DK151272B - Process for immobilizing microorganisms with a glucose isomerase activity - Google Patents

Description

151272

The invention relates to a method for immobilizing microorganisms which exhibit glucose isomerase activity, in which a glutaraldehyde-induced polymerization process is carried out in the presence of a protein-containing carrier.

In the specification of GB Patent No. 1,401,946, a crosslinking polymerization of cells is disclosed in the presence of a diazotized diamino compound such as 2,6-diaminopyridine-3,6-diaminoacridine which contains from 1 to 3 heterocyclic rings. .

In the disclosure of U.S. Patent No. 3,694,319, a continuous process for the isomerization of glucose to fructose is disclosed using thermally treated whole cells of Arthrobacter.

According to U.S. Patent No. 3,980,521, whole cells were treated by Bacillus sp. NRLL 5656 with glutaraldehyde and the polymerization product thus obtained was used to isomerize glucose to fructose, this isomerization being carried out continuously in a tank.

Whole cells of Streptomyces have been immobilized by ion exchange, e.g. on DEAE cellulose or ECTEOLA-Cellulose (U.S. Patent Nos. 3,788,945 and No. 3,708,397). Immobilization can also be achieved on a collagen gel by subsequent coating of the thus obtained mixture with glutaraldehyde to insolubilize the mixture (Kolarik, MJ et al. In "Immobilized Enzymes in Food and Microbial Processes." Pergamon Press (1974) p.71 -83).

Direct thermal treatment of the cells, which treatment is performed to decrease the permeability of the cell membrane, usually results in a product which has both low activity and short life under the conditions of the isomerization process. Immobilization of cells using ion exchangers such as DEAE cellulose results in a gradual leaching of enzyme activity with the substrate solution.

The same effect is observed if the cells are immobilized in a gel structure such as polyacrylamide or collagen.

The most stable preparations of immobilized enzymes are usually obtained by a covalent bond of reactive groups on the surface of the cell membrane. Such methods have also been used to immobilize glucose isomerase active cells. Polymerization using diazotized diamines leads to a good recovery of cell activity, but the method is limited due to the high toxicity and carcinogenic effect of the reagents used, namely the 2,6-diaminopyridine-3,6-diaminoacridine. The direct polymerization of the cells by glutaraldehyde treatment leads to a low degree of polymerization due to the lack of free reactive groups on the cell surface. Therefore, it is not possible to obtain the sufficient cross-linked structure necessary for better contact between the substrate and the enzyme to be immobilized. The method of the patent mentioned above does not guarantee an opportunity for standardization of the final product.

Attempts have been made to immobilize glucose isomerase-10 active cells on blood fractions, namely the 5th fraction after Kon-fibrin, but no preparations with good activity were obtained (US Patent No. 3,788,945 and No. 3,705,084).

The object of the present invention is to provide a method for immobilizing glucose isomerase-active microorganisms using an readily available proteinaceous carrier material, which provides preparations that are both highly active and stable, necessary for the production of high fructose syrups. by a continuous technical process.

During the development of the present process, glutaraldehyde-induced polymerization of glucose isomerase-active cells was used. It was found that when the glutaraldehyde-induced polymerization was carried out in the presence of blood serum or blood plasma as a protein-containing carrier, a glucose isomerase active preparation with high activity per grams of immobilized preparation (150-200 Glu / g), favorable pH optimum (7.0), high temperature optimum (70-75 ° C) and considerable thermal stability at temperatures of 45 to 65 ° C. The conversion efficiency of the preparation obtained is 1.5 GIU per liter. g of glucose at a conversion rate of 45 to 50%.

Accordingly, the method of the invention is characterized in that the protein-containing carrier is blood serum or blood plasma.

Blood serum or blood plasma taken from healthy slaughter animals, namely sheep and cattle flocks, pigs, etc., must meet the veterinary sanitary requirements and must be used in a cooled, preferably frozen, freeze-dried or dried, preferably spray-dried state. In the latter two cases, the blood serum or plasma must initially be dissolved in water and adjusted to a concentration of 10-12% solids. The polymerization product is washed in water. The product obtained after washing 3 or after being frozen to a temperature of from -5 to -10 ° C for 12 hours is then pressed, granulated and dried at a temperature not exceeding 50 ° C under vacuum or in a dry air stream.

In addition to the product being usable in the granular form, the dried product can be used milled and screened, selecting the fraction capable of passing a mesh of 10-30 mesh.

The thus obtained immobilized preparation is loaded onto a reaction column or a series of columns, where it is treated with a 2-3 M glucose solution containing activators, namely Mg and 10 Co-ions in optimal concentration according to the enzyme used, at a temperature of from 45 to 75 ° C. The substrate filling rate is adjusted such that a 40-50% conversion of glucose to fructose is achieved, i.e. the equilibrium of the isomerization reaction.

The glucose isomerase activity in the column or row of columns and the life of the enzyme preparation depend on the duration of contact between the enzyme and the substrate, ie. column flow rate, temperature, activity and the amount of preparation used, as well as the presence of activators, viz. magnesium and cobalt ions. The glucose isomerase activity is determined in a tank by the method proposed in the specification of U.S. Patent No. 3,788,945. The amount of glucose converted to fructose is determined colorimetrically by the method described by Dische et al., Or by poliometry. This determination is performed repeatedly at specified times during the operation of the column. The column activity index 25 is determined using the term (US Patent No. 3,980,521): A = RIE x log [0.504 (0.505-1)] where A represents the column's efficiency I denotes the amount of glucose converted to fructose (in grams) 3 30 R denotes the flow rate in cm / min and E denotes the amount of glucose isomerase activity in international units contained in the column or row of columns.

