DD149874A3 - PROCESS FOR THE PREPARATION OF 3-HYDROXYBUTYRATE DEHYDROGENASE - Google Patents

Abstract

The invention relates to a process for the preparation of 3-hydroxybutyrate dehydrogenase. The object of the invention is to provide a cost effective method which provides an enzyme of high specific activity. The object is seen to produce the desired enzyme by culturing a new strain on previously unused culture substrates. The essence of the invention consists in cultivating the strain Pseudomonas putida either on culture substrates which decompose acetoacetate or hydroxybutyrate or on substrates which consist of straight-chain hydrocarbons C & ind6! to C & ind10! consist.

Description

Process for the preparation of 3-hydroxybutyrate dehydrogenase

jftnweiidungsgebie _i t der_ invention

The invention relates to a process for the preparation of 3-hydroxybutyrate dehydrogenase, which is used in the clinical-chemical diagnostics for the quantitative determination of ketone bodies *.

^ PP foi ^sci ! Solutions

It is already known to produce 3-hydroxybutyrate dehydrogenase _o For this purpose, it is customary to breed the bacterial strain Rhodopseudomonas spheroides on 3-hydroxybutyrate as a carbon source, to separate the bacteria by centrifuging * and the cell-free protein extract, which has an initial activity of approx "0.1 / umol / min / mg protein has to undergo protamine precipitation to remove the nucleic acids". After fractionated ammonium sulphate precipitation, the appropriate fraction is separated off, washed, dissolved in buffer and purified several times or chromatographed to an activity of about 17 μg / mmol / mg protein (Literature _{c c} B _c H U * Bergmeyer et al., Biochenu J · ^ ⁹⁶ ?)? The described method has the drawback that the end product is very expensive "especially if it concerns strongly enriched 3-Hydroxybutyratdehydrogenase", The cause is not only due to the elaborate cleaning operations, but also in the high price of Culture substrate. Another disadvantage is the fact that the activity of the her-

  - 2 - 2 2 0 0 6 0

enzyme does not exceed 17-20 / umol / min / mg protein. The reason is that the strain Rhodopseudomonas spheroides used is incapable of producing an enzyme having higher starting activity than described above.

The object of the invention is to provide a process for enzyme production which is considerably more cost-effective than previously known and which, moreover, makes it possible to produce an enzyme with a significantly higher specific activity.

Presentation of the essence, the invention

The object of the invention is to modify essential features of the previously known procedure and to provide the desired enzyme cheaply and with high specific activity by using a different strain and other culture substrates in conjunction with a new technology.

The object is achieved according to the invention by producing the enzyme 3-hydroxybutyrate dehydrogenase from the strain Pseudomonas putida. The strain is preferably carried out on 2% bouillon agar at 4 C in the dark. "Cultivation of Pseudomonas putida occurs after inoculation from a preculture temperatures of 20 ~ 40 ⁰ C, preferably at 30 ⁰ C, in submerged culture in a liquid minimal medium "

Culturing is inventively done on C sources, is formed during biodegradation acetoacetate or hydroxybutyrate "Such carbon sources are," as D, _L-lysine} D, L-leucine, D! "Carnitine Ue a., In addition, straight-chain hydrocarbons with a chain length Cg to Cq or fatty acids from C to C ₁ g _e In the cases KH ₄ Cl, (HH ^) ₂ SO ₄ Oc dgl * is used as the IT source, in which the C source is not at the same time as a C and N source can be utilized _e Wach a growing time of 5 - 40 hours - depending on physiological condition and amount of vaccine »

, .- 3 - 220060

The bacteria are harvested (eg by centrifugation), washed with phosphate buffer and then transformed into cell-free crude extract, which can be carried out, for example, by ultrasound treatment at temperatures close to freezing with subsequent centrifuging. The enzyme contained in the cell-free crude extract Depending on the C-source, _s is the specific activity shown in Table 1.

By fractionated salt precipitation or extraction, ζ. Β. with ammonium sulfate, the enzyme is enriched to 10 to 15 times. For this purpose, the crude protein extract is expediently diluted and precipitated with 70 % of saturated ammonium sulfate, the majority of the protein. The entire activity remains in the sediment. For further enrichment of the 3-hydroxybutyrate dehydrogenase, the residue is stirred successively with ammonium sulfate solution containing in each case 0.05 mol / l Tris at 4 ^° C. and decreasing concentration of (UEL ² SO 4 for 5 min The bulk dissolves in the concentration range between 35 % and 20 % saturated ammonium sulfate solution · The enzyme is reprecipitated by addition of ammonium sulfate to a final concentration of $ 50.

Further enrichment of the enzyme is on a hydrophobic support material such. B. lO-carboxydecyl-Sepharose as follows possible. On a column filled with 10-Oarboxydecyl Sepharose enzyme is added in 20 % saturated ammonium sulfate solution. Wax washing with ammonium sulphate solution of this concentration is fractionally eluted with 17% saturated ammonium sulphate. "In the first fraction, the specific activity increased to 4 times, in the second 7 times." Adsorption is favored by structuring ions such as phosphate, citrate, sulphate. "

The enzyme produced according to the invention is readily storable. For specific activities of 10-20 yumol / min / mg protein, at most the following foreign activities are detectable:

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It is advantageous to work with a graded oxygen deficit in the method step of bacterial growth.

