DD211933A3 - PROCESS FOR THE PREPARATION OF ISOCITRATE DEHYDROGENASE - Google Patents

Abstract

The invention relates to a process for the preparation of isocitrate dehydrogenase. Its purpose is to provide a method for the production of this enzyme, in the course of which an activity of the Isocitratdehydrogenase is already achieved in the crude protein extract of 1 to 3 U / min. The object is seen to provide the desired enzyme with high activity by using a microbial enzyme source in conjunction with a new technology. The object is achieved by preparing the enzyme isocitrate dehydrogenase from the strain Pseudomonas putida ATCC 17633.

Description

- Title of the invention

Process for the preparation of isocitrate dehydrogenase

Field of application of the invention

, The invention relates to a process for the preparation of isocitrate dehydrogenase, which is used in the clinical-chemical diagnostics -4 and in food analysis for the quantitative determination of isocitrate.

j Characteristic of known technical solutions

'{It is already known to produce isocitrate. The most widely used method is the isolation of porcine heart (S.Ochoa in Methods in Enzymology, ed. SP Colowick and NDKaplan, Vol. I, p.699-704, Academic Press New York 1955); WWCle-Iand, VW Thompson and REBarden in Methods in Enzymology, ed. JMLowenstein, Vol. XIII, pp. 30-33, Academic Press New York 1969).

For the preparation of isocitrate dehydrogenase, the procedure is generally such that the starting material porcine heart is first mechanically comminuted, from which a cell-free protein extract is obtained, for example by centrifugation.

At an acceleration of 4000Ox g, the activity of the obtained enzyme is up to 0.4U / mg, at low numbers of turns it is lower. If the extract is obtained via the acetone dry powder, the initial activity is about 0.7 U / mg. By multiple ammonium sulfate or ethanol precipitations, the enzyme may be added to a specific activity of about 2-4U / mg, followed by chromatography on e.g. Sephadex G25 and CM-cellulose can be enriched up to 10-20U / mg. Commercially available are preparations with an activity of 2-3U / mg (MDH less than 3%).

It is also known that in the metabolism of numerous microorganisms isocitrate dehydrogenase is produced in small amounts. Examples are Salmonella typhimurium, Halobacterium salinarium, Acinetobacter calcoaceticus, Pseudomonas putida, Citrobacter fremdii u. v. a. It is not known to commercially produce isocitrate dehydrogenase using one of the above-mentioned or using other strains.

^; ! _Zie! the invention

The invention aims to provide a method for enzyme production, in the course of which an activity of the Isocitratdehydrogenase is already achieved in the crude protein extract of 1-3 U / mg, so that the production can be designed much cheaper than in previously known methods.

_JDurchung the nature of the invention ., ~

The object of the invention is to provide the desired enzyme with high specific activity by using a microbial enzyme source in conjunction with a new technology.

The object is achieved in that the enzyme isocitrate dehydrogenase (NADP-dependent) from the strain Pseudomonas putida ATCC 17633 (also deposited as ZlMET 10947) is produced. The stock is preferably on 2% agar. At 4 ° C in the dark using decane as C source and (NH ₄ J ₂ SO ₄ as N source.

The cultivation of Pseudomonas putida is carried out after inoculation from a preculture at temperatures of 20 to 40 ⁰ C,

preferably from 30 ^c C, in submerged culture in a liquid minimal medium.

Culturing is performed under aeration conditions in which the formation of isocitrate dehydrogenase is not inhibited by an intracellular NADH excess. Since this is apparently the case both at the beginning of the growth and after reaching the stationary phase, the activity of the enzyme undergoes a steep maximum, which is reached toward the end of the logarithmic growth phase.

This behavior is detectable in principle with the use of all C sources that the strain can utilize. However, the absolute values that can be maximally achieved for enzyme activity depend on both the air input and the C source. Substances which turn out to be particularly advantageous are larger amounts of such intermediates (for example acetoacetate) in the course of the oxidation, which, in particular in the case of limited O ₂ supply during their further reaction, have NADH-consuming and thus NAD ₂ -regenerating action. A selection of such species is given in Table 1.

