DD264230A1 - PROCESS FOR OBTAINING BETA GALATOSIDASE FROM ESCHERICHIA COLI - Google Patents

Abstract

The invention relates to a method for obtaining highly purified b-galactosidase from Escherichia coli, which is suitable for use as a marker enzyme in enzyme immunoassay and other analytical and preparative applications. Starting from the crude extract obtained after cell disruption of a cultured Escherichia coli bacterial mass, a fractionated PEG precipitation is first carried out. The fraction containing the enzyme is distributed in an aqueous two-phase system consisting of PEG and a hydrophilic second component. In contrast to the other proteins contained in the mixture, the b-galactosidase is almost quantitatively contained in the PEG phase and is therefrom finely purified and isolated in a subsequent ion exchange step. The process provides an electrophoretically uniform enzyme at 30-40 fold enrichment in an overall yield of 60-70%.

Description

field of use

The invention relates to a process for obtaining β-galactosidase from Escherichia cdi in a highly pure form. This enzyme is preferably used as a marker enzyme in cnzymimmiinologische measurement methods in medical diagnostics, in microbiology and in food analysis. It is also used for enzymatic cleavage reactions in the food industry and in biochemical research for various purposes.

Characteristic of the known state of the art

β-galactosidase is an enzyme found in many microorganisms and numerous animal tissues. Their content depends on the type of tissue or on the type and method of cultivation of the microorganisms. The recovery of the enzyme is preferably carried out in the described methods of microorganisms. The following procedures were followed: G.R. Graven, E.J. Steers, G.B. Anfinsen, J. Biol. Chem. 240, 2468-2477 (1965) purified the enzyme from Escherichia coli by ammonium sulfate precipitation, ion exchange chromatography and gel filtration.

E. Jr. Steers, P. Cuafecasas, H. B. Pollard, J. Biol. Chem. 246, 1996-200 (1971) used specific affinity sorbents as an essential step for the isolation of the enzyme from the same material.

The disadvantages of the beschrieoenen method consist in eintm high time (column chromatography), the principal difficulties of gel chromatography under preparation conditions on a larger scale and the low yields, which are usually below 50%.

The advantage of selectivity of affinity adsorption is limited by the relatively high time and expense involved in making the affinity sorbent and its limited reusability. The described methods are consistently designed as laboratory methods and can not be scaled up without a methodical change. An A.Veide.A.-L.Smeds, S.-O.Enforsin Biotechnol. Bioeng. 25, 1789-1800 (1983), an aqueous two-phase system for enrichment of the enzyme from extracts of Escherichia coli did not serve the purpose of high purification of the enzyme.

The processes described in the patent literature for isolating and purifying β-galactosidase from various microorganisms all use well-known conventional methods for protein purification, such as precipitation, ultrafiltration, gel and ion exchange chromatography with different combinations of the traversing steps (eg.

DE-PS 2904225, DD-PS 2558012, GB-PS 2038837).

Object of the invention

The aim of the invention is to obtain high purity β-galactosidase according to a simple process which is to be carried out under both laboratory and pilot plant conditions and which provides the enzyme in high yield with low material and time expenditure.

Explanation of the essence of the invention

It is an object of the present invention to obtain high-grade β-galactosidase in a quality which is suitable for use as a marker enzyme in the enzyme immunoassay and moreover as an analytical reagent in microbiology and food chemistry as well as in enzymatic cleavage reactions in the food industry and in biochemical research , The object is achieved according to the invention in that the source of the extraction is a Zßllmasse of fc'scherichia coli is used, from which the enzyme is obtained as follows:

The cells are in a conventional manner mechanically, osmotic or c'urch ultrasound with the addition of a suitable buffer, such as. B. Tris / HCl buffer pH 7.6, digested and the soluble cell content v / ird obtained as a supernatant by centrifugation. A subsequent fractional precipitation with Polyethyler.glykol odor oxide wax A is designed so that the enzyme is in the first concentration range of precipitation in the supernatant and in the second concentration range of precipitation in the sediment. It is separated by another centrifugation and dissolved in a buffer such as phosphate buffer, pH 7.0. Thereafter, the dissolved protein mixture in an aqueous two-phase system, which is formed from a solution of polyethylene glycol and salt or of polyethylene glycol and another suitable ¹ !, Water-soluble polymer of certain concentration, distributed, the ß-galactosidase, in contrast to the other, in a mixture contained proteins is contained almost quantitatively in the upper phase.

