EA017266B1 - Biocatalyst, method of its preparation and method of obtaining glucose-fructose syrups - Google Patents

Abstract

The invention relates to biotechnology, in particular to methods of preparing biocatalysts for obtaining glucose-fructose syrups from renewable starch-containing natural sugar substitutes - glucose-fructose syrups. A biocatalyst is described containing microbial biomass with glucose isomerase activity, silicon dioxide and insoluble hydro compounds Co (II). The microbial biomass is used as a paste with humidity 75-80%, silicon dioxide is used as hydro gel with humidity 60-95%, hydro compounds CO (II) are used as wet sediment washed by water. For increasing biocatalyst stability a cross link of the microbial mass is performed by a bifunctional reagent, for example, by glutaric dialdehyde in a concentration not exceeding 100 mg of reagent per 1 g of dry cells. A method is also described for preparing the biocatalyst and a method for obtaining glucose-fructose syrups. The technical result - increase of stability and glucose isomerase activity of biocatalyst.

Description

The invention relates to biotechnology, in particular, to methods for the preparation of biocatalysts for the production of renewable starch-containing raw materials of natural sweeteners for glucose-fructose syrups.

It is known that glucose-fructose syrups are widely used in the food industry in the production of soft drinks, confectionery and diet foods, as well as in the preservation of fruits and vegetables. The use of these syrups in the recipes for the preparation of various products significantly improves their consumer properties, slows down the drying and hardening (staling) processes.

The heterogeneous (multiphase) mode of biocatalytic conversion of natural substrates with the participation of solid biocatalysts loaded into a flow reactor is the most economically advantageous. Inorganic matrices (metal oxides, silicates, carbon-mineral carriers) as carriers for immobilizing enzymatically active substances (enzymes or microbial cells containing them) are characterized by inertness, high stability in aqueous media, and resistance to chemical and microbial destruction. The high mechanical strength of inorganic carriers allows their use in reactors of various types, including high-performance vortex reactors.

An urgent task in the preparation of highly stable and active biocatalysts is to optimize their composition and develop new methods of immobilization.

There is a method of immobilizing cells of actinobacteria Ar (11gbac (er chebobae, possessing glucose isomerase activity and catalyzing the enzymatic reaction of glucose isomerization to fructose by physical adsorption on inorganic carriers [G. Kovalenko, L. Perminova, T. Terentyeva, Sapunova L.I., Lobanok A.G., Chuenko T.V., Rudina N.A., Chernyak E.I. Glucose isomerase activity in suspensions of Ar cells (HbObacobacterium and adsorption immobilization of microorganisms on inorganic carriers // Prikl. Biochem Microbiol. 2008. - T. 44. - Issue 2. P. 193-201]. It has been established that the actinobacteria A. Sheo Bapae have a weak ability to adhere to the surface of solid carriers, which does not depend on the textural characteristics of the carrier, nor on the chemical nature, nor on the surface morphology The adhesion does not exceed 1.6 mg of dry cells per 1 g of carrier Maximum glucose isomerase activity (EA ^ μmol / min) of biocatalysts prepared by adsorption of bacterial cells on a macroporous carbon-mineral carrier Sapropel followed by joint height stallation of cell suspension and carrier is 2Ea / g. The maximum stability is possessed by biocatalysts obtained in the process of deep cultivation of microorganisms in the presence of carbon-containing foam ceramics; These biocatalysts retain their initial activity in the monosaccharide isomerization reaction for 10 hours at 70 ° C. The disadvantages of this method are:

1) low adsorption;

2) low biocatalytic activity (<2 EA / g);

3) low stability under reaction conditions (half-inactivation time 1 _1/2 <18 h).

Known is a method of immobilization of glucose isomerase extracted from cells 81ger1otuse5 tiYdtokik, by adsorption on the particles 8ίΘ ₂ followed by cross-linking with glutaric dialdehyde [Vyo8a1e 8.N., Kao MV, Ge ^ Erapbe ν.ν. Mo1essi1ag apb ίηάιΐ5ΐιύι1 acrea οί d1ieo8e schoteta8e // M1goyu1. Wow. 1996, wo. 60, No. 2, p. 280-300]. According to this technology, the company Mjek Ka11-Syet1e (I8) produces a commercial biocatalyst ORYA5 \\ 'ee1 2®, whose activity is 22 EA / g at 60 ° C. The disadvantage of this method is the low glucose isomerase activity of the biocatalyst.

