DD228974A3 - METHOD FOR PRODUCING CARNITINE DEHYDROGENASE - Google Patents

Abstract

The invention relates to a process for the preparation of carnitine dehydrogenase. The object of the invention is to provide a process for the production of carnitine dehydrogenase, in the course of which, after a simple cell disruption process, an activity of the enzyme is already achieved in the crude protein extract of at least 3 U / mg. The object of the invention is to provide the desired enzyme with high specific activity by using a microbial enzyme source in conjunction with an effective purification technology. The object is achieved by preparing the enzyme carnitine dehydrogenase from the strain Pseudomonas putida ATCC 17633.

Description

- 2 - 249 090

Enzyme prepared according to the invention is readily storable. For specific activities of 10-20 U / mg, the following maximum foreign activities are detectable:

MDH, LDH each <0.1%

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 which contains the following constituents per 1:

K ₂ HPO ,, 7.46 g; NaH ₂ PO ₄ , 0.54 g; MgSO ₄ .7H ₂ O, 20 mg;

CaCl ₂ 2H ₂ O, 20 mg; Fe (NH ₄ ) ₂ (SO ₄ ) ₂ .6H ₂ 0.10 mg;

The N source used is (NH ₄ J ₂ SO ₄ (1 g / l), as C source D, L-carnitine HCl (5 g / l).

The bacteria are pre-cultured on the same medium using gaseous hexane as C source (0.015% in the feed air). The inoculation takes place in the course of the logarithmic growth phase, the seed densities are at Ejoo ^m ~ 0.1. The cultivation is carried out in a 10 l fermentation vessel with a filling content of 7 l with intensive stirring and an air intake of 2-10 l / h. After a culture time of 20-30 h at 30 ⁰ C - the turbidity of the culture is then Ej ₀ J " ¹ = 1.5-2.5 - the bacteria are harvested by centrifugation and then in 0.05 mol / l phosphate buffer pH 8 resuspended

100 ml portions of this suspension are frozen at -80 ⁰ C and left for 60 min at this temperature. Subsequently, the samples are kept for 4 h at 30 ⁰ C. After centrifugation at 10,000 xg for 30 min, at least 75% of the carnitine dehydrogenase activity is in the cell-free supernatant. The specific activity of the enzyme under these conditions is between 2-4 U / mg, the protein content is about 5 mg / ml.

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 carnitine dehydrogenase is in the precipitate. After centrifugation, the sediment is resuspended in ammonium sulfate solutions (decreasing concentrations in each 0.05 mol / l Na ₂ HPO ₄ ) at 0 ⁰ C and centrifuged again. In this way 90% of the total activity of carnitine dehydrogenase in the supernatant can be detected in the concentration range between 50 and 45% saturation (see Table 2). The enzyme (specific activity 5-10 U / mg) can be reprecipitated by adding ammonium sulfate to a final concentration of 70% saturation. If the supernatant (T 45) obtained by ammonium sulfate extraction is applied to a column containing 10-carboxy-decyl-Sepharose and eluted at 22 ° C. with ammonium sulfate solutions (saturation 40, 35, 30%), the carnitine dehydrogenase becomes a specific activity of about 25 U / mg enriched (Table 3).

Table 1

Obtaining carnitine dehydrogenase from Ps putida using various digestion methods The bacteria are harvested after centrifugation on D, L-carnitine, resuspended in 0.05 mol / l phosphate buffer pH 8 (Ejojj " ¹ ~ 20) and then 20 ml of each by Sonication (4 min at 50 W / cm ² ) or freezing (30 min, -80 ° C) / thawing (1 h, 30 ° C) digested.

pulping Gesamtakti Gesamtaktivi total ing) spec. Act. method after in the over protein Got over cell disruption got up in the over (U / mg) (U) conclusion and centrifuge at 15,000 xg (U)

Ultrasonic 180 177 120 1.5 Freeze / re-thaw 177 183 80 2.3

Table 2

Fractionated ammonium sulfate extraction of carnitine dehydrogenase

The data refer to 320 ml of a protein extract (4.5 mg protein / ml) obtained by freezing / thawing.

(NH ₄ J ₂ SO ₄ volume activity protein spec. activity yield (%-Saturation) (Ml) (U) (Mg) (U / mg) (%) 60 (sediment) 3,950 1 700 2.3 60 (supernatant) 50 55 (LI 1) 57 35 0.8 55 (over 2) 50 105 2.6 55 (over 3) 50 40 120 0.3 1.0 45 (Ü 1) 54 1 144 178 6.4 29 45 (over 2) 52 1 548 210 7.4 39 45 (over 3) 42 670 110 6.1 17 35 (Ü 1) 48 220 585 0.4 5.5

Table 3

Purification of carnitine dehydrogenase on 10-carboxydecylsepharose

On a column containing 18 ml of 10-carboxydecyl-Sepharose and equilibrated with (NH ₄ ) ₂ SO ₄ solution (45% saturation), 106 ml of a carnitine dehydrogenase solution in 45% saturated ammonium sulfate solution are applied. The solution contains 2 080 U of the enzyme and 3,229 mg of protein. Elution is carried out at 22 ° C.

