WO1998048031A1 - Process for producing adenosine 5'-triphosphate and use thereof - Google Patents
Process for producing adenosine 5'-triphosphate and use thereof Download PDFInfo
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- WO1998048031A1 WO1998048031A1 PCT/JP1998/001711 JP9801711W WO9848031A1 WO 1998048031 A1 WO1998048031 A1 WO 1998048031A1 JP 9801711 W JP9801711 W JP 9801711W WO 9848031 A1 WO9848031 A1 WO 9848031A1
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
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- C12P19/32—Nucleotides having a condensed ring system containing a six-membered ring having two N-atoms in the same ring, e.g. purine nucleotides, nicotineamide-adenine dinucleotide
Definitions
- the present invention provides a method for producing adenosine 5′-triphosphate (ATP) by reacting adenosine 5′-monophosphate (AMP) with polyphosphate kinase, adenylate kinase and polyphosphate. It is about its application.
- ATP adenosine 5′-triphosphate
- AMP adenosine 5′-monophosphate
- ATP is required as an energy donor or a phosphate donor.
- ATP is supplied from the organism of the microorganism used, but in enzymatic methods, ATP must be added to the reaction system or an efficient ATP regeneration system must be developed. Is essential.
- ATP is synthesized from AMP or adenine using a chemical synthesis method or a microorganism or yeast.
- an inexpensive method for synthesizing ATP has not been established at present, and commercially available ATP is extremely expensive.
- an object of the present invention is to provide a method for producing or regenerating expensive ATP more efficiently than AMP. Disclosure of the invention
- the present inventors have conducted studies to achieve the above object, and found that the activity of synthesizing ATP from AMP by coupling to adenylate kinase in the presence of polyphosphate kinase And completed the present invention.
- the present invention provides a method for producing adenosine 5'-triphosphate, which comprises reacting adenosine 5'-monophosphate with polyphosphate kinase, adenylate kinase and polyphosphate. Is what you do.
- the present invention provides a method for producing a compound using an enzymatic reaction consuming adenosine 5'-triphosphate, wherein adenosine 5'-monophosphate is replaced by polyphosphate kinase, adenylate kinase and polylysine.
- a process for producing the compound characterized in that adenosine 5′-triphosphate is produced by the action of an acid and supplied to the enzyme reaction.
- the present invention provides a method for producing a compound using an enzymatic reaction consuming adenosine 5'-triphosphate, wherein the produced adenosine 5'-phosphate and Z or adenosine 5'-diphosphate are produced.
- a method for producing the compound characterized in that the enzyme reaction is carried out while adenosine 5'-triphosphate is regenerated by reacting polyphosphate kinase, adenylate kinase and polyphosphate with the enzyme. Is what you do.
- the present invention relates to an adenosine 5'-to adenosine 5'-triamine comprising a combination of polyphosphate kinase, adenylate kinase and polyphosphate.
- the present invention provides a system for synthesizing an acid.
- FIG. 1 shows changes in AMP, ADP, and ATP in the ATP synthesis system of the present invention.
- the polyphosphate kinase (EC 2.7.4.1) and adenylate kinase (EC 2.7.4.3) used in the present invention are both known enzymes, and are derived from animals, plants and microorganisms. Those of origin and the like can be used. Among them, microorganisms, particularly Escherichia coli-derived polyphosphate kinase and adenylate kinase, are advantageous in terms of ease of enzyme preparation and the like.
- the polyphosphate kinase gene or adenylate kinase gene is cloned using recent gene recombination technology, and polyphosphate kinase or adenylate kinase is mass-produced using E. coli or the like as a host. It is also possible to prepare the above two types of enzymes respectively (J. Biol. Chem., 267, 22556-22561 (1992), Nucleic Acids Res., 13, 7139-7151 (1985)).
- the polyphosphate kinase and adenylate kinase added to the reaction system may be in any form as long as they have the activity.
- the preparation of the cells of the c microorganism which can be exemplified by the cells of the microorganism, the processed product of the cell, or the enzyme preparation obtained from the processed product, is performed by using a medium in which the microorganism can grow. It can be carried out by a conventional method, followed by culturing by a conventional method, and collecting cells by centrifugation or the like.
- bacteria belonging to the genus Bacillus or E. coli are described as examples.
- the culture medium is bouillon medium, LB medium (1% tryptone, 0.5% yeast extract, 1% salt) or 2XYT A medium (1.6% tryptone, 1% yeast extract, 0.5% salt) can be used.
- LB medium 1% tryptone, 0.5% yeast extract, 1% salt
- 2XYT A medium (1.6% tryptone, 1% yeast extract, 0.5% salt) can be used.
- the treated cells of the microorganisms can be obtained by mechanically crushing the above microorganisms (using a ring blender, French press, homogenizer, mortar, etc.), freeze-thawing, self-digesting, drying (freeze-drying, air-drying, etc.) ), Enzyme treatment (eg, with lysozyme), ultrasonic treatment, chemical treatment (eg, with acid or alkali treatment), etc.
