WO1986000336A1

WO1986000336A1 - A stable glucose isomerase concentrate and a process for the preparation thereof

Info

Publication number: WO1986000336A1
Application number: PCT/FI1985/000057
Authority: WO
Inventors: Kalevi Juhani Visuri
Original assignee: Suomen Sokeri Oy
Priority date: 1984-06-25
Filing date: 1985-06-19
Publication date: 1986-01-16
Also published as: FI78117C; HU194937B; FI78117B; SU1575946A3; FI852270A0; HUT40463A; FI852270L

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

A stable glucose isomerase concentrate and a process for the preparation thereof

The present invention relates to a concentrate of a glucose isomerase enzyme which is chemically and microbiologically stable without addition of microbi- cides and which is very useful as such for the immobilization of the enzyme on the carrier material in a reactor column. The invention also relates to a process for the preparation of such a concentrate. The use of glucose isomerase for the isome- rization of glucose to fructose is a well known indus¬ trial process, in which the enzyme is always used in the immobilized form. In all of the technical processes that are in use, the immobilized enzyme preparation is prepared as a separate process, in which either the enzyme is immobilizied on the carrier by means of the adsorption technique or the entire microbial cell mass with the enzymes is immobilized to make a matrix. The reactor column is made up of ready immobilized enzyme. When the enzyme has been used up, the column is emptied and again made up of fresh immobilized enzyme preparation.

It is evident that if the enzyme inactivated in the column can be replaced by fresh enzyme by using a conventional regeneration technique, considerable economies can be obtained. The consumption of the carrier material is reduced and, moreover, the loss of activity taking place in connection with the prepa- ration of immobilized enzyme is avoided. When the enzyme is immobilized on the carrier directly in the column, neither drying nor mechanical handling is required, in which connection some enzyme is always destroyed. It is known that a purified enzyme is adsorbed on a carrier much better than a non-purified crude preparation (US Patent 4,347,322). The problem has been to prepare such a stable, easy-to-handle and easy-to-store enzyme preparation, which can be used directly for immobilizing the enzyme on the carrier in a column.

A precipitate or crystal mass obtained from glucose isomerase purification processes based on precipitation or crystallization contains at least 60 per cent by weight of water, which cannot be removed without destruction of the enzyme structure. The crystal mass as such is not microbiologically stable, it is difficult to handle and to dose. An alternative is dissolution of the crystal mass to a solution, the concentration of which should preferably be as high as possible, in view of storage, stability and transport. The most natural way is to dissolve the enzyme crystals in water or in a dilute salt solution. In practice, however, it has been noticed that, owing to the rela- tively low solubility, it is not possible to prepare a sufficiently concentrated solution of the isomerase by dissolving crystals in water. A diluted water solution of the isomerase is highly instable and loses its activity as a result of microbiological and chemical deterioration in a few days.

It has also been suggested that the enzyme could be dissolved in an organic solvent (US Patent 4,077,842) However, it is not advisable to use organic solvents in a process of food production, wherefore this technique is poorly suited for an enzyme that is used directly for the preparation of foods.

There is an abundance of literature concerning the preparation, purification and use of glucose iso¬ merase, because glucose isomerase is one of the most important enzymes at present in use. However, all the published purification processes are quite compli¬ cated and, as a rule, give rather low yields.

The purification of enzymes and proteins by means of crystallization is, in itself, well known, and, for example, in the manual Enzymes (Dixon, M. and Webb, E.C., Enzymes, 3rd ed. , Longman Group Ltd., Bungay 1979, 1116 p.) 192 photographs are shown on enzyme crystals. Moreover, it is known that ammonium sulfate is in several cases suitable as an agent that induces crystallization and reduces solubility. In addition to ammonium sulfate, crystallization can often be carried out by means of some other salt or organic solvent, such as acetone or alcohol. In this field of chemistry, it is, however, generally admitted that it is never self-evident that an enzyme or any other protein is crystallized by means of some, in itself well known, precipitant. Numerous proteins and enzymes are known whose crystallization has not been succesful as yet, even though the purification processes in biochemistry have undergone an immense development.

