CS236178B1 - Method of D-glucose isomerization by a microorganism with glucose isomerase activity - Google Patents

Abstract

The invention consists in that D-glucose isomerized by a microorganism in pellet form. After treatment of the pellets with heat shock, there is a further increase in glucose isomerase activity and at the same time its stabilization. A further increase in glucose isomerase activity can be achieved by partial destruction of cell walls by periodate oxidative cleavage of vicinal diols in the cell wall of occupied polysaccharides. The stability of glucose isomerase in pellets thus treated can be increased by cross-linking with bifunctional agents, preferably with glutaraldehyde.

Description

The invention relates to a process for the isomerization of D-glucose by a microorganism having glucosaisomerase activity for the preparation of fructose syrup.

Fructose syrup is obtained by the enzymatic isomerization of D-glucose to D-fructose. It can be advantageously used as a substitute for sucrose in almost all food products, in particular for the production of soft drinks and syrups, frozen creams, alcoholic beverages, confectionery, canning products, bakery products and all reduced calorie products. The sweetness of fructose syrup is 1.2 times higher than that of sucrose.

In addition to direct use as a sweetener, fructose syrup can be used to produce pure D-fructose, which can replace D-glucose as a sweetener in the. certain types of diseases (gallbladder diet, heart and circulatory diseases).

The raw material for the preparation of fructose syrup, i.e., D-glucose, can be obtained by hydrolysis / enzymatic or chemical / cheap starchy raw materials such as potato, corn, cereal, rice starch. Starting from starchy raw materials, fructose syrup contains 42 to 45% D-fructose, 50 to 52% D-glucose and 4 to 6% oligosaccharide. is D-fructose.

Industrial production of fructose syrup started in the US in 1967. It currently accounts for 50 to 40% of the total consumption of sweeteners in the US, while being 15 to 25% cheaper than sucrose.

Worldwide, production of fructose syrup is expected to increase from 5.2 million tonnes in 1981 to 5.6 million tonnes in 1985.

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On an industrial scale, D-glucose is carried out by heterogeneous catalysts prepared by immobilization of either the isolated enzyme or whole cells of the microorganisms producing giukdlaisomerism.

The source of the glucoeaeomeraey are mainly bacteria of the genera Bacillus, Streptomycee, Arthrobacter and others.

In whole-cell immobilization, the very expensive extraction and purification of the enzyme is often avoided, there is practically no loss of enzyme activity, the stability of the obtained preparations is high. Another advantage is the overall low cost of the catalyst.

The disadvantage of using immobilized isolated glucosaisomerase is, in addition to the already mentioned technical and economic demands of the isolation, the cost of the carrier and the technical demands of the immobilization itself.

When immobilized whole cells are administered, the limiting factor is the obtaining of e strains by overproduction of glucoeaomerase. However, in the case of microorganisms with a fibrous structure, the possibility of a method of immobilization by methods commonly used for the immobilization of unicellular microorganisms is limited.

By incorporating microorganisms into the gel matrix, the activity of the glucoeaomerase in the obtained biocatalyeator is also somewhat reduced due to the difficulty of the passage of the substrate through the gel layer.

The method according to the invention is D-glucose isomerized by a microorganism in pellet form which exhibits a significantly higher glucoaaomeromeric activity as compared to the same microorganism in fibrous form. The rigid structure of the pellets gives the possibility of use in a continuous process without further treatment.

The ionomer is carried out at 60 to 65 ° C at 50 to 40% by weight. substrate for 12 to 24 hours.

The pallet form is obtained by culturing Streptomycee bacteria at 25 to 50 ° C on a medium containing xylose and glucose at a concentration of 2-4% by weight. for 48 to 72 hours

Advantageously, the microorganisms in the form of pellets can be subjected to a heat shock at a temperature of 50 to 120 ° C, which further increases the glucose isomeric activity and at the same time stabilizes it.

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Further enhancement of the glucpsoisomeric activity contained in microorganisms in pellet form can be achieved by partial destruction of the cell walls by periodate oxidative cleavage of vicinal diols in the cell wall of the contained polysaccharides.

The stability of the glucosoisomerase in the treated pellets can be further increased by cross-linking with bifunctional reagents, preferably with glutaraldehyde. The use of microorganisms with glucose isomerase activity in pellet form in the production of fructose syrup eliminates the laborious isolation of the enzyme glucose isomerase and its immobilization onto carriers. Likewise, the activity of the glucosoisomerase is higher than that of pure enzyme, or cells bound to a carrier. The pallet form of microorganisms enables their easy separation after isomerization.

The invention is illustrated by examples.

Example 1

A 1.8 x 20 cm column was packed with 20 g of wet clean pellets showing a Glucosaisomerase activity of 4.25 ukat / g. of dry cells. A sample is applied continuously to the column in the form of a 40% D-glucose solution in Britton-Robinson buffer plus 0.1 mol / l MgSO4.7H2O. The column flow rate was 30 ml / h. 32% D-fructose was found in the eluate.

Example 2

Pure pellets were heated to 70 ° C for 10 minutes. 250 ml of a 40% solution of D-glucose in Britton-Robinson buffer pH 8.5 with 0.1 mol / l MgSO4 »7 H ^ was added to the obtained 5.4 g / ml dry cell preparation of 20 g. O. After centrifugation, a 35% D-fructose content was found in the supernatant.

Example 3

8 ml of 25% glutaraldehyde was added to the pure 10 g pellets. The obtained preparation showed an activity of 4.8 ukat / g dry matter. The thus prepared formulation was used to convert D-glucose to D-fructose as described in Example 1. The D-fructose content was determined to be 35% in the eluate.

To pure heat shock treated pellets (see Example 2) in an amount of 10 g, 8 ml of 25% glutaraldehyde was added. The biocatalyst obtained was found to have an activity of 7.33 ukat / g dry carrier. The thus prepared preparation was used to convert D-glucose according to the procedure of Example 2. The D-glucose content was found to be 38%.

Example 5

0.05 mol / l NaIO 4 was added to the clean pellets at a ratio of 0.5 ml / 1 g. After 6 h oxidation at 4 ° C, 100 mg albumin and 1 ml 25% glutaraldehyde were added per 10 g cells. The resulting 8.3 µKat preparation was used to convert D-glucose as described in Example 1. The D-fructose content was found to be 39% in the eluate.

Example 6

- Pure pellets were heat shock treated (see Example 1) and then processed according to Example 5. The obtained biocatalyser of activity

12.5 UKat / g dry matter is used for the conversion of D-glucose as described in point 2. A 41% content of D-fructose was determined in the supernatant

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Claims (4)

SUBJECT OF THE INVENTION 1. A method of isomerization of D-glucose with glukosoisomerósovou microorganism activity, characterized in that the isomerization of D-glucose is carried out in a microorganism of the genus Streptomyces pellet form, which is obtained by culturing at 25 to 30 ° ^C on medium containing xylose and glucose at a concentration of 2 to 4% by weight for 48 to 72 hours, the isomerization being carried out at 60 to 65 ° C at 50 to 40% by weight. substrate for 12 to 24 hours. 2. A process according to claim 1, wherein the microorganism pellets are heat-treated at a temperature of from 50 to 120 [deg.] C. 3. A method according to claim 1 or 2, characterized in that the microorganism in pellet form is subjected to partial destruction of the cell walls, for example by periodate oxidative cleavage. 4. The method according to claim 1, 2 or 3, characterized in that the microorganism in pellet form is treated with bifunctional agents, for example glutaraldehyde.