- Enzyme treatment eg, with lysozyme
- ultrasonic treatment eg, with acid or alkali treatment
- chemical treatment eg, with acid or alkali treatment
- a fraction having polyphosphoric acid kinase activity or adenylate kinase activity from the above treated cells is purified by a conventional enzyme purification method (salt-out treatment, isoelectric point precipitation treatment, organic solvent precipitation treatment). , Dialysis, various types of chromatography, etc.).
- AMP can be used for the present invention.
- concentration used eg if 1 to 2 0 O mM, preferably. 1 to 5 0 mM range c and may be appropriately set from, polyphosphate can be used a commercially available added.
- concentration used can be appropriately set, for example, in the range of 1 to 100 mM, preferably 10 to 100 mM in terms of inorganic phosphoric acid.
- ATP can be produced by, for example, adding AMP and polyphosphoric acid to an appropriate buffer having a pH in the range of 4 to 9, and further adding 0.001 units or more, preferably 0.01 to 1 unit. 0.1 units of polyphosphate kinase, and 0.01 units or more, preferably 0.01 to 100 units // of adenylate kinase, and added at 20 ° C or more. The reaction can be carried out preferably at 30 to 40 ° C. for about 1 to 50 hours with stirring as necessary.
- the ATP thus prepared can be isolated and purified by known methods.c Also, in a method for producing a compound using an enzyme reaction consuming ATP, The compound can be produced by reacting AMP with the above-mentioned polyphosphate kinase, adenylate kinase and ATP produced by reacting polyphosphoric acid to the enzyme reaction to supply the compound to the enzyme reaction. . In particular, in a method for producing a compound using an enzymatic reaction that consumes ATP, it is possible to carry out the reaction while regenerating ATP using AMP and / or ADP generated by the enzymatic reaction as a raw material.
- the target compound can be specifically produced, for example, galactose-1-phosphate synthesis system using galactokinase, UDP synthesis system using UMP kinase, phosphocollage using corin kinase. It can be applied to any enzymatic reaction that consumes ATP, such as a synthesis system.
- reaction conditions for such an ATP synthesis system and the enzymatic reaction may be appropriately determined by a small-scale test, and the target compound can be isolated and purified by a known method.
- Chromosomal DNA of E. coli K12 strain JM109 was prepared by the method of Saito and Miura (Biochim. Biophys. Acta., 72, 619 (1963)). This Using DNA as a template, the following two primers, DNA, were synthesized in a conventional manner, and the E. coli polyphosphate kinase (ppk) gene was amplified by PCR.
- Amplification of the ppk gene by PCR was performed in a reaction mixture of 10 (5 OmM chloride chloride, 10 mM Tris-HCl (pH 8.3), 1.5 mM magnesium chloride, 0.001% gelatin , Temperate DNA 0.1 lg, Primer DNA (A) (B) 0.2 ⁇ M each, Ampli Taq DNA polymerase 2.5 units) were replaced with Perkin-ElmerCetus Instrume. Thermal denaturation (94 ° C, 1 minute), annealing (55 ° C, 1.5 minutes), polymerase reaction (72, 1.5 Min) was repeated 25 times.
- the reaction solution was treated with a mixture of phenol Z-cloth form (1: 1), and the DNA was precipitated by adding twice the volume of ethanol to the water-soluble fraction.
- the DNA collected by precipitation was separated by agarose gel electrophoresis according to the method described in the literature (Molecular cloning. Supra), and a DNA fragment equivalent to Okb was purified.
- the DNA was cleaved with restriction enzymes NcoI and BamHI, and plasmid pTrc99A (obtained from Pharmacia Biotech), also digested with restriction enzymes NcoI and BamHI, and T4 DNA ligase Was connected using Escherichia coli JM109 was transformed using the ligation reaction solution, and plasmid pTrc-PPK was isolated from the obtained ampicillin-resistant transformant.
- pTr c-PPK is obtained by inserting an NcoI-BamHI DNA fragment containing the Escherichia coli ppk gene into the NcoI-BamHI cleavage site downstream of the trc promoter of pTrc99A.
- Escherichia coli JM109 carrying plasmid pTrc-Ppk was inoculated into 2XYT medium 30 containing 10OigZ ⁇ ampicillin, and cultured with shaking. Once at the 4 X 1 0 3 bacteria to a final concentration of 1 mM to the culture solution
- the specific activity of the polyphosphate kinase in the crude enzyme solution was 0.19 unit / mg protein, which was the specific activity of the control bacterium (E. coli JM109 bacterium harboring pTrc99A). 0.18 units (Zmg protein).
- the crude enzyme solution was fractionated using DEAE Toyopearl 6.5 M (Toy Corporation) with a concentration gradient of 0 to 0.5 M Na to obtain a polyphosphate kinase fraction. This fraction was used as a polyphosphate kinase enzyme preparation.
- the specific activity of polyphosphate kinase in this enzyme preparation was 0.6 unit Zmg protein.