The N source used is (NH ₄ J ₂ SO ₄ , NH ₄ Cl, etc.) After a growth time of 5 to 40 hours, depending on the physiological state and amount of inoculum, the bacteria are harvested (e.g. Centrifugation), washed with phosphate buffer and then transformed into cell-free crude extract, which can be done, for example, by sonication at temperatures near freezing with subsequent centrifugation.

The enzyme contained in the crude cell-free extract has the specific activities shown in Table 1, depending on the C source. These are Emers cultivations on 2% agar. Under Submersbedingungen such activity values are achieved after appropriate optimization of the air intake and the stirring speed (depending on the design parameters of the fermentation vessel). Particularly favorable results are achieved if the cultivations are carried out using hydrocarbons as C-Queile, which are supplied to the fermentation vessel in gaseous form.

From the cell free crude test extracts, isocitrate dehydrogenase may be obtained by fractional salt precipitation or extraction, e.g.

be enriched with ammonium sulfate to 5 to 10 times. For this purpose, solid ammonium sulfate is added to the crude protein extract up to a final concentration of 70% saturation, the entire activity reaching the sediment. For further enrichment of the isocitrate dehydrogenase, the residue is stirred with ammonium sulfate solution containing in each case 0.05 mol / l Na ₂ HPO ₄ at 0 ° C. and decreasing concentration of (NH ₄ J ₂ SO ₄ for 5 min in each case The majority dissolves in the concentration range between 40 and 50% of saturated ammonium sulfate solution and the enzyme is reprecipitated by the addition of ammonium sulfate to a final concentration of 70%.

The enzyme produced according to the invention is readily storable. At specific activities of 5-10μηιοΙ / ιτπη / ιτ ^ protein

the following external activities are provable:

Alcohol, malate, lactate dehydrogenase each less than 1%. .- ^ ~ - <rrSr -.- .. ·. """""'" _. - - "

Isocitrate dehydrogenase can be detected by using hydrophobic chromatography, e.g. to 10-Carboxydecylsepharose except for a spec. Activity of about 50 U / mg to be enriched.

The invention will be explained in more detail below using an exemplary embodiment.

embodiment

The cultivation of Pseudomonas putida ATCC 17633 is carried out in a medium containing per I the following components:

K ₂ HPO ₄ , 7.46g; NaH ₂ PO ₄ , 0.54g; MgSO ₄ .7H ₂ O, 20 mg; CaCl ₂ · 2H ₂ O, 20mg; Fe (NH ₄ J ₂ (SO ₄ J ₂ .6H ₂ 0.10 mg; NH ₄ J ₂ SO ₄ (I g / l) serves as N source.

For Emers cultivation of the strain on Slagagar tubes, a 2% agar (Difco agar) is used in the above nutrient medium. 6ml agar solution is used per tube. The solidification of the agar should take place in a largely horizontal position of the tubes. To stem the tubes after inoculation of the surface z. B. placed vertically with an eyelet. Subsequently, 0.1 ml of decane are introduced, the gel surface comes into contact only at the bottom of the vessel with the C source.

After incubation at 25 ° C. for about 60 h, the bacteria are washed off the agar surface with 2 ml of 0.05 mol / l phosphate buffer pH 7.5 (EgQQ ¹⁷¹ 2.5) and used to inoculate the submerged cultures. The inoculation densities are at EgQQ ^m 0.05. When culturing on hydrocarbons, for example η-hexane, the C source is introduced via the gas phase. Thus, in a 101-fermentation vessel with a Füllin stop of 71, the air supply (2-10 l / h) via a heated to 30 ⁰ C wash bottle containing hexane realized. The mean hexane entry into the fermentation vessel is about 5-10 ml / h under these conditions. The culture solution is stirred intensively (800 U / min.) After a cultivation period of 20-30 h at 30 ⁰ C - the culture turbidity after EQQQ is ¹⁷¹ = 2 to 4 - the bacteria are harvested by centrifugation, washed with phosphate buffer (0, 05mol / l, pH 7.5) and then destroyed by sonication at 4 ° C. The cell-free crude extract is obtained by centrifugation. The specific activity of isocitrate dehydrogenase under these conditions is between 1-2U / mg.