- 3 - 249 090 O

fraction

(No.)

ml

(NH ₄ I ₂ SCX ₁ (% saturation)

activity

(U)

protein spec. Act. Out prey (Mg) (U / mg) (%)

1. 106 45 Second 25 45 3rd · 25 45 4th 25 40 CJL 25 40 6th 25 35 7th 25 35 8th. 25 35 9th 25 35 10th 25 30 11th 20 30 12th 25 30

22 2.2 0.6 1.3 1.9 43

177

225

167

386

546

520

25 7 8.5 18 12.5 11 17.5 9.5 8th 19 20 18.5 22 25 26 46 11 25

Claims (3)

A process for the production of carnitine dehydrogenase by cultivating Pseudomonas putida in submerged culture and minimal liquid medium, characterized in that the strain ATCC 17633 is grown in a restricted oxygen supply. - 1 - 248 090 Invention claim: 2. The method according to item 1, characterized in that 0.3 to 1.5 l of air per hour and liter of nutrient medium are introduced. 3. The method according to item 1 and 2, characterized in that the cell digestion is carried out by freezing and thawing. Field of application of the invention The invention relates to a process for the preparation of carnitine dehydrogenase, which can be used in the clinical-chemical diagnosis for the determination of L-carnitine. Characteristic of the known technical solutions Carnitine dehydrogenase is not commercially produced yet. There are no studies on a use for quantitative determination of carnitine. L-carnitine is quantitatively enzymatically z. Z. with the aid of carnitine acetyltransferase (EC 2.3.1.7.) In combination with acyl-CoA synthetase (EC 6.2.1.2.) Or DTNB (Pearson, DJ, Tubbs, PK and Chase, JFA in methods of enzymatic analysis [ HU Bergmeyer, ed.] Academy publishing house Berlin 1970, S. 1710-1723). Both methods have a number of disadvantages, which must be mentioned in addition to the considerable cost of the relatively complicated handling and high susceptibility. After possibilities have been created for the quantitative determination of L-carnitine by means of the optical test (Schöpp, W., Analyt. Biochem., In preparation), the widespread use of carnitine dehydrogenase can also be expected. The enzyme is formed by various Pseudomonas species (Aurich, H., Kleber, H.-P., and Schöpp, W., Biochem., Biophys. Acta, 139, 505 (1976); Kleber, H.-P. Seim, H., Aurich, H. and Strack, E., Arch. Microbiol., 116, 213 [1978]). The determined specific activities in the cell-free extracts are between 1 and 1.5 U / mg. U.S. Patent No. 4,221,869 discloses a process for producing carnitine dehydrogenase by cultivating Pseudomonas strain CIP 52 191 under conventional culture conditions at 30 ° C for 24 hours. There is obtained enzyme with an activity of 0.55 U / mg. It can be seen that a product of such low activity is not commercially interesting. An effective enrichment process for the enzyme is not disclosed in the cited patent. Object of the invention The invention has the object to provide a method for the production of carnitine dehydrogenase, in the course of a simple Zellaufschlußverfahren an activity of the enzyme is already achieved in the crude protein extract of at least 3 U / mg and an enrichment with the help of less, but effectively acting Trenntehnikiken to values inexpensive at 25 U / mg In addition, a preparation is to be obtained, which is virtually free of malate and lactate dehydrogenase. This is essential because it fully demonstrates the considerable benefits of carnitine dehydrogenase L-carnitine determination by the optical test of W. Schöpp. Explanation of the essence of the invention The object of the invention is to provide the desired enzyme with high specific activity, free of interfering foreign activities, by selecting a microwave enzyme source and applying new cultivation conditions in conjunction with an effective purification technology. The object is achieved according to the invention in that the enzyme CaTnitindehydrogenase ~ by K-ultivieretv the strain Pseudomonas putida ATCC 17633 is produced. This strain, which has also been deposited in the collection of the Jena Central Institute for Microbiology and Experimental Therapy where it has been assigned the name ZIMET 10947, is cultured according to the invention in the case of limited oxygen supply. In detail, the following process steps are completed. The stock is preferably on 2% agar at 4 ° C in the dark using decane as the C source and (NH ₄ ) ₂ SO ₄ as the N source. The cultivation of Pseudomonas putida on carnitine is carried out by inoculation from a preculture using z. B. gaseous hexane was recovered. Culture is carried out at 20 ° -40 ° C. in submerged culture in a liquid minimal medium. According to the invention, the cultivation is carried out under aeration conditions which correspond to a severely restricted oxygen supply to the microorganisms Under these conditions, the activity of carnitine dehydrogenase also reaches maximum values (NH ₄ J ₂ SO ₄ , NH ₄ Cl or the like used.) After a growth time of 5-40 hours, depending on the physiological state and amount of inoculum, the bacteria are harvested (eg by centrifugation) and then without interposition The recovery of the cell-free crude extract is preferably carried out by freezing the suspension and re-thawing, followed by centrifugation The high performance of this simple digestion process can be demonstrated especially when compared to ultrasound treatment (Table 1). From the cell-free protein extracts carnitine dehydrogenase by fractional salt precipitation or extraction, z. B. be enriched with ammonium sulfate to 3 to 5 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 carnitine dehydrogenase, the residue is stirred for 5 min with ammonium sulfate solution containing 0.05 mol / l Na ₂ HPO ₄ at 0 ^° C. and decreasing concentration of (NH ₄ J ₂ SO ₄₎ The majority dissolves in the concentration range between 50 and 45% of saturated ammonium sulphate solution, carnitine dehydrogenase can be obtained directly from these solutions by using hydrophobic chromatography, for example on 10-carboxydecyl-Sepharose, to specific activities of about 25 U / mg be enriched. The technical and economic advantages of the proposed procedure are that the strain Pseudomonas putida ATCC 17633 already produces a multiple of enzyme activity primarily in comparison to known strains. This is due both to the peculiarity of the strain used, which differs from all other Pseudomonas putida strains investigated, as well as the special ventilation conditions, ie. H. the limited oxygen supply. Further advantages are provided by the digestion process by freezing and the accumulation of the enzyme on 10-carboxydecylsepharose. The