- the unit (unit) of polyphosphate kinase activity in the present invention is measured and calculated by the following method. That is, the enzyme preparation was added to a 25 mM Tris-HCl buffer (pH 7.8) containing 5 mM magnesium chloride, 10 OmM ammonium sulfate, 5 mM ADP, and polyphosphoric acid (100 as inorganic phosphoric acid). Te, 3 7 ° to carry out the reaction by incubating at C, and ⁇ in the reaction solution with 1 0 0 ° C, 1 minute annealing c high-performance liquid ⁇ Matogurafi one to stop the reaction by (HP LC) Quantify the activity to produce 1 mo ⁇ e of ATP per minute at 37 ° C. O
- Chromosomal DNA of Escherichia coli 12 strain JM109 was prepared by the method of Saito and Miura (Biochim. Biophys. Acta., 72, 619 (1963)).
- DNA was synthesized according to a conventional method, and the Escherichia coli adduct kinase (adk) gene was amplified by PCR.
- Amplification of the adk gene by PCR was performed in a reaction mixture of 100 mM (50 mM chloride, 10 mM Tris-HCl (pH 8.3), 1.5 raM magnesium chloride, 0.001% 0.1 g of gelatin and temperate DNA, 0.1 M of each of primer DNA (A) and (B), 2.5 M of Amp1i Taq DNA polymerase (2.5 units) were added to Perkin-Elmer C Thermal denaturation (94 min., 1 min.), annealing (56 ° C, 1.0 min.), polymerization (72 min.) were performed using DNA Thermal Cyc 1 er manufactured by etus International. And 3.0 minutes) were repeated 25 times.
- the reaction solution was treated with a mixture of funinol Z-cloth form (1: 1), and DNA was precipitated by adding twice the volume of ethanol to the water-soluble fraction.
- the DNA collected by precipitation was separated by agarose gel electrophoresis according to the method described in the literature (Molecular cloning. Supra), and a DNA fragment equivalent to Okb was purified.
- the DNA was digested with the restriction enzymes BamHI and HindIII, and the plasmid pUC18 (obtained from Takara Shuzo) and T4 DNA digested with the restriction enzymes BamHI and HindIII. Ligation was performed using ligase.
- pUC-ADK Escherichia coli JM109 using ligation reaction solution And the plasmid pUC-ADK was isolated from the resulting ampicillin-resistant transformant.
- pUC-ADK is obtained by inserting a BamHI-Hind UI DNA fragment containing the E. coli adk gene into the BamHI-HindII cleavage site downstream of the lac promoter overnight in pUC18.
- Escherichia coli JM109 carrying plasmid pUC—ADK was inoculated into 300 XYT medium containing 100 Ozg / ampicillin and cultured at 37 ° C with shaking. Once at the 4 X 1 0 8 bacteria Bruno, to a final concentration of 1 mM to the culture solution
- IPTG was added, and shaking culture was continued at 30 ° C. for 5 hours. After completion of the culture, the cells were collected by centrifugation (9,000 X g., 10 minutes), and the buffer of 60 ⁇ (50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0 mM 1% Triton X—100, 0.2 mg Z ⁇ rhizodium). After incubating at 37 ° C for 1 hour, the cells were sonicated to disrupt the cells, and the cells were removed by centrifugation (20,000 X g., 10 minutes).
- the supernatant fraction thus obtained was dialyzed against 5 OmM Tris-HCl (pH 7.8) containing 5 mM magnesium chloride and 1 mill 2-mercaptoethanol to obtain a crude enzyme solution.
- the specific activity of adenylate kinase in the crude enzyme solution was 134 units of Zmg protein, and the specific activity of the control bacterium (E. coli JM109 carrying pUC18) (1.9 units / unit). mg protein).
- the crude enzyme solution DEAE preparative Yono, 0 - fractionated by gradient 0 ⁇ 0. 5 M Na C _ ⁇ with Le 6 5 0 M (DOO one source i (Inc.)), Ade two rate Fractions with kinase activity were collected.
- This fraction was used as an adenylate kinase enzyme preparation.
- the specific activity of polyphosphate kinase in this enzyme preparation was 344 unit Zmg protein.
- the unit (unit) of the adenylate kinase activity in the present invention was also measured and calculated by the following method. That is, an enzyme preparation was added to 50 mM Tris-HCl buffer (pH 7.8) containing 5 mM magnesium chloride, 5 mM ATP, and 5 mM AMP. The reaction is carried out by keeping the temperature at 37 ° C, and the reaction is stopped by heat treatment at 100 ° C for 1 minute. Quantify ADP in the reaction solution using HP LC, and define the activity to produce 2 / mo ⁇ e of ADP per minute at 37 ° C as 1 unit (unit).
- PPK Polyphosphate kinase
- ADK Adenylate kinase
- AMP phosphorylation does not occur when a crude enzyme solution prepared from normal E. coli (JM109 CpUC18)) is mixed with polyphosphoric acid kinase, but adenylate kinase-producing strain ( When the crude enzyme solution prepared from JM109CpUC-ADK)) was mixed with polyphosphate kinase, a marked AMP phosphorylation reaction occurred. From the above, it is clear that co-presence of polyphosphate kinase and adenylate kinase causes AMP phosphorylation.
