JPS61124384A - Atp reproduction bioreactor - Google Patents

Abstract

PURPOSE:To provide the titled bioreactor, excellent in the continuity, operability and the cost of raw material, etc., by introducing an ATP-synthetase used as an ATP-producing enzyme into a flat lipid double-layered membrane, laminating the flat membranes in a reactor, and applying physical energy from an external source. CONSTITUTION:The plane membranes 8 integrated with an ATP-synthetase (ATPase) 12 are laminated in the reactor 1. An electrical potential (DC pulse or DC) is applied to the electrode 9 from an external power source 10 in a manner that the membrane potential optimum for the synthesis of ATP (about 200-250mV) is applied to both sides of each membrane 8. In the course of the above process, ADP (adenosine phosphate) and phosphoric acid are introduced (4) from the reservoir 4 to the reactor 1. An ATP-reproduction reactor is produced between the membranes 8 laminated with ATP by the membrane potential generated in the plane lipid double-layered membrane 11, by this process. The reaction proceeds continuously during the above process, and accordingly, a continuous ATP-reproduction can be carried out by combining the above reactor with a main reactor consuming ATP.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field 1] The present invention relates to a bioreactor that regenerates azo/syn triphosphate (ATP), a high-energy phosphoric acid compound essential for biological reactions.

[Prior Art] Conventionally, enzymatic methods, painting methods, and the like have been employed for the reproduction of ATP.

For example, in the enzymatic method, the formula (
7-denylate kinase, which catalyzes formula (1), and acetate kinase, which catalyzes formula (2), are commonly used.

^HP+ATP dooryo 2^OP (t) 80 P + 7 Se+ Le'J 7 tel = Niko A T P +n N (2) ATP reproduction bio+772 goo block using # identity in Figure 2 0 showing diagram
In the figure, (1) beam 7, one main body, (2) and (3
) are immobilized adenylate kinase and acetate kinase, respectively, and (4) are the raw materials 7 cetyl phosphate and 7 denosine monoline am! (＾HP) and 7denonnorin #
I! (^OP) and a reservoir, (5) a gas pump, (6) a separator that decomposes the product ATP from acetic acid, and (7) a glue reservoir for the recycled ATP.

Next, the actual operation will be explained.

To reproduce ATP, either introduce two phosphoric acids into HP, or
This is done by introducing one phosphoric acid into ^OP.

In the reactor, ^HP is converted to ^OP by ATP according to formula (1), and ^DP is further phosphorylated by 7-cetyl phosphate to regenerate ATP.

To explain based on Fig. 2, DP, HP, and 7 cetyl phosphate in the reservoir (4) are introduced into the 7 cell (1) by the pump (5), and the immobilized 7 denylate kinase (2) It is converted into ATP and acetate by both immobilized enzymes, and immobilized acetate kinase (3). A.T.
P is separated from acetic acid by a separator (6) and stored in a reservoir (7).

On the other hand, in the painting method, glucosin, etc. are added to a powder made by drying yeast sono, and the glycolytic enzymes remaining in the powder turn the glucose into a form of sugar, and the energy is used to produce ^HP,^ ATP is produced from OP. Generally, a batch type glue tank is used.

[Problems to be solved by the invention] Since the conventional enzymatic method ATP regeneration bioreactor has the above-mentioned configuration, a phosphoric acid compound typified by 7-cetyl phosphate is used as a raw material for ATP regeneration. It is necessary to input However, 7-cetyl phosphoric acid has problems such as being unstable and expensive.

In addition, in the case of the image-based method, the raw material glucose is similarly expensive, and it is costly and time-consuming to recover and purify ATP from the product, and it is difficult to control because it is a batch method. The present invention has been made to solve the problems of σ, which are the deficiencies of the conventional devices as described in 1-1.

Means for Solving Problems E The present invention utilizes ATP synthase (^d) as an ATP regenerating enzyme.
enosineLripl+ospl+alse :A
TPase), this enzyme is introduced into a planar lipid bilayer membrane, and the planar membranes formed are stacked together in a reactor, and an external physical engineering agent, Toluie, is used as energy to regenerate ATP. Regarding the ATP regeneration bioreactor, ATPa localized in biological membranes
Planar lipid bilayer membranes containing se are stacked in a membrane, external physical energy is applied as energy for ATP regeneration, and proton gradients and membrane potentials are generated on both sides of each membrane. and phosphoric acid and Gara A
ATP regeneration bioreactor that regenerates TI'
This invention provides an ATP regeneration bioreactor with a new concept compared to the conventional ATP regeneration bioreactor.