The use of fractional sulfate precipitation (ammonium and/or magnesium sulfate) as a purification process for glucose isomerases or as a part of such a process is known from several publications (US

Patent 4,237,231, US Patent 4,077,842, Agr. Biol. Chem. 45_ (1981) 619-627, id. _28 (1965) 1123-1128, id. 3_4 (1970) 1795-1804, id. 29_ (1965) 1129-1134, Biochem. Biophys. Acta 151 (1968) 670-680). The sulfate concentrations used in these purification processes for isomerases have usually been relatively high. For example, in the process in accordance with the US Patent 4,237,231, the unneces¬ sary proteins are first precipitated from the iso- merase solution at the saturation degree of 40 % of ammonium sulfate (about 240 g (NH^J-SO. in a litre of solution) , and then the isomerase itself is precipitated at a higher saturation degree (up to 60 %) . When high ammonium sulfate concentrations are used in fractional precipitation, an abundance of other proteins always precipitate along with the iso¬ merase, the more other proteins the higher the con¬ centration of ammonium sulfate used, unless the isomerase has already been prepurified in some way by some other methods.

In purification processes based on fractioning precipitation, the result is usually an amorphous precipitate, which is very difficult or even impossible to separate from the mother liquor in good yield by means of centrifuges or separators intended for indus¬ trial use.

The amorphous precipitate is, as a rule, a mixed deposit, which, besides the isomerase, also contains other proteins if the raw-material is a microbial-cell liquid which has not been prepurified. Such an amorphous precipitate is difficult to separate from the mother liquor, and precipitation does not give the desired purification effect with respect to the isomerase.

Crystallizations of glucose isomerases by means of ammonium sulfate, other salts, or organic solvents have been described in some publications (Biochem. Biophys. Acta 151 (1968) 670-680, Agr. Biol. Chem. 34 (1970) 1795-1804, id. _45_ (1981) 619-627, id. 33_ (1969) 1527-1534). Characteristic features of the crystal¬ lization processes described in said publications are low yield, very long crystallization time, or relati¬ vely high consumption of chemicals. In all of these cases, the aim has been to prepare a small quantity of pure isomerase for basic research purposes. Thereby, the isomerase has first been purified by means of some methods (extraction with an organic solvent, DEAE-Sephadex-column chromatograph , ammonium sulfate precipitation, dialysis) , whereupon crystallization of purified isomerase from pure water solution has been carried out with ammonium sulfate, phosphate buffer, or acetone.

None of the processes described in the^"above publications has been used on an industrial scale, because of their poor economy.

The present invention relates to a stable glucose isomerase concentrate, which is characterized in that it contains glucose isomerase dissolved in a concentrated carbohydrate water solution.

It has been found that the concentrate according to the invention is both chemically and microbiologi- cally stable and particularly useful in processes in which the immobilization of glucose isomerases on the carrier material is carried out by the regeneration technique in a reactor column. Suitable carbohydrates are all non-toxic carbohydrates readily soluble in water, such as sugars and polyols. Particularly suitable are sugars that are used as foods as such as well as sugar alcohols which are allowed as food additives. Since the end product of isomerization is a glucose-fructose syrup, the mixture of these sugars, i.e. invert sugar, is excellently suitable for the purpose. The total dry solids content in the concentrate must be such that it is in itself microbiologically stable, i.e. the dry solids content should be about 60 to 70 per cent by weight (the activity of the water must be sufficiently low) . The glucose isomerase concentrate according to the invention preferably contains 5 to 15 wt. % of glucose isomerase, 30 to 60 wt. % of a carbohydrate soluble in water, such as glucose, maltose, fructose, saccharose, sorbitol, xylitol, or of a mixture thereof, e.g. invert sugar, glucose syrup or isomerized glucose syrup, no more than 15 wt. % of an appropriate salt, such as ammonium and/of magnesiu 'sulfate, and/or a buffer (pH 6.0 to 8.0), e.g. sodiumpo^'tassium- phosphate buffer, carbonate buffer, or a buffer made of an organic salt (e.g. salt of an aminoacid) or a mixture of buffers, and balance water.

The pH of the concentrate is preferably 6.0 to 8.0, and the enzyme activity is 2000 to 5000 GlU/g concentrate (GIU = glucose isomerase units) . In the following, some examples are given of suitable enzyme concentrates according to the invention:

1. 50 wt. % glycerol 10 wt. % water

15 wt. % enzyme 5 wt. % ammonium sulfate

2. 50 wt. % invert sugar 30 wt. % water

15 wt. % enzyme 1 wt. % buffer /Na-K-phosphate) 4 wt. % ammonium sulfate

3. 50 wt. % sorbitol 40 wt. % water

10 wt. % enzyme

4. 50 wt. % saccharose 30 wt. % water

15 wt. % enzyme 5 wt. % buffer (sodium phosphate) The composition of the concentrate may, however, be varied as required, while taking care that the total concentration is sufficiently high and the solution, yet, in liquid form and easy to handle.

