CONTINUOUS PRODUCTION METHOD OF ADENOSINE TRIPHOSPHATE AND NICOTINAMIDE ADENINE DINUCLEOTIDE (PHOSPHATE) USING PHOTOSYNTHETIC MEMBRANE VESICLE

Abstract

The present invention relates to a composition for production of photosynthetic light-reaction products comprising photosynthetic membrane vesicles, and a production method for the photosynthetic light-reaction products by using the composition. In addition, the present invention relates to a preparation method for a photosynthetic light-reaction monomer comprising a step of isolating vesicles from the cell membrane of photosynthetic bacteria or algae.

Claims

1. A composition for producing a product of the light-dependent reactions in photosynthesis, comprising: a photosynthetic membrane vesicle, wherein the membrane is one or more selected from the group consisting of the thylakoid membrane (TM) of cyanobacteria or algae and the intracytoplasmic membrane (ICM) of purple non-sulfur bacteria.

2. The composition of claim 1, wherein the product of the light-dependent reactions in photosynthesis is one or more light-dependent reaction products selected from the group consisting of adenosine triphosphate (ATP), nicotinamide adenine dinucleotide (NADH) and reduced nicotinamide adenine dinucleotide phosphate (NADPH).

3. The composition of claim 1, wherein the cyanobacteria are selected from the group consisting of Synechocystis sp., Synechococcus sp., Nostoc sp., Anabaena sp., Gloeobacter sp. and Cyanobacterium sp.

4. The composition of claim 1, wherein the purple non-sulfur bacteria are selected from the group consisting of Rhodobacter sp., Rhodospirillum sp., Rhodopseudomonas sp., Roseobacter sp., Bradyrhizobium sp., and Rubrivivax sp.

5. The composition of claim 1, which includes a vesicle separated from the thylakoid membrane of cyanobacteria or algae and a vesicle separated from the intracytoplasmic membrane of purple non-sulfur bacteria.

6. The composition of claim 1, wherein the vesicle separated from the thylakoid membrane of cyanobacteria or algae and the vesicle separated from the intracytoplasmic membrane of purple non-sulfur bacteria absorb light in mutually different wavelength ranges.

7. The composition of claim 6, wherein the light in the mutually different wavelength bands is visible light and infrared light.

8. The composition of claim 1, wherein the thylakoid membrane of cyanobacteria or algae includes chlorophyll a (Chl a) in a concentration of 1 g chlorophyll a/ml to 1 mg chlorophyll a/ml.

9. The composition of claim 1, wherein the vesicle separated from the intracytoplasmic membrane of purple non-sulfur bacteria includes bacteriochlorophyll a (Bchl a) in a concentration of 1 g bacteriochlorophyll a/ml to 1 mg bacteriochlorophyll a/ml.

10. The composition of claim 1, wherein the vesicle separated from the thylakoid membrane of cyanobacteria or algae and the vesicle separated from the intracytoplasmic membrane of purple non-sulfur bacteria have a diameter of 1 to 500 nm.

11-13. (canceled)

14. A method of producing a product of the light-dependent reactions in photosynthesis using a photosynthetic membrane vesicle, the method comprising: a) separating a vesicle from one or more cell membranes selected from the group consisting of the thylakoid membrane (TM) of cyanobacteria or algae and the intracytoplasmic membrane (ICM) of purple non-sulfur bacteria; b) preparing a light-dependent reaction-performing unit including the separated vesicle; and c) applying light to the light-dependent reaction-performing unit.

15. The method of claim 14, wherein the product of the light-dependent reactions in photosynthesis is one or more light reaction products selected from the group consisting of adenosine triphosphate (ATP), nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH).

16. The method of claim 15, wherein the NADH is able to be converted into NADPH without additional enzyme treatment.

17. The method of claim 15, wherein the NADPH is able to be converted into NADH without additional enzyme treatment.

18. The method of claim 14, wherein the vesicle includes vesicles separated from the thylakoid membrane of cyanobacteria or algae and the intracytoplasmic membrane of purple non-sulfur bacteria together.