Closest to the claimed invention is a method of immobilization of microbial biomass by incorporating it into a xerogel of silicon dioxide [VI 2341560, С12Р 19/24, С12Ы 11/04, 12/20/2008]. To do this, use microbial biomass in the form of a paste with a moisture content of 75-80%. Silicon dioxide is used in the form of a hydrogel with a set of properties described in (VI 2341560), namely: humidity 60-95%, the specific surface area and bulk density of the product obtained after drying the hydrogel at 105-120 ° C, respectively 30-550 m ² / g and 0.1-0.7 g / cm ³ . Immobilization of bacterial cells is carried out by mixing microbial biomass in the form of a wet paste, silicon dioxide in the form of a hydrogel and magnesium salts and divalent cobalt in the form of aqueous solutions of sulfates, or nitrates, or chlorides, followed by drying the resulting mixture in air at a temperature of 50-70 ° C in no more than 2 hours, pressing and granulation into particles of 0.4-3.0 mm in size.

Significant disadvantages of this method are:

1) low enzymatic activity of the biocatalyst, component 25 EA / g at 70 ° C;

2) a relatively low resistance to destruction in aqueous buffer media at pH 7.8, due to the introduction of magnesium and cobalt ions in the form of soluble salts into the biocatalyst composition;

3) low stability of the biocatalyst, the half-inactivation time (1 _1/2 ) of which is 22 hours at 70 ° C.

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The invention solves the problem of optimizing the composition of the biocatalyst in order to increase its activity and stability and to develop an effective method for producing glucose-fructose syrups with the participation of the prepared biocatalyst.

The technical result of the invention is to increase the enzymatic activity, stability of the biocatalyst and its resistance to destruction in an aqueous reaction medium by optimizing its composition.

The problem is solved by using a solid biocatalyst prepared by immobilizing the biomass of microorganisms producing cell-bound glucose isomerase (microbial biomass) during the isomerization of monosaccharides.

Biocatalyst containing microbial biomass, silicon dioxide ₍₂ occupies 8) and gidroksosoedineniya insoluble cobalt (Co _x O _y), wherein the mixing ratio of the biocatalyst (biomass of microbial, silicon dioxide and insoluble cobalt gidroksosoedineny) depends on the keys for microorganisms producing glucose isomerase. For example, for actinobacteria of the genus Lg111goLae1sg, the optimal composition of the biocatalyst, in terms of dry weight (in wt.%), Is as follows: microbial biomass - 10-15; 8U ₂ - 45-60; So _x Oy - 20-40.

The proposed biocatalyst for producing glucose-fructose syrups in the process of isomerization of monosaccharides includes a microbial biomass with glucose isomerase activity, silicon dioxide and cobalt compound (P) in the form of insoluble hydroxy compounds and has a composition, in wt.%, On dry matter: microbial biomass - 10-60; silicon dioxide - 20-70; insoluble hydroxy compound of cobalt (P) - 20-40.

Silicon dioxide is contained in the form of a hydrogel with the following set of properties: the specific surface area of the product obtained after drying the hydrogel at 105-120 ° C is 30-550 m ² / g, bulk density of the product obtained after drying the hydrogel at 105-120 ° C equal to 0.1-0.7 g / cm ³ .

The finished (dry) biocatalyst has a specific surface area of 50-300 m ² / g, a pore volume of 0.3-0.9 cm ³ / g; pore diameter - 10-15 nm.

The problem is also solved by the method of preparation of a biocatalyst for the production of glucose-fructose syrups, in which microbial cells with glucose isomerase activity are immobilized into silicon dioxide by thoroughly mixing silicon dioxide in the form of a hydrogel, freshly precipitated cobalt hydroxocompounds in the form of a wet sediment, microbial biomass biomass a crosslinking reagent, followed by drying the resulting mixture to a dry air state, pressing at an excess pressure of 150 tm granulation and particle size of 0.2-4.0 mm obtained biocatalyst has the composition in wt% on dry substance: microbial biomass - 10-60;. silicon dioxide - 20-70; insoluble hydroxy compound of cobalt (P) - 20-40.

To increase the stability of the biocatalyst, they are treated with bifunctional cross-linking reagents - dialdehyde (glutaraldehyde, GA) or carbodiimide (1-cyclohexyl-3 (2-morpholinoethyl) carbodiimide meto-4-toluenesulfonate, CD), adding these reagents per 20-4 mg 1 g of dry microbial biomass. Since the crosslinking reagent reduces the glucose isomerase activity of microbial biomass, it is added at the last stage of preparation of the biocatalyst to reduce the negative effect. Therefore, the mixing order of the components is an essential sign of the preparation of biocatalysts: first, silica hydrogel and a wet precipitate of cobalt hydroxo compounds are thoroughly mixed, microbial biomass is added to this mixture and mixed until homogeneous, then a solution of a crosslinking reagent is added to this mixture, it is thoroughly mixed again and dried to dry air condition (humidity 10-12%).

To prepare the biocatalyst, a microbial biomass is used, which has a glucose isomerase activity of at least 600 EA per 1 g of dry cells, in the form of a paste with a moisture content of 75-80%.

