DE2224131C - Process for obtaining the enzyme cholesterol oxidase, which oxidizes cholesterol with O deep 2 to cholestenone and H deep 2 O deep 2 - Google Patents

Description

'S

active agent, but ultrasound precipitation with salts such as ammonium sulfate or organic

Extraction. Solvents such as methanol and the like. It can also be useful

Particularly good results are obtained if the exchange chromatography is moderate the microorganism is precipitated out of the solution before the digestion and before the enzyme in order to perform the solution extraction in the presence of the surface-active agent to lower the amount of agent. For the redissolution by means of a "washing with a butanol-water gel", a precipitated cholesterol oxidase is preferably mixed is subjected. Surprisingly, a buffer that uses a small amount wisely performs this washing with a butanol-water emulsion contains a surfactant. It didn't work out to the expected agglomeration or denaturation observed that occasional dissolution ration of the enzyme at interfaces, although a sol- ίο the precipitated enzyme causes great difficulties before action in the usual not for assembly but in the presence of the surface-active agent enzymes with a tendency to interface enrichment do not occur.

with the method according to the invention can be

Activity. In the course of the errindungsge- an approximately 3000-fold enrichment of the enzyme

However, a moderate method can be carried out by this Wa- 15. Another cleaning according to the usual

A further considerable enrichment of biochemical fine cleaning methods is possible beforehand,

aim. The butanol-water washing can be done in so far as the enzyme is precipitated or on surfaces

carried out in one or more steps. is adsorbed or bound, should such

The effectiveness of the method methods according to the invention in the presence of a surface-active one

steps can be carried out numerically by comparison with ao means.

a conventional ultrasound digestion demonstrates the method according to the invention according to the

Ren. Enzyme preparations obtained by ultrasound digestion of an anion exchange elution can thus be obtained

certain amount of the microorganism with 0.05 M can be used directly for the quantitative determination of cholesterol

Phosphate buffer pH 7.0 30 units of cholesterol oxygenation according to the reaction equation given above

obtained with a specific activity of 0.01. 35 insert.

If, on the other hand, the digestion of the organism is carried out with the Dk activity of the enzyme and thus the equally effective buffer to which about 0.5% nonionic surface-active agent has been added in the process according to the invention, the result obtained according to the above equation is obtained a total activity of 60 units of H ₈ O ₁ , according to known methods of H ₂ O ₂ determination and a specific activity of 1.0. This corresponds to 30 was measured.

a doubling of the yield and a 100-fold The following examples illustrate the invention

Enrichment. Will the latter procedure continue.

repeated, but before the digestion a washing with Example 1
Carried out butanol-water mixture, so one obtains

60 units of enzyme with a specific activity of 35 40 ml of a suspension of Nocardia erythropolis

3.0, so a total of 300-fold enrichment. ATCC 4277 (about 10 g dry weight) are with

The extract obtained according to the invention, which adds 40 ml of n-butanol and a few minutes for room cholesterol oxidase contains dissolved, is then stirred at temperature. The mixture is centrifuged Purified via an anion exchanger. It's got itself and both the butanol and the supernatural found that it is not possible to pour off 40 aqueous phase. The precipitation will he the 40 ml of water are added again to the usual methods of such cleaning and after preparation turn to. It turned out that the end of a homogeneous suspension of the cells again with enzyme no longer eluted from the exchanger, 40 ml of n-butanol were added, a few minutes at room temperature. Stirred and centrifuged even using a high ion concentration temperature. The lower rations the enzyme could no longer be recovered- 43 will hit in 40 m! 0.01 molar phosphate buffer, will be. It was also possible with solutions pH = 7.0, suspended, the 0.3% of a non-ionic Surfactants no longer have to recover the enzyme to surfactant (alkylaryl polyethylene glycol. col) are added, and at room temperature for 30 minutes

Surprisingly, however, it was possible to stir with a grain. Then it is centrifuged and the

The combination of buffer and non-ionic surface precipitate is discarded. The extract contains about

active agent of relatively low concentration containing 120 units of cholesterol oxidase with a specific

See enzyme almost completely from exchanger again activity of 3 units / mg protein,

to be washed down. This extract is made up to with solid ammonium sulfate

Of the anions with a concentration of 1.3 M in ammonium sulphate, the weakly basic types are particularly suitable as exchangers. 5.5 0 ⁰ C offset. Then the suspension is zen-Preference is given to exchangers which are subjected to a diethylamine in which the precipitate is due to the ethanol group. However, other exchangers with an obviously lipophilic component on the comparable basicity are also suitable. surface of the solution collects. The caked

The elution from the exchanger is preferably filtered with precipitate, with 0.01 molar phosphorus

a 0.01 to 0.2 molar buffer solution of the above phate buffer, pH = 7, dissolved and then against

given pH range. The concentration dialyzed the same buffer at 0 ⁰ C exhaustively.

