JPH078233B2 - Photosynthetic reaction unit Solubilization Purification and utilization - Google Patents

Description

TECHNICAL FIELD The present invention relates to purification and use of a photosynthetic reaction unit obtained from a photosynthetic organism.

[Prior Art] Photosynthetic organisms such as plants and photosynthetic bacteria have photosynthetic organs involved in photosynthetic reactions. Photosynthetic organs contain lipids, photosynthetic units, oxidoreductases, etc., and the photosynthetic granules obtained as fragments thereof are small, closed membranes composed of chromatophores, proteins such as spheroblast-derived vesicles, and lipids It is a cell. It is known that this type of membrane has a photosynthetic reaction center protein complex that carries out a photoelectric conversion reaction, and that it causes a potential difference across the membrane by photostimulation. For example, the fragment of the purple membrane, which is the membrane of the photosynthetic tissue of halobacterium halobium, which is a halophilic photosynthetic bacterium, is partially decomposed with a surfactant, and the charges on the front and back of the membrane differ greatly. Photoelectric conversion devices utilizing these photosynthetic reactions have been studied.

The photosynthetic organ of red-color photosynthetic bacteria has a protein complex called a photosynthetic reaction unit embedded in a vesicular, lamellar, or tubular membrane structure composed of a lipid bilayer. There are structures shown in FIGS. 1A and 2 as a structure in which this photosynthetic organ is taken out of the cell. FIG. 1A is called a chromatophore, which is a fragment of a vesicular photosynthetic organ obtained when a photosynthetic bacterium is disrupted by a method such as ultrasonication. FIG. 1B is an enlarged view of a part of FIG. 1A and schematically shows a state in which the photosynthetic reaction unit 2 is embedded in the lipid bilayer 1.

FIG. 2 shows a protein complex called a reaction center 4 obtained by treating the chromatophore with a surfactant. The photosynthesis reaction unit 2 converts the light harvesting protein complex 5 into a protein complex. It is a missing structure. A molecule 6 of a surfactant is attached around the reaction center protein complex 4.

The chromatophore is a structure having a complicated molecular composition in which, in addition to the photosynthetic reaction unit 2, proteins of the electron transfer system, oxidoreductase and the like are mixed. Since it is a vesicle with a diameter of about 60-100 nm,
It is considered that when a device applying the function of the photosynthetic reaction unit 2 is constructed, it is difficult to control the molecular orientation and it is not easy to control the molecular composition.

Photosynthetic bacteria skillfully carry out photosynthetic reactions by capturing light of various wavelengths from visible to near infrared. Antenna chlorophyll plays that role. Reaction center protein complex 4 is a light-trapping protein complex 5 containing antenna chlorophyll
It is a protein complex lacking γ, and only light of a specific wavelength can be used, and the utilization efficiency of light energy decreases. A surfactant called lauryldimethylamine oxide (LDAO) is generally used for solubilizing and purifying the reaction center protein complex 4, but this reagent is expensive.

Previously, the West were rhodospirill (rhodospirill)
um rubrum, ATCC11170), was reported to have been solubilized and purified with a mixture of sodium cholate and sodium deoxycholate (J. BioChem. 86 , 1211).
-1224 (1979)). However, the main fraction was an aggregate of photosynthetic reaction units, and the yield of monodispersed photosynthetic reaction units was not high. The aggregates also have photoactivity, but the mode of association and the size of the aggregates are considered to be irregular. There is a problem that it is more difficult to control the molecular orientation when used as a photoelectric conversion element than in a monodisperse photosynthesis unit.

[Problems to be Solved by the Invention] The present invention is mainly in a monodisperse state which has not been industrially obtained so far, can utilize light of various wavelengths, does not contain excessive components, and controls molecular orientation. It is intended to obtain a relatively easy photosynthetic reaction unit of.

[Means for Solving Problems] When the photosynthetic reaction unit is solubilized and purified, the red-color photosynthetic bacteria are treated with a surfactant in the presence of a salt having an ionic strength or higher corresponding to a physiological salt concentration.