19. The method of claim 14, wherein the light has a wavelength in the visible or infrared range.

20. A method of manufacturing photosynthetic light reaction-performing apparatus, comprising: separating a vesicle from one or more cell membranes selected from the group consisting of the thylakoid membrane of cyanobacteria or algae and the intracytoplasmic membrane of purple non-sulfur bacteria.

21. The method of claim 20, wherein the separating of a vesicle from the cell membrane includes: a) preparing a cell lysate by disrupting cyanobacteria, algae or purple non-sulfur bacteria; and b) separating a vesicle from the cell membrane contained in the cell lysate by centrifugation under a sucrose density gradient.

22. The method of claim 21, wherein the disruption is performed using glass beads or ultrasonic waves.

23. The method of claim 21, wherein the sucrose density is 5 to 50%.

24. The method of claim 21, wherein the centrifugation is performed at 50,000 to 500,000 g for 20 minutes to 24 hours.

Description

DESCRIPTION OF DRAWINGS

[0064] FIG. 1 shows levels of ATP generation investigated under a light-applied condition (light) and a dark condition (dark) using a thylakoid membrane (TM) vesicle or an intracytoplasmic membrane (ICM) vesicle. Under each condition, an ATP production rate was expressed in nmole/min.Math.mg protein.

[0065] FIG. 2 shows ATP production rates investigated under a light-applied condition (light) and a dark condition (dark) using a thylakoid membrane (TM) vesicle or an intracytoplasmic membrane (ICM) vesicle. The drawing on the upper panel shows the ATP production rate using the thylakoid membrane vesicle, and the drawing on the lower panel shows the ATP production rate using the intracytoplasmic membrane vesicle. Concentrations of the thylakoid membrane vesicle and the intracytoplasmic membrane vesicle were determined by concentrations of chlorophyll a (Chl a) and bacteriochlorophyll a (Bchl a), respectively. Under each concentration condition, the ATP production rate was expressed in nmole/min.

[0066] FIG. 3 shows the improvement of ATP production efficiency when the thylakoid membrane vesicle and the intracytoplasmic membrane vesicle, which have different absorption wavelengths, are used together. Concentrations of the thylakoid membrane (TM) vesicle and the intracytoplasmic membrane (ICM) vesicle were determined by concentrations of chlorophyll a (Chl a) and bacteriochlorophyll a (Bchl a), respectively. Under two conditions in which the thylakoid membrane (TM) vesicle was not used, or used at a fixed concentration of 40 g Chl a/ml, varying concentrations of the intracytoplasmic membrane (ICM) vesicles were added, and then the ATP production rates were measured when using incandescent light in a wide range of wavelengths at an intensity of 15 Watts/m.sup.2, and the values were expressed in nmole/min.

[0067] FIG. 4 shows the efficiency of reducing NAD.sup.+ and NADP.sup.+ under the light condition and the dark condition using a thylakoid membrane vesicle. NADH and NADPH production rates were expressed in nmole/min.Math.mg protein.

[0068] FIG. 5 shows efficiency of reducing NAD.sup.+ and NADP.sup.+ under the light condition and the dark condition using an intracytoplasmic membrane vesicle. NADH and NADPH production rates were expressed in nmole/min.Math.mg protein.

MODES OF THE INVENTION

[0069] Hereinafter, the present invention will be described in further detail with reference to examples. There examples are merely provided to explain the present invention in further detail, and therefore, according to the inventive concept, it should be obvious to those of ordinary skill in the art that the scope of the present invention is not limited by the examples.

