SU1731815A1 - Method for preparation of immobilized cells, showing glucoisomerase activity - Google Patents

Abstract

The image relates to the microbiological industry, in particular, to methods of immobilizing microbial cells with glucose isomerase activity, and 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 strength of the granules of the target product. The incorporation of Streptomyces albogriseolus 28-3 cells into an insoluble carrier is carried out by mixing cell biomass with a chitosan solution with a pH of 4.0-4.5 and then with an aqueous soluble trivalent cobalt acetate at a ratio of 1: (0.1-0.5) : (0.15-1.5), respectively. Next, granulation of the immobilized preparation is carried out by introducing the drops of the resulting mixture into a 5-10% aqueous solution of ammonia. The method allows to increase the specific activity of the immobilized preparation in comparison with the known one by 15-20%. 2 hp f-ly, 3 tab. with

Description

The invention relates to the microbiological industry, in particular, to methods for immobilizing microbial cells having glucose isomerase activity, and can be used in the food industry to produce a glucose syrup.

Methods for the preparation of immobilized preparations of glucose is known.

The disadvantages of these methods are the use of raw materials, mainly food grade, as carriers, the difficulty of obtaining immobilized preparations, since additional processing with a crosslinking agent is required to ensure mechanical and thermal strength and the activity of the biocatalysts is not high enough.

A method is known for immobilizing Streptomyces albogriseolus 28-3 cells with glucose isomerase activity into a cobalt (II) hydroxide gel, for which they are suspended in an aqueous solution of cobalt acetate with a dry / cobalt salt weight ratio of 1: (0.1-10.0) . Then a concentrated ammonia solution was added dropwise to a pH of 6.5-10.0. The resulting precipitate was filtered, washed with water and phosphate buffer (pH 7.0). dried and crushed. The finished immobilized preparation had an activity of 133 units / g of a wet preparation.

The disadvantage of this method is that. that the final product has the form of a powder, and

h |

°°

| 00

ate

The use of a biocatalyst in this form under industrial conditions is impossible.

Closest to the proposed technical essence is a method for producing a biocatalyst based on Streptomyces albogriseolus 28-3. whereby the biomass is pre-suspended in a solution of magnesium acetate, the suspension is mixed with chitosan dissolved in an aqueous solution of cobalt (II) acetate, the gel is granulated by introducing the mixture into an 8% aqueous solution of ammonia, with the final concentrations of the components in the preparation being , wt.%: the biomass of cells (by dry mass) of 3.0-10.0; cobalt (II) acetate 7.0-15.0; chitosan 1.0-4.0.

The disadvantage of this method of immobilization is. when using the obtained preparation in the column reactor for the process of isomerization of glucose syrup, which proceeds at a temperature of 60 ° C, an intense leaching of Co + ions from the biocatalyst is observed. As a result, cobalt ions contaminate the final product - glucose-fructose syrup; it is stained in pink color, which is absolutely unacceptable on demand for food products. Additional purification of glucose fructose syrup up to the content of permissible cobalt concentrations is associated with the complication of the process.

The aim of the invention is to increase the enzymatic activity and strength of the granules of the target product.

This goal is achieved by the fact that according to the method of obtaining immobilized cells with glucose isomerisa different activity, cell biomass is incorporated into an insoluble carrier. For this, Streptomyces albogriseolus 28-3 cells are mixed with chitosan solution (pH 4.0-4.5), an aqueous solution of a cobalt (III) salt is added to this mixture and granulation of the preparation is carried out by dropping in 5-10% - aqueous ammonia solution. In this case, the initial ratio of components taken to obtain the drug (calculated on dry substances) should be as follows: cell biomass: chitosan: cobalt (III) acetate as 1: (0.1-0.5) :( 0.15-1, five).

The choice of 5-10% ammonia solutions as a precipitator is due to the fact that it is in a solution of ammonia (and not some other alkaline solution) of this concentration (5-10%) that the biocatalyst granules are formed with the necessary mechanical strength. When conducting a granulation step in ammonia solution

the process of incorporation of cells into a double gel occurs: into chitosan gel, which changes its solubility at alkaline pH, and into a cobalt (III) hydroxide gel. A decrease in the concentration of ammonia leads to a decrease in the mechanical strength of the resulting granules and lengthening the process of their formation. An increase in the ammonia concentration above the indicated values also leads to a decrease in the mechanical strength due to an excessively rapid formation of the gel.