[Action] In the presence of a salt having an ionic strength equal to or higher than the physiological salt concentration, the ionic strength is strong. Therefore, when the chromatophore of purple photosynthetic bacteria is treated with a surfactant, the photocapture protein complex is retained. The reaction units are mainly obtained in a monodisperse state.

[Example] An example applied to rhodopseudomonas viridis (ATCC19567), which is a kind of red-light photosynthetic bacteria, will be described.

Rhodopseudomonas viridis has a lamellar photosynthetic organ 8 as shown in FIG. 3A. It is presumed that in this photosynthetic organ, the photosynthetic reaction unit protein complex 9 shown in FIG. 3B is regularly arranged in the lipid bilayer in which the cell membrane is invaded, and the structure 10 shown in FIG. 3C is taken. The state in which the photosynthetic reaction unit is embedded in the lipid bilayer is shown in FIG. 1B. The cylindrical object 11 at the center of the photosynthetic reaction unit shown in FIG. 3B is a reaction center (reaction center).
center, RC) protein complex, and six homologous structures 12 surrounding it are light-harvesting proteins (light-harvesting proteins).
protein, LH) complex. Fig. 3D shows the optical absorption spectrum in the visible and near-infrared region of a live bacterium of Rhodopseudomonas viridis.

The cells were disrupted by ultrasonic treatment or a method called French press, and fractionated by fractional centrifugation or density gradient centrifugation, resulting in vesicles of photosynthetic membrane fragments called chromatophores shown in Figure 1A. can get.

This chromatophore was added to a final concentration of 5-75% Triton X-100,
Tris-hydrochloric acid buffer containing 0.5-1.0 mol (M) NaCl (pH
It was suspended in 8.0) and subjected to ultracentrifugation at 400,000 xg for 15 minutes, and the resulting supernatant was collected as a solubilized fraction.

FIG. 4 shows the results obtained by fractionating this solubilized fraction by high performance liquid chromatography using Superose (Pharmacia). In this case, Triton X-
100 and NaCl are added. 20 after putting the sample
FIG. 5 shows the spectrum of the main peak component eluted in about a minute in the visible / near infrared region. This spectrum is very similar to the spectrum of live Rhodopseudomonas viridis shown in Figure 3D. From the elution position in Fig. 4, the molecular weight is 40
It was judged that about 000 protein-surfactant complexes had been fractionated, and the analysis by SDS-polyacrylamide gel electrophoresis and the knowledge of the spectral change associated with redox, etc. revealed that the photosynthetic reaction unit retained the activity of monodisperse. It was suggested that it was solubilized in the state. The solubilized fraction thus obtained is assumed to have the structure shown in FIG. 6 and be monodispersed.

The salt is not limited to NaCl, and the cation may be a monovalent one such as K ⁺ and NH4 ⁺ , and the anion may be a polyvalent one. it is conceivable that. Most of the ions contained in the intracellular fluid, body fluid, or medium are monovalent cations and have a concentration of about 150-200 mmol. This is called ionic strength corresponding to physiological salt concentration.

Previously, it was reported that treatment of a Rhodopseudomonas viridis chromatophore with Triton X-100 yielded a reaction center protein lacking the light-trapping protein complex, similar to treatment with lauryldimethylamine oxide. This is probably because the solubilized solution and the separated solution had low salt concentration, that is, ionic strength. If the surfactant is effective for isolating the photosynthetic unit,
By performing the solubilization and purification steps under high ionic strength, the concentration of the surfactant used can be kept low or the yield of the photosynthetic reaction unit can be increased.

In addition, another red photosynthetic bacterium, rhodobactor spheroides (ATCC17023), was used to solubilize it with sodium deoxycholate under high ionic strength. Then, it was confirmed that the yield of the monodisperse photosynthesis reaction unit was improved.

As the surfactant, use an ionic surfactant such as Triton X, a surfactant having a steroid skeleton such as sodium deoxycholate, an amphoteric surfactant such as CHAPS, and an ionic surfactant. You can However, when an ionic surfactant is used, it is necessary to keep the salt concentration to coexist lower than when using a nonionic surfactant.