- the cells were collected by centrifugation (9,000 X g, 10 minutes), and a buffer of 6 ⁇ (50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0 mM 1% Triton X—100, 0.2 mg Z7 ⁇ ribzyme). After incubation at 37 ° C for 1 hour, sonication was performed to disrupt the cells, and centrifugation (20,000 X g,
- the unit (unit) of galactokinase activity was measured and calculated by the following method. Add enzyme preparation to 10 OmM Tris-HCl buffer (pH 7.8) containing 5 mM magnesium chloride, 1 OmM ATP, and 10 mM galactose.
- the reaction is carried out by keeping the temperature at 37 ° C, and the reaction is stopped by heat treatment at 100 T: 1 minute.
- the 1-phosphoric acid is quantified, and the activity to produce 1 mo ⁇ e of galactose- 1-phosphoric acid per minute at 37 ° C is defined as 1 unit (unit).
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Abstract
A process for producing adenosine 5'-triphosphate (ATP) by reacting adenosine 5'-monophosphate (AMP) with a polyphosphate kinase, an adenylate kinase and a polyphosphoric acid. ATP can be easily produced at a low cost by this process.
Description
明 細 書 アデノシン 5 ' —三リ ン酸の製造法及びその応用 技術分野 Description Method for producing adenosine 5'-triphosphate and its application
本発明は、 アデノシン 5 ' —一リン酸 (AMP) にポリ リン酸キナーゼ、 アデ 二レー トキナーゼ及びポリ リ ン酸を作用せしめ、 アデノ シン 5 ' —三リ ン酸 (ATP) を製造する方法及びその応用に関するものである。 背景技術 The present invention provides a method for producing adenosine 5′-triphosphate (ATP) by reacting adenosine 5′-monophosphate (AMP) with polyphosphate kinase, adenylate kinase and polyphosphate. It is about its application. Background art
近年の遺伝子操作技術の進展により、 さまざまな酵素の安価な大量調製が可能 となり、 従来、 微生物菌体を用いた微生物変換あるいは発酵生産により合成され てきた有用生理活性物質が直接酵素反応により安価に製造することが可能となつ てきている。 Recent advances in genetic engineering technology have made it possible to prepare inexpensive large quantities of various enzymes.Useful bioactive substances that have been conventionally synthesized by microbial conversion or fermentation using microbial cells can be produced at low cost by direct enzymatic reactions. It is becoming possible to manufacture.
ところで、 リン酸化反応、 アミノ化反応などの高エネルギーを必要とする酵素 反応には、 AT Pがエネルギー供与体あるいはリン酸供与体として必要である。 従来の微生物変換あるいは発酵生産においては、 AT Pは用いた微生物の生体内 より供給されるが、 酵素法においては AT Pを反応系に添加したり、 効率的な ATPの再生系を開発することが不可欠である。 現在、 ATPは化学合成法ある いは微生物もしくは酵母菌体を用いて AMPもしくはアデニンから合成されてい る。 しかしながら、 ATPの安価な合成法は現時点で確立されておらず、 市販さ れている ATPは極めて高価である。 また、 ATPの再生系としてはホスホクレ ァチンとホスホクレアチンキナーゼとの組み合わせが実験室レベルで使用される 力、 基質、 酵素とも極めて高価であるため実用的ではない。 また、 ポリ リ ン酸キ ナ一ゼとポリ リ ン酸の組み合わせも検討されているが、 依然高価な ATPあるい は AD Pの使用は不可欠であり、 実用化には至っていない。
このように AT Pは極めて高価であるのに対し、 AMPは比較的安価に製造さ れうる。 そのため、 ATPを使用する酵素反応系において、 高価な ATPを添加 するのではなく、 安価な AMPから ATPを製造する方法、 あるいは消費された AT Pを効率的に再生する方法の開発が望まれていた。 By the way, for enzymatic reactions requiring high energy such as phosphorylation and amination, ATP is required as an energy donor or a phosphate donor. In conventional microbial conversion or fermentation production, ATP is supplied from the organism of the microorganism used, but in enzymatic methods, ATP must be added to the reaction system or an efficient ATP regeneration system must be developed. Is essential. At present, ATP is synthesized from AMP or adenine using a chemical synthesis method or a microorganism or yeast. However, an inexpensive method for synthesizing ATP has not been established at present, and commercially available ATP is extremely expensive. In addition, as a system for regenerating ATP, a combination of phosphocreatine and phosphocreatine kinase is not practical because the power, substrates, and enzymes used at the laboratory level are extremely expensive. Also, a combination of polyphosphate kinase and polyphosphate is being studied, but the use of expensive ATP or ADP is still indispensable, and has not yet been put to practical use. Thus, while ATP is extremely expensive, AMP can be produced relatively cheaply. Therefore, in an enzyme reaction system using ATP, it is desired to develop a method for producing ATP from inexpensive AMP or a method for efficiently regenerating consumed ATP, instead of adding expensive ATP. Was.
