FI78117C - Process for preparing a stable glucose isomerase concentrate - Google Patents

Description

1 78117

Method for preparing a stable glucose isomerase concentrate

The invention relates to a process for the preparation of a stable glucose isomerase concentrate in the form of a clear solution. The concentrate obtained according to the invention is chemically and microbiologically stable without microbicides and as such is well suited for use in binding the enzyme to a support material in a reaction column.

The use of glucose isomerase to isomerize glucose to fructose is a known industrial process in which the enzyme is always used in bound form. In all of the technical processes in use, the bound enzyme preparation is prepared as a separate process in which either the enzyme is bound to the support by an adsorption technique or the whole microbial cell with its enzymes is bound into a matrix. The reaction column is packed with the finished bound enzyme. When the enzyme is consumed, the column is emptied and refilled with fresh bound enzyme preparation.

20 It is clear that if the enzyme inactivated in the column can be replaced by a new enzyme using a standard regeneration technique, significant savings can be achieved. The consumption of the carrier is reduced and, in addition, the loss of activity during the preparation of the bound enzyme is avoided. When the enzyme is bound to the support directly in the column, no drying or mechanical treatment is required, in which the enzyme is always somewhat destroyed.

It is known that a pure enzyme binds to a carrier much better than a crude crude preparation (U.S. Pat. No. 4,347,322). The problem has been to prepare a stable, easy-to-handle and storable enzyme preparation that can be used directly to bind the enzyme to the support in the column.

The precipitate or crystalline mass obtained from Glu-35 co-isomerase purification processes based on precipitation or crystallization contains at least 60% by weight of water, which cannot be removed without destroying the enzyme structure. Kidemas as such is not microbiologically stable, it is difficult to handle and administer. An alternative is to dissolve the crystalline mass into a solution which, for storage, shelf life and transport, is advantageous to prepare as concentrated as possible. The most natural way is to dissolve the enzyme crystals in water or dilute saline. In practice, however, it has been found that due to the relatively low solubility, it is not possible to prepare a sufficiently concentrated solution of the isomerase by dissolving the crystals in water. A dilute aqueous solution of isomerase is very poorly preserved and loses its activity due to microbiological and chemical contamination within a few days.

It has also been proposed to dissolve the enzyme in an organic solvent (U.S. Patent 4,077,842). However, the use of organic solvents in the food production process is not recommended, so this technique is ill-suited to an enzyme used directly in food production.

There is a wealth of literature on the preparation, purification and use of glucose isomerase due to the fact that glucose isomerase is one of the most important enzymes in use today. However, all published purification methods are quite complex and lead to general; relatively low yields.

Purification of enzymes and proteins by crystallization is well known per se and, for example, the manual Enzymes (Dixon, M. and Webb, E.C., Enzymes, 3rd ed., Longman Group Ltd., Bungay 1979, p. 1116) presents 192 photographs of enzyme crystals. In addition, it is known that ammonium sulfate 30 is in many cases suitable as a crystallization solubilizer. In addition to ammonium sulfate, the crystallization can often be performed with another salt or organic solvent such as acetone or alcohol. However, it is generally recognized in this field of chemistry that it is never self-evident that an isomerase or any other protein crystallizes well in itself.

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3 78117 with a known precipitator. Numerous proteins and enzymes are known that have not yet been successfully crystallized, although methods of purification in biochemistry have evolved tremendously.

The use of fractional sulfate precipitation (ammonium and / or magnesium sulfate) as a method or part of a purification of glucose isomerases is known from several publications (U.S. Patent 4,237,231, U.S. Patent 4,077,842, Agr. Biol. Chem.

45 (1981) 619-627, same 28 (1965) 1123-1128, same 34 (1970) 1795-1804, same 29 (1965) 1129-1134, Biochem. Biophys. Acta 10 151 (1968) 670-680).

The sulfate concentrations of the solutions used in these isomerase purification methods have usually been relatively high. For example, in the process of U.S. Patent 4,237,231, unnecessary proteins are first precipitated from an isomerase solution at a saturation rate of 40% ammonium sulfate (about 240 g (NH 2 SO 2 per liter of solution)), followed by precipitation of the isomerase itself at a higher saturation rate (up to 60%).