The phases are separated and the β-galactosidase is recovered from the polyethylene glycol-rich phase by adsorption to a weak Απί inaustauschers (eg., DEAE-cellulose) and subsequent desorption by increasing the ionic strength. For storage, the enzyme is concentrated by known methods. The invention will be explained with reference to the following implementation examples:

Au & führungsbeisplele

Example I

50g bacterial mass Escherichia coli K12SA288 (M. Kaasch, J. Kaasch, Diploma thesis Martin-Luther-University Halle, 1986) with 100g Alcoa with the addition of 50ml Tris / HCl buffer pH7.6 (Tris-HCl, 200mmol / l, NaCl , 200 mmol / l, magnesium acetate, IOmmol / 1, mercaptoethanol, 10 mmol / l, glycerol, 50g / l) 10 min intensivveiriebbn at 4 ° C and centrifuged after addition of 150ml woiteren Tris / HCl buffer 30min (K23,4000xg, 4 ⁰ C). The supernatant (200 ml) is added with stirring to 12 g of solid PEG 6000, stirred for 1 h at room temperature, the precipitate was centrifuged off in the cold at 4000 × g and discarded. From the supernatant is reprecipitated by adding another 12g PEG, as described above, and

centrifuged. The supernatant is discarded and the precipitate taken up in 50 ml potassium phosphate buffer pH 7.0 (potassium phosphate, 10 mmol / l, MgCl ₂ , 1 mmol / l, mercaptoethanol, 50 mmol / l).

The dissolved sediment is in a two-phase system PEG / phosphate (5% PEG 6000.12% KaüUiTiphosphat pH7.0, MgCl ₂ , 1 mmol / l, mercaptoethanol, 50mmol / l) by brief gentle shaking for distribution (2.5 mg Protein / g system) and the enzyme-containing upper phase separated after centrifugation.

The upper phase is diluted 1: 5 with Tris / HCl buffer pH 7.5 (Tris-HCl, 10 mmol / l, MgCl ₂ , 1 mmol / l, mercaptoethanol, 10 mmol / l and with DEAE-cellulose (10 mg protein / g cellulose [Wet weight]), which is equilibrated with the above Tris / HCl buffer.

Subsequently, unbound protein with Tris / HCl buffer (Tris-HCl, 10 mmol / l, MgCl ₂ , 1 mmol / l, mercaptoethanol, 10 mmol / l) pH 7.5 was batch-washed over a G3 frit and then triturated with KCI-containing Tris / HCl. Buffer pH7.5 (Tris-HCl, 10 mmol / l, KCl, 750 mmol / l, MgCl ₂ , 1 mmol / l, mercaptoethanol, 10 mmol / l) the pure β-galactosidase eluted. For stable storage at 4 ⁰ C, the enzyme-containing eluate is concentrated by ultrafiltration and dialyzed against saturated ammonium sulfate solution.

Based on 50 g of bacterial mass about 5100OU electrophoretically uniform ß-galactosidase with a specific activity of 180U / mg (determined at 20 ° C and with p-nitrophenyl galactopyranoside as a substrate conversion factor for comparison with literature data at 37 ⁰ C and with o-nitrophenyl-galactopyranoside as substrate is 4.3).

The overall yield of the process is thus between 60 and 70%. Based on an average specific activity of the crude extract of U / mg, the purification factor is 36.

Example Il

Bacterial digestion and fractionated PEG-FSIIung values as described in Example I performed.

For phase distribution, a PEG / ammonium sulfate system (5% PEG 6000.14% (NH ₄ I ₂ SO ₄ , MgCl ₂ , 1 mmol / l, mercaptoethanol, 50 mmol / l) is used (1.5 mg protein / g system). and the upper phase containing the enzyme wje further processed under Example I.

Example IM

Bacterial digestion and fractionated PEG precipitation are carried out as described in Example I.

For the phase distribution, a system PEG / AQUAPHASE PPT (6.5% PEG 6000.14% AQUAPHASE PPT [partially hydrolyzed hydroxypropyl starch, Perstorp AB, Sweden], KCl, 75 mmol / l, MgCl ₂ , 1 mmol / l, mornaptoethanol, 50 mmol / l ) (1 mg protein / g system) and the upper phase containing the enzyme further processed as in Example I.

Example IV

50 g bacterial mass Escherichia coli are digested and purified as in Example I.

Claims (5)

1. A process for the recovery of highly purified β-galactosidase from cultured Eschericnia coli strains after digestion of the cells and separation of cell debris, characterized in that a fractional precipitation with polyethylene glycol performed, the fraction containing the enzyme again dissolved and in an aqueous two-phase system, which is formed from a solution of polyethylene glycol and salt or polyethylene glycol and another suitable water-soluble polymer, distributed and the pure B-galactosidase from the polyethylene glycol-rich phase by adsorption to a weak anion exchanger and subsequent desorption is obtained by means of increased ionic strength. 2. The method according to item 1, characterized in that the precipitation of the enzyme-containing fraction from the material of Zbllaufschlusses in a concentration range of 6% to 12% PEG. 3. The method according to item 1, characterized in that the aqueous two-phase system consists of PEG and a polyvalent soluble inorganic salt such as phosphate or sulfate, or of PEG and a polar organic polymers such as Kydroxypropylstärke with the addition of KC or comparable salts. 4. The method according to item 1 and 3, characterized in that the quantitative composition of the two-phase system pairs of values corresponds to the binodial curvature of the respective system. 5. The method according to item!, Characterized in that the sorption of the enzyme takes place on a weak ion exchanger at pH 7-8 and low ionic strength and desorption in the presence of 0.6-1.0 mmol / l of a monovalent inorganic anion.