JPS62138186A - Atp-nad(p)h reproduction bioreactor - Google Patents

Abstract

PURPOSE:To enable simultaneous production of adenosine triphosphate and a coenzyme, by introducing adenosine triphosphatase and hydrogenase into a plane lipid double-layer membrane, placing the membrane in a reactor and feeding the reactor with hydrogen. CONSTITUTION:Adenosine triphosphatase (ATPase) as an adenosine triphosphate (ATP)-producing enzyme and a hydrogenase (H2ase) as a nico tinamide.adenine.dinucleotide (phosphate)[NAD(P)H]-producing enzyme are introduced together with an electron transfer system into a plane lipid double- layer to obtain a plane membrane 8. The membrane 8 is laminated in a reactor 1. ATP and NAD(P)H can be produced by supplying the reactor 1 with hydrogen (H2) as an energy source. The hydrogen (H2) used in the above process is prefer ably produced by the electrolysis of water with electricity generated from a solar battery.

Description

Detailed Description of the Invention [Field of Industrial Application] The present invention is directed to the use of adenosine triphosphate (ATP), a high-energy phosphate compound essential for biological reactions, and the coenzyme NA.
This relates to a bioreactor that produces D(P)H, and is a bioreactor that can be used not only on the ground, but also on space bases.

[Conventional technology]

Conventionally, enzymatic methods, bacterial cell methods, and the like have been used to reproduce ATP.

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

^HP+^■P=Go2 ADP [1
) ADP + acetyl phosphate = = ATP decacetic acid (2) Figure 2 shows a block diagram of a ^TP reproducing bioreactor using an enzymatic method. In the figure, (1) the main body of the beam acter, (2) and (3) immobilized adenylate kinase and acid-resistant kinase, respectively, and (4) the raw materials acetyl phosphate, adenosine monophosphate (AMP), and adenosindiline. (5) is the pump, (6) is the separator that decomposes the product P from acetic acid, (7) is the recycled A
TP glue+, r-bar.

Next, the actual operation will be explained.

Reproduction of ATP introduces two phosphates into AMP, or
This is done by introducing phosphoric acid into ADP.

In the reactor, 8HP becomes ADP with ATP according to formula (1), and 8DP is further phosphorylated with acetyl phosphoric acid to regenerate ATP.

To explain based on FIG. 2, ADP, 8HP, and acetyl phosphate in the reservoir (4) are transferred to the pump (5).
Immobilized adenylate kinase (2) and immobilized acetate kinase (3) are introduced into the reactor body (1) by
is converted into ATP and acetic acid by both immobilized enzymes. ATP is separated from acetic acid by a separator (6) and stored in a reservoir ('71).

On the other hand, in the J3 bacterial cell method, glucose, etc. is added to a powder made by drying yeast, and the glycolytic enzymes remaining in the powder oxidize the liglucose, and the energy is used to produce AHI. ', ATP is produced by ADP. Batch-type glue acu is generally used.

Currently, there is no established method for producing MAD (PJll) coenzyme, and it is produced in small quantities by combining extraction methods, chemical synthesis methods, fermentation methods, etc.

(Problems to be solved by the invention) Since the conventional enzymatic ATP regeneration bioreactor has the above configuration, ATP regeneration requires the following:
It is necessary to input a phosphoric acid compound such as acedelphosphoric acid as a raw material. However, acetyl phosphoric acid has problems such as being unstable and expensive.

In addition, when using the bacterial cell method, the raw material glucose is similarly expensive, and recovering and purifying ATP from the product is costly and time-consuming, and it is difficult to control because it is a batch method. There are problems such as: Also,
The coenzyme NAD(P)H is also expensive because it is produced in small quantities, and a continuous regeneration reactor has not yet been developed.

In addition, in conventional bioreactors, ATP and 14A
There is no actor who can continuously produce D(P)H at the same time.

-, j - The present invention is a newly created bioreactor to eliminate the drawbacks of the conventional devices as described above and to continuously produce both ATP and NAD(P)11. It was designed for use at space bases, etc.

