JPH07274955A - Isolation and stabilization of membrane bound oxidoreuctase - Google Patents

Abstract

PURPOSE:To facilitate the solubilization and isolation of membrane-bound oxidoreductase which is useful in production of a variety of compounds, particularly the oxidoreductaste for aromatic compounds by allowing a PEG ether-type nonionic surfactant to act on the cell membrane fraction of microorganism cell bodies. CONSTITUTION:A polyethylene glycol ether-type nonionic surfactant is allowed to act on the cell membrane fraction of the cell bodies of a gram-positive microorganism, for example, in Rhodococcus to solubilize the membrane-bound oxidoreductase from the cell membrane for isolation. The membrane-bound oxidoreductase isolated here can be kept stable in the presence of a surfactant similar thereto.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for separating a membrane-bound oxidoreductase from a cell membrane and a method for stabilizing the same.

[0002]

BACKGROUND OF THE INVENTION Membrane-bound enzyme is a generic term for enzymes that are bound to biological membranes with various strengths. The main ones of this membrane-bound enzyme are various redox enzymes, which are located in the first step of redoxing various harmful chemical substances (eg aromatic compounds) into harmless ones. It is greatly involved in the initial decomposition of petroleum. Therefore, the enzyme is widely used not only as a detergent enzyme, but also in the field of “bioremediation (environmental restoration by biotechnology)”, which has become a big problem recently, and in the field of manufacturing various compounds. Be expected.
As a method of solubilizing a membrane-bound enzyme, a physical method such as ultrasonic treatment or a method of treating with a chemical reagent such as KCl or EDTA or a surfactant such as Triton X-100 has been conventionally known. . However, depending on these methods, there is a problem that solubilization is not sufficient, or even if solubilization is possible, enzyme denaturation occurs and sufficient enzyme activity cannot be obtained. Also, among the membrane-bound enzymes,
In particular, aromatic compound oxidoreductase is extremely useful in that it directly introduces an oxygen atom into the aromatic ring of an aromatic compound to cause hydroxylation or aromatic ring cleavage, and that it can be applied to the production of useful compounds. However, it is said that the solubilization thereof is particularly difficult because the aromatic compound oxidoreductase itself is considerably unstable, and the present situation is that no specific attempt has been made yet. On the other hand, once solubilized, the membrane-bound enzyme has a decreased activity during storage for a long period of time, which is considered to be a major problem in industrial use. In contrast, SH such as mercaptoethanol
Various methods such as addition of reagents have been attempted, but none of them are sufficient for maintaining the enzyme activity after solubilization, and there is no effective method for stably maintaining the enzyme activity.

[0003]

The object of the present invention is to provide a method for easily solubilizing and separating a membrane-bound oxidoreductase, particularly an aromatic compound oxidoreductase, from the cell membrane of a microbial cell. is there. Another object of the present invention is to provide a method for stably retaining the activity of the enzyme that has been solubilized and separated.

[0004]

[Means for Solving the Problems] As a result of intensive studies to solve the above problems, the present inventors have found that a specific surfactant acts on the cell membrane fraction of microbial cells to produce a membrane. Ability to solubilize and separate bound oxidoreductase,
Further, they have found that the activity can be stably maintained by allowing a similar surfactant to coexist with the separated enzyme, and completed the present invention.

That is, the present invention provides a method for solubilizing and separating a membrane-bound oxidoreductase from a cell membrane by allowing a polyethylene glycol ether type nonionic surfactant to act on the cell membrane fraction of microbial cells, and In this method, a membrane-bound oxidoreductase that is solubilized from the cell membrane of a microbial cell and separated and a polyethylene glycol ether type nonionic surfactant are allowed to coexist to stabilize the enzyme.

The membrane-bound oxidoreductase used in the present invention may be of any type, but is preferably applied to an aromatic compound oxidoreductase which is said to be difficult to solubilize. As aromatic compound oxidoreductase,
Salicylic acid 5-oxidase, meta-hydroxybenzoic acid 6-oxidase, para-hydroxybenzoic acid 3-oxidase, phthalic acid 3,4-oxidase, benzene-1,2-oxidase, toluene-1,2-oxidase, phthalate-4,5-oxidase Etc.

In the present invention, the above-mentioned membrane-bound (aromatic compound) oxidoreductase is introduced into the microbial cells by growing the microbial cells in a completely synthetic medium in which each aromatic compound is the sole carbon source. After that, the microbial cells are sonicated to be divided into a cell-free enzyme solution and a membrane fraction, and the membrane fraction is suspended in phosphate buffer, Good buffer, etc., and a surfactant is added. By reacting, the membrane-bound oxidoreductase is solubilized outside the cell membrane.

The surfactant used here is a polyethylene glycol ether type nonionic surfactant, for example,
Examples thereof include polyethylene oxide condensates of higher alcohols and polyethylene oxide condensates of fatty acid esters of sorbitan.

Examples of polyethylene oxide condensates of higher alcohols include polyethylene glycol (8 to 30) condensates of lauryl alcohol, cetyl alcohol, oleyl alcohol and the like. Specifically, polyethylene (20) cetyl ether (trade name of Brij 58) is preferably used.