Tration of the surface-active agent is then available- The dialyzed solution is reduced to a 0.01 mol

preferably in the range from 0.05 to 2%. Especially for phosphate buffer, pH = 7, equilibrated column of a

It is preferred to use 0.03 to 0.1 molar commercially available anion exchanger with diethyl

Phosphate buffer with a content of 0.2 to 0.7% (»5 aminoethanol groups drawn on a dextran basis.

nonionic surfactant. at the enzyme is adsorbed. After washing the

From the eluate obtained in this way, the enzyme can be in a column with the same buffer is successively with Lö

isolate in the usual way, for example by dissolving 0.5%, 2% and 5% of the above surface area.

active agent washed in water. Large amounts of impurities are eluted in the process. Subsequently is successively with 0.01 M phosphate buffer, pH = 7.0.05 M phosphate buffer.% pH = 7.0.2 M phosphate buffer, pH = 7 and 0.5 M phosphate buffer, pH = 7, washed. Then again with 0.05 molar Phosphate buffer, pH = 7, washed and then the desired enzyme with 0.05 molar Phosphate buffer to which 0.5% nonionic surfactant has been added eluted. Find in the eluate about 80% of the enzymatic activity of cholesterol oxidase adsorbed on the column with a specific activity of 25 to 30 U / mg protein.

Example 2

The procedure of Example 1 was repeated, but the butylene-water wash was omitted. This gave an extract for ammonium sulfate precipitation which contained about 120 units of cholesterol oxidase with a specific activity of 1

unit / mg protein. The further processing in the manner described resulted in a product with a specific activity of 8 to 10 U / mg protein.

Example 3 (comparative example)

The procedure of Example 1 was repeated, but the exchanger was eluted with a aqueous solution containing 5% of the same surfactant dissolved. It couldn't Enzyme activity to be eluted again. The enzyme was irreversibly absorbed by the exchanger.

Example 4 (submitted later)

The procedure of Example 1 was repeated using Nocardia erythropolis NCIB 9158, Nocardia erythropolis ATCC17 895 and Nocardia formica ATCC 14 811 as microorganisms. The results obtained are summarized in the table below.

Microorganism Procedural stage E / g DM ¹ ) ΣΕ sA ² ) yield
% Nocardia erythropic.
NCIB 9158 extract
Exchanger EIuat 7.6 190
110 2.1
20th 100
60 Nocardia erythropic.
ATCC 17 895 extract
Exchanger eluate 1.5 42
24 0.41
2.2 100
58 Nocardia formica
ATCC 14 811 extract
Exchanger eluate 5.0 120
81 1.6
13th 100
65

') U / g DM = units per g dry matter.
^! ) sA = specific activity.

1 E is the activity at which 1 micromole of H ₈ O _{2 is formed} per minute under the following conditions:

2.31 ml potassium phosphate buffer (0.05 Μ, ρΗ 7.0),

containing 1 mg / ml 2,2'-azinodi- [ethyl

benzthiazoline sulfonic acid (6)] -

Ammonium salt,

0.05 ml cholesterol solution, 3.7 mg / ml in tertiary butanol,
0.6 ml tertiary butanol,
0.02 ml peroxidase (9 U),
0.02 ml bacterial extract,
T = 37 ° C.