従って、 本発明は高価な ATPを AMPより効率的に製造または再生する方法 を提供することを目的とするものである。 発明の開示 Therefore, an object of the present invention is to provide a method for producing or regenerating expensive ATP more efficiently than AMP. Disclosure of the invention
本発明者らは、 上記目的を達成すべく研究を重ねた結果、 ポリ リ ン酸キナーゼ 力 \ ポリ リン酸の存在下でアデ二レートキナーゼと共役することにより AMPか ら AT Pを合成する活性を有することを見出し、 本発明を完成させた。 The present inventors have conducted studies to achieve the above object, and found that the activity of synthesizing ATP from AMP by coupling to adenylate kinase in the presence of polyphosphate kinase And completed the present invention.
すなわち、 本発明は、 アデノシン 5 ' —一リ ン酸にポリ リ ン酸キナーゼ、 アデ ニレ一トキナーゼ及びポリ リン酸を作用せしめることを特徴とするアデノシン 5 ' —三リ ン酸の製造法を提供するものである。 That is, the present invention provides a method for producing adenosine 5'-triphosphate, which comprises reacting adenosine 5'-monophosphate with polyphosphate kinase, adenylate kinase and polyphosphate. Is what you do.
また、 本発明は、 アデノシン 5 ' —三リ ン酸を消費する酵素反応を利用した化 合物の製造法において、 アデノシン 5 ' —一リン酸にポリ リン酸キナーゼ、 アデ 二レートキナーゼ及びポリ リ ン酸を作用せしめてアデノシン 5 ' —三リ ン酸を生 成させ、 当該酵素反応に供給することを特徴とする当該化合物の製造法を提供す るものである。 Further, the present invention provides a method for producing a compound using an enzymatic reaction consuming adenosine 5'-triphosphate, wherein adenosine 5'-monophosphate is replaced by polyphosphate kinase, adenylate kinase and polylysine. A process for producing the compound, characterized in that adenosine 5′-triphosphate is produced by the action of an acid and supplied to the enzyme reaction.
さらに、 本発明は、 アデノシン 5 ' —三リ ン酸を消費する酵素反応を利用した 化合物の製造法において、 生成したアデノシン 5 ' ——リ ン酸及び Zまたはアデ ノシン 5 ' —二リ ン酸にポリ リ ン酸キナーゼ、 アデ二レートキナーゼ及びポリ リ ン酸を作用せしめてアデノシン 5 ' —三リ ン酸を再生しながら当該酵素反応を行 うことを特徴とする当該化合物の製造法を提供するものである。 Further, the present invention provides a method for producing a compound using an enzymatic reaction consuming adenosine 5'-triphosphate, wherein the produced adenosine 5'-phosphate and Z or adenosine 5'-diphosphate are produced. A method for producing the compound, characterized in that the enzyme reaction is carried out while adenosine 5'-triphosphate is regenerated by reacting polyphosphate kinase, adenylate kinase and polyphosphate with the enzyme. Is what you do.
さらにまた、 本発明は、 ポリ リ ン酸キナーゼ、 アデ二レートキナーゼ及びポリ リ ン酸を組み合わせてなるアデノシン 5 ' ——リ ン酸からアデノシン 5 ' —三リ
ン酸の合成系を提供するものである。 図面の簡単な説明 Furthermore, the present invention relates to an adenosine 5'-to adenosine 5'-triamine comprising a combination of polyphosphate kinase, adenylate kinase and polyphosphate. The present invention provides a system for synthesizing an acid. BRIEF DESCRIPTION OF THE FIGURES
図 1は、 本発明の ATP合成系における AMP、 ADP、 ATPの消長を示し たものである。 FIG. 1 shows changes in AMP, ADP, and ATP in the ATP synthesis system of the present invention.
発明を実施するための最良の形態 BEST MODE FOR CARRYING OUT THE INVENTION
本発明で使用するポリ リン酸キナーゼ (E. C. 2. 7. 4. 1 ) 及びアデ二 レートキナーゼ (E. C. 2. 7. 4. 3) はいずれも公知の酵素であり、 動物 由来、 植物由来、 微生物由来などのものを使用することができる。 このうち、 酵 素の調製の簡便さなどの点から微生物、 特に大腸菌由来のポリ リン酸キナーゼ及 びアデ二レートキナーゼが使用に好都合である。 また、 近年の遺伝子組み換え技 術を利用してポリ リン酸キナーゼ遺伝子またはアデ二レートキナーゼ遺伝子をク ローン化し、 大腸菌などを宿主としてポリ リン酸キナーゼまたはアデ二レートキ ナーゼを大量生産させ、 当該組み換え菌より上記 2種類の酵素をそれぞれ調製す ることも可能である (J. Biol. Chem., 267, 22556-22561(1992)、 Nucleic Acids Res. , 13, 7139-7151(1985)) 。 The polyphosphate kinase (EC 2.7.4.1) and adenylate kinase (EC 2.7.4.3) used in the present invention are both known enzymes, and are derived from animals, plants and microorganisms. Those of origin and the like can be used. Among them, microorganisms, particularly Escherichia coli-derived polyphosphate kinase and adenylate kinase, are advantageous in terms of ease of enzyme preparation and the like. In addition, the polyphosphate kinase gene or adenylate kinase gene is cloned using recent gene recombination technology, and polyphosphate kinase or adenylate kinase is mass-produced using E. coli or the like as a host. It is also possible to prepare the above two types of enzymes respectively (J. Biol. Chem., 267, 22556-22561 (1992), Nucleic Acids Res., 13, 7139-7151 (1985)).
