SU1634715A1 - Method for producing immobilized cells exhibiting glucosoisomerase activity - Google Patents

Abstract

The invention relates to biotechnology and m. used in the food industry in the production of glucose-fruiting syrup. The aim of the invention is to increase the enzymatic activity and stability of the target product. The method consists in carrying out the incorporation of Streptomyces albogrtseolis28-3 cells into an insoluble carrier by mixing the cell suspension in magnesium acetate with an aqueous solution of chitosan in a cobalt salt, followed by precipitation with aqueous ammonia and separating the resulting biocatalyst granules. The invention allows to increase the activity of the drug from 133 to 135.2-175 U / g and increase its stability from 42 to 46-55 days. In addition, the resulting product has a convenient for the production of granular form. 1 tab.

Description

The invention relates to biotechnology, in particular to methods for immobilizing microbial cells with glucose isomerase activity, which can be used in the food industry in the production of glucose fructose syrup.

The purpose of the invention is to increase the enzymatic activity and stability of the target product.

The method involves the incorporation of Streptomyces albogriseolls 28-3 cells into a carrier prepared from chitosan and cobalt acetate. Cells are pre-suspended in magnesium acetate solution. Cell suspension allows to obtain a biocatalyst with a uniform distribution of activity, and the use of 0.1% magnesium acetate stabilizes the desired product. Cell suspension mixed

with chitosan dissolved in an aqueous solution of cobalt acetate, and then the resulting mixture is instilled into an 8% aqueous solution of ammonia to form granules of the desired product. The granules are separated and washed with water. The final concentration of the components set as follows, wt.%:

Cell biomass by dry weight3-10

Chitosan1-4

Cobalt Acetate 7-15.

WaterEverything

Chitosan, a gel-forming polysaccharide, has a stabilizing effect on the enzyme. Chitosan is soluble in acidic media, so it is possible to use it in an aqueous solution of cobalt acetate having an acidic pH value.

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The application of the method allows to increase the activity of the target product (from 133 to 135.2-175 units / g) and its stability (from 42 to 46-55 days).

The method is illustrated by the following examples.

Example 1.9 g of wet cells (humidity 90%) Streptomyces albogrseolls 28-3, obtained after centrifuging the culture fluid and having an activity of 48.2 gels / g, are suspended in 10 ml of a 0.1% solution of magnesium acetate and mixed with 20 ml of solution 1 , 5 wt.% Chitosan in 13.5 wt.% Aqueous cobalt acetate (pH 4.0-4.5), the final concentrations of the components in the resulting mixture (30 ml) wt.%: Biomass (dry weight - s. m) 3, cobalt acetate 9, chitosan 1 (the mixture is instilled into an 8% aqueous ammonia solution (pH 11.0-11.2). The resulting granules are separated, washed with water and osfatnym buffer pH 7.0.

The activity of the wet immobilized drug is 135.2 units / r, stability is 46 days.

The amount of enzyme catalyzing the isomerization of 1 μmol of glucose to fructose at pH 7.0 and a temperature of 60 ° C for 1 min is taken as a unit of glucose isomerase activity. A 2M glucose solution with the addition of 10 M magnesium ions is used as a substrate.

The stability of the immobilized preparation is defined as the time of the half-inactivation of glucose isomerase at 60 ° C and pH 7.0.

The glucose isomerase activity of the control preparation obtained by a known method is 133.7 units / g of the wet preparation and taken as 100%, and the stability is 42 days.

Example 2. Cell immobilization is carried out analogously to example 1. Final concentrations of the components of the biocatalyst, wt%: biomass (cm) 10, cobalt acetate 8, chitosan 2.5. The activity of the wet immobilized drug is 137.1 units / g, stability is 46 days.

Example 3. Cell immobilization is carried out analogously to example 1. Final concentrations of the components of the biocatalyst, wt%: biomass (cm) 2, cobalt acetate 10, chittoen, 5. The activity of the wet immobilized drug is 81.2 units / g, stability is 13 days.

Example 4. Cell immobilization is carried out analogously to example 1. Final concentrations of the components of the biocatalyst,%: biomass (cm) 11, cobalt acetate 10, chitosan 2.5%. The activity of the wet immobilized drug 190.1 units / g, stability 25 days,

Example 5. Immobilization of cells is carried out analogously to example 1. Final

the concentration of the components of the biocatalyst, wt.%: biomass (cm) 7, cobalt acetate 15, chitosan 2. The activity of the wet immobilized preparation is 174.9 units / g, the stability is 55 days.

