KR100276837B1 - Separation Method of Bacteriodosin by Aqueous Phase Interface Concentration - Google Patents

Abstract

The present invention relates to a method for separating bacteriodrodopsine from a halobacterial cell lysate comprising concentrating a cell membrane of a halobacterial cell containing an aqueous bacteriodopsin at an interface of an aqueous two phase system composed of a polymer and an inorganic salt. . By using the bacteriorhodoxin separation method of the present invention, the cell membranes of the halobacteria containing the bacteriorodopsin can be efficiently concentrated and recovered, which in turn greatly improves the final separation yield of the bacteriododoxin. In addition, the bacteriorodoxin separation method of the present invention is cheaper and simpler than the conventional bacteriorodoxin separation method, and the mass production of the bacteriorodoxin is possible, so that the bacteriorodoxin separation method of the present invention is used for the development of biochips and three-dimensional memory devices. You will be able to contribute greatly.

Description

Separation Method of Bacteriododocin by Aqueous Phase Interfacial Concentration

The present invention relates to a method for separating bacteriorhodopsin from Halobacteria genus microorganisms. More specifically, the present invention includes the step of concentrating a cell membrane of halobacteria containing a bacteriododocin from a halobacterial cell lysate at an interface of an aqueous two phase system composed of a polymer and an inorganic salt. To a separation method.

Halobacteria are probiotics that survive in the Dead Sea. Under aerobic conditions, they produce ATP through aerobic metabolism, but under anaerobic conditions, they produce ATP through a cell membrane protein called bacteriorhodosin. Bacteriorhodoxin is a cell membrane protein synthesized in the cells of halobacteria under anaerobic conditions. It is a pigmented carotenoid retinal that is linked through an epsilon amino group of lysine residue and through Schiff's base. It has it as a chromophore. When the bacteriorhodosin having such a structure receives light, quantum escape occurs from the electric Schiff base due to isomerization of the retinal, and the released protons are released to the outside of the membrane, and the retinal is a Schiffbase. By re-absorption of protons within the membrane, a photocycle occurs back to the ground state (Henderson et al., Nature, 257: 28-32, 1975).

As such, the bacteriorhodoxin has a unique photochemical property such as color change depending on the wavelength of light, and a fast and variable optical cycle. While research in the field of bioelectronics is vigorous, it is drawing attention as a biomolecule that is expected to realize the development of biochips and three-dimensional memory devices. In particular, unlike most biomolecules, bacteriodhodopsin is stable in high temperature conditions and electrolyte solutions, and due to its useful properties that can be used in technical processes such as relatively wide pH range and stability in drying, It is more preferred than proteins.

As such, the demand for bacteriododocin has increased day by day, and the need for commercial mass production is urgent, while the development of the bacteriododosin separation method is still insignificant. Sucrose density gradient ultracentrifugation method (see Oesterhelt et al., Methods Enzymol) , 31: 667-678, 1974) and gel permeation chromatography methods (Neumann et al., German Patent, DE 3922133A1, 1989), and the like, and recently known ultracentrifugation methods A slightly modified method has been reported (Tan et al., Biophys. J., 70: 2385-2395, 1996).

However, in order to efficiently separate bacteriorhodoxin, a process of concentrating and recovering a cell membrane containing bacteriododocin is required. The conventional sucrose density gradient ultracentrifugation method has a low yield of the cell membrane concentration step, and is used to prepare a large amount of bacteriododocin. There are disadvantages such as impossible to apply and use of expensive equipment. On the other hand, the gel filtration chromatography method does not require an expensive device compared to the sucrose density gradient ultracentrifugation method, and can be separated at a relatively high yield, and the manufacturing scale can be expanded. The method also has the disadvantage that it is not suitable for the complete mass production of bacteriododocin because cell membrane recovery is by ultracentrifugation.

Therefore, when anticipating the production of biocomputers using biochips that will be realized in the near future and the industrial demand of bacteriodopsin, there is an urgent need to develop a method for mass production of bacteriodopsin more efficiently.

Therefore, the present inventors analyzed the problems of the conventional methods of separating bacteriodopsin, and the yield of the bacteriodopsin separation of the conventional methods was only 30 to 50%, which was caused by the step of recovering the cell membrane containing bacteriododocin from the cell lysate by ultracentrifugation. It is confirmed that the loss is very large. Therefore, the present inventors have made intensive studies to improve the yield in the cell membrane recovery process, which is an intermediate step of the bacteriododocin separation process, and replaced the ultracentrifugation process for cell membrane recovery with an aqueous two-phase interfacial condensation process. In this case, it was confirmed that the recovery rate of the cell membrane containing bacteriododocin can be significantly increased, and the present invention was completed.