Example 1

Separation of Thylakoid Membrane Vesicle of Cyanobacteria

[0070] To separate a thylakoid membrane vesicle including a light reaction apparatus of cyanobacterium, Synechocystis sp. PCC 6803 was used as a target strain. For growth of the strain, partially-modified BG11 minimal medium [18 mM sodium nitrate (NaNO.sub.3), 0.23 mM potassium monohydrogen phosphate (K.sub.2HPO.sub.4), 0.30 mM magnesium sulfate (MgSO.sub.47H.sub.2O), 0.24 mM calcium chloride (CaCl.sub.2.2H.sub.2O), 31 M citric acid, 23 M ferric ammonium citrate, 0.19 mM sodium carbonate (Na.sub.2CO.sub.3), 8.8 mM sodium thiosulfate, 46 M boric acid (H.sub.3BO.sub.3), 14 M manganese chloride (MnCl.sub.2), 0.77 M zinc sulfate (ZnSO.sub.47H.sub.2O), 1.6 M sodium molybdate (Na.sub.2MoO.sub.42H.sub.2O), 0.32 M copper sulfate (CuSO.sub.45H.sub.2O), 0.17 M cobalt nitrate (II) (Co(NO.sub.3).sub.26H.sub.2O) (Cratz and Myers. 1955. Am. J. Bot. 42: 282-287)] was used, wherein, for rapid growth of the strain, glucose sterilized using a filter was added to be 10 mM. The Synechocystis strain was inoculated into a 300 ml flask containing 50 ml of BG11 minimal medium, and then incubated at 30 C. with shaking at 100 revolutions per minute (rpm) under an aerobic condition. Here, white fluorescent light was used with an intensity of approximately 50 micro Einstein/m.sup.2.Math.s. When the optical density of the strain at 600 nm approached approximately 1.0, the strain was inoculated again into a 1 L flask containing 500 ml of BG11 minimal medium and then incubated under the above-described growth condition to have the optical density at 600 nm of approximately 2.0.

[0071] Cyanobacteria was cultured under a photosynthesis condition and centrifuged at 4 C. and approximately 7,000 g for 10 minutes to obtain cell pellets, which were suspended in approximately 10 ml of a 10 mM Tris-HCl (pH 7.5) buffer containing 1 mM EDTA and then placed on ice. The suspended cells were disrupted by glass beads, which were added to a buffer containing the cells to be full of the glass beads, followed by vigorously shaking for 2 minutes per each of 5 times (total 10 minutes) to disrupt the cells. Afterward, undisrupted cells, large cell fragments, and the glass beads were discarded after centrifugation at approximately 3,000 g and 4 C. for 10 minutes. Here, a supernatant obtained thereby included various water-soluble cell components and cell membranes (including vesicles), and were centrifuged at 100,000 g for 30 minutes to obtain a total cell membrane. Subsequently, to isolate the thylakoid membrane vesicle from the total cell membrane, the sample was centrifuged with a 10-50% sucrose density gradient at 130,000 g for 15 hours. From layers separated thereby, a part corresponding to 38 to 42% sucrose density was obtained, and concentrated by centrifugation again at 187,000 g for 45 minutes. The membrane vesicle located at the bottom layer after the centrifugation was dissolved in a 10 mM Tris-HCl (pH 7.5) buffer containing 0.25 M sucrose, and then subjected to phase partitioning (Norling et al. 1998. FEBS Lett. 436: 189-192) using dextran T-500 and PEG 3350, thereby obtaining parts showing a green color due to the presence of the thylakoid membrane vesicle of cyanobacteria from the fifth and sixth layers from the bottom.

