CS249325B3 - A method for strengthening and stabilizing biocatalyst particles for enzymatic inversion of sucrose - Google Patents

Abstract

The solution relates to a method of solidifying and stabilizing biocatalyst particles for enzymatic inversion of sucrose, prepared according to copyright certificate No. 231458, in which an aqueous solution of sucrose and its technical forms and intermediates of its production are brought into contact with biocatalyst particles. The essence of the solution lies in the fact that after adjusting the pH to a value between 6.0 and 8.0, either an aqueous solution of glutaraldehyde and/or an aqueous solution of technical or pure protein of animal, plant or microbial origin, preferably technical egg protein, and/or a disintegrated or autolyzed suspension of production cells containing, in addition to other soluble proteins, the invertase enzyme, is added to the stirred mixture of biocatalyst particles based on yeast cells containing the enzyme, then the continuously stirred reaction mixture is optionally supplemented with another addition of glutaraldehyde, the stabilized particles are separated from the reaction liquid by filtration, suspended with intensive stirring in ice-cold acetone, separated again by filtration and dried after homogenization in the open air at room temperature for 1 to 24 hours, then the particles are suspended in cold water, decanted several times with water and left to swell in water or in buffer at temperatures between 5 and 20 °C.

Description

The invention relates to a method of solidifying and stabilizing biocatalyst particles prepared according to basic copyright certificate No. 231 458 and stabilizing the enzyme -D-fructofuranosidase (invertase, EC 3.2.1.26) in these particles, which serve for the enzymatic inversion of pure sucrose, its technical forms and intermediate products of its production into a mixture of the respective monosaccharides, glucose and fructose, or glucose-fructose syrup.

The resulting enzyme inversion products created by single, multiple, repeated or continuous contact of these solidified and stabilized particles with aqueous solutions of pure sucrose (so-called raffinate), their technical forms, e.g. so-called affinate (which is raw sugar, not washed from mother liquors), claret (a highly viscous concentrated solution of raw sugar with a concentration of 60 to 70 percent by weight) and intermediate products of its production, especially molasses and heavy sugar juices, can either be used directly, e.g. in the food industry as glucose-fructose syrup, which has a higher degree of sweetness than sucrose, or both monosaccharides can be separated by isolation and further purification and thus pure or technical glucose and fructose can be prepared.

The obtained pure or technical monosaccharides can then be used in various sectors of the national economy, e.g. in the pharmaceutical industry, or they can be used as substrates for other enzymatic conversions, e.g. glucose can be used as a substrate for enzymatic conversion to D-gluconic acid.

For these purposes, in industrial practice, especially in the food and sugar industry, in the confectionery, lemonade and various other beverage industries, a single application of a soluble enzyme - invertase, isolated from various cells of microorganisms, is used. The source of the enzyme is mainly yeast. This enzyme is not yet produced in Czechoslovakia and for these purposes, i.e. for the preparation of so-called invert sugar, the enzyme preparation is imported from abroad. Sucrose can also be broken down non-enzymatically, either physically or chemically, using the so-called Finnish technology on ion exchangers at high temperatures with simultaneous isolation of both monosaccharides, or chemically, by acid hydrolysis.

However, the methods mentioned have their economic and other limits. In the case of a single application of soluble invertase, the limit is the price and import of the enzyme preparation, in the case of physicochemical technology, the limit is the initial concentration and quality of sucrose (35% mass/volume solution), energy requirements (digestion on ion exchange columns at 80 °C), the quality and price of ion exchangers and their regeneration. In the case of chemical digestion of sucrose, the limit is the high consumption of mineral acids, corrosion of the apparatus and, above all, the poor organoleptic and visual properties of the obtained glucose-fructose syrup (bitterness and color).

Recently, there has been a tendency towards a single application of live native brewer's or baker's yeast cells containing invertase for the enzymatic inversion of concentrated solutions of sucrose and its technical forms, especially raw sugar and sorghum. Enzyme hydrolysis is carried out by mixing whole live or partially autolyzed, or disintegrated yeast cells into concentrated sugar solutions with simultaneous mixing and heating to temperatures around 80 °C.

The limit of this technology is the import of yeast biomass from breweries or yeast houses to the place of application, the availability of biomass in the required quantity throughout the production period, its variable quality, turbidity in the obtained products and finally secondary contamination of yeast biomass mainly resulting from transport, storage and handling.

