DD274050A1 - METHOD FOR ISOLATING AND CLEANING DEHYDROGENASES - Google Patents

Abstract

The invention relates to a process for the isolation and purification of dehydrogenases. The aim of the invention is the economically and technologically advantageous recovery in particular of LDH and MDH from crude, enzyme-containing solutions on a large scale. The object of the invention is preferably to provide LDH and MDH with a chromatographic purification step in a quality which is suitable for use as auxiliary enzyme in the respective optical assay for the determination of alanine aminotransferase (EC 2.6.1.2.) And aspartate aminotransferase (EC 2.6.1.1 .) is suitable in serum. The object is achieved in that the enzymes are absorbed from the crude solution to a macroporous Massenadsorberharz in a mixed-ion loading and differentiated eluted. The eluate composition with respect to said dehydrogenases is controllable by differentiated washing of the loaded absorbent within limits. The preferred field of application of the invention is the isolation of dehydrogenases on a 100-liter scale in the pharmaceutical industry.

Description

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Field of application of the invention

The invention relates to a process for the isolation and purification of dehydrogenases, in particular lactate dehydrogenase (LDH) and malate dehydrogenape (MDH), from crude, enzyme-containing solutions, particularly advantageously applicable on a large scale.

Characteristic of the known state of the art

For the isolation and purification of enzymes, including dehydrogenases from crude solutions such. B. cell or tissue extracts, is carried out in a classical manner so that the enzymes are enriched by salting out and purified after separation and dialysis by ion exchange chromatography and by affinity chromatography (Paoli, C., Bardelli, F., Ital J. Biochem 1985 , 34 HJ, 11-18).

Functionalized cellulose, agarose or sepharose or crosslinked derivatives thereof are preferably used as ion exchangers. It is quite possible to isolate the target enzymes directly from the crude solution economically favorable with good purity (DD-PS 152359).

However, the use of such ion exchangers in large-scale chromatography columns does not guarantee a mechanically sufficiently stable chromatography bed with consistently good flow properties even when using granulated material or exchangers. In addition, this process requires two chromatography steps. Although enzyme isolation from the crude solution by affinity chromatography guarantees high purification effects, affinity gel and eluant are cost-limiting (Pettit, SM, Nealon, DA, Henderson, AR; Clin Chem 27 [1981188-9, Konecny, P .; Smrz, M., Borak, J., Slovakova, S .; J. of Chromatography 398 [1987] 387-390). For the isolation and purification of enzymes in general and of dehydrogenases such as LDH and MDH especially synthetic exchange resins are known (JP-PS 70598, US-PS 4560-661), which are characterized as strongly basic anion exchange resins with highly porous type with mass-related surfaces Ao / m> 5mVg dry resin and mass pore volumes V / m> 0.2cm ³ / g dry resin. They consist of a styrene-divinylbenzene polymer matrix, in which quaternary amino groups are inserted. The use of these resins is accomplished by placing them in contact with the crude enzyme-containing solution and then optionally washing with water or buffer solution to remove impurities. Subsequently, the adsorbed enzymes are eluted with buffer solution from the resin, the active enyme-containing fractions of the eluate collected and further processed by known biochemical methods. The special advantages of this solution are the increase in specific activity, the possibility of removal of foreign activities, the applicability to crude enzyme solutions, the high and stable flow rate also on an industrial scale and the large adsorptive capacity. The disadvantage is that the method requires the use of expensive specialty resins with the said high surface and porosity values. In view of the limited availability of such resins and their high price, multiple use of the resins is advised for economic reasons and thus requires regeneration.

This is labor-intensive and requires the use of considerable amounts of regeneration chemicals, the residues of which also represent a potential burden on the Umwek.

Object of the invention

The aim of the invention is the economically and technologically advantageous recovery of dehydrogenases - especially LDH and MDH - from crude, enzyme-containing solutions on a large scale in a quality that meets the requirements of clinical diagnostics.

Explanation of the essence of the invention The object of the invention is to provide an improved process for the isolation of dehydrogenases from crude enzyme solutions

develop.

This method is intended to eliminate the disadvantages of known methods resulting from the application of more highly porous and expensive ones Ion exchange resins result and consist in that for economically acceptable process management a regeneration

and multiple use of the resin is required, associated with considerable labor and material costs and a potential environmental impact.

By using a cheap adsorbent and the methods of its application, a low transaminase content in the Eluate be achieved safely in only one chromatography step, so that in the subsequent application of known

biochemical methods purified enzymes (e.g., LDH and MDH) that meet the conditions of clinical diagnosis for transaminase determination in the optical test.