Silicon dioxide is used in the form of a hydrogel with the following set of properties: humidity 60-95%, the specific surface area and bulk density of the product obtained after drying the hydrogel at 105-120 ° C, respectively 30-550 m ² / g and 0.1- 0.7 g / cm ³ .

Silicon dioxide hydrogel is obtained by reacting sodium or potassium silicate with a mineral acid selected from the range of sulfuric, nitric or carbonic acids, or by reacting with ammonium salts of sulfuric or nitric or carbonic acids, at pH 6.5-9.0, temperature 20-90 ° C, the deposition rate of 30-300 g 81O ₂ / l _susp- h, followed by washing and filtering the hydrogel [KI 234156020].

Insoluble cobalt hydroxyl compounds are obtained by precipitation from solutions of cobalt nitrate Co (IO3) ₂ with an ammonia solution with vigorous stirring at 20-22 ° C. A precipitate is formed for 20 hours at 22 ° C, then it is washed many times with distilled water.

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The problem is also solved by the method of producing glucose-fructose syrups in the process of isomerization of monosaccharides (glucose), which use the biocatalyst described above. The process is carried out in a flow reactor with a fixed bed at a temperature of (65 ± 5) ° C and at a continuous flow rate of the reaction medium through the biocatalyst layer not exceeding 2 ml / h. The biocatalyst is used in the form of particles with a size of 0.2-4.0 mm, to improve the hydrodynamic properties of the fixed layer of the biocatalyst, glass balls with a diameter of 2 mm are used as an inert filler,

The technical result is achieved due to the implementation of the following groups of distinctive features:

1) optimization of the composition of the biocatalyst by the mass content of the constituent components;

2) the introduction of Co (11) into the biocatalyst in the form of insoluble hydroxy compounds;

3) the order of mixing the constituent components of the biocatalyst;

4) by conducting the process of isomerization of monosaccharides (glucose) to glucose-fructose syrup in a flow reactor with a fixed bed of a prepared biocatalyst having an optimal composition.

The method of preparation of the biocatalyst is simple and versatile, all components are available and have a relatively low cost. The use of Co (11) in the form of insoluble hydroxo compounds leads to a significant decrease (by an order of magnitude) in the concentration of transition metal ions of Co ²⁺ in the final product, glucose-fructose syrup.

The biocatalyst for the production of glucose-fructose syrups contains the following components, in wt%, on dry matter: microbial biomass - up to 60; silicon dioxide - up to 70; From _x O _y - up to 40. The content of microbial biomass in the biocatalyst is determined by the taxonomic affiliation of the microorganism producing glucose isomerase. The silica hydrogel used to prepare the biocatalyst is prepared as described [KP 234156020]. The silica hydrogel is inert and has a high chemical and microbial stability. Hydroxy compounds Co (11) used to prepare the biocatalyst are obtained by precipitation from solutions of cobalt nitrate Co (IO ₃ ) ₂ with an ammonia solution with vigorous stirring at 20-22 ° С. A precipitate is formed for 20 hours at 22 ° C, then it is washed many times with distilled water. The use of Co (11) in the form of insoluble hydroxo compounds Co _x O _y increases the resistance of the prepared biocatalysts to destruction in an aqueous medium (compared to the prototype), ensures the entry of Co ²⁺ ions into the microenvironment of cells, which are necessary to increase the thermal stability of cell-bound glucose isomerase. Moreover, the addition of Co ²⁺ ions to the reaction medium is not required, which increases the quality and purity of the final product - glucose-fructose syrup. When using biocatalysts described in the scientific and technical literature, cobalt ions are added to the reaction medium at a concentration of not less than 1 mM, and then the final product is purified on ion-exchange resins.

A biocatalyst for glucose isomerization is prepared by mixing silicon dioxide hydrogel, insoluble Co _x O _y hydroxy compounds, microbial biomass until a homogeneous state, then a solution of a bifunctional cross-linking reagent is added to this mixture. Dialdehydes, preferably glutaraldehyde, are used as the bifunctional crosslinking agent. The resulting mixture is dried to a dry air state, for example, for 2-8 hours at 20-25 ° C. The drying temperature should not exceed 70 ° C, a further increase in temperature leads to a loss of enzymatic activity of the biocatalyst. The dried mixture is pressed at a pressure of 150 atm and mechanically crushed to particles with a size of 0.2-4.0 mm

The result is a granular solid biocatalyst with glucose isomerase activity equal to 100-350 EA / g dry biocatalyst (in the prototype 25 EA / g). The half-inactivation time of the prepared biocatalyst under the conditions of the glucose isomerization reaction at 70 ° C exceeds 500 hours (in the prototype 22 hours). Thus, the claimed composition of the biocatalyst, as well as the method of its preparation provide high activity and stability of the biocatalyst in the conditions of the glucose isomerization reaction to the final product - glucose-fructose syrup.

