DE1517749C3 - Process for the production of dry preparations of phosphotransacetylase from Escherichia coli which can be kept for a long time at room temperature - Google Patents

Description

Table I \ _;

Relationship between the cultivation time of Escherichia coli and the activity of phosphotransacetylase

■. ■ ··. in the cells

Breeding time (hours); ..

Specific activity of the enzyme ² )

2.6
4.0

■ 4.5

■ 4.7

6.6

5.3

22nd
0.5

Explanation of the table

!) B Erscherichia would cultured with shaking at 37 ° C in a medium having the above composition ■. The cells were collected after the times indicated and the cell suspension was exposed to 20 kHz ultrasound for 20 minutes. The phosphotransacetylase activity of the crude extract obtained by centrifugation at 14,000 rpm was determined.

² ) The activity of phosphotransacetylase is the amount of acetylphosphoric acid in μ-mol, which is decomposed in 15 minutes at 25 ° C in the presence of 5 units of coenzyme A according to the Stadtmann method; the specific activity is the activity per 1 mg enzyme protein:. ','''

B. Preparation of the raw extract

During the extraction, the cell membrane has to be broken because the substance is an intracellular component represents. In terms of the breaking effect and the time required, it is aiii most expedient to to treat the cell suspension with ultrasonic waves.

The phosphotransacetylase is not deactivated when treated with ultrasonic waves. the The cell membrane of Escherichia coli is almost completely broken when you get a suspension of 1 g of dry cells in 20 ml of a weakly alkaline buffer solution, for example a 0.02 molar Potassium bicarbonate solution, 5 minutes at low temperature with ultrasonic waves with one frequency from 10 to 20 kHz, preferably from 20 kHz, treated, the protein contained in the cells is extracted to a sufficient extent. The most appropriate time is during which the Material for extraction of the enzyme is treated with ultrasonic waves 15 to 20 minutes since the specific activity of the enzyme gradually increases up to 15 minutes. An example of the extraction is given in Table II.

Table II
Extraction of phosphotransacetylase

from Escherichia coli
by treatment with ultrasonic waves (raw extract)

Treatment time Protein * Specific with activity Ultrasonic waves 0 of the enzyme 0 minutes 21.3 mg / ml 0 5 minutes 22.0 mg / ml 3.9 10 mins 22.5 mg / ml 4.3 15 minutes 22.5 mg / ml 4.5 20 minutes 4.5

750 ηΐμ determined after the sample with an alkaline Copper sulfate solution and then with Folin-Ciocalteu reagent had been treated for color development. .-,. ... ■ -. ■ .-

C, cleaning

That in the one described above The crude extract contained in the procedure was obtained by adding proportionally with Sulfuric acid acidified saturated ammonium sulfate solution fractionated at low temperature precipitated and the precipitates were collected by centrifugation. The wet rainfall that containing a small amount of ammonium sulphate were dissolved in a weakly alkaline buffer solution (pH = 8), such as tris-hydroxymethylaminomethane or potassium bicarbonate solution.

The activity of phosphotransacetylase, which occurs when the ammonium sulfate is saturated of 0.3 to 0.35 precipitated protein fraction (Hoffmeister's method) and the specific activities are about three to four times that of the crude extract, as shown in Table III is specified. The yield of activity at this stage is about 80%.

45

Table III

Purification of phosphotransacetylase
by fractional precipitation of the crude extract

55

Explanation of the table

0 1g dry cells were 0.02 molar in 20 ml Potassium bicarbonate solution suspended, and the suspension was during the indicated times treated with ultrasonic waves at a frequency of 20 kHz and then at 10,000 for 15 to 30 minutes up to 14,000 rpm, centrifuged. In the protruding The amount of protein and enzyme activity were determined in the liquid.

² ) The protein was colorimetrically determined by the Lowry method at a wavelength of

Saturation level
of ammonium
sulfate protein activity
of the enzyme Specific
activity Crude extract 100 100 4.66 0 to 0.3 17.5 16.8 4.56 0.3 to 0.35 21.5 79.0 16.4 0.35 to 0.4 23.4 3.0 0.6 0.4 to 0.5 5.1 0 0 0.5 to 0.75 3.1 0 0

Explanation of the table

The protein and the activity of the enzyme were determined according to the method outlined in Tables I and II determined, the results represent relative values for each fraction, with the values for the crude extract were related to 100.

It is desirable that the enzyme preparation used to determine coenzyme A should not contain a co-

contains enzyme A. So far, the protein solution has generally been dialyzed to remove the low molecular weight To remove substances from it. However, the phosphotransacetylase is used in dialysis or in Filtering with a dextran gel (instead of dialysis) strongly deactivates, which is why these treatment methods cannot be used to remove coenzyme A. In the pre-published Work using Clostridium kluyveri as a starting material became coenzyme A. removed by treating the raw extract with activated charcoal. However, it is known that the coenzyme A content is very low in the cells of Escherichia coli, d. H. only about ve the content of Clostridium kluyveri is. For this reason there is no treatment to remove coenzyme A. necessary. It has been found experimentally that at the stage after fractionation with ammonium sulfate almost no coenzyme A was contained in the enzyme.

D. Preservation of the enzyme

If the precipitate obtained according to the measures of the above definition of the invention in a Dissolves buffer solution and this solution is finally subjected to freeze-drying, becomes a dry preparation received, which can be sent to remote areas without special precautions. If the dry enzyme preparation obtained in this way is stored at room temperature (approx. 25 ° C.), then finds the values given in Table IV are used for the enzyme activity.