Due to the variable quality of biomass, especially the enzyme content in the cells, the degree of inversion in the obtained products also fluctuates between 60 and 85%. Finally, certain cellular components (proteins, nucleic acids, lipids) remain in the final products, which increases the hygiene risk.

A certain progress in the field of modern biotechnology is the application of immobilized invertase or immobilized cells with invertase activity, which, unlike single applications, allows the use of immobilized biocatalyst particles many times over in batch stirred reactors or continuously in reactors with a buoyant or fixed bed or in stirred continuous reactors, which significantly reduces labor requirements, energy consumption and the quality of the conversion mixture is high, because the catalyst particles are insoluble, easily separable and do not pass into the final food products.

Certain methods of immobilization of enzymes, including invertase, have already been described in the patent literature. This is primarily the Czechoslovak patent certificate No. 214 096, which describes the use of microporous particles of organic polymers by sorption and subsequent covalent binding of enzymes using sorbed crosslinking agents, especially glutaraldehyde, or by sorption of solutions of various enzymes, including invertase, onto particles of polymer sorbents and subsequent covalent crosslinking of the sorbed enzymes with glutaraldehyde.

In the Czechoslovak patent certificate No. 209 743, a method of producing immobilized enzymes, including invertase, on microporous organic polymer sorbents based on poly-6-caprolactam and polycondensates of melanin with formaldehyde and urea with formaldehyde is described, whereby the principle of enzyme immobilization consists in the previous chemical reaction of particles of these synthetic amino polymers with free glutaraldehyde, the simultaneous chemical activation of these particles, washing out the excess crosslinker and the subsequent covalent binding of enzymes through chemically active aldehyde groups, with the formation of Schiff bases between the enzyme protein and particles of the sorbents activated in the manner specified and described.

However, the above-mentioned procedures require isolated technical or pure enzymes, i.e. invertase, which increases the costs of production and application of these catalysts. Significantly more economical immobilization procedures are those that do not require isolated enzymes, i.e. procedures using immobilized cells with the desired enzyme activity. Physical immobilization of yeast with invertase activity has been described, for example, by Toda and Shoda (Biotechnol. Bioeng., 17, 481, 1975) and D' Souza and Nadkarni (Hindustan Antibiotics Bull., 20, 68 1978). In the first case, the cells are embedded in agar pellets, in the second case, the cells are embedded in the network of the polymer formed by copolymerization of acrylamide with Ν,Ν'-methylene diacrylamide, with the formation of well-sedimenting catalyst particles.

However, repeated use of physically embedded cells results in leaching of cells from the particles, resulting in a decrease in specific activity and enzyme efficiency, as the natural or synthetic polymers used form very soft pellets.

A certain progress in the technology of enzyme transformations is, for example, the procedure described in the Czechoslovak patent certificate No. (PV 9573-82), which consists in the chemical activation of originally chemically inert particles of synthetic microporous polymers of Czechoslovak origin, with reactive water-soluble polymers and subsequent covalent binding of microbial cells, plant cells and enzymes to the activated particles. The cited patent certificate also describes a method of immobilizing whole intact yeast cells with invertase activity and isolated invertases onto particles of previously chemically activated organic and inorganic water-insoluble carriers.

This procedure provides very good results when using isolated enzyme, but when immobilizing whole cells, the resulting preparations have relatively low specific activity.

In the Czechoslovak patent No. 209 265, a method of producing mutually chemically bound microbial cells with different enzyme activities, including yeasts with invertase activity, is described. This method of immobilization is more advantageous compared to the previous one, as it does not require any water-insoluble carriers. The procedure consists in covalently crosslinking individual cells with glutaraldehyde, their subsequent permeabilization, after which individually crosslinked and permeabilized cells with different enzyme activities are covalently immobilized with each other to form stable cell aggregates under specific conditions, especially in a centrifugal field, by filtration pressure and freezing of the reaction mixture. This procedure can be used to obtain stable solid aggregates of immobilized cells, but the production technology requires special reaction conditions, which results in increased demands on the machine and technological equipment of the operation.

In an effort to remove this technological limit of production and use of cell catalysts in various industrial sectors, a significantly simplified and to a large extent universal technology for the production of various immobilized prokaryotic and eukaryotic cells has been developed, the particles of which contain various enzymes and are usable for various biotransformations, including those containing the enzyme invertase. These procedures are the subject of Czechoslovak Patent No. 231,458.