It is also desirable to have several dehydrogenases in the crude enzyme solution while maintaining the

principle procedure and easy to implement changes less process parameters the

To be able to control the composition of the eluates with regard to said dehydrogenases. Another object is to provide high stability, i. a slight decrease in the activity of the adsorbed dehydrogenases,

to ensure even with mehrtätiger storage of the loaded adsorbent.

The object is achieved in that the crude enzyme solution with strongly basic, macroporous Adsorbents of the type WOFATIT "EA60 (VEB Chemiekombinat Bitterfeld) with a mass-related surface of 3-5mVg

treated dry resin and a mass-pore volume of 0.05-O, 2cm ³ / g of dry resin, the adsorber resin, depending on the predominantly desired enzyme differentiated washed with differently concentrated buffer solution and then the enzymes eluted with buffer solution of higher ionic strength and optionally fractionated with ammonium sulfate.

Such adsorber resins, despite their low specific surface area and pore volumes, unexpectedly proved to be Enzyme isolation suitable. They consist of a styrene-divinylbenzene copolymer functionalized with quaternary amino groups. Corresponding resins are used according to the invention in a mixed ion assignment Ο ~ / Η ₂ Ρ0 ₄ ~ / ΗΡ04 ² ~ for adsorption. As a result, compared to the Cl ~ form of the resin, the enzyme stability and the selectivity of the purification are favorably influenced. For adsorption, it is advantageous if the crude enzyme solution prepared z. B. by extraction of crushed Cattle heart muscle tissue with water and subsequent coarse clarification, at 2-8 ° C in contract with a column bed of the Harz is brought. The average residence time in the column bed should be 30-60 minutes. Following this, the column bed is filled with either distilled water or distilled water and buffer

low ionic strength at 2-8 ⁰ C washed.

The elution with phosphate buffer 50mmol / l at pH = 5.5, containing sodium chloride in a concentration of 300mmol / l takes place

at 20-25 ⁰ C and provides an MDH fraction in which the ratio of still contained LDH activity to simultaneously pro

Volume unit contained MDH activity is 0.3. If the column bed is washed with phosphate buffer 50 mmol / l at pH = 5.5, the elution with the o.g. NaCI buffer system at 20-25 ⁰ C an LDH fraction in which the ratio of catalytic activities of MDH to LDH still

0.2. Elution may occur at 2-8 ° C at a later time when the loaded column is stored in a cool place. After 5 days, no activity losses compared to an immediate elution are detectable.

It is advantageous in both variants to concentrate the resulting eluate by ultrafiltration. In the resulting eluate or Concentrate LDH and MDH can be separated by fractionated Ammoniumsulfatfällung. The majority of the LDH can be obtained by fractionation with (NH ₄ I ₂ SO ₄ in the concentration range 1.64-2.05 mol / l at ^{0 0} C and

pH = 5.3 are precipitated. Upon crystallization of the enzyme, a purified LDH results with a specific activity of 3.3-3.7 mmol / s g and low ALAT and ASAT foreign activity.

It is similarly possible to remove most of the MDH from the concentrate by fractionation with (NH ₄ I ₂ SO ₄ im Concentration range 2.58-2.87 mol / l at 0 "C and pH = 5.3 precipitate. After crystallization of the enzyme, a purified MDH having a specific activity of 3.3 mmol / s · g and lower Received ASAT foreign activity. The invention will be explained in more detail on subsequent embodiments. embodiments example 1 Primary isolation of LDH A coarse-filtered extract prepared from 3 kg crushed beef heart with 7.51 distilled water is passed through with a

surface-related volume flow of 30 cm / h at 2-8 ⁰ C a column as described below.

For column preparation, add 1.51% WOFATIT "EA60 (Cl" form) for 30 minutes in 4.51 K phosphate buffer 100 mmol / l at pH = 6.0

suspended. From the mixed-ion adsorbent a column (0 5 cm, h - 75 cm) luggage, this with 31 distilled

Washed water and used for adsorption. After completion of adsorption with 101 K phosphate buffer 50mmol / l pH = 5.5 at 4 ⁰ C with a surface area Volume flow of 60cm / h washed. Thereafter, for elution in an analogous manner at 20-25 ⁰ C with K-phosphate buffer 50mmol / l at pH = 5.5, the 300mmol / l NaCl

contains, ancestors.

The fractions with high LDH activity, identical to the first protein peak of the elution chromatogram, become

collected. In this way, an eluate having an LDH activity of 6.7-7.5 mmol / s and a specific activity of 0.7-0.8 mmol / s · g was obtained (foreign activity levels, LDH activity «1: MDH 0, 2, AIAT, ASAT2 x 10 ^-4 ).