The glucose isomerase concentrates according to the invention are pure, stable, and easy to handle and dose. The enzyme activity can be adjusted. In view of the use, a suitable range of activity is 2000 to 5000 GlU/g preparation.

The sugars, sugar alcohols and salts to be used are preferably of food grade (e.g., Food Chemicals Codes standard) . The enzyme shall be purified adequate- ly by crystallization or by any other method.

The glucose isomerase can be readily adsorbed from the concentrate directly onto the carrier material placed in the reactor column. The concentrate is diluted with water and the dilute solution is fed into the column, whereby the enzyme is adsorbed onto the carrier. The technique resembles the regeneration of an ion-exchange column. Alter the enzyme has been in use and thus inactivated, the protein is washed off the column by means of an alkali, whereinafter fresh enzyme can be fed into the column so as to regenerate it.

A suitable carrier material is a material with anion-exchange capacity, e.g. glass beads, ion- exchange resin or a silica-based carrier. Particularly suitable are diethylaminoethyl (DEAE) derivatives. which are known to adsorb proteins, such as DEAE-cellu- lose and DEAE-dextran. There is a large number of carrier materials described in the literature.

When DEAE cellulose is used as carrier, it is easy to attain an activity higher than 1000 IGIU/g immobilized enzyme in an activity column (IGIU = immobilized glucose isomerase activity) .

The invention also relates to a process for the preparation of the stable glucose-isomerase concentrate described above. The process is characterized by a) adding a suitable salt to a ultrafiltered glucose isomerase solution obtained from fermentation, so as to crystallize the glucose isomerase, b) cooling the solution so as to promote crystallization of the glucose isomerase, and separating the crystal mass formed and then if desired, effecting one or several recrystallizations, and c) adding a carbohydrate or a concentrated water solution thereof to the obtained crystal mass, which dissolves, whereby a stable glucoseisomerase concentrate is obtained.

According to the present invention, the isomerase enzyme can be purified highly efficiently by crystal¬ lizing it from a salt solution. Suitable salts are all such non-toxic salts, which do not inactivate the enzyme. In the process according to the present invention, ammonium and/or magnesium sulfate is used. Precipitation of glucose isomerase by means of a salt is in itself a conventional procedure, being described, e.g., in Agr. Bio. Chem. 29 (1965) pp. 1129-1134 (Isumure and Sato) .

However, no such process has earlier been described in which the formation of crystals proper takes place. In the suggested processes, an amorphous precipitate is formed.

Depending on the conditions, either an amorphous precipitate or a crystalline precipitate can be obtained from the isomerase by using the same chemical, ammonium sulfate or magnesium sulfate. It is generally known hat numerous enzymes behave in the same way.

A characteristic and surprising feature of the process according to the invention is that the iso¬ merase precipitates as a crystalline substance and before all other substances that may precipitate. In the process, precisely selected conditions and such a low ammonium sulfate concentration are used that no other substances precipitate from the solution. In this respect, the process differs essentially from what has been described in the literature in this, field. Earlier isomerase has not been crystallized directly and alone as the only precipitating component from a cell liquid or a cell liquid concentrate of a production microbe. The process also gives a very high yield, which differs essentially from what has been earlier stated in the literature.

It is generally known that the storage quality and stability of enzyme preparations can be increased, e.g., by means of glycerol, polyalcohols and sugars. Such enzyme preparations are usually prepared by adding the said substances to a concentrated enzyme solution.

It has surprisingly been found that, when dry anhydrous polyalcohol or sugar (preferably glucose) is mixed into a concentrated isomerase crystal suspension or into a solid crystal mass having a highest possible activity of about 10,000 GlU/g, the isomerase crystals are dissolved and a genuine clear solution is produced. Such a solution is stable when its water concentration is sufficiently low and when its isomerase activity is sufficiently high. The solution may contain salts derived from the crystallization process or salts that have been added afterwards, which salts in themselves have an effect increasing the microbiological stability. The polyalcohol or sugar, however, has an essential importance in view of the dissolution of the isomerase crystals in order that an isomerase activity as high as possible could be attained for the solution.