The cited procedure, which is based on mutual chemical immobilization of various cells carrying various enzymes using reactive water-soluble polymers (hereinafter referred to as RRP), allows the creation of biocatalyst particles not only by using the freezing technique of the reaction mixture, but also by simply mixing the suspended cells in the reaction mixture at normal temperatures, preferably at room temperature. The resulting catalyst particles have high enzyme activity and good sedimentation properties, but they are soft and must be mechanically strengthened in various ways, either by using adhesives dissolved in organic solvents, e.g. acetone, or by post-forming voluminous particles into the form of granules or cylinders and their subsequent drying.

The first method of consolidation results in the creation of a diffusion barrier for the substrate and products into and out of the particles by reducing their porosity (partially sealing the pores of the particles), the second method leads to the formation of large particles, of the order of 1 to 10 mm, thus eliminating the initial advantage of the high specific activity of the originally formed smaller particles (the dependence of particle size on the specific surface).

If cross-linked and permeabilized cells were not used as the starting material for the production of catalysts according to Czechoslovak Patent No. 231,458, i.e. if whole intact native cells were used, the obtained particles showed lower stability of the monitored enzyme activity, i.e. invertase activity, both during storage and during repeated or continuous use.

This fact is due to the fact that RRP, due to its high molecular weight, does not penetrate native cells, which is an advantage for some enzymes contained in cells that are extremely sensitive to free low-molecular crosslinkers, especially glutaraldehyde. Enzymes that are not extremely sensitive to the otherwise relatively aggressive effects of bifunctional crosslinkers such as glutaraldehyde include the enzyme invertase.

This fact has already been exploited in the past, in various ways of immobilizing the enzyme and cells with this activity in the cited author's certificates, and these procedures are summarized in a recent article on immobilized cells (Folia Microbiol., 28,

309-340, 1983).

Now, it has been found that, depending on the method of production of immobilized cells described in Czechoslovak Patent No. 231,458, it is possible to strengthen and stabilize biocatalyst particles with invertase activity by chemical means in combination with mechanical means, and the obtained material is usable for the enzymatic inversion of sucrose, its technical forms and intermediates of its production on an industrial scale.

The technology of solidification and stabilization of particles according to the present invention is simple, does not require any water-insoluble carriers and, above all, does not require special technological and mechanical equipment. For the production of the catalyst, solidification and stabilization of particles, only a mixing boiler and a filtration vacuum device are needed.

and

The above-mentioned disadvantages were eliminated by developing a method for solidifying and stabilizing biocatalyst particles, prepared according to copyright certificate No. 231,458, for enzymatic inversion of sucrose, in which an aqueous solution of sucrose, its technical forms and intermediates of its production are brought into contact with biocatalyst particles, the essence of which consists in adding, after adjusting the pH to a range of 6.0 to 8.0, an aqueous solution of glutaraldehyde and/or an aqueous solution or suspension of technical or pure protein of animal, plant and/or microbial origin, such as egg protein, disintegrated or autolyzed microbial cells, preferably cells of the production microorganism used, to a continuously stirred mixture of biocatalyst particles based on yeast cells containing the invertase enzyme, preferably Saccharomyces cerevisiae or Saccharomyces uvarum, after which the mixture is optionally finished with a further addition of glutaraldehyde, the stabilized particles are separated from the reaction liquid separated by filtration, suspended with vigorous stirring in ice-cold acetone, after which they are separated again by filtration and dried after homogenization in the open air at room temperature for 1 to 24 hours, then suspended in cold water, decanted several times with water and finally allowed to swell in water or in buffer at temperatures between 5 and 20 °C.

The solidified and stabilized biocatalyst particles obtained in this way are brought into contact with aqueous solutions of sucrose, its technical forms, preferably raw sucrose or claret, and intermediate products of its production, or mother liquors, especially molasses and heavy sugar syrup, and at a pH of the reaction mixture of 4.0 to 5.0, discontinuous repeated or continuous hydrolysis is carried out at temperatures of 10 to 80 °C, after which, after the reaction, glucose and fructose are optionally isolated from the filtered liquid or syrup, separated from the catalyst particles, or both in the mixture or one of the products are subjected to the action of other enzymes, or the obtained mixture of both monosaccharides is used for food or other purposes.