Example 2

Separation of Intracytoplasmic Membrane Vesicle of Purple Non-Sulfur Bacteria

[0072] To separate an intracytoplasmic membrane vesicle including a light reaction apparatus of purple non-sulfur bacteria, Rhodobacter sphaeroides 2.4.1 (ATCC BAA-808, Cohen-Bazire et al. 1956. J. Cell. Comp. Physiol. 49: 25-68) was used as a target strain, and for growth of the strain, Sistrom's minimal medium [20 mM potassium dihydrogen phosphate (KH.sub.2PO.sub.4), 3.8 mM ammonium sulfate ((NH.sub.4).sub.2SO.sub.4), 34 mM succinate, 0.59 mM L-glutamate), 0.30 mM L-aspartate, 8.5 mM sodium chloride, 1.05 mM nitrilotriacetic acid, 1.2 mM magnesium chloride (MgCl.sub.2 6H.sub.2O), 0.23 mM calcium chloride (CaCl.sub.2 7H.sub.2O), 25 M ferrous sulfate (FeSO.sub.4.7H.sub.2O), 0.16 M ammonium molybdate ((NH.sub.4).sub.6Mo.sub.7O.sub.24.4H.sub.2O), 4.7 M EDTA, 38 M zinc sulfate(ZnSO.sub.47H.sub.2O), 9.1 M manganese sulfate (MnSO.sub.4.H.sub.2O), 1.6 M copper sulfate (CuSO.sub.4.5H.sub.2O), 0.85 M cobalt nitrate(II) (Co(NO.sub.3).sub.2.6H.sub.2O), 1.8 M borate (H.sub.3BO.sub.3), 8.1 M nicotinic acid, 1.5 M thiamine hydrochloride, 41 nM biotin (Sistrom. 1962. J. Gen. Microbiol. 28: 607-616)] was used. The Rhodobacter sphaeroides strain was inoculated into a 300 ml flask containing 30 ml of Sistrom's minimal medium and incubated with shaking at 250 rpm and 30 C. under an aerobic condition. When the optical density of the strain at 660 nm reached approximately 1.0, the strain was inoculated into a 1 L transparent container filled up with Sistrom's minimal medium to prevent input of oxygen, and grown under an anaerobic condition using fluorescent light with an intensity of 15 Watts/m.sup.2 until an optical density at 660 nm became approximately 2.0.

[0073] The Rhodobacter sphaeroides cells incubated under the photosynthesis condition were centrifuged at approximately 7,000 g and 4 C. for 10 minutes to obtain cell pellets, which were suspended in approximately 10 ml of a 10 mM Tris-HCl (pH 7.5) buffer containing 1 mM EDTA, and placed on ice. To disrupt the suspended cells, a cell disrupting method using ultrasonic waves (sonication) was performed for 5 minutes per each of four times (total 20 minutes). Afterward, undisrupted cells and large cell fragments were discarded after centrifugation at approximately 10,000 g and 4 C. for 30 minutes. Here, a supernatant obtained thereby included various water-soluble cell components and cell membrane (including vesicles), and to isolate an intracytoplasmic membrane, centrifugation was performed at 96,000 g for 4 hours with a 5 to 35% sucrose density gradient. Subsequently, parts showing a brown color, which contain the intracytoplasmic membrane vesicle containing a photosynthetic apparatus of Rhodobacter sphaeroides, were obtained from several layers separated from each other.

Example 3

Confirmation of ATP Generated Using Photosynthetic Membrane Vesicle

[0074] The thylakoid membrane vesicle containing the light reaction apparatus of cyanobacteria generated a proton concentration gradient between the inner and outer spaces of the membrane by transferring protons while electrons from water were transported under a light condition, and converted ADP and inorganic phosphate into ATP by the action of ATP synthase using kinetic energy derived from the proton concentration gradient as a driving force. Likewise, in reaction centers of Rhodobacter sphaeroides, a photochemical reaction by light energy was induced, resulting in induction of cyclic electron transfer, and ATP synthesis by ATP synthase using the proton concentration gradient generated thereby as a driving force. Accordingly, in this example, to investigate whether the separated thylakoid membrane vesicle and intracytoplasmic membrane vesicle had an activity of performing a light-dependent reaction under the light condition, ATP synthesis efficiency was tested. The ATP synthesis efficiency may be assessed by various methods that can selectively detect ATP (generally using an ATP quantification method performed on a final product having an optical density with respect to a specific wavelength or exhibiting fluorescence, which is generated by an enzyme reaction consuming ATP and converting a transparent substrate with the ATP as a driving force), and an optimal method for evaluating the efficiency may be determined by one of ordinary skill in the art according to the purpose of research and development. In this example, as a method for quantifying ATP, an ATP assay kit (BioVision) was used.