Table IV

Conservation status of a dry preparation

of phosphotransacetylase
of Escherichia coli at room temperature

Days after manufacture

Specific activity
of the enzyme

Degree of maintenance of activity

Explanation of the table

11.3 11.3

100 100

13 35
11.3 10.9 100 96.5

The degree of maintenance of the activity is obtained if one immediately afterwards for the activity _ the production of the dry enzyme preparation assumes the value 100.

Claims (25)

1 2 Draws between the abnormality of the coenzyme A Claim: Metabolism and various diseases. Were it is possible to easily use phosphotransacetylase Process for the production of room temperatures, so could the field of biological, bioperatur long-lasting dry preparations of the 5 chemical and medical research on the Phosphotransacetylase from Escherichia coli, since coenzyme A can be expanded, which is of great help characterized by having cells for the advancement of medical research of Escherichia coli in a weakly alkaline one. Buffer solution for breaking cell membranes It is known that phosphotransacetylase except treated with ultrasonic waves so that in the _i0 in Clostridium kluyveri also in Escherichia coli, Protein contained in crude extract, partly due to Lactobacillus delbrueckii and Bacillus megatherium Addition νρη acidified with sulfuric acid, ge ^ (Nature, Vol. 179, 1957, pp. 103 and 104) included saturated ammonium sulfate solution at lower. Escherichia coli was found to be . Temperature falls fractionally and the source for phosphotransacetylase is best suited that the _15, since these organisms are very easily handled and Only a small fraction of protein that can ultimately be cultivated in one for cultivation The degree of saturation of the solution with ammonium sulfate is required, the enzyme is easily removed from the cost from 0.3 to 0.35 precipitates, wins that these cells can be extracted and the purification moist, the phosphotransacetylase and an extremely simple one. In the present. Fall succeeded small amount of ammonium sulphate containing _ao extraction and purification of Phosphotrans- Precipitate dissolves in a buffer solution and this acetylase from Escherichia coli and the manufacture Finally, the solution is freeze-drying using an enzyme preparation that is used in the dry state throws - room temperature could be stored for a long time. The so-called - Enzyme preparation can be used immediately to determine 25 mung of coenzyme A can be used, namely simply by placing it in a method prescribed buffer solution, which is why it is a very convenient substance for the knowledge-die Invention relates to a method for producing represents. position of a dry preparation of phosphotrans- _{3o D} i _e invention now consists in a process for acetylase that can be kept for a long time at room temperature, production of Escherichia coli from Escherichia coli that can be kept for a long time at room temperature. Dry preparations of phosphotransacetylase from Hitherto coenzyme A was prepared according to methods loading Escherichia coli, and being characterized in that true where pigeon liver extracts from Clostridium _be cells of Escherichia coli in a weakly dium kluyveri extracted phosphotransacetylase, Ge ₃₅ alkaline buffer solution for breaking Cell membrane purified "ketoglutarate dehydrogenase and succinyl". membranes with ultrasonic waves treated in that the coenzyme-A deacetylase purified citrate cleavage in the crude extract protein contained by partial enzyme and 'Maleatdehydrogenase or purified addition of acidified with sulfuric acid, fatty acids sown activating enzymes from calf liver comparable _t i _gter ammonium sulfate solution were turned at low temp. In all of these methods, 40 _ra ture falls and the resulting enzymatic reactions are carried out, in which the precipitates are removed, so that the protein fraction, coenzyme A, takes part. Among these methods, which is _{the case} finally auswird at a saturation degree of the method used in the phosphotransacetylase solution Ammöniumsulfat 0.3 to 0.35, because of the high specificity to the falls to wins that. to damp these, the Phösphobestimmenden coenzyme A, transacetylase because of easy manufacturing 45 and a small amount of ammonium position of the enzyme used and because of the _su if _{at-containing} precipitate in a buffer simplicity of the method of determination on advanta- solution and this solution eventually triggers the Gefrierhaftesten . To apply this method of determination, subject to drying,
to be able to, one has phosphotransacetylase up to now extracted from Clostridium kluyveri and obtained 50 samples (J. Biol. Chem., Vol. 191, 1951, pp. 365 to 576). . _. . ..,. ,. However, Clostridium kluyveri is not a source of enzyme ^A · Eschenchia coil as a starting material favorable as it belongs to a group of bacteria, Escherichia coli B is grown in a medium that which is a nutrient medium with complicated composition- χο / ₀ glucose, 0.4% yeast extract, 0.4% peptone and Settling and completely anaerobic growth conditions 55 0.8% contains dipotassium hydrogen phosphate, approximately need. In the analytical method, grown for 6 hours at 37 ° C with shaking. One This enzyme is very difficult to produce; too long cultivation leads to a lowering of the and the applicability of the method is largely enzyme yield as shown in Table I. due to the instability inherent in the enzyme proteins. After cultivation, the cells are limited. Therefore the enzyme has to be trifuged at the moment, etc. collected and distilled Manufactured by specialists whose job is water washed. From the thus obtained fresh research in the field of Coen cells can direct the enzyme through ultra- zymsAist. sound waves are extracted; the cells can Coenzyme A is a substance that all species, but also over a long period of time, as a starting point of living things, including humans, a 65 material for the enzyme to be stored after it important role in sugar and fat metabolism in a suitable amount of distilled water sus- plays. Many biochemists and clinical physicians pend and work at low temperatures, for example are concerned with investigations into the evidence that has been dried by freeze-drying.