After all, it is an object of the present invention to provide a method for separating bacteriodopsin comprising the step of concentrating a cell membrane of a bacteriodopsin-containing halobacterial at an interface of an aqueous phase from a halobacterial cell lysate.

1 is a schematic view showing the concentration of the cell membrane of the halobacteria at the interface of the aqueous phase.

The present invention is characterized in that the step of separating the bacteriorodoxin from the microorganism of the genus Halobacteria, comprising the step of concentrating the cell membrane of the halobacteria containing the bacteriorodoxin from the halobacterial cell lysate at the interface of the aqueous phase.

Hereinafter, the separation method of the bacteriododocin by the aqueous phase-based interface concentration of the present invention will be described in more detail.

Step 1: Preparation of Halobacterial Cells

Inoculate the halobacterial microorganisms into the medium, shake culture for 60 to 80 hours at the conditions of 400 to 600rpm at 35-40 ℃, centrifugation of halobacterial culture at 5,000 to 6,000 × g 30 to 50 minutes The cells are recovered. The microorganisms of the genus Halobacterium are not particularly limited, for example, Halobacterium salinarium, Halobacterium halobium, Halobacterium cutirubium or Halobacterium trat. Palladium (Halobacterium traparium) can be used.

Second step: disruption of halobacterial cells

The cells recovered were sequentially washed with 20 to 30% (w / v), most preferably 25% (w / v) aqueous sodium chloride solution and distilled water, resuspended in distilled water, and then added with DNase I, followed by 5 hours to Stir for 7 hours to disrupt the cells.

Step 3: Aqueous Phase Interfacial Concentration

The cell lysate obtained from the foregoing is applied to an aqueous phase system composed of a polymer and an inorganic salt, and centrifuged at 1,500 to 2,500 × g, most preferably at 2,000 × g, 30 to 50 minutes, most preferably 40 minutes. The cell membrane containing bacteriododocin is concentrated at the interface of the aqueous phase system and recovered. The polymer is a polyelectrolyte (polyelectrolyte), but is not particularly limited to its kind, it is most preferred to use polyethylene glycol (polyethyleneglycol), polyethylene glycol may be preferably used polyethylene glycol having a molecular weight of 1450 to 8000, the molecular weight Most preferably, 8000 polyethylene glycol is used. In addition, it is preferable to use the salt of the ion in which pK _a does not deviate significantly from 7, and it is most preferable to use a phosphate salt as an electric inorganic salt. In addition, as long as the composition ratio of the polymer and the inorganic salt of the electro-aqueous phase system is not particularly limited, the aqueous phase system having a content ratio of polymer to inorganic salt of 8:11 by weight ratio is most preferred, and the electro-aqueous The pH of the ideal system is preferably 6.8 to 7.4, most preferably 7. On the other hand, in order to efficiently concentrate the cell membrane by using an aqueous aqueous phase system, an aqueous phase system in an amount of preferably 0.1 to 0.5 is required as a mass ratio of the aqueous phase system to the volume of the halobacterial culture.

Fourth Step: Purification of Bacteriododoxin from Concentrated Cell Membrane

The recovered halobacterial cell membrane is suspended in a small amount of distilled water, then loaded into a gel filtration chromatography column and eluted to purify the bacteriododocin.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

Example 1: Selection of Optimum Polymer for Aqueous Phase Interfacial Concentration

Example 1-1: Separation of Cell Membrane by Cultivation of Halobacteria and Aqueous Phase Interfacial Concentration

Halobacterium salinarium SP9 was added 25% (w / v) sodium chloride (NaCl), 2% (w / v) magnesium chloride 6-hydrate (MgCl ₂ · 6H ₂ O) g, 0.02% (w / v) medium containing calcium chloride dihydrate (CaCl ₂ .2H ₂ O), 0.2% (w / v) potassium chloride (KCl) and 1% (w / v) peptone, Oxoid, UK ) Was inoculated at 1 L, shaken incubated at 37 ° C. and 500 rpm for 70 hours, and then the cells were recovered by centrifuging the electroculture at 5,700 × g for 40 minutes. Then, cells obtained above were washed sequentially with 25% (w / v) aqueous sodium chloride solution and distilled water, resuspended in 70 mL of distilled water, 0.5 mg of DNase I was added, and stirred for 6 hours to disrupt the cells. . Subsequently, the cell lysate was applied to an aqueous phase system containing polyethylene glycol as a polymer and potassium phosphate as an inorganic salt, and centrifuged at 2,000 x g for 40 minutes, and the cell membrane containing bacteriodopsin was separated from the aqueous phase system. Concentrated at the interface and recovered. The molecular weight of polyethylene glycol and the composition ratio of polyethylene glycol: phosphate in the aqueous aqueous phase are shown in Table 1 below.