The prior art, no examples have been given of isomerase solutions having as high as an enzymatic activity as the product according to the present invention, nor have methods been described for the preparation of such solutions.

The preparation of the glucose isomerase con- centrate is preferably carried out as follows: a) A cell liquid is prepared from the organism Streptomyces rubiginosus by means of lysis (US Patent 4,410,627), and from the cell liquid, by ultrafiltration, a concentrate containing isomerase is prepared for raw-material for the crystallization process,* a preferred isomerase concentration of the concentrate is 200 to 800 GlU/g. b) The pH of the isomerase solution is adjusted to the range of 5.7 to 8.0, preferably pH 7.0. c) The solution is cooled to 16 C or below. d) Ammonium and/or magnesium sulfate is added to the solution, preferably 50 to 170 g per litre of solution. The sulfate quantity to be added depends on the original isomerase concentration and on the final temperature of crystallization. The most preferable quantity of sulfate to be added is such a quantity with which only the isomerase is crystallized but the other proteins do not yet start precipitating. e) The addition of the sulfates is preferably effected gradually so that the addition of the whole quantity takes 2 to 4 hours, even though a acceptable result may also be obtained by adding the whole quantity all at once, but in such a case the size of the isomerase crystals remains unfavourably small, f) The solution is cooled preferably during several hours, preferably close to the freezing point of the mixture concerned, the freezing point being at the lowest tested sulfate concentrations about -2 C and at the highest ones -6°C; the cooling may be started either simultaneously with the beginning of the sulfate addition or only upon completion of the sulfate addition; by means of gradual cooling, a cooling-crystallization effect is obtained that increases the size of the isomerase crystals advantageously, besides the fact that cooling has a solubility lowering effect, which again increases the yield. g) The isomerase crystals are separated from the solution by allowing them to settle to the bottom of the vessel, by filtering them or, on a large scale most preferably by centrifuging them by means of a continuous separator. h) The separated crystal mass is dissolved by adding to it a dr carbohydrate or its concentrated water solution, whereby the isomerase crystals are dissolved and a stable glucose-isomerase concentrate is produced.

If desired, the crystallization may be repeated, in which case the crystal mass must be dissolved after the separation (step g) into an abundant quantity of water at a relatively high temperature (20 to 30°C) . In this connection, a suitable quantity of water is such that the isomerase activity of the solution is 500 to 2000 Glϋ/ml, in other words, the weight of the quantity of water used is typically 4 to 10 times the weight of the crystal mass.

The following examples will illustrate the invention. Example 1

A batch of about 40 cubic metres of Streptomyces rubiginosus microbe was fermented in a way known per se (reference US Pat. 4,410,627). The cell mass was lysed in a known way (same reference) . The cell residues and the other solid matter were removed by filtration by means of a conventional siliceous- earth drum filter, whereby 32 tons of isomerase-con- taining filtrate was obtained. This filtrate was filtered by means of a PCI (Patterson-Candy Inc.) ultrafilter, whereby 3000 kg of isomerase-containing concentrate was obtained, the activity of which was 960,000,000 GIU. The permeate that had passed through the ultrafiltration membrane was removed.

120 kg magnesium sulfate and 300 kg ammonium sulfate (MgS0,7H 0 and (NH₄)-S0₄, food grade) were added to the concentrate. The mixture was cooled to 10 C in order to promote crystallization. The crystals formed were separated by decanting, and the crystallization was repeated by adding 411 kg water as well as 17 kg magnesium sulfate and 41 kg ammonium sulfate. The crystals were again separated by decanting. The crystal mass was dissolved by adding 402 kg water, and the pH of the solution was adjusted, by means of a 1 M ammonia solution, to 6.5. The solution was filtered with a plate filter, and the crystallization was repeated once more by using 16 kg of magnesium sulfate and 40 kg of ammonium sulfate.

A yield of 90 kg of crystal mass, of which 29 kg was enzyme, 3.7 kg salts (MgSO,, (NH_,) -SO,) , and balance water, was obtained. To the crystal mass 45 kg of glucose and 45 kg of fructose as well as 20 kg of invert sugar having a" dry solids content of 70 per cent by weight were added. In this way, 200 kg of an enzyme preparation was obtained, the composition of which was as follows: 29.2 wt. % water 52.0 wt. % sugars 14.5 wt. % glucose isomerase 4.3 wt. % salts (magnesium-ammonium sulfate) The glucose-isomerase activity of the enzyme concentrate was 4500 GlU/g.