The invention will be explained in more detail below with reference to an embodiment example.

embodiment

Pseudomonas putida (PPG 6) is grown at 30 ⁰ C in submerged culture in a liquid α Minimalrnedium The medium contains per liter: 6.97 g K ₂ HPO _4, 1.5 g Voh ₂ PO _4, 0.2 g MgSO _{4 β} 7H ₂ O, 7.5 mg PeSO ₄ . 7 K ₂ O, 0.75 mg ROiSO _{4 β} 4 H ₂ O, 0.75 mg ZnSO ₄ . 7 H ₂ O, 0.15 mg CuSO ₄ .beta. 5 H ₂ O, 0.15 mg CoCl ₂ . 6 H ₃ O and 0.15 mg boric acid "The vaccine suspensions are precultures in which yeast extract (0.4 g / l) and glucose (10 g / l) were added to the liquid minimal medium when cultivated on D, L-car Nitin is used as the sole C and IT source at a concentration of 2 g / l. When cultivated on other C sources (10 g / l), NH ₄ Cl (3 g / l) is used as the N source . After a growth time of 15 hours, the bacteria are harvested by centrifugation, repeatedly with phosphate buffer (0.067 mol / 1, pH 7 $ 5) and then the cell-free crude extract is collected by centrifugation destroyed by sonication at 4 ⁰ C ·.

This crude protein extract is (pH 8), diluted with 0.067 mol / 1 phosphate buffer until a Pr.oteingehalt of 5-20 mg / ml is reached * From this solution, at 4 ⁰ C by the addition of ammonium sulfate to a final concentration of 70% saturation After centrifugation, the sediment is suspended in ammonium sulfate solution (45/3 saturation in 0.05 mol / l Tris) at 0 ^° C. and then centrifuged again «The gesarate

• - 5 - · 2 2 C

Activity remains in the sediment The residue is stirred for further white enrichment with ammonium sulphate solutions (0.05 mol / l Tris / HCl, pH 8, containing) of decreasing concentration at 4 ^° C. for 5 min. The enzyme is thereby increasingly dissolved, preferably in the concentration range between 35 % and 20 % of saturated ammonium sulfate solution (see Table 2). The enzyme is reprecipitated by addition of ammonium sulfate to a final concentration of 50 % saturation. After centrifuging, the enzyme is suspended in 3.2 mol / l ammonium sulfate solution, pH 7, and stored at 4 ° C.

To further enrichment vjerden applied to a 2 ml column of 10-carboxydecyl-Sepharoee at 25 ⁰ G 1 ml of the enzyme solution in 20% saturated ammonium sulfate solution. The specific activity is 11 / umol / min / mg protein; Total protein 1.5 mg · All activity remains on the carrier. After washing with. In each case, 2 ml of ammonium sulphate solution (17 % saturation) are added to the column successively with 10 ml of ammonium sulphate solution (20 % saturation). In the 2 fractions obtained, the total activity is found, but only 25 % of the protein. The specific activity in the first fraction is 41 / umol / min / mg protein (0.3 mg protein), in the 2 × fraction 80 / umol / min / mg protein (0 ₅ 1 mg protein).

The Enzymsusρensionen can be stored in saturated ammonium sulfate "After 6 months when stored near 4 ⁰ C no loss of activity of 3-hydroxybutyrate detectable"

2 20 0

T off. 1

Specific activity of 3-hydroxybutyrate dehydrogenase in the crude extract from Pseudomonas putida after growth on acetoacetate-forming culture substances

C source 3-hydroxybutyrate dehydrogenase

(umol / min / mg protein)

DjL hydroxybutyrate 3,5

D, L-carnitine, 2.0

i), L ~ leucine 3.6

D, L-lysine 0.8

loud

Purification of 3-hydroxybutyrate dehydrogenase (culture substrate D, L-garnetin) from PseuGomonas putida

For preparation, 17 ml of crude extract (7.5 mg / ml of protein) were used. The sediment remaining after treatment with 45% ammonium sulfate solution was treated with 5 ml each of ammonium sulfate and S = sediment treated with decreasing concentration s U = supernatant

Total protein Total activity Specific activity Purification

(Mg)

(/ Pmol / min)

(/ umo 1 / min / mg)

(-subject)

Yield (56)

crude extract 70 % (1.) 228 S 45 % (2.) 202 Ü 45 % ü 35 % Ü 33 % Ü 30 % Ü 27 % ü 25 % , 7 ü 22 % 8th ü 19 % 7, ü

470

490 2.6 3.2 2.6 4.2 58

136

160

102 34

2.06 2.3

19

20 14

1.1

9.2

9.7 6.8

29

34 V 85 22 i

Claims (2)

claims 1. A process for the preparation of 3-hydroxybutyrate dehydrogenase, characterized in that the strain Pseudomonas putida is grown and thereby C sources are used in the degradation of acetoacetate or hydroxybutyrate is formed. 2. The method according to item 1, characterized in that the strain Pseudomonas putida is cultivated 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 separated -iert, in cell-free protein extract on The enzyme can be separated by fractional ammonium sulfate precipitation and, if appropriate, subjected to purification and / or enrichment. 3 · Method according to item 1 and 2, characterized in that as C sources, the degradation of which acetoacetate or hydroxybutyrate arise, D, L-leucine, D, L ~ lysine, D, L-camitine, straight-chain hydrocarbons of chain length Or to C ^ q, fatty acids from C to C ₁ g are used « 4 * Process according to point 1 to 3 », characterized in that an enzyme of specific activity 10 20 / umol / min / mg protein is produced from cell-free crude extract by fractionated ammonium sulphate precipitation alone · Method according to items 1 to 4, characterized in that an enrichment of the enzyme is carried out chromatographically on a hydrophobic carrier material such as "10-carboxydecyl-Sepharose" or the like ", 6e method according to item 5 *, characterized in that the enzyme is adsorbed at high concentrations of sulfate, phosphate or citrate, is eluted with decreasing concentration of these ions. 7 · Method according to items 1 to 3, characterized in that. In the process step of the bacterial culture with a graduated oxygen deficit is used.