The biocatalyst has the following set of properties: specific surface - 50-300 m ² / g; the total pore volume is 0.6-0.9 cm ³ / g; the average pore size is 10-15 nm. As can be seen from the above texture parameters, the biocatalyst has a mesoporous structure, which ensures its operation in the monosaccharide isomerization reaction in the kinetic region (without diffusion restrictions). The initial glucose isomerase activity of the biocatalyst is 100-350 EA / g dry biocatalyst.

Thus, the salient features of the inventive biocatalyst and the method of its preparation, in comparison with the prototype, are the following:

1) high glucose isomerase activity of the biocatalyst (> 10 times higher than in the prototype);

2) higher stability of the biocatalyst in the process of isomerization of monosaccharides at 6070 ° C (> 20 times higher than in the prototype);

3) higher quality of the final product (glucose-fructose syrup) due to a significant (70-fold) decrease in the content of transition metal ^ions Co ^{2+ in it} .

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The claimed set of properties of the biocatalyst (composition, preparation method, enzymatic activity and stability) ensure the high efficiency of this biocatalyst in the reaction of glucose isomerization into glucose-fructose syrups in various types of flow reactors (with a fixed biocatalyst layer, vortex reactors).

The following physicochemical methods are used to characterize the prepared biocatalysts.

The texture parameters of the biocatalyst are measured by the following methods. The specific surface area is determined by thermal desorption of argon (BET method). Pore volume and pore diameter are determined by mercury porosimetry.

The enzymatic activity of the prepared biocatalysts is determined by the rate of conversion (in μmol / min ^ EA) of 2 M (44% by dry matter) solution of monosaccharide (glucose or fructose) in 0.02 M phosphate buffer pH 7.0-7.8 to glucose-fructose syrup in the presence of magnesium ions MD ²⁴ (1 mm) at a temperature of 60-75 ° C. The enzymatic activity of microbial cells in suspension and in an immobilized state is expressed either in EA / g dry cells or EA / g dry biocatalyst.

The thermal stability of biocatalysts is evaluated under the conditions of periodic testing of the biocatalyst in the reaction of isomerization of monosaccharides at a temperature above 60 ° C according to the time of its half-inactivation (1 _1/2 ). The half-inactivation time is the time at which the ratio of the amount of activity in the current time (A ₁ ) to the initial activity (A _o ) of the biocatalyst is 0.5.

The operational stability of the biocatalyst is evaluated by the half-inactivation time of the biocatalyst (T _1/2 ) as follows. A biocatalyst with a granule size of 0.2–4.0 mm and an inert filler (glass beads) in a volume ratio of 1: (1-3) are loaded into a flow-through column reactor; a glucose / fructose solution is continuously pumped through this fixed bed at a rate of 1.8 ml / h The concentration of the substrate is 2 M (44% by dry matter) in 0.02 M phosphate buffer, pH 7.0-7.8. The process temperature is maintained in the range of 65 ± 5 ° C using a thermostat.

The invention is illustrated by the following examples.

Optimization of the biocatalyst composition according to the content of microbial biomass.

Example 1. Preparation of a biocatalyst.

As the microbial biomass, the biomass of actinobacteria Lg111b0ac1eaaaaaaaa producing glucose isomerase is used [patent of RB No. 11170, 10.30.2008]. Actinobacteria are grown on growth-friendly media containing sucrose or any other carbon source, and collected in the late logarithmic growth phase by centrifugation at 3-5 thousand rpm. Microbial biomass is a beige paste with a moisture content of 75-80%. The glucose isomerase activity of microbial biomass is on average 600 EA / g dry cells at 70 ° C.

Silicon dioxide hydrogel is obtained by reacting sodium silicate with ammonium nitrate at a temperature of 70 ° C, pH 7.5, the deposition rate of silicon dioxide in the form of a hydrogel of 300 g §1O ₂ / l-h.

Preparation of the biocatalyst is carried out as follows: 10 g of a wet silica hydrogel and 1.0 g of freshly precipitated Co _x O _y hydroxyl compounds are mixed to a homogeneous mixture, then 0.5 g of wet biomass is added, and mixed again to a homogeneous mixture. The resulting mixture is dried to dry air state - for 8 hours at 20-25 ° C. The dried mixture is pressed at a pressure of 150 atm. The tablets obtained after pressing are mechanically crushed and the fractions of a certain particle size distribution are sieved on the appropriate sieves. Get the biocatalyst of the following composition, in wt% on solids: microbial biomass - 10; silicon dioxide - 50; So _x O _y - 40.