When high concentrations of am-20 magnesium sulphate are used in fractional precipitation, the higher the ammonium sulphate content, the higher the ammonium sulphate content is always precipitated with the isomerase, unless the isomerase has already been pre-purified in some way by another method.

Purification methods based on fractional precipitation usually result in an amorphous precipitate which is very difficult or even impossible to separate from the mother liquor in good yield by centrifuges or separators for industrial use.

An amorphous precipitate is usually a mixed precipitate containing proteins other than isomerase if the raw material is a crude microbial cell fluid. Such an amorphous precipitate is difficult to separate from the mother liquor and the precipitation does not achieve the desired purification effect with respect to the isomerase.

Crystallizations of glucose isomerases with ammonium sulfate, other salts or organic solvents have been reported in some publications (Biochem. Biophys. Acta 151 (1968) 670-680, Agr. Biol. Chem. 34 (1970) 1795-1804, ibid.). 45 (1981) 619-627, same 33 (1969) 1527-1534). The crystallization methods disclosed in these publications are characterized by low yield, very long crystallization time or relatively high chemical consumption. In all cases, the goal has been to prepare a small amount of pure isomerase for basic research purposes. In this case, the isomerase is first purified by one of the methods (extraction with an organic solvent, DEAE-Sephadex column chromatography, ammonium sulfate precipitation, dialysis), followed by crystallization of the purified isomerase from pure aqueous solution with ammonium sulfate, phosphate buffer or acetone buffer.

None of the methods described in the above-mentioned publications have been used on an industrial scale due to their disadvantage.

It has been found that the concentrate obtained according to the invention is both chemically and microbiologically stable and is very well suited for use in processes in which the binding of glucose isomerases to the support material takes place by a regeneration technique in a reactor column. Particularly suitable sugars and sugar alcohols are sugars used as such in foodstuffs and sugar alcohols permitted in foodstuffs. Since the end product in the isomerization is glucose-fructose syrup, a mixture of these sugars, i.e. inverter sugar, is excellently suitable for the purpose.

The total dry matter contained in the concentrate should be such that it is itself microbiologically stable, i.e. the dry matter content should be about 60-70% by weight (the water activity should be sufficiently low).

The Gluose isomerase concentrate prepared by the process of the invention preferably contains 5-15% by weight of glucose isomerase, 30-60% by weight of sugar or sugar alcohol such as glucose, maltose, fructose, sucrose, sorbitol, xylitol or the like. a mixture, e.g. invert sugar, glucose syrup or isomerized Gluose syrup, up to 15% by weight of ammonium and / or magnesium sulphate and the remainder 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).

The following are examples of suitable enzyme concentrates prepared according to the invention: 1. 50% by weight of invert sugar 30% by weight of water 15% by weight of enzyme 10 1% by weight of buffer (Na-K-phosphate) 4% by weight of ammonium sulphate 2. 50% by weight -% sorbitol 40% by weight water 10% by weight enzyme 15 However, the composition of the concentrate can be varied as necessary, taking care that the total concentration is sufficiently high and that the solution is fluid and easy to handle.

The glucose isomerase concentrates obtained according to the invention are pure, stable and easy to administer.

: Enzyme activity is adjustable. A suitable range of activity for use is 2000-5000 Glu / g preparation.

The sugars, sugar alcohols and salts used are preferably food grade (e.g. Food Chemicals Codes 25 standard). The enzyme should be sufficiently purified by crystallization.

Glucose isomerase is readily bound from the concentrate directly to the support in the reactor column. The concentrate is diluted with water and the dilute solution 30 is fed to the column, whereupon the enzyme binds to the support. The technique is similar to the regeneration of an ion exchange column.

After the enzyme has been used and thus inactivated, the protein is washed out of the column with alkali, after which fresh enzyme can be fed to the column to regenerate it.

Suitable carriers are materials having an anion exchange capacity, for example glass beads, an ion exchange resin or a silica-based support. Particularly suitable are protein-absorbing diethylaminoethyl (DEAE) derivatives such as DEAE pulp and DEAE-dextran. Several suitable carrier materials have been presented in the literature.