[Example 1 The principle of the bioreactor of the present invention will be explained.

ATP is regenerated from ^OP* or ^HP in the living body by the energy conversion lIf system, which is represented by glycolysis and oxidative phosphorylation, but when comparing the amount of ATP produced per glucose, oxidative phosphorylation Oxidation is more efficient than glycolysis. The enzyme that generates ATP from directly seeded DP and phosphoric acid through this oxidative phosphorylation is ATPage. The second ATPage is localized in the cell membrane of living organisms and
In addition to the synthesis of ATP, it can also provide energy for the active transport of ions, substrates, etc. by hydrolyzing ATP.

Energy for synthesizing ATP requires a difference in proton ions (ΔpH) or a potential difference (membrane potential) between the inside and outside of the membrane. In living cells, ΔpH and membrane potential are supplied by the oxidation of organic substances, the decomposition of H and 0 by photoreactions, and the electron transport system. It is also known that ATP is generated in A dormitory 8 (Hatao Kagawa: Biomembranes and bioenergy, University of Tokyo Irukai).

The present invention applies such a principle, and instead of biomembranes, ATPase is introduced and reconstituted fat If! t
A double layer membrane is used. In order to increase ATP regeneration efficiency and provide strength, a porous polymer sheet is used as a carrier, and lipid 2tf
LMm is constructed in a planar manner, and the planar membranes are stacked and used in a reactor. By doing this, 'iI&
Compared to biological membranes and liposomes, external energy from fields is applied to each membrane more efficiently, and ATP
Continuous ATP because regeneration increases and is fixed
becomes possible to play.

Next, one embodiment of the bioreactor of the present invention will be described based on a block diagram (FIG. 1).

In Figure 1, (1) beam actor 1 (8) is ATPa
A planar membrane consisting of a planar lipid 2I single-layer membrane into which se has been introduced and a porous polymer sheet that holds this membrane, (9) is an electrode for applying an electric field, (10) is its power source, and (4) is a raw material. A reservoir for OP and puric acid, (5) is a pump that introduces raw materials into the reactor, and ()) is a reservoir that stores ATP.

Figure 3 is a microscopic diagram of the planar membrane (8) into which ATPase has been introduced. (11) is the planar lipid bilayer membrane, (1)
) is ATPase, and (13) is a porous polymer sheet that holds a flat membrane.

The planar lipid bilayer membrane used in the present invention is a bilayer membrane formed from lipids such as natural lipids or synthetic lipids, and usually has a thickness of about 1 to 50 nz. The planar lipid bilayer membrane retains ATPase and A
It functions to form the proton gradient and membrane potential necessary for TP synthesis.

ATPase may be extracted from mitochondria, microwigs, industrial bodies, etc., but ATPase from thermophilic white rice has the highest stability.

As the material of the porous polymer sheet for holding the planar lipid bilayer membrane, a 7-carbon ik resin sheet such as Teflon is used, but the thickness of the sheet is not limited to these. A pore size of about 1G-1000μ2 is used, and the sheet has a pore size of 0.01 to 5.5mm in order to hold a planar lipid bilayer membrane. Holes about the size of Ox shoes are provided at about 0.01 to 0.8 mm 2/layer 2. In the above explanation, the case was explained using a porous molecular sheet, but a planar lipid bilayer membrane may be held in a porous microcapsule as shown in Fig. 4. AT with more stable operation
P can be played. In addition, in Figure 4 (14
) is a planar lipid bilayer membrane into which ATPase has been introduced, ' (
ts) is the microcapsule body.

In the present invention, the porous polymer sheet as described above)
A planar membrane (8) may be formed from a planar lipid bilayer m (11) into which ATPase (13) and ATPase (1)) have been introduced, and the planar membrane (8) thus formed is usually An ATPase reproducing bioreactor is fabricated by stacking about 2 to 100 WL layers in the reactor so that the spacing between the planar membranes is about 0.1 to 511.