反応系に添加するポリリン酸キナーゼ及びアデ二レートキナーゼは、 当該活性 を有する限りどのような形態であってもよい。 具体的には、 微生物の菌体、 該菌 体の処理物または該処理物から得られる酵素調製物などを例示することができる c 微生物の菌体の調製は、 当該微生物が生育可能な培地を用い、 常法により培養後、 遠心分離等で集菌する方法で行うことができる。 具体的に、 バシラス属または大 腸菌類に属する細菌を例に挙げ説明すれば、 培地としてはブイヨン培地、 L B培 地 ( 1 %トリプトン、 0. 5 %イーストエキス、 1 %食塩) または 2 X YT培地 ( 1. 6 %トリプトン、 1 %イーストエキス、 0. 5 %食塩) などを使用するこ とができ、 当該培地に種菌を接種後、 3 0〜5 0°Cで 1 0〜5 0時間程度必要に
より攪拌しながら培養し、 得られた培養液を遠心分離して微生物菌体を集菌する ことによりポリ リ ン酸キナーゼ活性またはアデニレ一トキナ一ゼ活性を有する微 生物菌体を調製することができる。 The polyphosphate kinase and adenylate kinase added to the reaction system may be in any form as long as they have the activity. Specifically, the preparation of the cells of the c microorganism, which can be exemplified by the cells of the microorganism, the processed product of the cell, or the enzyme preparation obtained from the processed product, is performed by using a medium in which the microorganism can grow. It can be carried out by a conventional method, followed by culturing by a conventional method, and collecting cells by centrifugation or the like. To be more specific, bacteria belonging to the genus Bacillus or E. coli are described as examples. The culture medium is bouillon medium, LB medium (1% tryptone, 0.5% yeast extract, 1% salt) or 2XYT A medium (1.6% tryptone, 1% yeast extract, 0.5% salt) can be used.After inoculating the medium with the inoculum, 30 to 50 ° C, 10 to 50 hours About need It is possible to prepare microbial cells having polyphosphoric acid kinase activity or adenylate kinase activity by culturing with further stirring and centrifuging the obtained culture solution to collect microbial cells. it can.
微生物の菌体処理物としては、 上記微生物菌体を機械的破壤 (ヮ一リ ングブレ ンダ一、 フレンチプレス、 ホモジナイザー、 乳鉢などによる) 、 凍結融解、 自己 消化、 乾燥 (凍結乾燥、 風乾などによる) 、 酵素処理 (リゾチームなどによる) 、 超音波処理、 化学処理 (酸、 アルカリ処理などによる) などの一般的な処理法に 従って処理して得られる菌体の破壊物または菌体の細胞壁もしくは細胞膜の変性 物を例示することができる。 The treated cells of the microorganisms can be obtained by mechanically crushing the above microorganisms (using a ring blender, French press, homogenizer, mortar, etc.), freeze-thawing, self-digesting, drying (freeze-drying, air-drying, etc.) ), Enzyme treatment (eg, with lysozyme), ultrasonic treatment, chemical treatment (eg, with acid or alkali treatment), etc. A modified product of the above can be exemplified.
酵素調製物としては、 上記菌体処理物からポリ リ ン酸キナーゼ活性またはアデ 二レートキナーゼ活性を有する画分を通常の酵素の精製手段 (塩析処理、 等電点 沈殿処理、 有機溶媒沈殿処理、 透析処理、 各種クロマトグラフィー処理など) を 施して得られる粗酵素または精製酵素を例示することができる。 As the enzyme preparation, a fraction having polyphosphoric acid kinase activity or adenylate kinase activity from the above treated cells is purified by a conventional enzyme purification method (salt-out treatment, isoelectric point precipitation treatment, organic solvent precipitation treatment). , Dialysis, various types of chromatography, etc.).
本発明で使用する A M Pは、 市販のものが使用できる。 使用濃度としては、 例 えば 1〜2 0 O mM、 好ましくは 1〜 5 0 mMの範囲から適宜設定することができる c また、 添加するポリ リン酸も市販のものが使用できる。 使用濃度としては、 例え ば無機リン酸に換算して 1〜 1 0 0 0 mM、 好ましくは 1 0〜 1 0 0 mMの範囲から 適宜設定することができる。 Commercially available AMP can be used for the present invention. The concentration used, eg if 1 to 2 0 O mM, preferably. 1 to 5 0 mM range c and may be appropriately set from, polyphosphate can be used a commercially available added. The concentration used can be appropriately set, for example, in the range of 1 to 100 mM, preferably 10 to 100 mM in terms of inorganic phosphoric acid.