[Means for solving and overcoming problems]

The present invention uses adenosine triphosph 7tase (ATPasc) as an ATP-generating enzyme, hydrogenase (lI2ase) as a di]dinamide adenine dinucleotide (phosphate)-generating enzyme, and an electron transport system in a planar lipid 2f1 layer. The planar membranes formed by introducing ATP into the membrane are stacked and constructed in a reactor, and ATP, NA[
Hydrogen (H2
) ATP-HAD(P)H characterized by using
Regarding regenerative bioreactors, AT localized in biological membranes
Pase and a planar lipid bilayer membrane into which an electron transport system related to 12as (!) was introduced in a reactor, and AT
Hydrogen (H2) is added from the outside as energy for P regeneration, and a proton gradient and membrane potential are generated on both sides of each membrane, thereby converting ATP from ADP and phosphate, and from NAD(P)'. ATP that reproduces NAD(P)H
-NA[)(P)H reproducing bioreactor,
A new concept ATP-NAD(P)H reproducing bioreactor is provided to replace the conventional ATP reproducing bioreactor.

[For production]

The principle of the bioreactor of the present invention will be explained.

ATP is regenerated from ADP or 8HP in the body by the energy metabolic system represented by glycolysis and oxidative phosphorylation, but when comparing the amount of ATP produced per glucose, oxidative phosphorylation is more efficient than glycolysis. ATPage is an enzyme that directly generates ATP from ADP and phosphoric acid through this oxidative phosphorylation. This AT
Pages are localized in the cell membranes of living organisms, and can supply energy for active transport of ions and substrates by hydrolyzing ATP rather than synthesizing ATP.

The energy for synthesizing ATP requires a difference in protons (hereinafter referred to as ΔpH) or a potential difference (hereinafter referred to as membrane potential) between the inside and outside of the membrane.

In living cells, Δpi+ and membrane potential change due to the oxidation of organic matter,
It is supplied by decomposition of H2O by photoreaction, electron transport chain, etc., but it has been observed that ATP is generated by artificially applying Δpl+ and membrane potential to the cell membrane (t).
Hatao Kagawa: Biomembrane and Bioenergy, published by the University of Tokyo Press)
.

On the other hand, some microorganisms can create a Δroll across this membrane or a membrane potential from H2.

Among these, methane bacteria, which ferment methane, are a representative species. To create ΔpH from this H2, hydrogenase bound to the cell membrane and an associated electron transport system are required. Due to the action of hydrogenase, H2 molecules in an aqueous solution generate ■'' on the outside of the cell and electrons (hereinafter referred to as
(referred to as O).

This H+ enters the inside of the cell via ATPase, and as a result, ATP is synthesized. Further, the e discharged inside finally reduces NAD(P)+ to generate HAD(P)I+ via the electron transport chain related to H2ase. The latter reaction route is shown in the formula below. Here X G, t H
This is an electron transport system related to 2ase.

The present invention applies such a principle, and uses a lipid bilayer membrane in which ATPase, 12ase, and an electron transport system are introduced and reconstituted instead of a biological membrane. ATP and N
In order to increase the regeneration efficiency of A D (P) H and provide strength, a lipid bilayer membrane was constructed in a planar manner using a porous polymer sheet 1- as a carrier, and this planar membrane was placed in a reactor. Used in layers. By doing this, hydrogen (H2
) is added to each membrane more efficiently than biological membranes or liposomes, increasing the amount of ATP and HAD(P)H regenerated, and since it is a fixed type, continuous ATP and MAD(P)H can be generated. becomes possible to play.

〔Example〕

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

In Figure 1, (1) the main body of the beam actor, (5) the pump, ('7) the ATP-NAD(P)H reservoir, and (8) the introduction of -7=ATPase, H2ase, and electron transport system. A planar membrane consisting of a flat lipid bilayer membrane and a porous polymer sheet that holds this membrane, (9) is ADP-HAD (P).
+Reservoir (Foundation) is a ■2 generator and reservoir that supplies H2.

Figure 3 is a microscopic diagram of a planar membrane (8) into which ATPaseQl) and H2aS (!02) and related electron transport systems are introduced. It is a sheet and holds a (to) planar lipid bilayer membrane.

The planar lipid 2tfi and layer membrane used in the present invention are bilayer membranes formed from lipids such as natural lipids or synthetic lipids, and the thickness of the bilayer membrane is usually about 1 to 50 nm. The planar lipid bilayer membrane contains ATPase and H2
It functions to maintain asa and form the proton gradient and membrane potential necessary for hetl' synthesis.

ATPase can be extracted from any of the following sources, such as mido]ndria, bacterial membranes, and chloroplasts, but ATPase derived from thermophilic organisms has the highest stability.

H2ase and related electron transport systems may be extracted from methane bacteria, but it is not necessary to completely purify them. Furthermore, H2ase derived from hyperthermic methane bacteria is highly safe.