The activity of the prepared biocatalyst with a granule size of 0.2-4.0 mm is 11 and 25 EA / g at 60 and 70 ° C, respectively. The biocatalyst _half- inactivation time (1 _1/2 ) is 45 hours at 75 ° C.

Example 2. Preparation of a biocatalyst.

Similar to example 1, characterized in that the composition of the prepared biocatalyst is as follows, in wt% on dry matter: microbial biomass - 15; silicon dioxide - 45; So _x O _y - 40.

The activity of the prepared biocatalyst with a grain size of 0.2-4.0 mm is 16 EA / g at 6 ° C. For this biocatalyst, 1 _1/2 = 22 hours at 75 ° C.

Example 3. Preparation of a biocatalyst.

Similar to example 1, characterized in that the composition of the prepared biocatalyst is as follows, in wt% on dry matter: microbial biomass - 20; silicon dioxide - 40; So _x O _y - 40.

The activity of the prepared biocatalyst with a granule size of 0.2-4.0 mm is 21 EA / g at 60 ° C. For this biocatalyst a 1 _1/2 = 2 hours at 75 ° C due to the low stability of the biocatalyst to degradation in the reaction medium. After 2 hours, the biocatalyst turns into an amorphous mass, losing its mechanical strength and the shape of the granules.

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Example 4. Preparation of a biocatalyst.

Similar to example 1, it differs in that for the preparation of the biocatalyst, the microbial biomass of the recombinant Exjepsyasoy strain, a producer of glucose isomerase obtained by genetic engineering, is used. The glucose isomerase activity of microbial biomass is on average 1000 EA / g dry cells at 70 ° C.

Get the biocatalyst of the following composition, in wt% on solids: microbial biomass 20; silicon dioxide - 60; So _x O _y - 20.

The activity of the prepared biocatalyst with a granule size of 0.2-4.0 mm is 70 EA / g at 70 ° C. For this biocatalyst, 1 _1/2 = 8 hours at 70 ° C.

Example 5. Preparation of a biocatalyst.

Similar to example 4, characterized in that the composition of the prepared biocatalyst is as follows, in wt% on dry matter: microbial biomass - 40; silicon dioxide - 40; So _x O _y - 20.

The activity of the prepared biocatalyst is 560 EA / g at 70 ° C. For this biocatalyst, 1 _1/2 = 2 hours at 70 ° C.

Example 6. Preparation of a biocatalyst.

Similar to example 4, characterized in that the composition of the prepared biocatalyst is as follows, in wt% on dry matter: microbial biomass - 60; silicon dioxide - 20; So _x O _y - 20.

The activity of the prepared biocatalyst with a granule size of 0.2-4.0 mm is equal to 310 EA / g at 70 ° C. For this biocatalyst, 1 _1/2 = 2 hours at 70 ° C.

Example 7. Preparation of a biocatalyst.

Similar to example 4, characterized in that the composition of the prepared biocatalyst is as follows, in wt% on dry matter: microbial biomass - 70; silicon dioxide - 10; SokhU - 20.

The activity of the prepared biocatalyst with a granule size of 0.2-4.0 mm is 260 EA / g at 70 ° C. For this biocatalyst, 1 _{1/2 is} less than 2 hours at 70 ° C, because after 2 hours of functioning in the reaction medium (pH 7.8) the biocatalyst swells, the structure of its granules is destroyed.

From a comparison of examples 1-3, it can be seen that with an increase in the content of microbial biomass of actinobacteria Lg111goBac1cg soyoiae, the enzymatic activity of the biocatalyst increases proportionally and linearly, and its stability decreases sharply due to loss of stability and mechanical strength of the biocatalyst in the reaction medium. Thus, with the use of actinobacteria L11bObac1e2 schoiae, the optimal content of microbial biomass in the biocatalyst is 1015 wt.%. From a comparison of examples 4-7, it is seen that the enzymatic activity of the biocatalyst prepared using the recombinant Exjeyssoysoi strain does not increase with an increase in the amount of microbial biomass above 60 wt.%, And its stability is relatively low. Thus, when using the recombinant strain Exexcisoy, the optimal content of microbial biomass in the biocatalyst is 40-60 wt.%.

The results of optimizing the composition of biocatalysts according to the content of microbial biomass are presented in table. one.

Optimization of the composition of biocatalysts by the content of Co _x O _y .

Example 8. Preparation of a biocatalyst.

Similar to example 1, characterized in that the composition of the prepared biocatalyst is as follows, in wt% on dry matter: microbial biomass - 15; silicon dioxide - 80; So _x O _y - 5.

The activity of the prepared biocatalyst with a granule size of 0.2-4.0 mm is 23 EA / g at 60 ° C. For this biocatalyst, ΐ _1/2 = 22 hours at 75 ° C.

Example 9. Preparation of a biocatalyst.