0 Example 6. Cell immobilization is carried out analogously to example 1. Final concentrations of components of the biocatalyst, May,%: biomass (cm) 5, cobalt acetate 7, chitosan 1.5%. Activity

5 wet immobilized drug 168.8 units / g, stability 48 days.

Example 7. Cell immobilization is carried out analogously to example 1, Final concentrations of the components of the biocatalyst, wt.%: Biomass (cm) 5, cobalt acetate 6, chitosan 1. The activity of the wet immobilized preparation is 118.3 units / g, stability 42 days.

Example 8. Immobilization of cells

5 is carried out analogously to example 1. The final concentrations of the components of the biocatalyst, wt.%: Biomass (cm,) 7, cobalt acetate 16, chitosan 1%. The activity of the wet immobilized drug

0 142.8 units / g, stability for 37 days.

Example 9. Cell immobilization is carried out analogously to example 1. Final concentrations of the components of the biocatalyst, wt.%: Biomass (cm) 8, co5 acetate of balt 10, chitosan 1.5. The activity of the wet immobilized drug 140.2 units / g, stability 47 days.

Example 10. The immobilization of cells is carried out analogously to example 1. The final

0 the concentration of the components of the biocatalyst, wt.%: Biomass (cm) 9, cobalt acetate 7, chitosan 4. The activity of the wet immobilized preparation is 150.2 units / g, stability - 51 days.

5Example Cell immobilization

carried out analogously to example 1. The final concentration of the components of the biocatalyst, wt.%: biomass (cm) 10, cobalt acetate 8, chitosan 4.5%. Activity

0 wet immobilized drug

130.3 units / g, stability 40 days. Example 12. Immobilization of cells

carried out analogously to example 1. The final concentrations of the components of the biocatalyst 5 wt.%: biomass (cm) 7%, cobalt acetate 10%, chitosan 0.5%. The activity of the wet immobilized drug

188.4 units / g, stability 21 days. Example 13. Immobilization of cells

carried out analogously to example 1, but instead

0.1% magnesium acetate solution using distilled water. The final concentration of the components of the biocatalyst; wt.%: biomass (dl) 8, cobalt acetate 14, chitosan 3. The activity of the wet immobilized preparation is 131.8 units / g, the stability is 29 days.

Example 14. Immobilization is carried out analogously to Example 1, except that a 0.05% solution of magnesium acetate is used instead of a 0.1% solution. The final concentration of the components of the biocatalyst, wt.%: Biomass (RV) 8, cobalt acetate 14, chitosan 3.

The activity of the wet immobilized preparation is 143 units / g, the stability is 34 days.

Example 15. Immobilization was carried out analogously to example 1, but instead of a 0.1% solution, a 0.5% solution of magnesium acetate was used. The final concentrations of the components of the biocatalyst, wt.%: Biomass (dl) 5, cobalt acetate 14, chitosan 3. The activity of the wet immobilized preparation is 140 units / g, stability 32 days.

From these examples, it can be seen that the positive effect of magnesium acetate is to increase the stability of the drug.

Comparative results of the known and proposed methods are presented in the table.

The choice of an aqueous solution of ammonia as a precipitator, as well as the choice of its concentration (8%), is due to the fact that it is in an ammonia solution of such a concentration that granules are formed with the formation of hydrated cobalt oxide of the required mechanical strength.

A decrease in ammonia concentration leads to a decrease in mechanical strength.

formed granules and to the lengthening of the process of their formation.

An increase in ammonia concentration also leads to a decrease in mechanical strength due to excessively rapid gel formation.

Other precipitators (alkalis) do not give the granules the necessary strength.

Thus, the proposed L chosob compared with the known allows to increase the activity of the immobilized drug by 26-30% (from 133 to 135.2-175 units / g), stability by 14-30% (from 42 to 44-55 days) and also to obtain the target product in granular form. The proposed method is simple to implement, cheap, does not require the use of complex, expensive equipment.

Claims (1)

The invention of the method for producing immobilized cells with glucose isomerase activity consists in incorporating Streptomyces albogriseolis 28-3 cells into an insoluble carrier based on cobalt acetate, characterized in that, in order to increase the enzymatic activity and stability of the target product, the cells are previously suspended in a magnesium acetate solution. , then the suspension is mixed with chitosan dissolved in an aqueous solution of cobalt acetate, and the granulation of the gel is carried out by introducing the mixture into 8% aqueous ammonia solution, while the final concentration of the components in the preparation is, wt.%: Cell biomass by dry weight3-10 Cobalt Acetate 7-15 Chitosan1-4 WaterEverything