As the material of the porous polymer sheet for holding the planar lipid bilayer membrane, a fluororesin sheet such as Teflon is used, but the material is not limited thereto.

The thickness of the sheet used is about 10 to 1 mm, and the sheet has pores with a pore diameter of about 0.01 to 5.0 M in order to hold the planar lipid bilayer membrane.
．． 01-0.8TIi/TI1. There are certain degrees.

In the above explanation, the case was explained using a porous polymer sheet, but a planar lipid bilayer membrane may be held in a porous microcapsule as shown in FIG. vesicles) with similar behavior,
ATP can be regenerated more stably. In FIG. 4, (c) is a planar lipid bilayer structure into which ATPase and H2ase have been introduced, and 0e3 is the main body of a one-microphone cell.

Similar results can also be obtained by using hollow fibers instead of microcapsules.

In the present invention, the above-mentioned porous polymer sheet 0
A planar membrane (8) may be formed from a planar lipid bilayer membrane (to) into which ψ, ATPaseoll, and H2ase (12J) are introduced, and the planar membrane (8) formed in this way
This bioreactor is usually fabricated by laminating two to several hundred layers in the reactor so that the distance between the planar legs is about 0.1 to 5 layers.

Next, the operation of the bioreactor of the present invention will be explained based on FIGS. 1 and 3. Planar membranes (8) incorporating ATPaseoll and H2ase and related electron transport systems (12) are stacked in a reactor, and an aqueous solution saturated with H2 and an aqueous solution saturated with H2 are placed between each plane membrane.
An aqueous solution containing OP-NAD(P) was placed in the first stage with H
In the second stage, the aqueous solution saturated with 2 was ^DP-NAD (
An aqueous solution containing P) and an aqueous solution saturated with 1 (2) are alternately flowed into the third stage. ■ The aqueous solution saturated with 2 is recirculated for reuse. is separated into ■1 and e by 82ase in the planar membrane and the related electron transport chain, and the generated H+ synthesizes ATP from ^DP via ATPase, and e is NAD(P)+
is reduced to produce NAD(P)H. The reaction progresses continuously, and by combining the main reactor that consumes ATP and MAD(P)H, continuous ATP/NAD(
It is possible to generate P)H.

In the above explanation, a planar membrane was formed using a lipid bilayer membrane, but the invention is not limited to a planar membrane, and it goes without saying that liposomes may also be used. FIG. 5 schematically shows a lipid bilayer membrane liposome 07) incorporating ATPase (II), H2ase, and an electron transport system, and its operation 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 6 schematically shows the formation of a polymerized lipid bilayer membrane liposome.
Lipid bilayer membrane liposome 09 is a polymerized lipid bilayer membrane formed by polymerizing the lipid bilayer membrane formed by ultraviolet rays etc.
is formed. The properties of the polymeric lipid bilayer membrane are similar to those of the lipid bilayer membrane in terms of improved stability.

In the above explanation, [(2 energy was used as the energy for synthesizing ATP and N A D (P) +1, but it is not limited to H2, and other physical energies such as electric fields can also be used in the same way. I can do it.

Although the embodiments related to the bioreactor of the present invention have been described, it is possible to use regenerated ATP and NAD(P)H, or to combine them with various enzyme systems in the living body to achieve higher levels of bioreactors. It may also be used to produce trace actors.

FIG. 7 shows a system diagram of the ATP-NAD(P)H regeneration actor for space bases. Possible main reactors include immobilized enzyme reactors that produce high value-added substances and cell culture reactors. Hydrogen is generated by electrolysis of water using electricity from solar cells, and the generated 02 is used for respiration purposes such as cell culture. (1) is the ATP/NAD(P)H bioreactor, (5) is the pump, (to) is the H20 electrolytic cell, ■ is the solar cell, and (2v is the main reactor.) To briefly describe the operation, The power generated by the battery is decomposed by H20 using an H20 electrolysis cell.
H2 and 02 are generated, and H2 is A saturated in an aqueous solution.
TP-NAD(P)H bioreactor (1). This bioreactor (1) is heated by the above operation.
ATP and NAD(P)H are synthesized using the energy of 2 and supplied to the main reactor. H2 is recycled in the form of a saturated aqueous solution. AT consumed in main reactor
P and NAD(P)H are converted into ATP and NAD(P)'' and are recycled and enter the ATP-MAD(P)H bioreactor (1) again as feedstock.

