SU1575946A3 - Method of manufactg concentrate of clucosoomerase - Google Patents

Abstract

A stable glucose isomerase concentrate, in which the glucose isomerase is dissolved in a concentrated carbohydrate water solution. The invention also relates to a process for the preparation of such a glucose isomerase concentrate, whereby a) a suitable salt is added to the ultrafiltered glucose isomerase solution obtained from fermentation, so as to crystallize the glucose isomerase, b) the solution is cooled so as to promote crystallization of the glucose isomerase, and the crystal mass formed is separated, whereupon, if desired, one or several recrystallizations are performed, and c) a carbohydrate or a concentrated water solution of same is added to the crystal mass obtained, which said mass is dissolved, whereby a stable glucose isomerase concentrate is obtained. The glucose isomerase concentrate according to the invention is used for immobilizing the glucose isomerase on a carrier material in a reactor column.

Description

This invention relates to biotechnology and concerns a method for producing a glucose isomerase concentrate.

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

The method of producing glucose isomerase is carried out as follows.

From the producing microorganisms Streptoj.yces rubiginosis ATCC 21175, by the geysis of bacterial cells, a cell-free extract is obtained. its ultrafiltration on a membrane with a permeability of 100,000 dalton., a concentrate with glucose isomerase activity of 200 about .800 IHE / ml is obtained.

The pH value of the solution is set to B within 5, 7-8, preferably pH 7.

Cool the solution to 16 ° C or below.

Ammonium and / or magnesium sulphate is added to the solution in a colic from 50 to 170 g / l. The amount of ata depends on the initial concentration of the glucose isomerase, as well as on the final crystallization temperature. It is preferable that the amount of sulfate added at which the crystallization of glucose isomerase occurs occurs without the oxidation of other proteins.

The addition of sulphate is preferable to be carried out gradually, so that

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The addition of the whole salt takes 2-4 hours. The desired result can be obtained by adding the total amount of salt in one portion, but in this case the size of the isomerase crystals remains large.

The solution is cooled for several hours to a temperature approaching the freezing point of the particular solution. With the lowest tested sulphate concentration of about + -2 ° C and at the highest concentration, the freezing temperature Delivers + 16 ° C9 cooling can be started either with simultaneous addition of sulphate or after addition of sulphate6 By gradual cooling, the crystallization-cooling effect is achieved. the size of the crystals and in TOMS that cooling reduces the solubility of u, which leads to an increase in yield

Crystals of the glucose isomerase are separated by precipitating them at the bottom of the tank by filtration or centrifugation in continuous separators.

A certain crystalline mass is mixed with a dry carbohydrate or its concentrated aqueous solution. At the same time, YaAl glucose isomerase is dissolved to obtain a stable concentrate.

If desired, crystallization can be repeated and in this case the crystalline mass should be dissolved in a large amount of water with a temperature of 20-30 ° C.

The amount of water should be such that the activity of the enzyme in the solution is 500-2000 IHE / ml, that is, the amount of water is 4-10 times the crystalline mass.

The resulting glucose isomerase concentrates are pure, stable, easy-to-use and easily dosed preparations. Their activity can be regulated in the range from 2000 to 5000 IHE / g-preparation.

The glucose isomerase concentrate preferably contains 30-60 wt.% Water-soluble carbohydrate, 5-17 wt.%, Glucose isomerase, not more than 15 wt.% Ammonium sulfate and / or magnesium and / or buffer pH 6-8 and water.