Example 1-2 Measurement of Interfacial Concentration Yield of Halobacterial Cell Membranes Containing Bacteriododoxin

The total protein concentration of halobacterial cells was measured using the Bio-Rad DC Protein Assay Kit (Bio-Rad Pacific Ltd., USA), where bovine gamma globulin was used as the standard protein. ) Was used. Meanwhile, the concentrations of total bacteriododocin in the halobacterial medium of Example 1-1 and the concentration of bacteriododocin in the cell membrane layer recovered by the aqueous phase interfacial concentration were measured by the following methods: First, in a space where light was blocked. 4N ammonia water (NH ₄ OH) and 4N sodium hydroxide (NaOH) solutions were added in a volume ratio of Sample: NH ₄ OH: NaOH = 9: 0.5: 0.5 to measure the absorbance at 568 nm, and then the retinal was removed from the bacteriodopsin. It was exposed to light for 24 hours, and the absorbance was measured again at 568 nm. Subsequently, bacteriododoxin was quantified by the difference in absorbance before and after retinal removal (Shand, RS and Betlach, MC, J. Bacteriol., 173: 4692-4699, 1991). As a result of comparing the total bacteriododocin concentration in the halobacterial culture medium and the bacteriododocin concentration of the cell membrane layer recovered by the aqueous phase interfacial concentration, as shown in Table 1, polyethyleneglycol having a molecular weight of 8000 (hereinafter referred to as 'PEG 8000') is weak. ), The interfacial concentration yield of halobacterial cell membranes containing bacteriododocin was found to be the best.

Interfacial Concentration Yield According to Molecular Weight and Composition Ratio of Polymer and Inorganic Salt Polymer Composition ratio of polymer and inorganic salt (PEG%: Phosphate%) Interfacial Concentration Yield (%) PEG 600 16: 20 54% PEG 1450 14: 17 86% PEG 3350 10: 12 88% PEG 8000 8: 11 89% PEG 20000 10: 8 0%

Hereinafter, in Examples described later, only the cell membrane concentration yield measured in the same manner as described above will be disclosed.

Example 2 Determination of Optimal Composition Ratio of Aqueous Phase System

PEG 8000 is used as the polymer, and the composition ratio of polymer: inorganic salt (% PEG (w / v):% phosphate (w / v)) is increased by 8:11, 9:13 or 10:14 or each aqueous phase system prepared to be 13: 8.5, 10:10, 5:12 or 3:13 on the same connecting line, respectively. Cell membranes were concentrated and recovered in the same manner as in Example 1-1. Subsequently, the interfacial concentration yield of the halobacterial cell membrane containing bacteriododocin was measured, and in each case, the interfacial concentration yield was found to be almost constant at 89 to 90%.

Hereinafter, in the examples described below, only the result of using an aqueous phase system in which the polymer is PEG 8000 and the composition ratio of the polymer: inorganic salt is 8:11 by weight ratio will be described for convenience.

Example 3: Effect of Salt Addition on Interfacial Concentration Efficiency

Concentrate the cell membrane in the same manner as in Example 1-1, adding sodium chloride to 0.1% (w / v), 1% (w / v), or 10% (w / v), respectively, in addition to the aqueous phase system. And recovered. Subsequently, as a result of measuring the interfacial concentration yield of the halobacterial cell membrane containing bacteriododocin, it was confirmed that the interfacial concentration yield decreased to 80% or less when sodium chloride was added at a concentration of 1% or more. These results suggested that complete removal of medium containing salts at high concentration and suspension of cell debris in an appropriate amount of distilled water in the step of recovering the cells from the culture can directly affect the interfacial concentration yield.

Example 4 Determination of Optimal pH for Aqueous Phase Systems

By adjusting the ratio of potassium phosphate and potassium dihydrogen phosphate in the phosphate contained in the aqueous phase system, the cell membrane was concentrated and recovered in the same manner as in Example 1-1 while changing the pH of the aqueous phase system from 5.0 to 8.0. . Subsequently, the interfacial concentration yield of the halobacterial cell membrane containing the bacteriodopsin was measured. As a result, the degeneration of the bacteriodopsin occurs at pH 8, the loss of bacteriodopsin in the phosphate buffer at pH 6.5 and below, and the phase separation at pH 5, while the pH is separated. In 6.8 to 7.4, the interfacial concentration yield was found to be almost constant, 89 to 90%.

