FI88510B - Method for crystallization of glucose-isomerase with the aid of high pressure - Google Patents

Description

1 38510

Method for crystallization of glucose isomerase by high pressure

The invention relates to a new method for crystallizing proteins. The invention is based on the fact that a supersaturated solution of the protein is crystallized by means of a high pressure, which is preferably between 1000 and 3000 bar.

Crystallization of proteins requires some general conditions that are not necessarily required for crystallization of small molecule substances. The detailed conditions guaranteed to lead to crystallization for the various proteins are not known. In most cases, the crystallization of proteins is slow, the appearance of the first crystals can take weeks or months.

Supersaturation can also be achieved by increasing the pressure, and it is known to apply high pressure crystallization in difficult separations, for example 13 when the substances to be separated have very close melting points or when the temperature of the mixture could not be raised or lowered. However, experimental pressure crystallization has only been used in the separation of relatively small molecule substances (see Moritoki M, Kitagawa K, Nishiguchi N, Chem Econ. & Eng. Review, Vol. 16, pp. 30-35, December 1984). No attempt has been made to isolate high molecular weight substances such as proteins by high pressure crystallization because proteins such as enzymes are easily denatured under high pressure (see Moorild E, Advances in Protein Chemistry, Vol. 34, p. 93 (1981)).

It has now been found that at high pressure the rate of protein shrinkage 25 increases sharply. For example, glucose isomerase crystallizes at a pressure of 2000 bar in a few minutes after several hours at normal pressure. However, very high pressures should not be used because the rate of inactivation and denaturation of the proteins is then too high; for example, glucose isomerase forms an amorphous, sparingly soluble mass at a pressure of more than about 3000 bar.

It is surprising that high pressure results in high molecular weight. crystallization of the native substance. After all, a protein molecule is mechanically quite •; "weak", i.e., it deforms easily, and as mentioned above, it may even irreversibly change, i.e., be denatured. Thus, it was not expected that permanent crystals would form with the high pressure of 2 88510 k

The general filtration principle of the method is as follows. A supersaturated solution of the protein is prepared in a manner generally known per se. This supersaturated solution is then transferred to a pressure chamber and pressed under high pressure, whereby part of the protein crystallizes.

Based on the experimental runs performed, it can be concluded that the rate of crystallization of the protein is a constant function of the pressure applied. However, as the pressure rises above 10,000 bar, an amorphous precipitate begins to appear in addition to the crystals.

The appearance of an amorphous precipitate and obvious damage to the structure of the protein molecule is considered disadvantageous in this context. Thus, a pressure range favorable for crystallization is observed, which in the exemplary cases is between 1000 and 2500 bar.

15

The crystals obtained under high pressure were similar in shape to the crystals prepared from the corresponding solution at normal pressure. The crystals prepared under high pressure were stable and grew at normal pressure if the surrounding solution was supersaturated.

20

The method used in the exemplary embodiments for glucose isomerase was as follows.

First, glucose isomerase was prepared as follows. This enzyme, 25 D-xylose isomerase (D-xylose ketol isomerase, EC 5.31.1.5), is known in the industry as glucose isomerase and is produced on an industrial scale by fermentation using Streptomyces rubiginosus. After fermentation, the solution is filtered clear, and concentrated by ultrafiltration. Ammonium sulfate (10 g / cubic decimeter) is added to crystallize glucose isomerase. The crystals are separated by centrifugation.

Such a glucose isomerase is used in industry under the trade name Spezyme. The exact method of preparation is disclosed in "Visuri K, Stable glucose isomerase Concentrate and a process for the preparation thereof, U.S. Patent No. 4,699,882,1987".

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Glucose isomerase was dissolved in an amount of 45-55 grams of dry protein per liter of 10% aqueous ammonium sulfate at 50 ° C. the solution was filtered through a 0.45 micron filter with filter paper or a membrane filter to make it as clear as possible so that it did not contain crystalline embryos or particles that could act as crystallization centers. The solution was cooled to 15 ° C and part of it was transferred to a pressure chamber. The solution was compressed in a chamber at a pressure of 500-4000 bar for 2-30 minutes. At the end of the compression, the chamber was opened and the crystals were immediately separated from the solution by filtration or centrifugation. The residual protein content of the solution was determined to be 10, and the Iddesa yield was calculated from the decrease in porosity. The quality of the crystals was determined by microscopy and photography with a microscope camera. The results of the experiments are summarized in the example table. For comparison, the part of the solution left at normal pressure was examined in the same way.

The method according to the invention is illustrated in more detail by the following embodiments.