Claims (1)

'L ren for obtaining the enzyme cholesterol oxidase, claims: which cholesterol according to the equation Chclesterin + O ₂ -> Cholestenone + H ₁ Oj
1. A method for obtaining the enzyme cholesterol oxidase, which oxidizes cholesterol according to the equation 5, which is characterized in that a chung cholesterol-converting microorganism by decomposition rh _n \ ^ u. _r ; _n j- η _ ^ n, _n i.ct. _nnn _l η γ »disturbance of the cell wall with a non-ionic Cholesterol + O ₂ ^ cholestenone + H, 0 ₂ surfactant-containing buffer solution oxidized, characterized, digested and extracted, the extract after it that a cholesterol-converting microorganism io centrifugation and the rejection of the by destroying the cell wall with a non-existent precipitate via an anion exchanger given containing ionic surface-active agent, the enzyme with a non-ionic surface Buffer solution digested and extracted, the surface-active agent-containing buffer solution is eluted After centrifuging the extract and isolating it from and from the eluate in a manner known per se the precipitate obtained in this way is thrown over is. given an anion exchanger, the enzyme with in the case of the cholesterol produced according to the invention the nonionic surfactant oxidase is a new enzyme which containing buffer solution and eluted from the one hand in the cell membrane of the microorganism Eluate is isolated in a manner known per se. is bound and on the other hand an extremely hydrophobic 2 The method according to claim 1, characterized in that ge ao substance, namely the cholesterol, has to convert. indicates that the microorganism in front of the enzymes of this kind show in the classic gain destruction the cell wall with a butanol watering process creates a particularly unpleasant intrinsic mixture is washed. shaft. They are extremely difficult to get out of the 3. The method according to Arspruch 1 or 2, thereby releasing the membrane and inclining, even if the release indicates that the enzyme from the extract has succeeded in settling, always pulling itself together Addition of ammonium sulphate precipitates and clumps and on vessel walls, phase boundary layers after being taken up again in buffer solution. or adsorbents and exchange materials 4. Procedure to subscribe to one of the foregoing supplies. It is therefore not possible to meet the usual claims, characterized in that for EIu- chen methods of enzyme recovery and isolation tion of the exchanger a 0.01 to 0.2 molar to apply the 30. There is a particular difficulty here Buffer solution containing surfactants in that the activity and specificity of the enzyme do not is used. may be changed. As a microorganism is suitable for implementation of the method according to the invention as mentioned in 35 Principle of every microorganism that converts cholesterol. Particularly good results have been obtained with bacteria of the genus Proactinomyces obtained, and preferred The invention relates to a method for obtaining the bacteria ATCC 17 895, ATCC 4277 and of the enzyme cholesterol oxidase, which cholesterol Nocardia formica ATCC 14 811. The erfindungsgegemäß However, the method according to equation 40 is also based on in the same way Cholesterol + O ₂ - Cholestenone - * H ₂ O ₂ ^3ηαε ? Microorganisms, bacteria such as fungi, which are able to convert cholesterol, can be used, oxidized. The breakdown of microorganisms by decomposition It is known that a number of cell wall microorganisms are disrupted by detergents per se men are able to convert cholesterol. So describes 45 known, and basically all of the this for example T u r f i 11 in J. Bacteriol. 47,487 to 493 described and put into practice (1944) 188 strains of bacteria using cholesterol as the only method. Could exploit carbon source. About the Me- The microorganism is in the invention mechanism of cholesterol utilization and the processes involved in it with a non-ionic surface However, nothing was known about enzymes. The buffer solution containing the active agent was digested however, there is interest in finding a chole and extracted. Preferred nonionic surface sterols converting enzyme, since it is expected that an active agent are the alkylarylethylene glycols and poder-like Enzyme for a specific cholesterol ethylene oxide-polypropylene oxide adduct, in particular method is of interest. Such a special its ester. The concentrate-specific to be used Cholesterol determination method lacks up to 55 tion of the surface-active agent in the solution, but in medical diagnostics it would be conclusive and the buffer solution used for extraction great importance. depends to some extent on the particular use From the German Offenlegungsschrift 2 021 465 th surface-active agents can be obtained by also already a sterol dehydrase known, in which it can be determined. Generally suitable is a dehydrogenase, which unspecific concentrations between about 0.01 and 3%, preferably Fish hydroxyls in the 3- and 17-position of sterols are between 0.1 and 1%. The buffer solution can implements. For a specific determination of cholesterol, pH values between approximately 5 and 9 are preferred If this unspecificity is a serious handicap, a pH between 6 and 8. through the described combined with the digestion The object of the invention is therefore to provide 65 nated extraction in the presence of the surface-active a method for obtaining a cholesteroloxy agent can be the specific activity of the sought that which does not have these disadvantages. Dissolved enzyme increase 100-fold compared to one According to the invention, the object is achieved by a method with the same buffer in the absence of the surface