Similar to example 1, characterized in that the composition of the prepared biocatalyst is as follows, in wt% on dry matter: microbial biomass - 15; silicon dioxide - 65; So _x O _y - 20.

The activity of the prepared biocatalyst with a granule size of 0.2-4.0 mm is 16 EA / g at 60 ° C. For this biocatalyst, 1 _1/2 = 37 hours at 75 ° C.

Example 10. Preparation of a biocatalyst.

Similar to example 1, characterized in that the composition of the prepared biocatalyst is as follows, in wt% on dry matter: microbial biomass - 15; silicon dioxide - 45; So _x O _y - 40.

The activity of the prepared biocatalyst with a granule size of 0.2-4.0 mm is 18 EA / g at 60 ° C. For this biocatalyst, 1 _{! / 2} = 37 h at 75 ° C

Example 11. Preparation of a biocatalyst.

Similar to example 4, characterized in that the composition of the prepared biocatalyst is as follows, in wt% on dry matter: microbial biomass - 15; silicon dioxide - 25; So _x O _y - 60.

The activity of the prepared biocatalyst with a granule size of 0.2-4.0 mm is 21 EA / g at 60 ° C. For this biocatalyst, ΐ _1/2 = 16 hours at 75 ° C.

A comparison of examples 8-11 shows that with an increase in the content of Co _x O _{y the} activity of the biocatalyst practically does not change, and its stability increases. The optimal content of Co _x O _y is 20-40 wt.%.

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The results on the optimization of the composition of biocatalysts according to the content of cobalt hydroxo compounds are presented in table. 2.

Optimization of the composition of biocatalysts by the content of bifunctional crosslinking agent.

Example 12. Preparation of a biocatalyst.

Similar to example 5, it differs in that for the preparation of the biocatalyst, a bifunctional crosslinking reagent, glutaraldehyde (HA), is used. To do this, the silica hydrogel, insoluble Co _x O _y compounds, HA solution and microbial biomass are thoroughly mixed, then dried, pressed and granulated as described in Example 1. The cross-linking reagent solution is used in such an amount that in terms of 1 g of dry cells accounted for 20 mg of HA.

The activity of the prepared biocatalyst is 300 EA / g at 70 ° C. For this biocatalyst, 1 _1/2 = 20 hours at 70 ° C (cf. with example 8 1 _1/2 = 8 hours).

Example 13. Preparation of a biocatalyst.

Similar to example 5, characterized in that the amount of HA used for crosslinking microbial biomass is 50 mg / g of dry cells.

The activity of the prepared biocatalyst is 110 EA / g at 70 ° C. For this biocatalyst, 1 _1/2 = 65 hours at 70 ° C.

Example 14. Preparation of a biocatalyst.

Similar to example 5, characterized in that the amount of HA used for crosslinking microbial biomass is 100 mg / g of dry cells.

The activity of the prepared biocatalyst is equal to 100 EA / g For this biocatalyst, ΐι _{/ 2} = 25 hours at 70 ° C.

Example 15. Preparation of a biocatalyst.

Similar to example 5, characterized in that the amount of HA used for crosslinking microbial biomass is 160 mg / g of dry cells.

The activity of the prepared biocatalyst is 81 EA / g For this biocatalyst, ΐ _1/2 = 18 hours at 70 ° C.

Example 16. Preparation of a biocatalyst.

Similar to example 5, characterized in that the amount of HA used for crosslinking microbial biomass is 240 mg / g of dry cells.

The activity of the prepared biocatalyst is 12 EA / g For a given biocatalyst 1; _{/ 2} = 22 h at 70 ° C.

Example 17. Preparation of a biocatalyst.

Similar to example 5, it differs in that carbodiimide (CD) - 1-cyclohexyl-3- (2-morpholinoethyl) carbodiimide meto-4toluenesulfonate is used as a bifunctional crosslinking reagent. The amount of CD used for crosslinking microbial biomass is 400 mg / g of dry cells.

The activity of the prepared biocatalyst is equal to 72 EA / g For this biocatalyst, 1 _1/2 = 8 hours at 70 ° C.

A comparison of examples 12-17 shows that to increase the stability of biocatalysts prepared by immobilizing a recombinant producer strain, it is necessary to use bifunctional crosslinking reagents (dialdehydes, diimides). Glutaraldehyde exhibits a maximum stabilizing effect and is relatively cheap, affordable and, as a result, the most widely used reagent. A comparison of examples 12-16 shows that with an increase in the amount of glutaraldehyde, the enzymatic activity of the biocatalyst (by 2–3 times) decreases, while the dependence of stability on the concentration of HA has an optimum in the range of 20–100 mg HA / g of dry cells.

The results for optimizing the composition of biocatalysts for the content of glutaraldehyde are presented in table. 3.