[0075] A reaction was performed using a 50 mM PBS buffer (phosphate buffered saline, pH 7.4) at 30 C. while ADP was added as a substrate to a concentration of 1 mM. Levels of proteins respectively containing the thylakoid membrane vesicle and the intracytoplasmic membrane vesicle were quantified, thereby determining concentrations of the proteins. Values of ATP production rates measured several times were normalized to a concentration of the protein contained in each of the two types of cell membrane vesicles. A degree of ATP synthesis was assessed by a change in optical density at 570 nm, and a standard curve was plotted through an experiment performed as described above using various concentrations of ATP, and thereby the measured value of the optical density can be converted into an amount of ATP generation. The amount of ATP production determined thereby was expressed as a value over time, and the ATP production rate was expressed as nmole ATP-generating values per unit protein and per minute. FIG. 1 shows ATP synthesis efficiency by the thylakoid membrane vesicle and intracytoplasmic membrane vesicle determined by the above-described method. Both of the cell membrane vesicles exhibited considerably lower ATP synthesis rates under a dark condition in which light was not applied than those under the light condition in which light was applied, and from this result, it can be considered that most of ATP generated in this example were products of the light-dependent reaction in photosynthesis. In this example, to measure the ATP synthesis efficiency of the thylakoid membrane vesicle, fluorescent light with an intensity of 50 micro Einstein/m.sup.2.Math.s was used, to assess ATP synthesis efficiency of the intracytoplasmic membrane vesicle, incandescent light with an intensity of 15 Watts/m.sup.2 was used.

Example 4

Experiment on Efficiency of ATP Generation According to Concentration of Photosynthetic Cell Membrane Vesicle

[0076] Optimal concentrations of the thylakoid membrane vesicle and the intracytoplasmic membrane vesicle to produce ATP by a light-dependent reaction were to be determined. To this end, the separate thylakoid membrane vesicle and intracytoplasmic membrane vesicle were prepared at various concentrations, and ATP synthesis rates were measured in the same manner as described in Example 3. In this example, the thylakoid membrane vesicle was quantified based on the concentration of chlorophyll a, and the intracytoplasmic membrane vesicle was quantified based on the concentration of bacteriochlorophyll a. A part of the separated thylakoid membrane vesicle or intracytoplasmic membrane vesicle was extracted with a mixed solvent containing acetone and methanol (in a ratio of 7:2), resulting in extraction of the chlorophyll a or bacteriochlorophyll a, which is a pigment, from the vesicle. Here, optical densities were measured at approximately 660 nm for the chlorophyll a and at approximately 780 nm for the bacteriochlorophyll a, and applied to the standard curve plotted with the previously-known concentration of chlorophyll a or bacteriochlorophyll a, and the concentration of the separated thylakoid membrane vesicle or intracytoplasmic membrane vesicle was assessed.

[0077] FIG. 2 shows ATP synthesis efficiency according to the concentrations of the thylakoid membrane vesicle and intracytoplasmic membrane vesicle determined by the above-described method. As a result, both of the cell membrane vesicles showed increased ATP synthesis rates according to the increase in concentrations under the light condition, and low degrees of ATP synthesis under the dark condition. In the thylakoid membrane vesicle, the ATP synthesis rate was saturated at a concentration of approximately 80 g chlorophyll a/ml, and in the intracytoplasmic membrane vesicle, similarly, the ATP synthesis rate was saturated at a concentration of approximately 100 g bacteriochlorophyll a/ml. This result shows that in the presence of a high concentration of a photosynthetic apparatus, the photosynthetic apparatus absorbs light, thereby decreasing light permeability, and therefore, the use of the photosynthetic membrane vesicle at the suitable concentration or more may be inefficient. As light intensity is increased, an optimal concentration of the added photosynthetic membrane vesicle may also be increased, by taking the conditions for providing light energy into consideration, a concentration of the photosynthetic membrane vesicle used herein may be determined by those of ordinary skill in the art according to the purpose of research and development.

