CS233867B1 - A method for oxidizing D-glucose to D-gluconic acid - Google Patents

Abstract

The invention relates to a method for the oxidation of D-glucose to D-gluconic acid using the enzyme glucose oxidase contained in the mycelium of a microorganism cultivated in the production of this enzyme. Centrifuged disintegrated mycelium, i.e. a waste product of the production of the enzyme glucose oxidase, which is isolated from the separated supernatant, is preferably used for the oxidation. The oxidation of D-glucose can be carried out either directly in the form of a fine suspension of mycelium or preferably using a heterogeneous biocatalyst prepared by embedding mycelium in a natural or synthetic gel. In addition to pure D-glucose, glucose syrup or invert sugar can also be used to produce D-gluconic acid or its salts. In the case of invert sugar, especially in combination with the ingestion of a heterogeneous biocatalyst, it is possible to obtain D-fructose, which is very valuable for food purposes, after the oxidation of D-glucose and the isolation of sodium δ-gluconate.

Description

The invention relates to a process for the oxidation of D-glucose to D-gluconic acid by the enzyme glucose oxidase.

D-gluconic acid, respectively. its salts have found wide application in a number of industries (dye components, enzyme stabilizers in biodetergents, concrete setting retarders, galvanizing bath components for surface cleaning of metallurgical products, etc.) and in health care (calcium application in the form of calcium D-gluconate).

The oxidation of D-glucose from which D-gluconic acid is produced can be accomplished chemically, electrolytically or enzymatically. A number of oxidizing agents have been used in chemical oxidation, in particular halogens and their oxygenated compounds (bromine, chlorine lime etc.). Oxygen oxidation has also been applied under the catalytic action of a platinum or palladium catalyst, most recently. are introduced microbial and enzyme production processes which are in. compared to chemical or electrolytic oxidation more energy, technologically and, most importantly, economically advantageous.

According to the method of the invention, the oxidation of D-glucose is carried out by a disintegrated mycelium of a microorganism grown in the production of the enzyme glucose oxidase. Preferably, centrifuged disintegrated mycelium is used for the oxidation, i.e. the waste product of glucose oxidase production, which is isolated from a separate supernatant. In the waste mycelium, a significant proportion of both glucoseoxidase and catalase activity remains usable for the intended application.

The oxidation of D-glucose according to the invention can be carried out either directly with a fine suspension of mycelium. or preferably using a heterogeneous biocatalyst prepared by incorporating mycelium into a natural or synthetic gel. The reaction conditions are analogous to enzymatic

233 867 catalytic utilizing pure or immobilized glucose oxidase, i.e. at normal temperature and pressure at a maintained pH preferably of about 6 to 7. The oxygen consumed, which is 50% regenerated by operation of the catalase / decomposing reaction mixture present, can be supplemented by aeration with plain air .

The produced glucose oxidase can be used in other areas of the national economy, where the application of isolated enzyme is necessary (especially in clinical biochemistry laboratories for the selective determination of D-glucose, for the preservation of foodstuffs and other materials, etc.).

In addition to pure D-glucose, glucose syrup / amylases and amyloglucosidase-cleaved starch / or invert sugar can also be used to produce the D-gluconic acid of the invention. In the case of invert sugar, in particular, in combination with the use of a heterogeneous biocatalysator, D-fructose, which is of high food value, can be obtained from the foodstuff after D-glucose oxidation and isolation of D-gluconic acid.

The invention is illustrated by examples of use.

Example 1

To 150 L of Aspergillus niger waste mycelium (with a 10% wet insoluble fraction) was added 50 kg of 60% glucose syrup and the reaction mixture was stirred at 25 ° C with an open 6-blade turbine of 1/5 reactor diameter at 400 rpm. The reaction mixture was aerated at 80 L air / min. with inlet under the mixer. The pH of the reaction mixture was maintained by the addition of 16% sodium hydroxide to pH 6.0 with a total consumption of 24 L of solution over 6 hours. No chemical antifoam was used for antifoaming; The reaction mixture was worked up by centrifugation, ¥ the supernatant protein present was denatured by heating to 80-90 ° C for 0.5 h, and the resulting precipitate was filtered off after the addition of carboraffin. The filtrate was concentrated to crystallize, and after addition of methanol, 18 kg of sodium D-gluconate was obtained.

Example 2

To 500 g of wet disintegrated mycelium of Aspergillus niger (after separating the supernatant - intermediate of glucose oxidase production) in 2 L of saline was added 40 g of acrylamide, 2.2 g of R, N ^from -methylenebisacrylamide, 25 ml of 5% 5-dimethylaminopropionitrile solution. and 25 ml of a 2.5% potassium persulfate solution. After solidification, the resulting gel was tempered for 50 minutes at 57 ° C in a water bath and then blended. The obtained immobilized preparation had a glucoseoxidase activity of 0.5 ukat / g dry matter of the formed biocatalyst.

The obtained biocatalyst was added to 2500 ml of 20% D-glucose. The reaction mixture was stirred vigorously and aerated at 5 L of air per minute. The pH of the reaction mixture was maintained at 6.0 by addition of 16% sodium hydroxide solution. After 650 ml of caustic was consumed, the reaction mixture was filtered and the filtrate was concentrated on a vacuum rotary evaporator. After addition of methanol, 550 g of sodium L-gluconate were obtained.

Example 5

A solution of 0.5 kg of water glass and 2.1 water was acidified with 6 M formic acid to pH 5.9. After the addition of 120 g of wet centrifuged disintegrated Aspergillus niger mycelium, the mixture was stirred at 25 ° C for 5 h. The formed mycelium silica hydrogel was washed 5 times with 5 L of water. The obtained biocatalyst exhibited a glucoseoxidase activity of 0.7 ukat / g dry matter. The obtained biocatalyst was added a pound of invert sugar obtained by hydrolysis of a 40% sucrose solution. Oxidation of the present D-glucose was carried out as described in Example 2.

After consuming 290 mL of 16% sodium hydroxide, the reaction mixture was filtered and concentrated to an approximately 65% solution. After addition of methanol. and seeding was obtained by crystallization overnight at 4 ° C. 250 g of sodium D-gluconate. The mother liquors were concentrated on a rotary evaporator under a water pump vacuum.

Chromatography (in conjunction with flow-through refractometer detection) on a 500 x 55 mm glass column packed with Ostion LG-KS 0806 in the sodium cycle at a flow rate of 5 ml deionized water per minute and 24 g of compound mixture in 25 ml water was fed onto the column. a total of 204 g of D-fructose and 7.6 g of the remaining sodium D-gluconate.

Claims (3)

SUBJECT OF THE INVENTION A method of enzymatic oxidation of D-glucose to D-gluconic acid by a glucose oxidase, characterized in that the oxidation is carried out by a disintegrated mycelium of a microorganism cultivated in the production of glucose oxidase. 2. The process of claim 1, wherein the oxidation is carried out by sedimenting the disintegrated mycelium after separation of the supernatate used to isolate the enzyme glucose oxidase. 3. The method of item 1; characterized in that the oxidation is carried out by a disintegrated mycelium or a sediment thereof incorporated into a natural or synthetic gel.