Optimization of the mixing order of biocatalyst components.

Example 18. Preparation of a biocatalyst.

Similar to example 13, differs in that the mixing order of the components of the biocatalyst is as follows. First, silica hydrogel, insoluble cobalt hydroxyl compounds (11) and microbial biomass are mixed until homogeneous, then an HA solution in the optimal amount (40 mg HA / g dry cells) is added to the resulting mixture.

The activity of the prepared biocatalyst is 120 EA / g at 70 ° C. For this biocatalyst, 1 _1/2 = 100 hours at 70 ° C.

Example 19. Preparation of a biocatalyst.

Similar to example 18, characterized in that the mixing order of the components of the biocatalyst is as follows. First, silica hydrogel and insoluble cobalt hydroxyl compounds are mixed (11). Microbial biomass is crosslinked with a GA solution in the optimal amount (40 mg HA / g dry cells). Then, a silica hydrogel containing Co _x O _y and a microbial biomass crosslinked by HA are mixed.

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The activity of the prepared biocatalyst is 140 EA / g at 70 ° C. For this biocatalyst, ΐι / 2 = 45 h at 70 ° C.

From the above examples it is seen that the mixing order of the components that make up the biocatalysts plays a significant role in increasing the stability of the biocatalyst. The optimal order is when glutaraldehyde is added to the mixture of components (silicon dioxide + cobalt hydroxo compound + microbial biomass) last.

Example 20. Obtaining glucose-fructose syrup.

Use the biocatalyst in example 1. The prepared dry biocatalyst with an activity of 22 EA / g at 70 ° C in the form of granules with a size of 0.2-4.0 mm, mixed with glass balls with a diameter of 2 mm in a volume ratio of 1: 1, is loaded into a column flow reactor. The reaction medium is pumped through this fixed bed at a space velocity of 1.8-2.0 ml / h. The composition of the reaction medium is as follows: 0.02 M phosphate buffer, pH 7.8; glucose concentration - 44 wt.%; the concentration of MD ²⁺ in the form of sulfate is 1 mm. The temperature is maintained in the range of 65 ± 5 ° C. An inert filler (glass beads) is introduced to reduce the hydrodynamic resistance of the biocatalyst layer to the flow.

Example 21. Obtaining glucose-fructose syrup.

Use the biocatalyst in example 1. The prepared dry biocatalyst with an activity of 22 EA / g at 70 ° C in the form of granules with a size of 0.2-4.0 mm (without inert filler) is loaded into a column flow reactor. The reaction medium is pumped through this fixed bed at a space velocity of 1.8-2.0 ml / h. The composition of the reaction medium is as follows: 0.02 M phosphate buffer, pH 7.8; glucose concentration - 44 wt.%; the concentration of MD ²⁺ in the form of sulfate is 1 mm. The temperature is maintained in the range of 65 ± 5 ° C. After 2 hours, the hydrodynamic resistance of the biocatalyst layer to the flow of the substrate solution begins to increase, which, ultimately, after 2-3 days leads to the fact that glucose-fructose syrup stops flowing out of the reactor.

A comparison of examples 20, 21 shows that the inert filler can improve the hydrodynamic parameters of the fixed layer of the biocatalyst.

For the above-described conditions of the process of isomerization of a glucose solution into a glucose-fructose syrup, the half-inactivation time of the T _1/2 biocatalyst is more than 500 hours. As noted above, cobalt (11) is introduced into the composition of the biocatalysts in the form of insoluble hydroxy compounds. In this case, Co ^{2+ ions are} not added to the reaction medium. Chemical analysis of samples taken at the outlet of the reactor shows that during 820 hours of biocatalyst testing at 65 ± 5 ° С, the Co content in glucose-fructose syrup practically does not change and amounts to (1-2) -10 ^-5 M. From the scientific and technical The literature knows that the content of Co ²⁺ in glucose-fructose syrup is 1 -10 ^-3 M. Thus, the process of glucose isomerization using the prepared biocatalysts reduces the content of Co ²⁺ in the final product by more than 50 times.

As can be seen from the above examples, biocatalysts having an optimal composition have high glucose isomerase activity (14 times higher than in the prototype), high operational stability (23 times higher than in the prototype), good resistance to destruction in the reaction medium with a value pH 7.0-7.8, high mechanical strength for use in flow reactors. During the glucose isomerization process with the participation of the prepared biocatalysts, the content of Co ^{2+ ions} in glucose-fructose syrup decreases by more than 50 times, which increases its quality and does not require additional purification.