A T Pの製造法は、 例えば pH 4〜 9の範囲の適当な緩衝液中に A M P及びポリ リ ン酸を添加し、 さらに 0 . 0 0 1ユニッ ト/ 以上、 好ましくは 0 . 0 0 1〜 1 0ュニッ ト ^ のポリ リ ン酸キナーゼ、 及び 0 . 0 1ュニッ ト 以上、 好ま しくは 0 . 0 1〜 1 0 0ユニッ ト/ / のアデ二レートキナーゼを添加し、 2 0 °C 以上、 好ましくは 3 0〜4 0 °Cで 1〜5 0時間程、 必要により攪拌しながら反応 させることにより実施できる。 ATP can be produced by, for example, adding AMP and polyphosphoric acid to an appropriate buffer having a pH in the range of 4 to 9, and further adding 0.001 units or more, preferably 0.01 to 1 unit. 0.1 units of polyphosphate kinase, and 0.01 units or more, preferably 0.01 to 100 units // of adenylate kinase, and added at 20 ° C or more. The reaction can be carried out preferably at 30 to 40 ° C. for about 1 to 50 hours with stirring as necessary.
このようにして調製した A T Pは、 公知の方法にて単離精製することができる c また、 A T Pを消費する酵素反応を利用した化合物の製造法においては、
AMPに上記ポリ リ ン酸キナーゼ、 アデ二レートキナ一ゼ及びポリ リ ン酸を作用 せしめて生成させた AT Pを当該酵素反応に供給しながら反応を行うことにより、 当該化合物を製造することができる。 特に、 AT Pを消費する酵素反応を利用し た化合物の製造法においては、 当該酵素反応により生じた AMP及び/または ADPを原料として AT Pを再生しながら反応を行うことが可能なため、 効率的 に目的とする化合物を製造することができ、 例えば、 ガラク トキナーゼを用いた ガラク ト一ス— 1—リン酸合成系、 UMPキナ一ゼを用いた UD P合成系、 コリ ンキナーゼを用いたホスホコリ ン合成系など AT Pを消費するあらゆる酵素反応 に応用することができる。 The ATP thus prepared can be isolated and purified by known methods.c Also, in a method for producing a compound using an enzyme reaction consuming ATP, The compound can be produced by reacting AMP with the above-mentioned polyphosphate kinase, adenylate kinase and ATP produced by reacting polyphosphoric acid to the enzyme reaction to supply the compound to the enzyme reaction. . In particular, in a method for producing a compound using an enzymatic reaction that consumes ATP, it is possible to carry out the reaction while regenerating ATP using AMP and / or ADP generated by the enzymatic reaction as a raw material. The target compound can be specifically produced, for example, galactose-1-phosphate synthesis system using galactokinase, UDP synthesis system using UMP kinase, phosphocollage using corin kinase. It can be applied to any enzymatic reaction that consumes ATP, such as a synthesis system.
このような A T P合成系と酵素反応との反応条件は、 小規模試験にて適宜決定 すればよく、 また目的化合物の単離精製も公知の方法により行うことができる。 実施例 The reaction conditions for such an ATP synthesis system and the enzymatic reaction may be appropriately determined by a small-scale test, and the target compound can be isolated and purified by a known method. Example
以下、 実施例を示し、 本発明を具体的に説明するが、 本発明がこれに限定され ないことは明らかである。 また、 実施例における DNAの調製、 制限酵素による 切断、 T4DNAリガ一ゼによる DNA連結、 並びに大腸菌の形質転換法は全て 「Moiecular cloningj (Maniatisら編、 Cold spring Harbor Laboratory, Cold Spring Harbor, New York(1982)) に従って行った。 また、 制限酵素、 Amp 1 i T a qDNAポリメラ一ゼ、 T 4 DNAリガーゼは宝酒造 (株) より 入手した。 さらに、 反応液中のヌクレオチド類の定量は H PLC法により行った c 具体的には、 分離には YMC社製の 0DS— AQ 3 1 2カラムを用い、 溶出液と して 0. 5M リン酸一カリウム溶液を用いた。 Hereinafter, the present invention will be described in detail with reference to Examples, but it is apparent that the present invention is not limited thereto. In the Examples, DNA preparation, restriction enzyme digestion, DNA ligation using T4 DNA ligase, and transformation of E. coli were all described in `` Moiecular cloningj (Maniatis et al., Edited by Cold spring Harbor Laboratory, Cold Spring Harbor, New York ( 1982)) Restriction enzymes, Amp1i TaqDNA polymerase and T4 DNA ligase were obtained from Takara Shuzo Co., Ltd. Quantification of nucleotides in the reaction solution was performed by the HPLC method. the c specifically went, the separation using 0DS- AQ 3 1 2 column manufactured by YMC was used 0. 5M monopotassium phosphate solution as an eluent.