When DEAE pulp is used as a carrier, more than 1000 IGIU / g of bound enzyme per activity column (IGIU = immobilized glucose isomerase activity) is easily obtained.

The precipitation of glucose isomerase by salt is conventional per se and is described, for example, in Agr. Biol. Chem. 29 (1965) pp. 1129-1134 (Isumure and Sato).

However, no method has previously been described in which the actual formation of crystals takes place. In the methods shown, an amorphous precipitate is formed.

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

It is characteristic and surprising of the process according to the invention that the isomerase precipitates as a crystalline substance and above all other possible precipitating substances. The method uses carefully selected conditions and such a low ammonium sulphate content that nothing:. No other substances precipitate from solution. In this respect, the method differs substantially from anything previously described in the literature. Previously, isomerase has not been crystallized directly and alone as the sole precipitating component from the production microbial cell fluid or cell fluid concentrate. The method also achieves a very good yield, which differs substantially from that previously published.

It is well known that the shelf life and stability of enzyme preparations can be increased with, for example, glycerol, polyalcohols and sugars. Such V 78117 enzyme preparations are usually made by adding these substances to a concentrated enzyme solution.

Surprisingly, it has been found that mixing dry anhydrous sugar alcohol or sugar (especially glucose) in a concentrated isomerase crystal slurry or solid crystal mass with a maximum possible activity of about 10,000 GlU / g dissolves the isomerase crystals to form a true clear solution. Such a solution is stable when its water content is low enough and its iso-10ase activity is high enough. Sugar alcohol or sugar is essential for the dissolution of isomerase crystals in order to achieve the highest possible isomerase activity in solution.

No examples of isomeric-15 silicon solutions with enzymatic activity as high as po have been previously reported. in the product of the invention and no methods for preparing such solutions.

The preparation of the glucose isomerase concentrate is preferably carried out as follows: a) Prepared from Streptomyces rubiginosus by lysing the cell fluid (U.S. Patent 4,410,627) and ultrafiltrating the isomerase-containing concentrate as a raw material for the crystallization process; the preferred isomerase content of the concentrate is 200 to 800 Glu / g.

B) The pH of the isomerase solution is adjusted to 5.7 to 8.0, preferably to pH 7.0.

c) Cool the solution to 16 ° C or below.

d) Add ammonium and / or magnesium 30 sulphate to the solution in 50-170 g per liter of solution. The amount of sulfate to be added depends on the initial isomerase content and the crystallization temperature. The most preferred amount of sulfate to be added is one in which only the isomerase crystallizes, but the other proteins do not yet begin to precipitate.

E) The addition of sulphates is preferably carried out by slowly increasing the amount so that it takes 2-4 hours to increase the total dose, although even a sudden addition can give a useful result, but then the size of the isomerase crystals remains unnecessarily small.

5 f) The solution is preferably cooled over a period of several hours, preferably close to the freezing point of the mixture in question, which at the lowest sulphate concentrations tested is about -2 ° C and the highest -6 ° C, cooling can be started either at the same time as the sulphate addition 10 or only gradually. cooling provides a cooling crystallization effect which advantageously increases the size of the isomerase crystals in addition to cooling has a solubility lowering effect, which in turn improves the yield.

G) The isomerase crystals are separated from the solution by settling to the bottom of the vessel, filtering or, most preferably, centrifuging on a continuous separator.

h) The separated crystalline mass is dissolved by adding dry sugar or sugar alcohol or a concentrated aqueous solution thereof to sii-20, whereby the isomerase crystals dissolve and a stable glucose isomerase concentrate is formed.

If desired, the crystallization can be repeated, in which case the crystalline mass must be dissolved after separation (or -25 he g) in a large amount of water at a higher temperature (20-30 ° C). A suitable amount of water is then such that the isomerase activity of the solution is 500 to 2,000 GIU / ml, i.e. water is typically used in an amount of 4 to 10 times the weight of the crystal mass.

The following examples illustrate the invention.