Example 5

Experiment on Efficiency of ATP Generation Using Two Types of Photosynthetic Cell Membrane Vesicles with Different Absorption Wavelengths

[0078] The thylakoid membrane vesicle absorbs a short wavelength, which is shorter than and around the wavelength of 660 nm, but the intracytoplasmic membrane vesicle directly uses light energy at wavelength in a range of 800 to 900 nm in photosynthesis. Accordingly, when the two types of photosynthetic membrane vesicles were used at the same time to induce the light-dependent reaction, they will absorb light energy respectively at corresponding wavelengths, and thus one vesicle will not affect the photosynthesis efficiency of the other. Therefore, it was expected that ATP can be synthesized in a limited space with relatively higher efficiency. To confirm this, while the concentration of the separated thylakoid membrane vesicle was fixed, the intracytoplasmic membrane vesicle was added at varying concentrations, followed by measuring an ATP synthesis rate using the same method as described in Example 3. To simultaneously measure the ATP synthesis efficiency of the two types of photosynthetic membrane vesicles, incandescent light having a wide wavelength range of a light source was applied at an intensity of 15 Watts/m.sup.2. FIG. 3 shows ATP synthesis efficiency measured when ATP is produced only using the thylakoid membrane vesicle and when ATP is produced using the thylakoid membrane vesicle and the intracytoplasmic membrane vesicle together. From the conditions in which the concentration of the intracytoplasmic membrane vesicle is beyond 60 to 80 g bacteriochlorophyll a/ml, the ATP synthesis rate is no longer increased according to the concentration of the added photosynthetic apparatus, and thus its value was saturated. However, under all the concentrations at which the ATP synthesis rate is not saturated and saturated, an experiment was conducted by the addition of the two types of photosynthetic membrane vesicles, resulting in relatively higher ATP synthesis efficiency. This result shows that the two types of photosynthetic membrane vesicles absorb light at different wavelengths. Moreover, when the saturation of the ATP synthesis rate is saturated by the use of a high concentration of either one of the photosynthetic membrane vesicles, the two types of photosynthetic membrane vesicles can be used together, thereby synthesizing ATP with higher efficiency.

Example 6

Experiment on Reduction of NADP Using Thylakoid Cell Membrane Vesicle

[0079] The electron flow taking place in the thylakoid membrane vesicle during light irradiation is transferred to ferredoxin, and finally generates NADPH by reduction of NADP.sup.+. The NADPH generated thereby may be converted into NADH by the action of pyridine nucleotide transhydrogenase (Pnt) expected to be present in the cell membrane vesicle. Such NADH and NADPH are necessarily required for various types of biosynthesis in vivo, and have become critical products of the light-dependent reactions in photosynthesis as well as ATP. Accordingly, in this example, to investigate whether the separated thylakoid membrane vesicle also enables reduction to NADH and NADPH as well as ATP synthesis activity under a light condition, efficiency of reduction to NADH and NADPH was assessed. The efficiency of reduction to NADH and NADPH may be assessed by various methods capable of selectively detecting the two substances (mostly by using a method of quantifying NADH and NADPH by oxidizing NADH and NADPH to induce an enzyme reaction for converting a transparent substrate using the oxidized substances as a driving force, resulting in a final product having an optical density with respect to a specific wavelength or fluorescence, and then measuring the final product), and the optimal method can be determined by those of ordinary skill in the art depending on the purpose of research and development. In this example, as the method of quantifying NADH and NADPH, an NADH assay kit (BioVision) and an NADPH assay kit (BioVision) were used. Both types of kits were used to selectively measure NADH and NADPH.

[0080] A reaction was conducted with a 50 mM PBS buffer (phosphate buffered saline, pH 7.4) and a solution containing 1 mM NAD and 1 mM NADP as substrates at 30 C. To assess NADPH synthesis efficiency, only NADP was added to a reaction solution, but to assess NADH synthesis efficiency, both of NAD and NADP were added thereto. By using the quantification of a protein contained in the thylakoid membrane vesicle used herein, the protein concentration was determined, and values obtained by several measurements were normalized to a value of the concentration of the protein contained in the thylakoid membrane vesicle. A degree of NADH and NADPH synthesis was assessed by a change in optical density at 450 nm, and a standard curve was plotted through an experiment performed as described above using various concentrations of NADH and NADPH, and thereby the measured value of the optical density can be converted into amounts of NADH and NADPH generation. The amounts of NADH and NADPH generation determined thereby were expressed as values over time, and the NADH and NADPH production rates were expressed as nmole values of NADH and NADPH produced per minute. FIG. 4 shows NADH and NADPH synthesis efficiency using the thylakoid membrane vesicle determined by the above-described method by applying fluorescent light with an intensity of 50 micro Einstein/m.sup.2.Math.s. As a result, the NADH and NADPH synthesis rates under the dark condition in which light is not applied were considerably lower than those under the light condition, and from this result, it can be considered that most of NADH and NADPH generated in this example were products of the light-dependent reaction in photosynthesis using the light. As a result, the NADPH production efficiency was higher than that of NADH, and thus the method of generating a light-dependent reaction product using the thylakoid membrane vesicle may be useful to induce a biosynthetic reaction that consumes ATP or NADPH. However, from the result shown in FIG. 4, it was also identified that a considerable level of NADH is also generated by the action of a nucleotide transhydrogenase, and may also be employed to induce biosynthesis consuming NADH by using the activity of the nucleotide transhydrogenase present in the thylakoid membrane vesicle.