Example 2

An ultrafiltered fermentate was prepared in the way described in Example 1. To 4000 kg of ultra- filtered fermentate, 244 kg of crystalline ammonium sulfate was added, followed by an ammonium sulfate solution in which 600 kg of salt had been dissolved into 900 kg of water. The solution was cooled to 13°C and kept at this temperature for 20 hours. The crystal mass formed was separated by means of a

Westfalia NA 7 separator. The crystals were dissolved into water, and the solution was filtered. The quantity of filtrate was 2000 litres. The crystal¬ lization was repeated by using 122 kg of crystalline ammonium sulfate and ammonium sulfate solution that contained 300 kg of ammonium sulfate as dissolved into 460 litres of water. The crystal mass obtained was again separated by means of a separator. To the crystal mass (525 kg) , the same quantity of crystalline fructose was added, whereby the mass was dissolved and the fructose was partly isomerized to glucose. In this way, a stable enzyme concentrate was obtained, the composition of which was as follows:

Sugars (glucose + fructose) 50.0 wt. % Enzyme 11.2 wt. %

Ammonium sulfate 3.5 wt. %

Water 35.3 wt. %

The glucose-isomerase activity of the concentrate was 3000 GIU per gram. Example 3

An ultrafiltered fermentate was prepared in the way described in Example 1. For the crystallization of the glucose isomerase 4000 litres of ultrafiltered fermentate having an activity of 2,400,000,000 GIU was used. The pH of the solution was adjusted by means of a 5 % NaOH solution to pH 7.0, and the temperature of the solution was adjusted to 12 C. For the crystal¬ lization of the glucose isomerase, 500 kg of ammonium sulfate dissolved in 750 litres of water was added to the solution during two hours with an even rate of feed. Then the solution was cooled to -2 C, and the solution was stirred for 24 hours. The glucose isomerase crystals were separated by means of a Westfalia NA-7 separator. The yield was 390 kg of crystal mass having a dry solids content of 23.6 per cent by weight and an activity of 2,300,000,000 GIU. 30 kg of sodium chloride and 180 kg of glucose were added to the crystal mass, and the pH of the enzyme concentrate obtained was adjusted, by means of a 5 % NaOH solution, to 7.0 (the glucose was partly isomerized) . In this way, 600 kg cf stable enzyme concentrate was obtained, the composition of which was as follows:

49.7 wt. % water 30.0 wt. % sugars

12.8 wt. % enzyme

2.5 wt. % ammonium sulfate 5.0 wt. % sodium chloride. The glucose-isomerase activity of the enzyme concentrate was 3400 Glϋ/g. Example 4

An ultrafiltered fermentate was prepared in the way described in Example 1. For the crystallization of the glucose isomerase 4000 litres of ultrafiltered fermentate, having an activity of 2,400,000,000 GIU was used. The pH of the solution was adjusted by means of a 5 % NaOH solution to pH 7.0. The temperature of the solution was adjusted to 12°C. For the crystallization of the glucose isomerase, 500 kg of ammonium sulfate dissolved in 750 litres of water was added to the solution during two hours with an even rate of feed. The solution was then cooled to -2°C, and stirred for 24 hours. The glucose isomerase crystals were separated by decanting. The yield was 230 kg of a crystal mass having a dry solids content of 40.0 per cent by weight and an activity of 2,300,0Cα000 GIU. To the crystal mass 115 kg of glucose and 115 kg of fructose as well as 50 kg of invert sugar having a dry solids content of 70 % were added. In this way, 510 kg of an enzyme preparation was obtained, the composition of which was as follows:

30.0 wt. % water

52.0 wt. % sugars (glucose, fructose) 15.1 wt. % enzyme

2.9 wt. % ammonium sulfate. The glucose-isomerase activity of the enzyme concentrate was 4500 GlU/g. Example 5 An ultrafiltered isomerase concentrate was prepared in the way described in Example 1. 50 g of ammonium sulfate was added to 0.95 litre of an isomerase concentrate having an activity of 600 GlU/ml and a temperature of 25 C. No precipitate was formed in the solution at this stage. The solution was cooled during 16 hours to 0 C, and it was kept at that tem¬ perature under gentle stirring constantly.