Example 1, Streptomyces rubiginosis ATCC 21175 Sowing Culture

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fermented in a known manner in a 40cc fermenter. The cell mass is lysed, the cell residue is removed by filtration through conventional silica gel drum filters5 to give 12 tons of filtrate containing gzzhopzoprezomerezu, the filtrate obtained is subjected to ultrafiltration through a PCI ultrafilter PCI (Patterson - Candy Inc) with a permeability of 100,000 daltons to obtain 3000 kg a glucose isomerase concentrate with an activity of 960,000,000 IHE; the filtrate that has passed through the membrane is removed,

To the concentrate obtained, 120 kg of magnesium sulfate and 300 kg of ammonium sulfate are added. To accelerate the crystallization, the mixture is cooled to 10 ° C. The crystals formed are separated by decantation and the crystallization is repeated by adding 411 kg of water, as well as 17 kg of magnesium sulfate and 1 kg of ammonium sulfate. The crystals are again separated by decanting. By adding 452 kg of water, the crystal mass is dissolved and by adding 1 M ammonia solution the pH of the solution is adjusted to 6.5. The solution is filtered through a plate filter and the crystallization is repeated again by adding 16 kg of magnesium sulfate and 40 kg of ammonium sulfate.

90 kg of crystalline mass containing 20 kg of enzyme, 3.7 kg of salts, the rest is water, 45 kg of glucose and 45 kg of fructose are added to the crystalline mass, and 20 kg of inverted sugar having a dry matter content of 70 wt.%. As a result receive 200 kg of an enzyme preparation having a composition, wt.% g water 29.2, sugar 52 glucose isomers 1495, magnesium sulfate and ammonium salt 4, 3 „

Glucose activity in concentrate 4500 IHE / g.

Example 2. According to the procedure of Example 1, an ultrafiltered enzyme was prepared. To 4000 kg it was added 244 kg of crystalline ammonium sulfate, and then a solution of ammonium sulfate containing 600 kg of salt in 900 liters of water. The solution is cooled to 13 ° C and kept at this temperature for 20 hours. The crystalline mass formed is separated in a Westfalia-Na- separator. The crystals are dissolved in water and the resulting solution is filtered. 51575946

This gives a 20 L liter filtrate. The crystallization is repeated using 122 kg of crystalline ammonium sulfate in an ammonium sulfate solution containing 300 kg of ammonium sulfate in 460 l of water. The resulting crystalline mass is again separated in the separator. To the 525 kg crystalline mass, the same amount of fructose is added crystalline, then the resulting mass is dissolved with partial isomerization of fructose to glucose. Get concentrate containing

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wt.%: sugar 50; enzyme 11,2; ammonium sulfate 3, 5; water 35.3. The activity of the glucose isomerase in the concentrate is 3000 IHE / g.

PRI me R 3. According to the method of Example 1, 4000 l of ultrafiltered enzyme with an activity of 240,000,000 IHE is prepared. The pH was adjusted to 7.0 with a 5% NaOH solution, the temperature was set to 12 ° C.

To crystallize the glucose omerase in 750 l of water, 500 kg of ammonium sulfate is dissolved and the resulting solution is added over 2 hours at a uniform rate of addition. The resulting solution is cooled to -2 ° C and stirred for 24 hours. In the Westphali separator a-7, glucose isomerase crystals are separated. The yield of crystalline mass is 290 kg with a content of 23.6% by weight of dry matter and an activity of 23,000,000 IHE. 30 kg of sodium chloride and 180 kg of glucose are added to the crystalline mass, the pH of the resulting enzyme concentrate is adjusted to 7.0 by adding a 5% aOH solution, and the glucose is partially insulated.

Get 600 kg of a stable concentrate of the enzyme, containing, wt.%: Ode 49,7; sugar 30; enzyme 12.8; ammonium ulfate 2, 5 and potassium chloride. The concentration of glucose isomerase in the concentrate is 3400 IHE / g.

Example 4. According to the method of Example 1, 4000 l of fermentate is obtained with an alcohol content of 240,000,000 IHE. The pH of the solution is adjusted to 7.0, the temperature is 12 ° C by adding the% solution. To fix the glucose isomerase, 50 kg of mmonium sulfate is dissolved in 50 l of water and the resulting solution is added within 2 hours with a uniform feed rate. Crystals of glucose omerase are decanted.

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Get 230 kg of crystalline mass containing 40 wt.% Dry

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substances with an activity of IGU 2300000000.