The equilibrium of the isomerization reaction could only be achieved in the presence of at least 2000 international glucose isomerase units.

35 The by-products of the reaction, as well as the cobalt and magnesium ions in the product, were removed by treatment with activated charcoal and cation exchange using resins of types such as vofatite, duolite, amberlight, dowex, etc. The color index of the high fructose syrup was determined spectrophotometrically using an o 3 4 151272 1 cm spectrophotometric cell at wavelengths of 450 and 600 nm, respectively. Color units of the fructose-rich syrup were determined by the term (U.S. Patent No. 3,980,521): 109 (A450-A600)

and C

where B represents color index.

A450 09 A600 is, the ice absorption at wavelengths of 450 and 600 nm, respectively.

3 C is the concentration of the solution in grams per gram. 100 cm.

10

The following examples will illustrate the invention in more detail:

EXAMPLE I

Growth medium obtained after fermentation of microbial cells of strains producing glucose isomerase enzyme was centrifuged or filtered. The biomass thus filtered was washed once with water. The wet cells obtained after centrifugation or filtration must have a dry matter content of 10-12%. When deviations from these values were obtained, the amount of blood serum or blood plasma was appropriately adjusted. The fresh biomass was mixed with freshly obtained blood serum or blood plasma in a weight / pn volume ratio of from 1: 1 to 1:10 such that the preparation after polymerization had a glucose isomerase activity of 150-200 Glu / g dry matter. If deviations from these values occurred, the amount of blood serum or blood plasma was corrected. 380-400 cm of an aqueous solution containing 25% glutaraldehyde per ml was added. to the reaction mixture, and the mixture was stirred for approx. 1 hour with a mechanical stirrer at room temperature. The polymerization product thus obtained was subjected to filtration and repeated washing until no yellow color could be observed in the rinse water. Excess water was removed by pressing and the preparation was frozen ΛΛ 0 for 12 hours at a temperature of -5 to -10 ° C. Then the preparation was thawed, the water removed by pressing, and the thus obtained porous mass was granulated and dried at a temperature not exceeding 50 ° C under vacuum or in a dry air stream. In addition to the preparation being usable in the granular form, it can also be used after grinding and sieving to a grain size of 10-30 mesh.

5 151272

EXAMPLE II

The polymerization of whole cells with glucose isomerase activity together with blood serum or blood plasma was performed as in Example I.

After pressing, the preparation thus obtained was not frozen but directly granulated and dried as in Example I.

EXAMPLE III

In the polymerization of glucose isomerase-active cells, a freeze-dried or spray-dried blood serum or blood plasma was used. This serum or plasma was previously dissolved in water to a solids content of 10 to 12%. The subsequent steps are the same as in Examples I and II.

EXAMPLE IV

The polymerization of glucose isomerase-active whole cells was carried out as in Examples I, II and III except that the blood sera or blood plasma had been previously frozen. In this case, the blocks of blood serum or plasma were thawed at a temperature not exceeding 55 ° C. The dry matter content was then determined and, after appropriate correction, the steps were performed as in Examples I and II.

EXAMPLE V

The dried preparation obtained according to one of Examples I-IV

was soaked for 12 hours either in a glucose syrup with a concentration of 2 to 3 molar containing optimum concentration of cobalt and magnesium ions depending on the enzyme used or in water in a 1:10 w / v ratio. One column or row of columns 25 was filled with the swollen enzyme preparation, providing a good and uniform filling so that no air was entrapped in the column. The amount of enzyme preparation needed for the reaction was determined by the expression I.

A glucose syrup with a concentration of 2 to 3 molar was passed continuously through the column. The fructose content of the column outlet was determined colorimetric or polariometric. The conversion of glucose syrup to fructose was maintained at a level p from 40 to 50% using a series of columns, so that when the activity of a given column decreased to a value below 20%, the column was replaced with a new 35. It is recommended that the temperature during the isomerization be kept in the range of 60 to 65 ° C to avoid any microbial contamination of the column and that the syrup be preheated to the operating temperature. The flow rate in the column or row of columns is controlled by an appropriate pump and depends on the amount and activity of the preparation used (cf. the term for A).

The operation time of a single column (i.e., a column not disposed in a row) depends on the initial activity of the preparation to be immobilized and on the thermal stability of the enzyme as well as the flow rate of the substrate.

Claims (3)

151272 Patent Claims. A method of immobilizing glucose isomerase-active microorganisms in which a glutaraldehyde-induced polymerization process is carried out in the presence of a proteinaceous carrier, characterized in that the proteinaceous carrier is blood serum or blood plasma. Method according to claim 1, characterized in that the blood sera or blood plasma is used in a spray-dried, freeze-dried or frozen state. Method according to claim 1 or 2, characterized in that the ratio of the weight of microorganisms to the volume of the blood serum or blood plasma is in the range of 1: 1 to 1:10.