The biocatalyst particles obtained according to the present invention are solid, very rapidly sedimenting formations of irregular size and shape with high invertase stability, which is demonstrated in the following examples. Their strength, good sedimentation properties, easy and rapid separability from the reaction mixture and high specific activity and efficiency of enzyme conversion allow their multiple repeated or continuous use in common types of reactors, at high initial substrate concentrations.

Commercial brewer's or baker's yeast were used as the starting enzymatically active cell material. However, the method of solidification and stabilization of biocatalyst particles according to the present invention does not exclude the use of other strains of microorganisms containing intracellular invertase, or strains of yeast and other microorganisms obtained, for example, by selection and accumulation or gene manipulation or by other breeding methods.

To separate the particles of the obtained biocatalyst, metal sieves for sieve analysis with mesh sizes of 0.8; 0.5 and 0.315 mm were used, while the particles were separated by floating on these sieves with water. To evaluate the activity of the particles, both enzymatic methods (glucose oxidase test) were used to determine the reaction rate of glucose concentration increases according to Jérgensen and Anderson (Anal. Biochem., 53, 141-145, 1973) and spectrophotometrically. The reference methods were the spectrophotometric method for determining the concentration increase of reducing sugars according to Janíček (Rukovět potravinářské analýzy, p. 381, 1962, SNTL), at high initial sucrose concentrations, the substrate concentration at the beginning was evaluated refractometrically and gravimetrically (dry matter), and the course and degree of enzyme inversion were evaluated polarimetrically.

The unit of enzyme activity of invertase in biocatalyst particles is the amount of enzyme that liberates one ^jmol of glucose in one milliliter in one minute under conditions of zero-order enzyme reaction kinetics, at a temperature of 30 °C, an initial concentration of sucrose of 0.25 M dissolved in Oj 05 M acetate buffer of pH 4.6 in a stirred reactor. The specific activity of biocatalyst particles has the dimension «mol glucose . min ^- ^ . mg ^- ''' dry matter.

The sedimentation rate of the undivided catalyst particles and the divided fractions was determined by the time it takes for 1 ml of the given catalyst fraction to settle; the dimension ml. s ^- ^. The specific resistance (R) was calculated from the time it takes for a given amount of substrate, under a given pressure, to flow through a certain column of catalyst particles, _2

R=p.S/v.d Pa.s.m p - substrate pressure

S - column cross-section v - flow rate d - column height

Specific resistance measured for water at room temperature. Particle size was determined both by optical microscope and built-in measuring eyepiece, and by dividing on sieves.

Particle porosity was assessed indirectly by measuring the reaction rate as a function of substrate concentration and was also determined by scanning electron microscopy.

The catalyst particles were suspended (about 0.5 kg/1 liter) in 0.05 M acetate buffer pH 4.6 containing 0.1% (w/v) sorbic acid and stored in screw-capped polyethylene bottles at 5-10°C. Under these storage conditions, the catalyst is stable for 6 months to 1 year, with sorbic acid preventing the growth of any secondary contamination during long-term storage. The catalyst particles can also be treated with 0.5 to 1.0% formaldehyde for sanitization without significant loss of specific activity.

In the following, the invention is explained in more detail in the examples, without being limited thereto.

Examples of implementation

Example 1

200 g of wet mass of Saccharomyces uvarum yeast with invertase activity was suspended in 500 ml of reactive water-soluble polymer, hereinafter referred to as RRP, obtained 24 h prior polymerization of 4% flocculant containing polyethyleneimine (Sedipur CL-930 BASF,

NSR) and 2% glutaraldehyde according to the procedure of Czechoslovak author's certificate No. 231 458; the pH of the suspension was adjusted to 7.0 with 40% NaOH solution and the highly viscous suspension containing voluminous particles of aggregated cells was continuously stirred for one hour. Then 28 ml of 25% aqueous glutaraldehyde solution was added and stirred intensively again for one hour.

After this time, the pH of the suspension was adjusted to 8.0 with continuous stirring and the particles were sucked off through a textile material (milling nylon cloth). The material, which had been well sucked off and squeezed free of excess reaction mixture, was suspended in 500 ml of pure acetone pre-cooled to -20 °C. The particles were mixed in ice-cold acetone for about 3 min using a high-speed stirrer and then passed through a textile film, the lumps were homogenized and the material was left to dry for 24 h in the air at room temperature.