9,781 1 7

Example 1

The Streptomyces rubiginosus microbe was fermented in a previously known manner (ref. US 4,410,627) in a batch of about 40 m3. The cell mass was lysed in a known manner (same reference). Cell-5 debris and other solids were removed by filtration through a conventional diatomaceous earth drum filter to give 32 tons of filtrate containing isomerase. This filtrate was filtered through a PCI (Patterson-Candy Inc.) ultrafilter to give 3,000 kg of an isomerase-containing concentrate with an activity of 960,000,000 GIU. The permeate passed through the ultrafiltration membrane was removed.

To the concentrate were added 120 kg of magnesium sulfate and 300 kg of ammonium sulfate (MgSO 4 VI 2 O and (NH 4 O 2 SO 4, food grade). The mixture was cooled to 10 ° C to promote crystallization. The formed crystals were separated by decantation and the crystallization was repeated by adding 411 kg of water and 17 kg of magnesium sulphate and 41 kg of ammonium sulphate, the crystals were again separated by decantation, the crystalline mass was dissolved by adding 402 kg of water and the pH of the solution was adjusted to 6.5 with 1 M ammonia solution, filtered through a plate filter and repeated once using 16 kg of magnesium. - and 40 kg of ammonium sulphate.

90 kg of crystalline mass were obtained, of which 29 kg were enzyme, 3.7 kg salts (MgSO 4, (NH, j) 2 SO 4) and the rest water. 45 kg of glucose and 45 kg of fructose and 20 kg of invert sugar with a dry matter content of 70% by weight were added to the crystal mass. This gave 200 kg of an enzyme preparation having the following composition: 29.2% by weight of water 30 52.0% by weight of sugars 14.5% by weight of glucose isomerase 4.3% by weight of salts (magnesium ammonium sulphate)

The glucose isomerase activity of the enzyme concentrate was 4,500 Glu / g.

10,781 1 7

Example 2

The ultrafiltered fermentate was prepared as described in Example 1. To 4,000 kg of ultrafiltered fermentate was added 244 kg of crystalline ammonium sulfate followed by an ammonium sulfate solution in which 600 kg of salt was dissolved in 900 kg of water. The solution was cooled to 13 ° C and maintained at this temperature for 20 hours. The formed crystal mass was separated on a Westfalia ΝΑ 7 separator. The crystals were dissolved in water and the solution was filtered. There were 2,000 10 liters of filtrate. The crystallization was repeated using 122 kg of crystalline ammonium sulfate and ammonium sulfate solution / containing 300 kg of ammonium sulfate dissolved in 460 liters of water.

The resulting crystalline mass was again separated by a separator. An equal amount of crystalline fructose was added to the crystalline mass (525 kg), whereupon the mass dissolved and the fructose was partially isomerized to glucose. This gave a stable enzyme concentrate having the following composition:

Sugars (glucose + fructose) 50.0% by weight

Enzyme 11.2% by weight 20 Ammonium sulphate 3.5% by weight

Water 35.3% by weight

The glucose isomerase activity of the concentrate was 3,000 GIU per gram.

Example 3 An ultrafiltered fermentate was prepared as described in Example 1. 4,000 liters of ultrafiltered fermentate with an activity of 2,400,000,000 GIU were used to crystallize glucose isomerase. The pH of the solution was adjusted to 7.0 with 5% NaOH solution and the temperature of the solution was adjusted to 12 ° C. To crystallize glucose isomerase, 500 kg of ammonium sulfate dissolved in 750 liters of water was added to the solution over a period of two hours at a constant feed rate. The solution was then cooled to -2 ° C and the solution was stirred for one day.

35 Glucose isomerase crystals were separated on a Westfalia NA-7 separator. 390 kg of crystalline mass with a dry matter content of 23.6% by weight and an activity of 2,300,000,000 GIU were obtained. To the crystal mass were added 30 kg of sodium chloride and 180 kg of glucose, and the pH of the obtained enzyme concentrate was adjusted to 7.0 with 5% NaOH solution. (glucose was partially isomerized) 5 In this way, 600 kg of a stable enzyme concentrate having the following composition were obtained: 49.7% by weight of water 30.0% by weight of sugars 12.8% by weight of enzyme 10 2.5% by weight of ammonium sulphate 5.0% by weight of sodium chloride

The glucose isomerase activity of the enzyme concentrate was 3,400 Glu / g.

