalescens Asp 24 with aspartatammonium lyase activity of 28.1 ukat / g wet cells was mixed in physiological saline to form a 20 wt% suspension. of wet cells. An equal amount (v / v) of a 5% solution of gene-carrageenan 55 ° C warm was added to the suspension heated to 50 ° C. After thorough mixing, the mixture was cooled and overlaid with a 0.5 M ammonium chloride solution cooled to 10 ° C. After 24 hours, the cured gel was mixed in a mixer and filtered. The obtained biocatalyst was found to have an aspartase activity of 2.17 µm / g wet cured gel.

Example 2

To a wet cell suspension of 40 wt. cells of the same strain as in Example 1, heated to 45 ° C, were added three times the volume (v / v) of a 4% 50% C-carrageenan gene solution warm. The mixture was thoroughly mixed and dripped warm to a cold (10 ° C) 0.3M potassium chloride solution through a paraffin oil layer. The resulting gel beads with immobilized cells were left in 0.3 M potassium chloride solution (10 ° C) for 24 h.

After cooling to 5 ° C and filtering, a 1: 1 (v / v) cooled (5 ° C) solution containing 0.5 M phosphate buffer pH 7.0, 0.3 M chloride was added to the 2.5 mm diameter beads. potassium and 0.085M 1,6-diaminohexane. After stirring for five minutes, a solution containing 0.5 M phosphate buffer pH 7.0, 0.3 M potassium chloride and 0.085 M glutaraldehyde was added to the mixture in a ratio of 0.5: 1 (v / v beads). After an additional 30 minutes of stirring at 5 ° C, the obtained beads were washed with a cold 0.3 M potassium chloride solution. The thus prepared heterogeneous biocatalyst was found to have an aspartase activity of 1.7 ukat / g wet biocatalyst.

Example 3

The wet paste of Streptomyces nigrificans cells with a glucose-isomerase activity of 160 ynkat / g wet cells was mixed in physiological saline to form a 30 wt% suspension. of wet cells. To the suspension heated to 45 ° C was added twice the volume (v / v) of a 5% solution of gene-carrageenan 55 ° C warm. After thorough mixing, the mixture was poured hot onto a dish so that the gel layer was 5-6 mm high. The solidified gel was overlaid with a solution of 0.3M potassium chloride and 0.085M ethylenediamine. After standing for 18 h, the cured gel was cut into approximately 5x5x5 mm particles, overlaid with a solution of 0.3 M potassium chloride and 0.08514 glutaraldehyde and after 30 min. the stirring was aspirated and washed with 0.3M KCl. The obtained biocatalyst was found to have a glucosaisomerase activity of 10 yukat / g wet biocatalyst.

Example 4

A gel layer was prepared from the wet mass of Aspergillus niger cells exhibiting a glucoseoxidase activity of 120 µm (ikat / g wet cells) according to Example 3, which was overlaid with a solution of 0.2M potassium chloride, 0.2M ammonium chloride and 0.0514 histamine. The gel was cut into 5x5x5 mm particles, to which was added a solution containing 4% by weight of acrylamide, 0.8% by weight of methylene-bis-acrylamide and 0.5% by weight of 3-dimethylaminopropionitrile (equal proportions of solution volume and gel weight) After 15 minutes, the gel particles were transferred to a 0.5% potassium persulfate solution, and after 30 minutes of stirring, the gel particles were filtered and washed thoroughly with water, and the obtained heterogeneous biocatalyst was found to have a glucose oxidase activity of 5 yUkat / g wet material.

Example 5

To the wet cell wall suspension of yeast Saccharomyces cerevisiae containing 60 wt. dry matter having an invertase activity of 850-yakat / g wet material heated to 55 ° C, an equal amount (volume / volume) of 5% carrageenan 60 ° C warm gene was added. After mixing, a solution of 1% by weight of tannin in saline was added to the mixture so that the final concentration of tannin in the gel was 0.1%. The mixture was again mixed and then hot dropped through a layer of paraffin oil into a cold (10 ° C) solution of 0.314 potassium chloride. The formed 2 mm diameter beads were left in 0.3 M potassium chloride for 24 h. Upon removal from this solution, a biocatalyst having an invertase activity of 225 yukat / g wet mass was obtained.