Example I

20 Crystallization of glucose isomerase.

A crystallizer according to Figure 1 was used, the main components of which were a cylindrical steel tank (inner diameter 40 mm and volume 50 cc), a piston going into the tank and a water jacket surrounding the tank, by means of which the tank was thermostated to 15 ° C. In addition, heat and pressure sensors were connected to the tank, the readings of which were fed to a plotter.

A glucose isomerase solution was prepared by dissolving 11 g of glucose .; : isomerase in 200 ml of a diammonium sulphate solution with a concentration of 10 to 30% by weight at 50 [deg.] C. The glucose isomerase content of the solution was then about 55 g / l). The solution was rapidly cooled to 15 ° C, and a batch of about 50 ml was charged to a steel tank of the crystallizer, vented, and pressurized hydraulically. which were between 1000 and 4000 bar. Crystallization times were between 2 and 30 'I minutes.

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The results are shown in Table I.

It was found that the activity of the enzyme was not impaired.

Table I Crystal yield as a percentage of test solution glucose isomerase S at the end of each experiment. The glucose isomerase concentration at the beginning of the experiment was about SS g / l.

Crystallization time Isomerase Pressure in test solution ____ ___1 bar 1000 bar 1500 bar 2000 bar min__gΛ __% __% __% __% 2 51.91 2.0 3 52.32 1.1 9.6 5 56.45 1.7 38.6 5 56 , 49 54.5 7__50.76__U ____ 56.4 10 48.65 78.9 10 52.51 84.6 12 54.43 1.4 77.2 17 51.86 3.5 73.2 17__54.70__y) ____ 82, 4 25 52.91 80.7 30 52.46 2.5 77.9 30 52.11 1.3 57.1 10 53.20 0.1 0.8 15__45.09__1.3 13.0 ___ 20 50.50 1.3 30.1 20 55.79 3.8 53.55 10 53.40 14.35 15 55.03 0.0 30__52.59__08 ____ 15 55.48 1.8 30 56.73 0.3 80.65 15 50.63 1.4 _ 16.95 _ 5 88510

Example 2

The procedure was as in Example 1, but using a glucose isomerase solution with an enzyme content of about 70 g / l (prepared by dissolving 14 g of glucose-5 isomerase in 200 ml of diammonium sulfate). The crystallization temperature was 23 ° C. Crystallization times were between 2 and 20 minutes.

The results are shown in Table Π.

It was found that the activity of the enzyme was not impaired.

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Table Π Crystal yield as a percentage of test solution glucose isomerase at the end of each experiment. The glucose isomerase concentration at the beginning of the experiment was about 70 g / l.

_Pressure__Crystallization time__Crystal yield _bar__mm __% _ 1000 20 17 2000 20 70 2500 20 49 _1 20 _0 15

Example 3

Use of high pressure crystals as seed crystals.

f \: 20

1 liter of isomerase solution was prepared with an isomerase content of 35: g / l calculated as dry matter. A batch of 50 ml was taken from the solution and pressed in a pressure chamber at 2000 bar for 5 minutes at 15 ° C. The batch was removed from the pressure chamber and it was found that 20 g of the isomerase contained therein had crystals averaging 10 micrometers in diameter. 1 ml was taken from this batch and added to 950 ml of isomerase solution under normal pressure. This mixture was stirred with a propeller stirrer so that no crystals settled to the bottom of the vessel (7 cm propeller, 80 rpm). Crystallization in solution began immediately due to the seed crystals, and after 5 hours crystallization was almost complete (4 g / l of isomerase remained soluble in solution, i.e. the remaining 31 gA was crystalline). The crystals were examined by microscopy and it was found that most of the crystal mass was in the form of crystals with a diameter of 95-120 micrometers.

Claims (6)

7 88510 A process for the crystallization of glucose isomerase, characterized in that a supersaturated aqueous solution of the substance is transferred to a high-pressure vessel and the solution is allowed to act under a pressure which is preferably between 1000 and 3000 bar. Method according to Claim 1, characterized in that the supersaturation is 5 to 20 and the pressure 1500 to 2500 bar. 10 Process according to Claims 1 and 2, characterized in that the crystallization time is from 30 seconds to 30 minutes. Process according to Claims 1 and 2, characterized in that the crystallization is carried out in two or more stages, in which case seed crystals are first prepared at high pressure, and the actual crystallization is carried out at lower pressure by means of said seed crystals. Process according to Claim 4, characterized in that the pressure in the first stage is 1500 to 3000 bar and the crystallization time is 30 seconds to 15 minutes. Process according to Claims 4 and 5, characterized in that the actual crystallization is carried out at atmospheric pressure. * 88510 Patentkraven