As the polyethylene oxide condensate of sorbitan fatty acid ester, sorbitan fatty acid ester, for example, sorbitan lauric acid monoester,
Sorbitan palmitate monoester, sorbitan
Stearic acid monoester, sorbitan oleic acid
Polyethylene glycol such as monoester (10-2
0) condensates may be mentioned, and specifically, polyethylene (2
0) Sorbitan oleic acid monoester (trade name: Tw
een 80) is preferably used.

Further, the surfactant is added at a concentration of 0.5 to 2%, preferably around 1%, and the addition amount is 50 with respect to the membrane fraction.
0 to 2000% (w / w), preferably 1000% (w
/ W) is exemplified. The surfactant is 0 to 10 ° C,
Preferably, the membrane fraction is allowed to act with stirring at about 5 ° C. for 1 to 10 hours, preferably about 2 hours. Then, by centrifugation, the supernatant and the membrane fraction are separated, and the enzyme can be separated and obtained in the supernatant.

The applied microorganism may be of any genus or species,
Rhodococcus, Micrococcus
ccus), Staphylococcus, Staphylococcus, Streptococcus, Leuco stock (Leuco)
nstoc), Ruminococcus, Bacillus (Bac
illus), Clostridium, Lactobacillus, and other Gram-positive bacteria, with Rhodococcus being particularly preferred. These Gram-positive bacteria are pseudomonas (P
seudomonas), Salmonella, Escherichia
(Escherichia), Acinetobacter, and other Gram-negative bacteria have a different cell surface structure, and Gram-positive bacteria have a stronger cytoplasmic membrane, resulting in a clearer effect.

The membrane-bound oxidoreductase solubilized and separated by the above method is subjected to hydrophobic chromatography, gel filtration chromatography, ion exchange chromatography as in the case of the crude enzyme solution obtained by ultrasonication. It can be purified by means such as chromatography.

The membrane-bound oxidoreductase thus obtained is the corresponding enzyme present in the membrane fraction or the corresponding enzyme purified from a cell-free enzyme solution and its physical properties (optimal p
H, optimum temperature, enzyme activity, amino acid composition, etc.) and does not undergo any modification by the solubilization treatment of the present invention.

According to the present invention, the membrane-bound oxidoreductase solubilized from the cell membrane of a microbial cell by the above-mentioned method is allowed to coexist with a polyethylene glycol ether type nonionic surfactant, whereby the enzyme is obtained. Can be stably maintained for a long period of time.

The surfactant used here is a polyethylene glycol ether type nonionic surfactant, for example,
Examples thereof include polyethylene oxide condensates of fatty acid esters of sorbitan.

Examples of the polyethylene oxide condensate of sorbitan fatty acid ester include those similar to those used in the above solubilization. Specifically, the same polyethylene (20) sorbitan oleic acid monoester (trade name: Tween 80) is used. ) Is preferably used.

The surfactant is added at a concentration of 0.5 to 2%, preferably around 1%, and is added in an amount of solubilized and separated enzyme of 500 to 2000% (w / w), preferably. Is about 1000% (w / w).

[0019]

According to the method of the present invention, a membrane-bound oxidoreductase, particularly an aromatic compound oxidoreductase, which is widely used in the field of producing various compounds, can be easily applied to the cell membrane of a microbial cell. It can be solubilized and separated, and the activity of the separated enzyme can be stably maintained for a long period of time.

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these.

[0021]

【Example】

[Example 1] (1) Rhodococcus eryth
culture of ropolis and inducible production of aromatic compound oxidase Rhodococcus erythropo
lis) KR-S-1 (FERM P-3530) was cultivated at 30 ° C. in a completely synthetic medium containing an aromatic compound as the sole carbon source. Four kinds of salicylic acid, meta-hydroxybenzoic acid, para-hydroxybenzoic acid and phthalic acid were used as aromatic compounds, and each enzyme was induced in the bacterial cells.

(2) Distribution of aromatic compound oxidase in the membrane fraction of enzyme-induced microbial cells The microbial cells in which aromatic compound oxidase was obtained in (1) were sonicated to obtain a cell-free enzyme solution. It was divided into membrane fractions. The oxidase activity of each of the prepared membrane fraction and cell-free enzyme solution was measured. The results are shown in Table 1.

[0023]

[Table 1]

As shown in Table 1, about 1/4 to 1/3 of the total activity remained in the membrane fraction in all cases.
This result suggests that these oxidases that mediate the early oxidation pathway of aromatic compounds are somehow bound to the membrane.

(3) Solubilization and Separation of Membrane-Bound Oxidases Next, various chemical reagents and surfactants were used to try to solubilize the enzymes remaining in the membrane fraction. The membrane fraction prepared by sonication was suspended in 10 mM phosphate buffer (pH 7.1) (containing 10% glycerol) to 10 (mg / ml), and the prescribed reagent or surfactant was added. The mixture was stirred at 4 ° C for 2 hours.
After that, centrifugation was performed to separate the supernatant and the membrane fraction, and the respective enzyme activities were measured. The results are shown in Tables 2-5.