115 kg of glucose and 115 kg of fructose are added to the crystalline mass, as well as 50 kg of inverted sugar with a content of 70% by weight of dry matter. Get it. 510 kg of gluco-eisomerase concentrate with content, wt.%: Water 30; sugar (glucose, fructose) 52; enzyme 15.1; ammonium sulfate 2.9.

Glucose isomerase activity in concentrate is 4500 IHE / g.

Example 5 According to the procedure of Example 1, an ultrafiltered glucose concentrate is obtained. At 25 ° C to a 0.95 L end of the waste of an isomerase with an activity of 600 IHE / ml, 50 g of ammonium sulfate are added. No precipitate formation is observed at this stage. The solution is cooled for 16 hours at 0 ° C and maintained at this temperature with gentle constant stirring. After two days the crystals begin to precipitate. form and only 2.5 wt.% all further in dissolved form, in the mother liquor. Using a laboratory centrifuge, the crystals are separated from the solution, yielding 56 g of a wet crystalline mass.

As can be seen from the example, it is possible to crystallize the isomerase at a very low concentration of ammonium sulphate in comparison with the amounts recommended by the literature. At the same time, this example is an example of crystallization only under the action of cooling. In the light of this example, it is easy to understand that if the precipitation under the action of ammonium sulfate is carried out quickly and thereafter immediately, for example after 15 minutes, the

sediment in the centrifuge, the isomerase remains completely in solution when using a low concentration of ammonium sulphate, but even if the isomerase is precipitated,

its crystallization is not observed and the benefits of crystallization remain unused. The crystalline mass prepared in accordance with the present example can be dissolved in the same manner as in the other examples.

The glucose isomerase concentrate produced according to the invention is used to immobilize glucose isomerase on the carrier material of the reactor.

1575946

IN THE COLUMN

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Claims (2)

Invention Formula H one . A method for producing a glucose isomerase concentrate comprising a culture | stimulation of the glucose-producing m. of the microorganism Streptomyces rubiginosis ATCCY27575 on a nutrient medium until the maximum activity has been reached, the separation of biomass, the lysis of the cell mass with the production of a cell-free extract, purification and concentration of the extract, about a liter and a fora; and with the fact that, in order to increase the activity and stability of the target product, ultra- filtering on a membrane with a permeability of 100,000 daltons to produce a concentrate with an activity of 200–800 IHE / ml. The enzyme is isolated by precipitation with a water soluble salt of magnesium and / or ammonium sulfate in an amount of 50–170 g / l of the concentrate, the mixture is cooled to minus 2 up to plus 13 C, which promotes crystallization, the crystalline mass obtained is separated by decanting, the crystals are dissolved in water and recrystallized one or more times; carbon is added to the crystalline mass about 0 eight 2. Pop-up method 1, characterized in that the enzyme is isolated from the concentrate by first adding crystalline ammonium sulfate and then a solution of ammonium sulfate as a separator followed by cooling, the resulting crystalline mass is separated and recrystallized by dissolving in water, the resulting solution is filtered and crystalline ammonium sulfate is added and its solution, the resulting crystalline mass is separated and mixed with Lructose, The method according to claim 1, wherein the enzyme is extracted from the concentrate by adding magnesium sulfate, ammonium sulfate and cooling, the resulting crystals are separated by decanting and recrystallized by adding water, magnesium sulfate and ammonium sulfate, the pH is adjusted to: 6 , 5 by adding diluted ammonia -. and recrystallization is carried out by adding magnesium sulfate and ammonium sulfate, and then glucose, fructose and water are added to the crystalline mass to form the desired product. Priority points and features: 06.25.84 pp. 1-3 06.06.85po n, 1 06/25/84 - the enzyme is precipitated out crystal water and / or Concentration with a water soluble sulfate salt solution in an amount that ensures its content in the target product is from 30 to 60 wt.% magnesium and / or ammonium in the amount of 50 to 170 g / l, and the mixture is cooled from -2 to m3 ° s water soluble sulphate salt magnesium and / or ammonium in the amount of 50-170 g / l, and the mixture is cooled from -2 to m3 ° s