For further preparation, the major part of the LDH can be obtained by (NH 2 SC 4fractionation {1.64-2.05 mol / l, O ⁰ C, pH = 5.3).

be precipitated. After dissolution of the enzyme and crystallization, 1.7-2.5 mmol / s LDH with a specific activity of 3.3-3.7 mmol / s · g and the following foreign activity components are obtained (LDH activity = 1)

Alanine aminotransferase (ALAT; EC 2.6.1.2.): 2 x 10 ^"4 Aspartate aminotransferase (ASAT; EC 2.6.1.1.): 2 x 10 ~ ⁴ Malate dehydrogenase (. MDH; EC 1.1.1.37): 5 x 10 ~ ⁴ Pyruvate kinase (PK; EC 2.7.1.40): 1 x 10 ^'4

By further ammonium sulfate fractionation (2.56-2.87 mol / l, O ⁰ C, pH = 5.3) and crystallization, 500 μηιοΙ / s of MDH with a specific activity of 3.3 mmol / s · g and Frsmdaktivitatsonteilen ( MDH = 1) fürASATvon1 x 10 ^"4 and for LDH be isolated from 1 x ^10-3.

Belspie '2

Priority insulation MDH

21 of a prepared as described in Example 1, extract of bovine heart muscle tissue are prepared

Resin column (0 2.6 cm, h = 75 cm) added (400ml WOFATIT "EA60, prepared with 1.21 K phosphate buffer 100mmol / l at

pH - 6.0, V / A = 30 cm / h; 2-8 ⁰ C). Then it is washed with 11 distilled water and 21 K phosphate buffer 10 mmol / l at pH = 5.5 (V / A = 60 cm / h, 4 "C).

The elution is carried out as described in Example 1. In the eluate, 1.7 mmol / s of MDH (foreign activity components MDH = 1: LDH 0.3; ALAT, ASAT: 2 - 10 ~ ⁴ ). For further purification of the MDH can proceed as in Example 1. you

obtains 0.6-0.85 mmol / s MDH with the quality features mentioned under Example 1.

The thus isolated LDH and MDH is without affinity chromatography or other post-purification as Hiifsenzym in the respective optical test for the / laninaminotransferase or aspartate aminotransferase in serum

suitable.

The use of cheap, in sufficiently large quantities ζ η available macroporous Adsorberharzen allowed

on the one hand, the use of the chromatography described ^; in scales of 1001 crude solution and beyond.

A mechanically sufficiently stable chromatography bed with good flow properties is guaranteed. In addition, from an economic point of view, a single use of the adsorbent is advantageously possible. Expensive and

laborious regeneration steps and associated wastewater are avoided.

The invention makes possible the LDH and MDH, which are required to a greater extent for clinical diagnostics, in an extremely economical way

favorable way to win in a chromatographic plant from a crude solution with a purity suitable for use as

Hiifsenzym sufficient for Transaminasonbestimmung. The stability of the LDH and MDH in the adsorbed state over several days opens up the possibility in the technical case of havera

the limited interruption of the procedure.

Claims (5)

1. A process for the isolation and purification of Drehydrogenasen, in particular LDH and MDH, by treating a crude enzyme-containing solution with a macroporous, strongly basic Anionenaustausch ^ with styrene-divinylbenzene polymer matrix and quaternary amino groups, characterized in that the crude enzyme solution with strong batschen , macroporous adsorbents of the type WOFATIT ^R EA60 massobezogenen with a surface area of 3-5 MVG dry resin and a mass-based pore volume of 0,05-0,2cm ³ / g dry resin treated, the adsorber depending mainly differentiated from the desired enzyme with different concentrations Wash buffer solution and then the enzymes eluted with buffer solution of higher ionic strength and optionally fractionated with ammonium sulfate. 2. The method according to claim 1, characterized in that the macroporous adsorbent resin in a mixed ion occupation of the functional groups with Cl ", H ₂ P (XT and HP (V" is used. 3. The method according to claim 1, characterized in that one selectively controls the ratio of LDH and MDH in the active fraction of the eluate by varying the ionic strength of the wash buffer within wide limits. 4. The method according to claim 1 and 3, characterized in that one carries out for the separation of the LDH and MDH washing and elution of the loaded adsorbent with buffer solution in the pH range of 5-7. 5. The method according to claim 1, characterized in that the crude enzyme solution is used after coarse clarification without further mechanical purification.