JPH01235591A - Production of catalase - Google Patents

Abstract

PURPOSE:To mass produce catalase in high yield and in high purity, by treating an aqueous solution containing both catalase and accompanying protein by chromatography of hydrophobic mutual action. CONSTITUTION:A blood cell, etc., is mixed with equivalent distilled water, hemolyzed and hemoglobin is removed to give an aqueous solution (A) containing catalase and protein. Then a functional group such as phenyl Sepharose CL-4B is supported on a carrier such as cellulose to give a chromatography resin (B) of hydrophobic mutual action. Then the resin B is equilibrated with 5-10 times as much a buffer solution (C) having a given pH as the resin, the solution A is adjusted in such a way that total protein in the solution A is <=500mg and total catalase is <=250mg based on 100ml resin and the solution A is passed through the resin B to adsorb catalase on the resin B. Then the resin B is washed with 3-10 times as much as the solution C and developed with a buffer solution at pH 6-9 to recover catalase.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a method for producing catalase, and more particularly to a method for producing catalase from an aqueous solution containing both catalase and associated proteins.

(Prior art and problems to be solved by the invention) Catalase is an enzyme that decomposes hydrogen peroxide, which is involved in the generation of active oxygen such as hydroxyl radicals, into harmless water and oxygen in living organisms.
2 Therefore, catalase is involved in tissue damage caused by active oxygen, such as inflammation, osteoarthritis, rheumatoid arthritis, oxygen retinopathy of prematurity, cataracts, side effects of anticancer drugs such as adriamycin, and the resumption of blood flow to ischemic areas. It is attracting attention as an effective therapeutic agent for disorders.

Conventionally, a method for purifying catalase is a method that combines a catalase-containing fraction with ammonium sulfate fractionation, anion exchange chromatography, cation exchange chromatography, and gel permeation chromatography [Journal of Biological φ Chemistry, 240]. ．． (11), 4299
(1965)] is known.

In the method using gel filtration chromatography, proteins with large molecular weights such as catalase (molecular weight 240.00
It is difficult to industrially process large amounts of 0) in terms of gel strength and separation ability. In addition, in the method using ion exchange chromatography, the elution becomes broad due to the heterogeneity of the charge of catalase itself.
As a result, there are problems such as an increase in the amount of eluate and insufficient separation from impurities.

(Means for Solving the Problems) Therefore, the present inventors have conducted extensive research with the aim of developing a method that can obtain catalase with high purity and high yield, and can also be processed in large quantities. As a result, by treating an aqueous solution containing both catalase and accompanying proteins by hydrophobic interaction chromatography, catalase can be recovered in good yield, and impurities can be effectively removed. Furthermore, we have discovered that this method is suitable for mass processing, and have arrived at the present invention.

That is, the present invention is a method for producing catalase, which is characterized by purifying catalase from an aqueous solution containing both catalase and accompanying proteins by hydrophobic interaction chromatography.

The aqueous solution containing both catalase and associated proteins used in the present invention can be obtained from animal-derived tissues such as red blood cells, liver, and placenta, plant tissues, or microorganisms. .1~0.5%
After hemolyzing by adding (W/V) distilled water containing a nonionic surfactant, both fractions containing catalase can be used by removing hemoglobin by a known method. Preferably, both components are further purified by a known method such as ammonium sulfate fractionation, anion exchange chromatography, cation exchange chromatography, zinc-supported metal chelating affinity chromatography, and more preferably. It is preferable to use a fraction with an increased content of catalase by combining these known methods. Animal-derived tissues such as liver and placenta or plant tissues are disrupted by a known method using a device such as a homogenizer. It is preferable to use the aqueous extract obtained by purifying the obtained water extract by the above method to increase the catalase content. In the use of these catalase-containing fractions, for example, it is preferable to use both fractions as a buffer component.
Contain 50mM phosphate or 10-50mM amine hydrochloride such as triethanolamine hydrochloride, add an electrolyte such as sodium chloride so that the ionic strength is 1-4, preferably 2, and then adjust the pH. Adjust to 6-9, preferably 6-7.

Examples of resins for hydrophobic interaction chromatography used in the present invention include resins in which hydrophobic functional groups such as phenyl, octyl, and butyl groups are supported on carriers such as cellulose, agarose, and synthetic polymers. It will be done. As a specific example, Octyl Sepharose CL-4B
(Pharmacia), Phenyl Sepharose CL-4
B (Pharmacia), Butyl Sepharose 4B (Pharmacia), and Butyl Toyopearl 650 (Tosoh).

In the method of the present invention, the catalase-containing solution obtained by the above method and adjusted in ionic strength and pH is usually mixed with a buffer solution (with the same composition and pH) using the kl# component and electrolyte used for preparing the catalase-containing solution. This is done by passing buffer A) through a resin equilibrated with 5 to 10 times the amount of resin.