Next, the operation of the bioreactor of the present invention will be explained.

A planar membrane (8) incorporating ATPase (1)) is
They are stacked in the reactor (1) and are placed on both sides of each planar membrane at an optimal membrane potential for ATP synthesis (approximately 200-250).
mV) is generated from the electrode (
A voltage (DC pulse or DC) is applied to 9), and the 80P and phosphoric acid are pumped from the reservoir (4) into the pump (5) and introduced into the planar lipid bilayer membrane (11).
Based on the membrane potential generated at
By combining a main reactor that consumes TP, continuous ATP regeneration is possible.

In the above explanation, a planar membrane was formed using the lipids 2i1 and 1lffl, but it is not limited to a planar membrane, and it goes without saying that liposomes (closed vesicles) may also be used. FIG. 5 shows an accidental lipid bilayer membrane liposome (16) incorporating ATPase (1), and its behavior is similar to that of a planar membrane.

Further, by forming a lipid bilayer membrane using a polymerizable lipid and polymerizing the lipid bilayer membrane, the formed planar membrane or liposome can be further stabilized. Figure v & 6 shows the composition of the formation of polymerized lipid double waste imitation liposomes.
The lipid bilayer membrane formed from step 17) is polymerized by ultraviolet rays or the like to form a polymerized lipid bilayer membrane liposome (18). The properties of the polymeric lipid bilayer membrane are similar to those of the lipid bilayer membrane, except that the stability is improved.

In the above explanation, energy generated by an electric field was used as the energy for synthesizing ATP, but it is not limited to energy generated by an electric field, and other physical energy (such as light) can be used.
can also be used.

When bacteriorhodopsin extracted from highly halophilic bacteria is added to a 21-layer lipid membrane, a proton gradient (ΔpH) is formed on both sides of the membrane by light irradiation, and ATP is similarly regenerated.

Planar lipid bilayer membrane (11) incorporating ATPase (1)) and bacteriorhodopsin (19) in IJ&7
), when this membrane is irradiated with light, bacteriorhodopsin acts as a photoproton pump, pushing protons to one side of the membrane, creating a proton gradient (
ΔpH) is formed. After the proton gradient is formed, the behavior is similar to that when an electric field is applied.

Furthermore, by using hydrogenase (6=ase), an enzyme that catalyzes the reaction shown in formula (3), and its electron transport chain, ΔpH is formed with hydrogen as the starting point, and ATP is synthesized, Ht ==: Yo2 H÷ + 2e (3
)l! Figure 118 shows ATPage (1)), Hzase (
20) and a planar lipid bilayer IK (11) incorporating an electron transport system (21). When this membrane is used, hydrogen molecules are converted to protons (I) by Il□ase (20).
t”) and electrons (e), and form an electron transport chain (2
1) is transmitted separately to both sides of the membrane, resulting in the formation of a proton gradient (ΔpH) on both sides of the membrane. ΔP
The operation after it is formed is the same as when a field is added.

Although the embodiment of the bioreactor of the present invention has been described, it may also be an actor that utilizes regenerated ATP. It's okay to frown.

Next, the bioreactor of the present invention will be explained based on Examples.

Example 1 A tear sheet with a thickness of 0.5 m+a was made with holes of 1-diameter and a porosity of 50%, and cut into pieces of 2 cm x 2 e- to serve as a support for a bilayer membrane. A extracted from thermophilic bacteria
TPase(Method in Enzysolog)
y) was prepared by the method described in R. 5ehindler et al. (Proc, Natl, ^aad.

Sci, US^, 71'l 2302 (1980))
A planar lipid bilayer membrane incorporating ATPase was obtained by forming a bilayer membrane on the above-mentioned Teflon sheet support.

A summary of the method of H. 5ehindler et al.
The endoplasmic reticulum formed of bacterial phospholipids containing E is destroyed on the water surface by surface tension, and the resulting monolayer film on the water surface is brought together from both sides in a pinhole of a Teflon sheet. All operations were performed underwater.