Then, the larger hard lumps were mechanically ground in a mortar, the dry material was homogenized and suspended in 1 liter of distilled water. After about 1 hour of swelling, the material was decanted 3 times with 1 liter of water each time, then 2 times with 1 liter of 0.05 M acetate buffer pH 4.6 and left to swell in this buffer for 24 hours at a temperature of 10 °C. The swollen catalyst particles were well sucked off through a textile material and weighed. 220 g of wet mass of solidified and stabilized biocatalyst particles with an average size distribution of 0.76 mm and a specific activity of 0.721 μmol glucose . min . mg dry matter were obtained.

g of wet catalyst mass was suspended in 45 ml of 1 M sucrose solution (raffinate), dissolved in acetate buffer pH 4.6. The suspension was placed in a Dewar flask.

The container was heated to 30 °C and at selected time intervals, during continuous stirring of the suspension with a steel propeller stirrer at a speed of 100 rpm, the increase in the concentration of one or both monosaccharides was monitored by the analytical procedures described in the description of the present invention.

It was found that the enzyme inversion is quantitative (100% of theory, i.e. 360.4 mg glucose and fructose . ml ^) in 90 min. This procedure with the same batch of biocatalyst was repeated a total of 20 times, always so that after the end of the reaction in each cycle (after 90 min) after quantitative sedimentation of the particles, the reaction mixture was vacuum-aspirated with a Pasteur pipette from the upper part of the reactor and the particles were suspended again in a fresh batch of substrate under otherwise identical conditions and sucrose concentration. The average degree of enzyme inversion was 99.9% of theory/20 cycles of repeated use of the same batch of biocatalyst.

200 g of wet mass of solidified and stabilized biocatalyst particles with an average size distribution of 0.76 mm was further physically and biochemically characterized by the procedures described in the description of the present invention, in particular with regard to particle size distribution, specific activity of individual size classes, sedimentation rate and specific resistance of particles in the slurry.

The results are presented as follows.

Particle fraction Particle size (mm) Amount of wet matter íg) Representation of units, size classes <»> A 0.8 103.3 51.5 B 0.8 - 0.5 57.5 28.8 C 0.5 - 0.315 15.3 7.7 D less than 0.315 23.9 12.0

Particle fraction of particles Sedimentation velocity (s) Specific resistance (Pa.sm ² ) Specific activity (μmol glucose .mg ^-3 ) A 4.0 19,69.10 ⁵ 0.161 B 8.0 40.78.10 ⁵ 0.473 C 9.6 - 0.565 D 21.7 289.02.10 ⁵ 1,524

g of undivided catalyst particles were suspended in 100 ml of acetate buffer in which sorbic acid was dissolved at a concentration of 0.1%? The pH of the buffer was 4.6. Under the storage conditions specified in the description of the present invention, the specific activity of the particles was monitored at one-week time intervals. After 192 days of continuous monitoring, no decrease in the specific activity of the particles was recorded.

Example 2 kg of wet mass of native Saccharomyces cerevisiae cells was suspended in 10 liters of distilled water using a high-speed stirrer and the suspension containing 0.5 g of wet cell mass/ml was cooled to 5 °C. Then the cells were disintegrated by passing through a slotted mechanical disintegrator four times at a maximum overpressure of 6.10 Pa. Between individual disintegration cycles, the suspension was always cooled to 10 to 15 °C. The degree of cell disintegration was monitored both microscopically and by the total amount of released proteins in the supernatant, the centrifuged suspension.

According to the procedure of Czechoslovak Patent No. 231 458, 15 liters of water were introduced into two 35-liter stainless steel fermentation tanks and 750 g of flocculant containing polyethyleneimine (Sedipur CL-930) was mixed into each of the fermenters and after its dissolution, 825 ml of a 46% aqueous solution of glutaraldehyde was added. The resulting concentration of both recreational components in each of the fermenters was 5% (mass/volume) of Sedipur and 2.5% (volume/volume) of glutaraldehyde.