Example 6

To 10 parts of a solution containing 4 wt. % of carrageenan gene heated to 50 ° C containing 0.1 wt. 1 part (v / v) of 3-galactosidase of 2 mkat / ml activity was added to tannin. A heterogeneous biocatalyst of 2 mm diameter spherical particles with a 3-galactosidase activity of 97 µm / g wet biocatalyst was prepared according to the procedure of Example 5.

Example 7

Dílu 1 part wet cell walls of yeast Saccharomyces uvarum was added 10 parts (volume / weight of cell walls) of 0.05 M phosphate buffer pH 6 and 3 parts of 0.0514 sodium periodate.

After shaking for 6 h at 4 ° C in the absence of light, the oxidized cell walls were centrifuged and washed with 0.05 M phosphate buffer pH 7.5. Κ 1 part oxidized cell walls were added 10 parts (v / v) of a solution of glucosoxidase in 0.05 M phosphate buffer pH 7.5 containing 2.4 mg protein per ml. After shaking for 16 h at 4 ° C, the enzyme-bound cell walls were centrifuged and resuspended in 3 parts (v / v) saline. After warming to 40 ° C, the suspension was added to an equal volume of gel containing 4 wt. 50 ° C heated gen-carrageenan. After stirring, the mixture was cooled and overlaid with 0.314 potassium chloride solution cooled to 10 ° C. After 24 h, the cured gel was mixed in a mixer and filtered. The obtained heterogeneous biocatalyst was found to have an invertase activity of 128 µg / g per g wet material and a glucoseoxidase activity of 36 µg / g wet biocatalyst.

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Example 8

To a slurry containing 50 wt. 3 times (v / v) of a 4% w / w solution of Saccharomyces cervisiae wet cells heated to 45 ° C were added. Gene-Carrageenan 50 ° C warm. After thorough mixing, the mixture was dropped warm in a cold (10 ° C) solution of 0.25M calcium chloride. The formed yeast-mobilized gel particles were left in a 0.25 M calcium chloride solution for 24 h. The produced biocatalyst produced 10 mg of ethanol per 1 g of wet gel.

Claims (6)

Heterogeneous biocatalysts for biotransformation with enzymatically active biological material, characterized in that they consist of enzymatically active biological material, such as prokaryotic or eukaryotic cells of organisms, in particular bacteria, yeasts, actinomycetes and other microscopic fungi, or fragments thereof carrying enzyme activities, optionally whole sequences of the logical sequence of the metabolic pathway or a mixture of whole native cells with their fragments or from whole cell conjugates or fragments thereof and added enzymes, or from enzymes themselves, enzyme conjugates or synthetic glycoproteins incorporated into gel-carrageenan. 2. A process for the preparation of heterogeneous biocatalysts according to claim 1, characterized in that the enzyme-active biological material is converted, after suspending in the sol, from 0.5 to 8% by weight of the carrageenan gene, preferably from 1 to 3% by weight. at a temperature of 20 to 70 ° C into a cold curing solution cooled to 4 to 15 ° C containing potassium, calcium, aluminum, ammonium ions containing an amino-containing substance or a mixture thereof, or a solubilized enzyme-active biological material overlaps with the enzyme carrageenan sol after cooling with curing solution. 3. A process according to claim 1, wherein the enzyme-active biological material is suspended in the carrageenan sol in the presence of hydroxyl-containing substances, preferably tannin, at a concentration of 0.1 to 5% by weight. 4. A method according to any one of claims 1 to 3, wherein the solute of the gene-carrageenan with the suspended enzyme-active biological material is dripped through a layer of liquid with a specific gravity lower than the cured solution, for example a paraffin oil layer. of potassium at a concentration of 0.3 mol / liter. 5. A process for the preparation of heterogeneous biocatalysts according to claims 1 to 4, characterized in that the particles of the gel-formed carrageenan gel with immobilized enzymatically active biological material are treated with 1,6-diaminohexane or polyethyleneimine or a substance containing two or more amino groups. is then treated with glutaraldehyde. 6. A process for the preparation of heterogeneous biocatalysts according to claims 1 to 4, characterized in that the gel-carrageenan gel formed particles are polymerized with acrylamide in admixture with N, N'-methylene-bis-acrylamide.