Table 1

Optimization of the composition of biocatalysts according to the content of microbial biomass

No. ra -------------------------------------------------- -------------------------------------------------- -------------------------------------------------- -------------------one The taxonomic affiliation of a microorganism - The content of microbial biomass, wt% The content of Sokh, wt% Enzymatic remy, semi-inactive activity, EA / g, II, hour (temperature _ζ 'ο / -Ί \ ι (temperature of determination, С) ^ν g / ^{g of} determination) one Arkkogasac sp. 10 40 11 (60) | 45 (75) 2 fifteen 40 16 (60) 1 22 (75) 3 twenty 40 21 (60) 2 (75) 4 sp twenty twenty 70 (70) 1 8 (70) 5 40 twenty 560 (70) 2 (70) 6 60 20 1 310 (70) 2 (70) 7 70 20 1 260 (70) <2 (70)

- 7 017266

table 2

Optimization of the composition of biocatalysts according to the content of cobalt hydroxyl compounds (P)

Example Number The taxonomic affiliation of a microorganism The content of microbial biomass, wt% The content of Co _x O _r wt% Enzymatic activity, EA / g (determination temperature, ’C) Half-inactivation time, hour (determination temperature) 8 AnthobaMeg 5p. fifteen 5 23 (60) 22 (75) nine fifteen twenty 16 (60) 37 (75) 10 fifteen 40 18 (60) 37 (75 eleven fifteen 60 21 (60) 21 (75)

Table 3

Optimization of the composition of biocatalysts according to the content of a bifunctional crosslinking agent - glutaraldehyde

Example Number The taxonomic affiliation of a microorganism Glutaraldehyde crosslinking reagent content, mg / g dry cells Enzymatic activity, EA / g (determination temperature, ° C) Activation half-time, hour (determination temperature) 12 EzskegmJaia sp. twenty 300 (70) 20 (70) thirteen fifty 110 (70) 65 (70) 14 one hundred 100 (70) 25 (70) fifteen 160 81 (70) 18 (70) sixteen 240 12 (70) 22 (70)

CLAIM

Claims (8)

CLAIM 1. A biocatalyst for producing glucose-fructose syrups during monosaccharide isomerization, including microbial biomass with glucose isomerase activity, silicon dioxide and cobalt compound (P), characterized in that the catalyst contains cobalt compound (P) in the form of insoluble hydroxo compounds and has a composition. % solids: microbial biomass 10-60; silicon dioxide - 20-70; the cobalt (P) hydroxo compound is 20-40, and also has a specific surface area of 50-300 m ² / g; pore volume 0.3-0.9 cm ³ / g; pore diameter - 10-15 nm. 2. The biocatalyst according to claim 1, characterized in that it contains silicon dioxide in the form of a hydrogel with the following set of properties: the specific surface area of the product obtained after drying the hydrogel at 105-120 ° C is 30-550 m ² / g, bulk density the product obtained after drying the hydrogel at 105-120 ° C. is 0.1-0.7 g / cm ³ . 3. A method of preparing a biocatalyst for the production of glucose-fructose syrups during the isomerization of monosaccharides, including mixing its components, drying, pressing and granulating, characterized in that the components of the biocatalyst are mixed in a specific order as follows: silicon dioxide hydrogel, insoluble cobalt hydroxyl compounds (P and the microbial biomass is mixed until homogeneous, then a bifunctional reagent for cross-linking the microbial biomass is added, mix again t all components, then dried to dry air condition, pressed and molded, resulting in a biocatalyst with the composition, in wt.% dry matter: microbial biomass - 10-60; silicon dioxide - 20-70; the cobalt (P) hydroxo compound is 20-40, which has a specific surface area of 50-300 m ² / g, a pore volume of 0.3-0.9 cm ³ / g, and a pore diameter of 10-15 nm. 4. The method according to claim 3, characterized in that they use microbial biomass in the form of a paste with a moisture content of 75-80%, a hydrogel with a moisture content of 60-95%, insoluble cobalt (P) hydroxyl compounds in the form of a wet cake. 5. The method according to claim 3, characterized in that dialdehydes, carbodiimide, preferably glutaraldehyde in an amount of 20-100 mg per 1 g of dry cells are used as a bifunctional reagent. 6. The method according to claim 3, characterized in that the insoluble hydroxyl compounds of cobalt (P) are obtained from solutions of cobalt nitrate (P) by precipitation with aqueous ammonia at pH 8-10, a temperature of 2025 ° C, followed by washing with water and filtering the precipitate. 7. A method for producing glucose-fructose syrups in the process of isomerization of monosaccharides, characterized in that they use a biocatalyst for the isomerization of monosaccharides according to claims 1, 2 or prepared according to claims 3-6 in a mixture with an inert filler. 8. The method according to claim 7, characterized in that the process for producing glucose-fructose syrups is carried out continuously in a flow reactor with a fixed bed of biocatalyst at a temperature of 60-70 ° C, the biocatalyst is used in the form of particles from 0.2 to 4.0 mm in size . Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky per., 2