Example 5 Determination of Minimum Requirements of Aqueous Phase Systems for Efficient Concentration

In order to determine the minimum requirement of the aqueous phase system necessary for the efficient separation and concentration of the bacteriododoxin-containing cell membrane from the large-scale cultured halobacteria, 50 g to 0.5 kg of the aqueous phase system was added to 1 L of the halobacterial culture medium. Cell membranes were concentrated and recovered in the same manner as 1. Subsequently, as a result of measuring the interfacial concentration yield of the halobacterial cell membrane containing the bacteriododocin, the interfacial concentration yield was obtained when 0.1 kg or more of the aqueous phase system was used for 1 L of culture medium, that is, when the mass ratio of the aqueous phase system to the volume of the culture medium was 0.1 or more. Was found to be about 90%, and when a smaller amount of the aqueous phase system was used, no loss to the phosphate layer occurred or no phase separation itself. Therefore, the minimum requirement of the aqueous phase system required to separate cell membranes from 1 L culture was determined to be 0.1 kg.

Example 6 Purification of Bacteriorhodoxin from Interfacially Concentrated Halobacterial Cell Membranes

Using 1 L of halobacterial culture medium and 0.1 kg of aqueous phase system, cell membrane concentration by aqueous phase interface concentration was carried out in the same manner as in Example 1-1, and the cell membrane concentration yield was about 90%. Cell membranes obtained from the above were suspended in a small amount of distilled water, loaded onto a gel filtration chromatography column using Sephacryl S1000 (Pharmacia, Sweden) as a filler, and eluted to obtain 17.5 mg of pure bacteriododocin, which was bacteriodopsin. The final yield of was found to be 70%.

Comparative Example 1: Isolation of Bacteriododoxin by Conventional Gel Filtration Chromatography

Halobacterium salinarium SP9 was cultured in the same manner as in Example 1-1, and cells were recovered from the culture. Then, cells obtained from the above were resuspended in 25% aqueous sodium chloride solution, added DNase I, and dialyzed in distilled water for 24 hours to disrupt the cells. Cell membranes were recovered by ultracentrifugation of the cell lysate obtained at 50,000 × g for 30 minutes, and the concentration yield of halobacterial cell membranes containing bacteriododocin was measured. It was%. The recovered cell membrane was resuspended in distilled water, loaded onto a gel filtration chromatography column using Sephacryl S1000 (Pharmacia, Sweden) as a filler, eluted to obtain 10 mg of pure bacteriododocin, and the final yield of bacteriodopsin was 40% was confirmed.

Comparison of Separation Yields of Bacteriododoxin by Aqueous Phase Interfacial Concentration and Conventional Gel Filtration Chromatography Aqueous phase interfacial concentration method Gel Filtration Chromatography Cell membrane concentration yield 90% 52% Separation yield of final bacteriodopsin 70% 40%

As described and demonstrated in detail above, the present invention provides a method for separating bacteriododocin comprising the step of concentrating a cell membrane of halobacteria containing a bacteriododoxin from an halobacterial cell lysate at the interface of an aqueous phase system. By using the bacteriorhodoxin separation method of the present invention, the cell membranes of the halobacteria containing the bacteriorodopsin can be efficiently concentrated and recovered, which in turn greatly improves the final separation yield of the bacteriododoxin. In addition, the bacteriorodoxin separation method of the present invention is cheaper and simpler than the conventional bacteriorodoxin separation method, and the mass production of the bacteriorodoxin is possible, so that the bacteriorodoxin separation method of the present invention is used for the development of biochips and three-dimensional memory devices. You will be able to contribute greatly.

Claims (4)

(i) inoculating the microorganisms of the genus Halobacteria into the medium, shaking culture for 60 to 80 hours at 400 to 600 rpm at 35 to 40 ° C, and then halobacterial culture solution at 30 to 50 at 5,000 to 6,000 × g. Recovering the cells by centrifugation for a minute; (ii) the cells were recovered by 20-30% (w / v) aqueous sodium chloride solution and distilled water sequentially, resuspended in distilled water, DNase I was added and stirred for 5 to 7 hours to disrupt the cells step; (iii) The cell lysate obtained from the foregoing is applied to an aqueous phase system having a pH of 6.8 to 7.5 and composed of polyethylene glycol polymer and an inorganic salt, and centrifuged at 1,500 to 2,500 × g for 30 to 50 minutes to contain bacteriododocin. Recovering the concentrated cell membrane at the interface of the aqueous phase system; And, (iv) a method of separating bacteriododocin, comprising: suspending the recovered halobacterial cell membrane in a small amount of distilled water, and then loading and eluting the gel filtration chromatography column. The method of claim 1, Microorganisms of the genus Halobacterium are characterized as Halobacterium salinarium, Halobacterium halobium, Halobacterium cutirubium or Halobacterium traparum. doing Bacteriorhodosin Isolation Method. The method of claim 1, The composition ratio of polyethylene glycol polymer and phosphate is 8:11, characterized in that Bacteriorhodosin Isolation Method. The method of claim 1, Polyethylene glycol is characterized in that the molecular weight of 1450 to 8000 Bacteriorhodosin Isolation Method.