Example 7

Experiment on Reduction of NADP Using Intracytoplasmic Membrane Vesicle

[0081] When, other than the photophosphorylation for generating ATP, reverse electron flow takes place in the intracytoplasmic membrane vesicle, succinate is converted into fumarate by the action of succinate dehydrogenase, which is complex II, and NAD is reduced into NADH by complex I. NADH generated thereby may be converted into NADPH by the action of pyridine nucleotide transhydrogenase (Pnt) like in the oxygenic photosynthesis. Accordingly, in this example, to confirm that the separated intracytoplasmic membrane vesicle enables reduction to NADH and NADPH as well as ATP synthesis activity under the light condition, the efficiency of reduction to NADH and NADPH was assessed by the method described in Example 6.

[0082] A reaction was conducted with a 50 mM PBS buffer (phosphate buffered saline, pH 7.4) and a solution containing 1 mM NAD and 1 mM NADP as substrates at 30 C. Unlike Example 6, 5 mM succinate was further added as an electron donor. To assess NADH synthesis efficiency, only NAD.sup.+ was added to a reaction solution, but to assess NADPH synthesis efficiency, both of NAD.sup.+ and NADP.sup.+ were added thereto. By using the quantification of a protein contained in the intracytoplasmic membrane vesicle used herein, the protein concentration was determined, and values obtained by several measurements were normalized to a value of the concentration of the protein contained in the intracytoplasmic membrane vesicle. FIG. 5 shows NADH and NADPH synthesis efficiency using the intracytoplasmic membrane vesicle determined by the above-described method by applying incandescent light at an intensity of 50 micro Einstein/m.sup.2.Math.s. As a result, similar to Example 6, NADH and NADPH synthesis rates under the dark condition in which light is not applied were considerably lower than those under the light condition, and from this result, it can be considered that most of NADH and NADPH generated in this example were products of the light-dependent reactions in photosynthesis using the light. Contrarily, in the intracytoplasmic membrane vesicle, different from the result shown in Example 6, the NADH production efficiency was considerably higher than that of NADPH, and thus the method of generating a light-dependent reaction product using the intracytoplasmic membrane vesicle may be useful to induce a biosynthetic reaction that consumes ATP or NADH. However, it was also identified that a considerable level of NADPH is also generated by the action of a nucleotide transhydrogenase, and therefore, the NADPH production efficiency can also be improved by various additional attempts to increase the activity of nucleotide transhydrogenase.

[0083] Moreover, a method of improving the NADH and NADPH synthesis efficiency using the thylakoid membrane vesicle and intracytoplasmic membrane vesicle together, each using light energy having different wavelength range, has not been described in detail, but may be easily achieved by those of ordinary skill in the art with reference to the method of improving the ATP synthesis efficiency using the thylakoid membrane vesicle and the intracytoplasmic membrane vesicle together, described in Example 5.

[0084] Hereinafter, exemplary embodiments of the present invention will be described in detail. The present invention can be modified and implemented in various forms, and therefore, only specific embodiments will be described in detail. However, the present invention is not limited to specific disclosures, and it should be understood that the present invention includes all modifications, equivalents and alternatives included in the technical idea and scope of the present invention.