The preparation of cobalt (III) acetate was carried out on the basis of aqueous solutions of cobalt (II) acetate during their oxidation using a 3% aqueous solution of hydrogen peroxide. The use of more concentrated solutions of hydrogen peroxide leads to a partial inactivation of the enzyme.

Example. 12 g of wet cells (90% moisture) of Streptomyces albogriseolus 28-3 actinomycetes obtained after centrifuging the culture fluid and having an activity of 48 units / g are mixed with 4 ml of 3% chitosan solution (pH 4.3 to 5% solution of acetic acid), add 4 ml of a previously prepared 15% solution of cobalt (III) acetate. The ratio of components taken to obtain the drug (on a dry matter basis) is as follows: Cell biomass: hitan: cobalt (III) acetate — 1: 0.1: 0.5. The resulting mixture is instilled into an 8% aqueous ammonia solution in order to produce a durable granular catalyst. The activity of the wet immobilized drug is 190.4 units / g.

The amount of enzyme catalyzing the isomerization of one micromole of glucose to fructose at pH 7.0 and a temperature of 60 ° C for 1 min was taken as a unit of activity of glucose isomerase. A 2 M glucose solution with the addition of M MD2 + ions was used as a substrate.

The glucose isomeric activity of the preparation prepared according to the prototype is 175 units / g of the wet preparation and taken over 100%.

The remaining examples of immobilization of cells characterizing this method are carried out analogously to example 1, changing only the ratio of the starting components; the concentration of the precipitating solution of ammonia and the pH of the chitosan solution. Their results are presented in Table. one.

The results of cell immobilization are given in table. 2

The results of thermomechanical studies of immobilized preparations are given in Table. 3

The biocatalyst granules obtained by the known and proposed methods were subjected to thermomechanical studies. To this end, the temperature dependence of the relative deformation of granules of the biocatalyst was studied using a thermomechanical method (using a load of 100 g).

Samples of catalysts obtained immediately after immobilization in two compared methods were tested for strength, and then the same samples, but after thermostating at 60 ° C in 0.01 M Na-maleate buffer (pH 6.8) - traditional conditions stability studies of drugs with glucose isomerase activity for 15 days. (tab. 3).

At a temperature of 60 ° C (optimal conditions for the isomerization process), the preparation based on chitosan and cobalt (III) hydroxide has the best strength characteristics (proposed version). The residual strain of such a sample is 16% less (at 60 ° C) than that which the preparation obtained by a known method has. Moreover, the mechanical properties of the catalyst, obtained by a known method, undergo changes in the direction of deterioration as a result of its testing in aqueous medium at 60 ° C due to the strong swelling of the gel granules. At the same time, Co + ions partially wash out of the carrier. Such a phenomenon is absent in the preparation obtained according to the proposed method. As a result, the strength characteristics of the biocatalyst (relative

five

0

five

0

five

0

The effective deformation of the sample is 28%) 2 times the same parameters for a known preparation (relative deformation of 53%).

Thus, the proposed method in comparison with the known allows to increase the specific activity of the immobilized drug by 15-20%, as well as to obtain the target product with improved mechanical properties. The reagents used in immobilization are cheap and available. To implement the method, it is not necessary to use complex or non-standard equipment.

F o rmula of the invention

Claims (3)

1. A method of obtaining immobilized cells with glucose isomerase activity, including preparing a mixture of biomass of Streptomyces albogriseolus 28-3 with chitosan and cobalt acetate solutions at pH 4.0-4.5, forming granules of the gel by introducing the resulting mixture dropwise into an aqueous solution of ammonia, characterized in that, in order to increase the enzymatic activity and strength of the granules of the target product, the mixture is prepared by introducing biomass into the chitosan solution, and then adding a solution of trivalent cobalt acetate. 2. A method according to claim 1, characterized in that the components necessary for the preparation of the preparation are taken in the following ratio for dry substances: cellular biomass: chitosan: cobalt (III) acetate, - 1: (0.1-0.5) : (0.15-1.5). 3. A method according to claim 1, characterized in that a 5-10% aqueous solution of ammonia is used to form gel granules. Table 1 Continuation of table 1 table 2 Table 3