The amount of catalase and accompanying protein to be passed through the resin includes both catalase and accompanying protein per 100 mm of resin. □ It is desirable that the total protein in the aqueous solution is 500 mg or less, of which catalase is 250 mg or less. The catalase that is
Wash with 1O times, preferably 5 times the amount of buffer A, and then add 5 to 1O times the amount of the buffer containing the electrolyte used to prepare the catalase-containing solution, the ionic strength of which is 0.5 to 1. The catalase preparation obtained by the method of the present invention can be obtained by developing with a buffer solution containing only buffer components, pH 6 to 9, preferably pH 7, after removing impurities. The following analysis confirmed that the purity was much higher than that obtained by previously known methods. That is, a single band was obtained in polyacrylamide electrophoresis, and a single band with a molecular weight of 59,000 was obtained in polyacrylamide electrophoresis in the presence of sodium dodecyl sulfate. In addition, gel filtration analysis using high-performance liquid chromatography can be performed using commercially available products (
A single peak was observed at the same position as the main peak of Sigma), and the molecular weight could be estimated to be 190,000. As a result of analysis using ion-coupled plasma spectroscopy, it was confirmed that the iron content was 4g atoms in one molecule of catalase.

(Examples) The present invention will be further explained by Examples and Reference Examples, but the present invention is not limited by these Examples.

Reference Example 1 Human CPD enriched red blood cells stored at 4°C for 3 weeks were centrifuged at 1500xc for 15 minutes at 4°C. After removing the supernatant, the precipitated red blood cells were washed three times with physiological saline, and one volume of cold water containing 0.2% (W/V) saponin was added to one volume of this red blood cell sediment, and the mixture was incubated at 4°C for 1 hour. The mixture was stirred for hours to cause hemolysis. Put the lysed hemolysate into a cellulose tube for dialysis, and place it in a 1 m tube at 4°C.
Dialysis desalting was performed against M phosphate buffer (pH 7, 2). Triton X-100 was added to the hemolysate after dialysis to a concentration of 0.1% (W/V), and the pH was adjusted to 7.2. Using DE-52 (Whatman) equilibrated with 1mM phosphate buffer (pH 7,2), the hemolysate subjected to the above treatment was treated batchwise, and the protein-adsorbed DE-52 was equilibrated. Washing was performed with the same buffer used to wash away non-adsorbed substances. DE with protein adsorbed after washing
-52 was packed into a column and developed with 50 mM phosphate buffer (pH 6, 8) in an amount three times the resin volume of DE-52 to obtain a fraction containing catalase.

Reference Example 2 70g of ammonium sulfate (
(for biochemistry) was added, dissolved, and left overnight at 4°C. The resulting precipitate was collected by centrifugation at 4°C, 7700XG, for 30 minutes. A buffer (p)17,0) consisting of 10mM) reethanolamine hydrochloride was added to the precipitate to dissolve the precipitate, and the precipitate was dialyzed against the same buffer for desalting. DEAE-Sepharose Fastflora (Pharmacia) was packed in a column, equilibrated with the same buffer as above, and both columns containing catalase after desalting were passed through and washed with the buffer used for equilibration. 5 times the resin capacity
It was developed with the above buffer containing 0 mM sodium chloride to obtain a fraction containing catalase.

Reference Example 3 0.0% was added to the catalase-containing fraction obtained in Reference Example 2.
Sodium chloride was added to make 5M to 1M degrees. Chelating Sepharose 6B (Pharmacia) Resin 1
00-, adsorbed 2.85 sol of zinc sulfate, washed with water, and prepared 10 mM containing 0.5 M sodium chloride).
Buffer solution consisting of reethanolamine hydrochloride (pH 7, 0
). Both portions containing the above catalase were passed through this and washed with the same buffer used for equilibration. Development of the adsorbed fraction was carried out by adding acetic acid to the same buffer used for equilibration to a concentration of 5 mM. High performance gel permeation chromatography of catalase in this fraction (
As a result of analysis using an FPLC system as an apparatus and a Superrose TM 12 column (Pharmacia), the content of catalase in both components was approximately 70%.

Example 1 Phenyl Sepharose CL-4B (manufactured by Pharmacia) resin 200t7 was packed in a column, and passed through 2 L of 10 mM) reethanolamine hydrochloride M solution (pH 7) containing 2 M sodium chloride to equilibrate the zero order. Sodium chloride was added to 1.5 grams of the crude catalase obtained in Reference Example 3 to give a concentration of 2M and dissolved. This solution was passed through the above-mentioned Phenyl Sepharose CL-4B to adsorb catalase. 141 equilibration buffer, then 0.05M
It was washed with 1 part of phosphate buffer + LM sodium chloride (pH 7,0) and then 1 part of 0.05 M phosphate buffer + 0.5 M sodium chloride (pH 7, 0). Development of catalase was performed by passing one portion of 0.05M phosphate buffer (pH 7.0) through. High-speed gel permeation chromatography of the obtained catalase fraction (FPLC system as device, Superrose TM 12 as column)
As a result of an analysis by a company (Pharmacia, Inc.), the content of catalase in both components was 98% or more, and polyacrylamide electrophoresis also showed a single band. Reference example 3
The recovery rate of catalase activity from the catalase fraction obtained was 52%. By purifying according to the Reference Examples and Examples, 550 mg of catalase could be obtained from 2.5 red blood cells. The activity of catalase was measured according to the method of Sinha [see Analytical Biochemistry, Sei 7, 389 (1972)].

(Effects of the Invention) As is clear from the above detailed description, according to the method of the present invention, impurities can be efficiently removed from an aqueous solution containing catalase and associated proteins, and catalase can be recovered with a higher yield. Moreover, since large-scale processing is done by rotation, high-purity catalase can be supplied in large quantities.

Claims (1)

[Claims] A method for producing catalase, which comprises purifying catalase from an aqueous solution containing both catalase and accompanying proteins by hydrophobic interaction chromatography.