Example 4 An ultrafiltered fermentate was prepared as described in Example 1. For crystallization of glucose isomerase, 4,000 liters of ultrafiltered fermentate with an activity of 2,400,000,000 GIU were used. The pH of the solution was adjusted to 7.0 with 5% NaOH solution. The temperature of the solution was adjusted to 12 ° C. To crystallize glucose isomerase, 500 kg of ammonium sulfate dissolved in 750 liters of water was added to the solution over a period of two hours at a constant feed rate. The solution was then cooled to -2 ° C and stirred for one day. Glucose-iso-25 merase crystals were separated by decantation. 230 kg of crystalline mass with a dry matter content of 40.0% by weight and an activity of 2,300,000,000 GIU were obtained. 115 kg of glucose and 115 kg of fructose and 50 kg of invert sugar with a dry matter content of 70% were added to the crystal mass. There were thus obtained 510 kg of ent-30 enzyme preparation having the following composition: 30.0% by weight of water 52.0% by weight of sugars (glucose, fructose) 15.1% by weight of enzyme 2.9% by weight of ammonium sulphate 35 The glucose isomerase activity of the enzyme concentrate was 4,500 GlU / g.

12,781 1 7

Example 5

The ultrafiltered isomerase concentrate was prepared as in Example 1. To 0.95 liters of isomerase concentrate having an activity of 600 GIU / ml and a temperature of 25 ° C was added 50 g of ammonium sulfate. No precipitate formed in the solution at this stage. The solution was cooled to 0 ° C over 16 hours and kept under constant stirring.

The isomerase began to crystallize after two days and continued to crystallize so that after five days 97.5% by weight of the isomerase was crystalline and 2.5% by weight was still soluble in the mother liquor. The crystals were separated from the solution by laboratory centrifugation. 56 g of wet crystalline mass were then obtained.

According to this example, it is possible to crystallize the isomerase at a very low ammonium sulfate content compared to typical amounts known in the literature. At the same time, this is an example of pure cooling crystallization. In the light of this example, it is readily apparent that upon rapid precipitation with ammonium sulfate, followed by separation of the precipitate by centrifugation immediately, e.g., 15 minutes, as in the process of U.S. Patent 4,237,231, the isomerase with low ammonium sulfate content remains in solution and if it precipitates, crystallization goes undetected and its benefits are not exploited. The crystalline mass prepared according to this example can be dissolved just as in the other examples.

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Claims (3)

781 1 7 13 A process for preparing a stable glucose isomerase concentrate in the form of a clear solution containing from about 30 to 60% by weight of sugar or sugar alcohol, from about 5 to 17% by weight of glucose isomerase, up to about 15% by weight of ammonium. and / or magnesium sulphate and the rest of the water, whereby the Gluose isomerase-producing microorganism is fermented in a manner known per se, the cell mass is lysed and the cellular debris and other solids are removed by filtration to give a glucose isomerase-containing filtrate with ammonium. and / or magnesium sulphate to precipitate glucose isomerase, after which the precipitate obtained is collected and stabilized, characterized in that the glucose isomerase-containing filtrate is ultrafiltered, ammonium and / or magnesium sulphate and optionally water are added to the obtained concentrate to form a mixture containing 50 - 170 g of ammonium and / or magnesium sulphate per liter, the mixture is cooled to In order to crystallize glucose isomerase at or below 16 ° C, the obtained crystalline mass is separated and optionally one or more recrystallizations are carried out, after which sugar or sugar alcohol is added to the crystalline mass. Process according to Claim 1, characterized in that glucose and / or fructose are added to the crystal mass. Process according to Claim 1, characterized in that the pH of the concentrate obtained after ultrafiltration is adjusted to 7.0 and the temperature of the concentrate is adjusted to 12 ° C, ammonium sulphate and water are added to the concentrate to form a mixture containing about 105 g of ammonium sulphate per liter. the mixture is cooled to -2 ° C to crystallize glucose isomerase, after which glucose is added to the crystalline mass. i4 781 1 7