The isomerase started crystallizing in two days, and the crystallization continued so that after five days, 97.5 per cent by weight of the isomerase was in crystalline form and 2.5 per cent by weight still in dissolved form in the mother liquor. The crystals were separated from the solution by means of a laboratory centrifuge. Thereby 56 grams of wet crystal mass was recovered. In accordance with this example, it is possible to crystallize the isomerase with a very low ammonium sulfate concentration as compared with the typical quantities that are known from the literature. At the same time, this is an example of pure cooling crystallization. In the light of this example, it is readily understandable that, if the precipitation by means of ammonium sulfate is carried out rapidly and, then, the precipitate is separated by means of a centrifuge immediately, e.g. in 15 minutes, as is the case in the process of the US Pat. 4,237,231, the isomerase remains completely in the solution, when ammonium sulfate concentration is low and, if it precipitates, the crystallization will not be observed and its advantages unutilized. A crystal mass prepared in accordance with the present example can be dissolved exactly in the same way as in the other examples.

Claims

Claims :

1. Stable glucose isomerase concentrate, c h r a σ t e r i z e d by containing glucose isomerase dissolved in a concentrated carbohydrate water solution.

2. Glucose isomerase concentrate as claimed in claim 1, c h a r a c t e r i z e d by containing about 30 to 60 per cent by weight of carbohydrate soluble in water, about 5 to 17 per cent by weight of glucose isomerase, no more than about 15 per cent by weight of a suitable salt, such as ammonium and/or magnesium sulfate, and/or a buffer having a pH of 6.0 to 8.0, and balance water.

3. Glucose isomerase concentrate as claimed in claim 1, c h a r a c t e r i z e d by the carbohydrate being glucose, maltose, fructose, saccharose, sorbitol, xylitol, or a mixture of same, such as invert sugar, glucose syrup, or isomerized glucose syrup.

4. Glucose isomerase concentrate as claimed in claim 1, c h a r a c t e r i z e d by containing 50 per cent by weight of glucose and fructose, 15 per cent by weight of glucose isomerase, 5 per cent by weight of ammonium and magnesium sulfate, and 30 per cent by weight of water.

5. Glucose isomerase concentrate as claimed in claim 1, c h a r a c t e r i z e d by containing 50 per cent by weight of glucose and fructose, 6.7 per cent by weight of glucose isomerase, 3.5. per cent by weight of ammonium sulfate, and 39.8 per cent by weight of water.

6. Process for the preparation of a stable glucose isomerase concentrate containing glucose isomerase dissolved in a concentrated carbohydrate water solution, c h a r a c t e r i z e d by a) adding a suitable salt to the ultrafiltered glucose isomerase solution obtained from fermentation, so as to crystallize the glucose isomerase, b) cooling the solution so as to promote crystallization of the glucose isomerase, and separating the crystal mass formed and then, if desired, effecting one or several recrystallizations, and c) adding a carbohydrate or a concentrated water solution thereof to the obtained crystal mass, which dissolves, whereby a stable glucoseisomerase concentrate is obtained.

7. Process as claimed in claim 6 , c h a r a c t e r i z e d by a) adding crystalline ammonium sulfate and then a water solution of ammonium sulfate to the ultra¬ filtered glucose isomerase obtained from fermentation so as to crystallize the glucose isomerase, b) cooling the solution obtained so as to promote the crystallization of the glucose isomerase, separating the crystal mass formed recrystallizing the crystals by dissolving them into water, filtering the solution formed, adding crystalline ammonium sulfate and a water solution of ammonium sulfate, and then separating the crystal mass formed, and c) adding crystalline fructose to the crystal mass obtained, which dissolves, whereby a stable concentrate of glucose isomerase is obtained.

8 . Process as claimed in claim 6 , c h a r a c t e r i z e d by a) adding magnesium sulfate and ammonium sulfate to the ultrafiltered glucose isomerase solution obtained from fermentation so as to crystallize the glucose isomerase, b) cooling the solution so as to promote the crystallization of the glucose isomerase, separating the crystals formed by decanting, recrystallizing, the crystals by adding water, magnesium sulfate and ammonium sulfate, separating the crystals by decanting, adjusting the pH to 6.5 by adding dilute ammonia, and crystallizing once more by adding magnesium sulfate and ammonium sulfate, whereby a crystal mass is obtained, and c) adding glucose and fructose as well as water to the crystal mass, which dissolves, whereby a stable concentrate of glucose isomerase is obtained.