Polymerization was carried out for 24 hours with continuous stirring at 20 °C. 10 kg of wet mass of whole cells of S. cerevisiae was homogenized in 10 liters of water in a polyethylene container using a high-speed stirrer. The cell suspension was divided into 2 equal volume parts and each part was poured into the RRP solution with increased stirring intensity. After homogenization of the suspension, the pH was adjusted from 5.2 to 7.5 with intensive stirring with a 40% NaOH solution and stirred for 1 hour at a speed of 800 rpm.

In 15 liters of disintegrated cell suspension, the preparation of which was described in the first paragraph, 374 g of technical egg protein were dissolved. The total protein concentration in the supernatant after centrifugation was 50 mg.ml ¹ . The suspension was divided into two equal volumes and each of them was poured into the stirred cell suspension in the RRP while stirring vigorously. After the highly viscous suspension thickened, the stirring intensity was reduced and stirred at approximately 300 rpm for 30 minutes.

Then the stirring intensity was increased again and 655 ml of 46% aqueous glutaraldehyde solution was added to each of the stainless steel tanks. The resulting concentration of free crosslinker was 1% (vol/vol). After intensive stirring of the suspension, the stirring was stopped and the reaction mixture was left to stand for 30 min, then gently stirred and, while stirring, gradually pumped onto a rotary vacuum filter on which a nylon mill cloth was stretched.

Particle filtration was performed under vacuum and the moist and well-sucked particles were cut in layers from the surface of the cloth from the drum of the rotary filter with a set stainless steel blade.

A total of 25.5 kg of wet mass of material was obtained after filtration.

The specified amount of wet material was divided into 25 parts (4 1 kg) and each part was gradually suspended in 1.5 liters of ice-cold acetone in a 5 liter plastic container using a high-speed stirrer equipped with a steel propeller, stirred intensively for about 3 minutes and then quickly and sharply sucked off through a textile material (milling cloth) using a vacuum suction and left to dry on plastic trays for 24 hours at room temperature without washing. All 25 parts by weight of the material were gradually processed in the described manner.

After drying, the material was mechanically homogenized and weighed. 6.045 kg of dry particle mass was obtained. The fraction with particles larger than 2 mm (lumps) was separated on a net in a dry state. The remaining amount of dry matter (5.5 kg) was suspended in 20 liters of drinking water in a polyethylene container and decanted several times with 20 liters of water, each time after quantitative sedimentation of the material. The material was left to swell overnight in a freezer at a temperature of 10 °C.

After swelling, the material was stirred intensively in water using a high-speed stirrer and washed many times with drinking water after quantitative sedimentation of the particles, while pouring off the upper cloudy layer and very fine particles. Decantation was continued until there was no turbidity in the washing water after intensive stirring. After quantitative decantation, the material was vacuum-drained through a mill cloth and weighed. The amount obtained was 7.5 kg of wet mass. This amount was suspended in 15 liters of 0.04 M acetate buffer with a pH of 4.6, which contained 0.1% sorbic acid, and left at a temperature of 10 °C until the next day.

The specific activity of the solidified and stabilized biocatalyst particles was 0.450 µmol.mg” of dry matter, the average particle size was 0.363 mm. With the obtained catalyst, 20 repeated conversions of the enzyme inversion of a 1.5 M sucrose solution (raffinate with a concentration of 51% mass/volume) were carried out using the procedure described in Example 1 with the difference that

That one cycle of enzyme inversion with a single batch of catalyst lasted 4 hours. Under the conditions given, the average degree of conversion was 96.8% of theory/20 cycles of repeated use of the solidified and stabilized particles. The specific activity of the catalyst did not change after its 20-fold use.

Example 3

With the solidified and stabilized biocatalyst particles, the preparation method and basic properties of which were described in Example 2, continuous enzymatic inversion of sucrose (raffinate) was carried out in three ways. (A) substrate conversion in a column with a fixed bed of biocatalyst, (B) substrate conversion in a column with a stirred bed of biocatalyst, (C, substrate conversion in a stirred single-stage continuous reactor.

(A) A glass column with an internal diameter of 16 mm and a length of 20 cm was filled to a height of 19.5 om with 20 g of wet catalyst particles. A 0.5 M sucrose solution in acetate buffer pH 4.6 was pumped onto the fixed catalyst bed, vertically placed column from top to bottom, at a rate of 5 ml.h at room temperature. After approximately two column volume changes (approximately 17 hours of continuous flow), the average degree of enzyme inversion at the column outlet was 90% of theory (162 mg.ml reducing sugars). The reactor was operated continuously for 140 hours, achieving an unchanged degree of enzyme inversion.