[0026]

[Table 2]

[0027]

[Table 3]

[0028]

[Table 4]

[0029]

[Table 5]

As shown in the above tables, it was found that all four enzymes were solubilized while retaining activity in Brij 58 and Tween 80, but were hardly solubilized in KCl and EDTA. Also, in the case of Triton X-100, there was almost no activity in the supernatant, which is considered to be due to the fact that the enzyme was denatured by the surfactant.

(4) Purification of solubilized and separated enzyme The solubilized and separated enzyme was converted into Butyl Toyopearl 650.
Purification by M, Toyopearl HWMF and then DEAE Toyopearl 650S gave electrophoretically single enzyme samples.

[Example 2] In Example 1, regarding the enzyme solubilized from the membrane fraction, separated and purified (hereinafter, purified enzyme) and the enzyme of the membrane fraction, taking parahydroxybenzoic acid trioxidase as an example, both The stability of the enzyme activity was compared. As a result, the purified enzyme decreased to about 40% after 3 months, while the enzyme in the membrane fraction retained about 70% activity even after 3 months. However, 1000% 1% Tween 80 was added to the purified enzyme.
When% (w / w) was added, almost the same activity retention as that at the time of membrane binding was observed (Fig. 1).

Test Example 1 (1) Identity of Purified Enzyme and Enzyme Obtained by Physical Method by Ultrasonic Treatment Purified enzyme obtained in Example 1 and cell-free enzyme solution were purified by the same method. When the two were compared and examined about the enzyme, the molecular weight of the subunit, the isoelectric point, the amino acid composition, the optimum pH, the optimum temperature, and the reaction product were almost the same.
Table 6 shows the results of comparing the amino acid compositions of both when taking para-hydroxybenzoic acid trioxidase as an example.
From the above, it was judged that the purified enzyme and the enzyme existing in the cell-free enzyme solution after sonication were the same.

[0034]

[Table 6]

(2) Identity of Purified Enzyme and Enzyme Present in Membrane Fraction The purified enzyme obtained in Example 1 and the enzyme present in membrane fraction were compared and examined. Approximately similar results were obtained with respect to points, amino acid composition, optimum pH, optimum temperature, reaction products, and the like. Therefore, it was judged that the purified enzyme and the enzyme present in the membrane fraction were the same and that they were not subjected to any denaturation by the solubilization treatment.

[Brief description of drawings]

FIG. 1 shows changes in the enzyme activity of the membrane-bound state, the purified enzyme, and the purified enzyme coexisting with Tween 80 during the storage period.

[Procedure amendment]

[Document name] Consignment number change notification

[Submission date] December 8, 1994

[Former name of depositary institution] Institute of Microbial Technology, Institute of Industrial Technology

[Old consignment number] FERM-P No. 3530

[Name of new depositary institution] Institute of Biotechnology, Industrial Technology Institute

[New contract number] FERM BP-4913

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Yoshihiro Nakamura Inventor Yoshihiro Nakamura, 1-3-1, Higashi Tsukuba City, Ibaraki Prefecture

Claims (12)

[Claims] 1. A membrane-bound oxidoreductase which is solubilized and separated from a cell membrane by allowing a polyethylene glycol ether type nonionic surfactant to act on the cell membrane fraction of microbial cells. Method for separating oxidoreductase. 2. The method according to claim 1, wherein the polyethylene glycol ether type nonionic surfactant is a polyethylene oxide condensate of a higher alcohol. 3. The method according to claim 2, wherein the polyethylene oxide condensate of higher alcohol is polyethylene (20) cetyl ether. 4. The method according to claim 1, wherein the polyethylene glycol ether type nonionic surfactant is a polyethylene oxide condensate of a fatty acid ester of sorbitan. 5. The method according to claim 4, wherein the polyethylene oxide condensate of sorbitan fatty acid ester is polyethylene (20) sorbitan oleic acid monoester. 6. The method according to claim 1, wherein a polyethylene glycol ether type nonionic surfactant having a concentration of 0.5 to 2% is allowed to act on the cell membrane fraction by 500 to 2000% (w / w). . 7. A method for stabilizing a membrane-bound oxidoreductase, which comprises coexisting a membrane-bound oxidoreductase solubilized from a cell membrane of microbial cells and separated and a polyethylene glycol ether type nonionic surfactant. . 8. The method according to claim 7, wherein the polyethylene glycol ether type nonionic surfactant is a polyethylene oxide condensate of a fatty acid ester of sorbitan. 9. The method according to claim 8, wherein the polyethylene oxide condensate of fatty acid ester of sorbitan is polyethylene (20) sorbitan oleic acid monoester. 10. A polyethylene glycol ether type nonionic surfactant having a concentration of 0.5 to 2% is solubilized, and 500 to 2000% based on the separated membrane-bound oxidoreductase.
(W / w) The method according to claims 7 to 9 by coexisting. 11. The method according to claim 1, wherein the membrane-bound oxidoreductase is an aromatic compound oxidoreductase. 12. The microorganism is Rhodococcu (Rhodococcu).
The method according to claim 1 or 7, which is a s) genus bacterium.