The tear sheets into which the planar lipid bilayer membranes prepared in this way were introduced were placed in water or stacked in a tank. Laminated darkness is 1+＊＋＊! O sheets were laminated. -J7 is made of lath or tear resin,
A water channel from the pump was formed to allow the substrate to flow evenly between the stacked Teflon sheets. Platinum electrodes with the same area as the Teflon sheet were attached to the upper and lower parts.

FIG. 9 is an explanatory cross-sectional view of the actor obtained as described above.

The test was carried out under conditions below which allow ATP regeneration.

The reservoir contained 50-M tris(hydroxymethylaminomethane) salt m1 (pH 7,5), 10mM HgC
l2.10mMADP, 505M! j :'el
f ) ') 'F A (pH 7,5) substrate is stored and sento pump is used for 50m! The liquid was delivered to 7 tanks at a flow rate of '/hr. The upper and lower electrodes of 1 no. 7 were placed so that a membrane potential of 200 to 250 mV was applied to each plane double layer membrane.
Apply a DC voltage of too-2000V every 30 seconds (30 seconds ON = 30 seconds 0FF).

As a result of establishing ATP under these conditions, 80P
ATP was able to rise to ^OP and Pi with a conversion rate of 80% or more.

[Effect of the invention 1] When the bioreactor of the present invention is used, electric field, light, H
By applying 2 or the like to a planar lipid double N membrane into which ATPage has been introduced, ATP is directly regenerated from 〇P and phosphoric acid. Therefore, it is recommended to switch to an ATP regeneration bioreactor that is superior in terms of continuity, operability, and raw material cost.
Furthermore, there are no by-products such as acetic acid, there is no need for a separator to separate ATP, and the device can be miniaturized and precisely controlled, allowing it to be used as an implantable device in a living body.

[Brief explanation of the drawing]

Fig. 1 is a block diagram of ATP regeneration by No. 177 Kugu by an embodiment of the bioreactor of the present invention, Fig. 2 is a block diagram of an ATP regeneration bioreactor using a conventional enzyme method, and Fig. 3 is a block diagram of ATP regeneration bioreactor using ATPase. Fig. 4 is a schematic diagram of a planar lipid bilayer membrane incorporated into a microcapsule, and Fig. 5 is a schematic diagram of a planar lipid bilayer membrane incorporating ATPase-incorporated lipid 2i1. A schematic diagram of a layered membrane liposome; Figure 6 is an explanatory diagram of the formation of a polymerized lipid bilayer membrane liposome; Figure 7 is a schematic diagram of a planar lipid bilayer membrane incorporating ATPage and bacteriorhodopsin; Figure 8 shows ATPa
The pI49 diagram, which is a schematic diagram of a planar lipid bilayer membrane incorporating a ge, H, ase, 'il transport system, is a cross-sectional WJ explanatory diagram of the bioreactor used in Example 1. In the figure, (1) Beam Actor 1 (8) is a planar membrane, (9) is an electrode, (11) is a planar lipid bilayer membrane, and (1)) is ATP.
age, (14) is ATPage-introduced planar lipid 2
Multilayer membrane, (16) is lipid bilayer membrane liposome, (17)
is a polymerizable lipid, (18) is a polymerized lipid bilayer membrane, (19) is bacteriorhodopsin, (20)
) is H-ase, and (21) is an electron transport chain. Note that the same reference numerals in each drawing indicate the same or corresponding parts.

Claims (5)

[Claims] (1) ATP synthase (Aden
osinetriphosphate: ATPase
), this enzyme is introduced into a planar lipid bilayer membrane, and the planar membranes formed are stacked together to construct a reactor, and external physical energy is used as energy to regenerate ATP. ATP regeneration bioreactor. (2) The ATP regeneration bioreactor according to claim (1), characterized in that a planar lipid bilayer membrane is formed in the pores of a porous polymer sheet. (3) Claim No. 1 (2) characterized in that energy from an electric field is used as external physical energy.
1) ATP regeneration bioreactor as described in section 1). (4) The ATP regeneration bioreactor according to claim (1), characterized in that optical energy is used as the external physical energy and bacteriorhodopsin is used as the optical proton pump. (5) Hydrogenase, an enzyme that uses external physical energy to separate hydrogen into protons (H^+) and electrons (e), and its electron transport system are incorporated into a planar lipid bilayer membrane together with ATPase. The ATP regeneration bioreactor according to claim (1), which is used.