This crude protein extract is diluted with 0.05 M phosphate buffer (pH 8) until a protein content of 5-20 mg / ml is reached. From this solution is precipitated at 4 ⁰ C by addition of ammonium sulfate to a final concentration of 70% saturation of the majority of the protein. The entire activity of isocitrate dehydrogenase is in the precipitate. After centrifugation, the sediment is suspended in ammonium sulfate solution (40% saturation in 0.05 mol / l Na ₂ HPO ₄ ) at 0 ° C and then centrifuged again. After repeating the procedure, more than 80% of the total activity of isocitrate dehydrogenase is usually found in the supernatant (see Table 2).

The enzyme is reprecipitated by adding ammonium sulfate to a final concentration of 70% saturation. If one carries the supernatant obtained by ammonium sulfate extraction (2nd supernatant 40%); 130mg protein in 15ml on a column containing 80ml 10-carboxydecyl sepharose and eluted at 22 ° C with 100-200ml ammonium sulfate solution (saturation 40%), the isocitrate dehydrogenase is enriched to a specific activity of 50 U / mg. Table 1

Specific activity of isocitrate dehydrogenase in the crude extract of Pseudomonas putida ATCC 17633 after growth on various C sources

The cultivations are carried out on slant agar, the C-Queilen are used in a concentration of 0.5%. The amount of hexane used is 0.5 ml. After incubation for 24 h at 30 ^° C., the bacteria are washed off. The activity is determined after freezing and thawing of the cells in the presence of 0.1% Triton X 100.

C source optical density enzyme activity

E ¹ ₈₀₀ (pmol / min / mg)

0.8 0.7 1.5 1.2 0.7 1.4 2.0 1.4 3.0 2.0 1.1

Table 2

Enrichment of Isocitratdehydrogenase from Pseudomonas putida by ammonium sulfate extraction

Total protein Total activity Specific yield

citrate 1.2 isocitrate 1.0 a-Ketoglutarate 3.0 fumarate 2.5 succinate 3.8 pyruvate 3.4 D, L-malate 1.9 leucine 1.5 GI uta rat 2.9 hexane 1.4 decan 1.5

(Mg) (ΜΓηοΙ / ηηίη) Activity (pmol / min / mg) (%) Crude extract or sediment 70% 5,800 6 060 1.04 1st supernatant 40% 1050 1650 1.5 27

2nd supernatant 40%

550

4040

7.3

Claims (6)

invention claims 1. Process for the preparation of isocitrate dehydrogenase, characterized in that the isocitrate dehydrogenase from the strain Pseudomonas putida ATCC 17633 is obtained by culturing in submerged culture in a liquid minimal medium. 2. The method according to item 1, characterized in that Pseudomonas putida ATCC 17633 is cultured at temperatures of 20-40 ⁰ C in Submerskultur in a liquid minimal medium, after a growth time of 5-40 hours, the bacteria are harvested, washed with phosphate buffer and in cell-free crude extract, the enzyme is enriched by fractional salt precipitation, with (NH ₄ J ₂ SO ₄ at 70% saturation leads to complete precipitation of the enzyme, while at 40-50% saturation almost everything dissolves. 3. The method according to item 1 and 2, characterized in that the carbon source citrate, isocitrate, a-ketoglutarate, Fumaraj succinate, pyruvate, DL-malate, leucine, glutarate, hexane, decane or the like. in any combination or used individually. 4. The method according to item 1 to 3, characterized in that 0.3-1.51 air / h.I nutrient medium are introduced. 5. The method according to item 1-4, characterized in that the hydrocarbons used as the carbon source hexane, decane or the like. be entered in gaseous form. 6. The method according to item 1 to 6, characterized in that the Isocitratdehydrogenase is enriched by hydrophobic chromatography on 10-Carboxyldecylsepharose up to 50μιηοΙ / ιτιίη / ιτ ^.