(B) The same glass column was filled with 18 g of wet catalyst mass and mounted in a horizontal position on a reciprocating shaker with a swing rate of 120.m”\ A 1 M sucrose solution in acetate buffer pH 4.6 was pumped into one end of the column at a rate of 10 ml.h and the reaction mixture was collected from the other end with continuous stirring at a temperature of 28 °C.

The column was operated continuously for 24 hours, achieving an average degree of theoretical enzyme inversion of 98%.

,C) 45 g of wet mass of catalyst particles was suspended in 450 ml of 1 M sucrose solution in buffer and the suspension was stirred slowly at room temperature. After reaching a steady state, after achieving quantitative conversion, a 1 M sucrose solution was pumped into the stirred vessel at a rate of 166 ml.h ¹ and the reaction mixture was aspirated at the same rate, using a second pump. In order to prevent particles from escaping (aspirating) from the reactor, the suction end of the tube was provided with a polyamide screen and wrapped with a miller's cloth. Under the above conditions, the single-stage stirred continuous reactor was operated for 24 hours with an average achievement of the theoretical inversion degree of 88.5%.

Example 4

The solidified and stabilized biocatalyst particles, the preparation method of which was described in Example 2, were used for the enzymatic inversion of technical forms of sucrose, in particular raw sugar (affinade) and kléré. The conversions of these forms of sucrose were carried out in a batch stirred reactor at a temperature of 40 °C, pH 4.6 (acidification of aqueous solutions of technical sucrose with acetic acid), catalyst concentration of 15 vol. % (150 g wet mass of particles + 850 ml solution or syrup). The only variable was the initial concentration of sucrose in their various technical forms. The initial sugar concentration in undiluted kléré determined by dry matter and refractometry was 66 g/100 ml, i.e. approximately 1.9 M sucrose solution. Kléré was diluted with acidified water. Raw sugar dissolved in acidified water. The results are summarized as follows.

Sucrose form kler afinada

sucrose conc. (wt. %) 66 56.5 max. achieved degree of inversion 78 87.6 (% theory) reaction time 4 4 <h>

51.4 46.3 41.3 60 65 91.0 96.0 98.0 88.3 81.2 3 3 3 4 4

Example 5

The catalyst, the method of solidification and stabilization of which was described in Example 2, was used for 10 repeated cycles of enzyme inversion of undiluted claret, in which the sucrose concentration was 67 wt. %. The conversions were carried out in a stirred batch reactor at a temperature of 60 °C and pH 4.6 in a Dewar flask in the manner described in Example 1. The only difference was the method of handling the catalyst. After each completed reaction cycle, the particles were filtered through a mill cloth and, without washing, put back into the reactor and poured over again with a fresh batch of undiluted claret. The pH of the claret for each repeated batch was adjusted to 4.6 with acetic acid. The duration of one cycle was standardly 4 hours.

Under the above conditions, the average degree of conversion was 83.6% of theory/10 repeated cycles of using the same catalyst batch. The specific activity of the particles did not change after their 10-fold use under the above conditions.

Claims (1)

Process for strengthening and stabilizing biocatalyst particles for sucrose enzyme inversion prepared according to the author's certificate No. 231 458, wherein an aqueous solution of sucrose, its technical forms and intermediates of its production is contacted with biocatalyst particles, characterized in that it is mixed into a continuously stirred mixture yeast cell biocatalyst particles containing an invertase enzyme, preferably Saccharomyces cerevisiae or Saccharomyces uvarum, add an aqueous solution of glutaraldehyde and / or an aqueous solution or suspension of technical or pure animal, vegetable and / or protein after adjusting the pH to 6.0 to 8.0. of microbial origin, such as egg protein, disintegrated or autolysed cells, preferably cells of the production microorganism used, after which the mixture is optionally sutured by further addition of glutaraldehyde, the stabilized particles are separated from the reaction liquid by filtration, sus pendant with vigorous stirring in ice-cold acetone, filter again to separate and dry after homogenization. freely in air at room temperature for 1 to 24 hours, then suspend the particles in cold water, decant several times with water and allow to swell in water or in a buffer at a temperature in the range of 5 to 20 ° C.