The basic process of photoreaction is that light energy reaches the reaction center protein, where electrons and holes cause charge separation. The photosynthetic reaction unit holding the light-trapping protein complex enhances the utilization efficiency of light energy.

Since the monodisperse photosynthetic reaction unit obtained in the above-mentioned examples is monodisperse and has excellent controllability, it is expected that the characteristics of the photoelectric conversion element will be improved by regularly aligning the molecules.

Example 7 of photoelectric conversion element using photosynthetic reaction unit
It will be described with reference to the drawings. The red-color photosynthetic bacteria are cultured under light irradiation, and the photosynthetic reaction unit in a monodispersed state is collected from the cultured red-color photosynthetic bacteria by the method described above. A monodispersed photosynthetic reaction unit is sealed between substrates with electrodes via a spacer 20. The terminals 25 and 26 are pulled out from the electrodes 21 and 23 on the substrate. Here, the material of the electrode includes indium tin oxide (ITO), tin oxide (NESA glass), metal, semiconductor and the like. The two electrodes preferably have different work functions. When such a photoelectric conversion element was irradiated with light, an electric output was generated between both electrodes.

If only the isolated photosynthetic unit is taken out, oriented and dried and solidified, a more efficient photoelectric conversion element will be obtained.

[Effects of the Invention] A solubilized photosynthetic reaction unit in a monodispersed state and a photoelectric conversion element using the same can be obtained.

This photosynthetic reaction unit shows a light absorption spectrum that is very similar to that of live bacteria, and can use light in a wide range of wavelengths as compared with the case where only the reaction center protein is used.

Furthermore, this photosynthetic reaction unit can also provide the following advantages.

In terms of molecular orientation, it is more controllable than vesicular chromatophores.

It is easy to add prosthetic molecules, stabilizers, etc., and the molecular composition can be changed according to various purposes of use.

[Brief description of drawings]

FIG. 1A is a schematic external view of the chromatophore, FIG. 1B is a partially enlarged view of FIG. 1A, FIG. 2 is a schematic explanatory view of the reaction center, and 3A.
Figures, 3B, and 3C are schematic views of Rhodopseudomonas viridis, a schematic diagram of its photosynthetic reaction unit, a schematic diagram of the sequence of the photosynthetic reaction unit, and 3D is a visible near-red color of live Rhodopseudomonas viridis. Light absorption spectrum in outer region, FIG. 4 is spectrum of liquid chromatograph, FIG. 5 is spectrum of absorbance, FIG. 6 is expected schematic diagram of reaction center obtained by the embodiment of the present invention, FIG. FIG. 4 is a schematic cross-sectional view of a photoelectric conversion element. Explanation of symbols 2 ... Photosynthetic reaction unit 4 ... Reaction center protein complex 5 ... Photocapture protein complex 6 ... Surfactant molecule 20 ... Spacer 22 ... Photosynthesis reaction unit layer 21, 23 ... Electrode

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location H01L 31/08 51/10 // (C12N 1/06 C12R 1:01) (72) Inventor Toyoda Hideki 5 5-9 Tokodai, Tsukuba, Ibaraki 5 Stanley Electric Co., Ltd. Tsukuba Research Center (72) Inventor Toshikazu Majima 1-4-1, Umezono, Tsukuba, Ibaraki Electronic Technology Research Institute (72) Invention Atsushi Miyake 1-3-1, Higashi, Tsukuba-shi, Ibaraki Institute of Industrial Technology, Institute of Microbial Technology (72) Inventor Masayuki Hara 1-3-1, Higashi, Tsukuba-shi, Ibaraki Institute of Microbial Technology, Institute of Industrial Technology (56) References Kai 64-110224 (JP, A)

Claims (2)

[Claims] 1. A method for solubilizing and purifying a photosynthetic reaction unit, which comprises treating red-color photosynthetic bacteria with a surfactant in the presence of a salt having an ionic strength or more corresponding to a physiological salt concentration. 2. A photoelectric conversion device comprising a photosynthetic reaction unit obtained by the method according to claim 1.