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

Example 1 A Teflon sheet with a thickness of 0.5all+ was punched with 1M diameter holes with a porosity of 50% and cut into 2m x 21 pieces to serve as a support for a bilayer membrane. ATPa extracted from thermophilic bacteria
se (prepared by the method described in Method in Enzymology) and H2a extracted from methane bacteria.
se and the related electron transport chain, H, sch in
The method of dl er et al. Proc, Nat1. Acad,
Sci, USA, 782302 (1980)) was formed as a bilayer membrane on the above-mentioned Teflon sheet support to obtain a planar lipid bilayer membrane incorporating ATPase.

A summary of the method of H. 5chindler 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 Teflon sheet containing the water surface lipid bilayer membrane obtained in this way was placed in water or exposed to water in advance.
Laminated inside. Ten sheets were laminated with a lamination interval of 1 m+. The reactor was constructed of glass or Teflon resin, and a water channel from the pump was formed to allow the substrate to flow uniformly between the stacked Teflon sheets. ＾TP-NAD(P)I
The regeneration of I was carried out in the system shown in FIG. 1 under the following procedure 1. The reservoir contains 50111H Tris-
(hydroxymethylaminomethane) hydrochloric acid (rll17.
5), 10mHHgC#2.10mHADP.

Store 1011 M NAD(P)+ in 50 mH sodium phosphate (pH 7, 5) and pump 5-20 d/min with a peristaltic pump.
The liquid was sent to the reactor at a flow rate of hr. Further, hydrogen (H2) generated by the 82 generator was saturated with water and sent to the bioreactor at the same flow rate as above.

As a result of regenerating TP and MAD(P)H under these conditions, ATP was regenerated from ADP with a conversion rate of 80% or more. NAI)(P)H was also regenerated in the same yield.

〔Effect of the invention〕

When using the bioreactor of the present invention, ATP.

As energy supplied from the outside to form NAD(P)H, H2 is supplied to a double-layered planar film into which ATPase, H2ase, etc. have been introduced, and ATP is directly generated from OP and phosphoric acid, and NAD(P) + is reduced to HA
D(P)H is reproduced. This bioreactor is
It is superior to conventional regeneration methods in terms of continuity and operability, and allows for precise control and ultra-miniaturization.In particular, since external energy is supplied by H2, it can be used for ATP in off-ground locations such as space bases. - It can be suitably used as a NAD(P)H regeneration bioreactor and has the advantage of being effective.

[Brief explanation of drawings]

Figure 1 is a block diagram of an embodiment of the bioreactor of the present invention, Figure 2 is a block diagram of an ATP regeneration bioreactor using an enzymatic method, and Figure 3 is a block diagram of an ATP regeneration bioreactor using an enzyme method.
Fig. 4 is a schematic diagram of a planar lipid bilayer membrane held in porous microcapsules, and Fig. 5 is a microscopic diagram of a planar membrane (8) containing ATPase and H2aSeQ2).
A schematic diagram of a lipid bilayer membrane liposome incorporating 2ase, Fig. 6 is a schematic diagram of the formation of a polymerized lipid bilayer membrane liposome, and Fig. 7 is an ATP-HAD (P) for space base use.
The system diagram of the H (re) generation bioreactor is shown. (Main symbol @ in the drawing) (1): Reactor main body (8): Planar membrane 01): ATPase 02): H2ase (131: Planar lipid bilayer membrane 0Φ : Porous polymer sheet CF3: Planar lipid bilayer membrane aa: Microcapsule body 0c: Lipid bilayer membrane Liposome 08 = Polymerizable lipid (to): Polymerized lipid bilayer membrane Baum ■ Two solar cells (2v: Main reactor (221: H20 electrolysis cell representative Yusai Oiwa 3 Figure 11: ATPase 12: H2ase

Claims (2)

[Claims] (1) Using adenosine triphosphatase (ATPase) as an ATP-generating enzyme, nicotinamide adenine dinucleotide (phosphate) (NAD(P)H
) Hydrogenase (H_2ase) as a producing enzyme,
A planar membrane formed by introducing an electron transport system into a planar lipid bilayer membrane is stacked to construct a reactor.
, hydrogen (
ATP・NAD(P
)H reproduction bioreactor. (2) Claim No. (2) characterized in that H_2 is supplied by electrolysis of water using power from a solar cell.
The ATP/NAD(P)H reproducing bioreactor described in section 1).