実施例 1 ; AT Pの合成 Example 1 Synthesis of ATP
( 1 ) 大腸菌ポリ リ ン酸キナーゼ遺伝子のクローニング (1) Cloning of Escherichia coli polyphosphate kinase gene
大腸菌 K 1 2株 JM 1 09菌 (宝酒造 (株) より入手) の染色体 DNAを斉藤 と三浦の方法 (Biochim. Biophys. Acta. , 72, 619(1963)) で調製した。 この
DNAをテンペレートとして、 以下に示す 2種類のプライマ一 DN Aを常法に従 つて合成し、 PCR法により大腸菌ポリ リ ン酸キナーゼ (p p k) 遺伝子を増幅 した。 Chromosomal DNA of E. coli K12 strain JM109 (obtained from Takara Shuzo Co., Ltd.) was prepared by the method of Saito and Miura (Biochim. Biophys. Acta., 72, 619 (1963)). this Using DNA as a template, the following two primers, DNA, were synthesized in a conventional manner, and the E. coli polyphosphate kinase (ppk) gene was amplified by PCR.
プライマー (A) 5 ' -TAC Primer (A) 5'-TAC
A - 3 , A-3,
プライマー (B) 5 ' ATGG ATC Primer (B) 5 'ATGG ATC
GAGTGA- 3 ' GAGTGA- 3 '
PCRによる pp k遺伝子の増幅は、 反応液 1 0 中 ( 5 OmM 塩化力リゥ ム、 1 0 mM ト リ ス塩酸 ( pH 8. 3 ) 、 1. 5 mM 塩化マグネシウム、 0. 0 0 1 %ゼラチン、 テンペレー ト DNA 0. l g、 プライマ一 DNA (A) (B) 各々 0. 2〃M、 Amp l i Ta q DNAポリメラ一ゼ 2. 5 ュニッ ト) を P e r k i n— E lme r C e t u s I n s t r ume n t社 製 DNA Th e rma l C y c 1 e rを用いて、 熱変性 ( 94 °C、 1分) 、 アニーリ ング (55°C、 1. 5分) 、 ポリメライゼ一シヨン ( 72で、 1. 5分) のステップを 25回繰り返すことにより行った。 Amplification of the ppk gene by PCR was performed in a reaction mixture of 10 (5 OmM chloride chloride, 10 mM Tris-HCl (pH 8.3), 1.5 mM magnesium chloride, 0.001% gelatin , Temperate DNA 0.1 lg, Primer DNA (A) (B) 0.2 、 M each, Ampli Taq DNA polymerase 2.5 units) were replaced with Perkin-ElmerCetus Instrume. Thermal denaturation (94 ° C, 1 minute), annealing (55 ° C, 1.5 minutes), polymerase reaction (72, 1.5 Min) was repeated 25 times.
遺伝子増幅後、 反応液をフエノール Zクロ口ホルム ( 1 : 1 ) 混合液で処理し、 水溶性画分に 2倍容のエタノールを添加し DN Aを沈殿させた。 沈殿回収した DNAを文献 (Molecular cloning. 前述) の方法に従ってァガロースゲル電気 泳動により分離し、 1. Okb相当の DNA断片を精製した。 該 DNAを制限酵素 N c 0 I及び BamH Iで切断し、 同じく制限酵素 N c o I及び BamH Iで消 化したプラスミ ド pT r c 9 9 A (Pharmacia Biotech 社より入手) と T 4 DNAリガ一ゼを用いて連結した。 連結反応液を用いて大腸菌 JM 1 0 9菌を形 質転換し、 得られたアンピシリ ン耐性形質転換体よりプラスミ ド pT r c一 PPKを単離した。 pTr c— PPKは、 pTr c 9 9 Aの t r cプロモーター 下流の Nc o I—BamH I切断部位に大腸菌 p p k遺伝子を含有する N c o I - B amH I DNA断片が挿入されたものである。
( 2) 大腸菌ポリ リン酸キナーゼの調製 After gene amplification, the reaction solution was treated with a mixture of phenol Z-cloth form (1: 1), and the DNA was precipitated by adding twice the volume of ethanol to the water-soluble fraction. The DNA collected by precipitation was separated by agarose gel electrophoresis according to the method described in the literature (Molecular cloning. Supra), and a DNA fragment equivalent to Okb was purified. The DNA was cleaved with restriction enzymes NcoI and BamHI, and plasmid pTrc99A (obtained from Pharmacia Biotech), also digested with restriction enzymes NcoI and BamHI, and T4 DNA ligase Was connected using Escherichia coli JM109 was transformed using the ligation reaction solution, and plasmid pTrc-PPK was isolated from the obtained ampicillin-resistant transformant. pTr c-PPK is obtained by inserting an NcoI-BamHI DNA fragment containing the Escherichia coli ppk gene into the NcoI-BamHI cleavage site downstream of the trc promoter of pTrc99A. (2) Preparation of Escherichia coli polyphosphate kinase
プラスミ ド p T r c— P PKを保持する大腸菌 JM 1 0 9菌を、 1 0 O i gZ ^のアンピシリンを含有する 2 XYT培地 3 0 に植菌し、 3 7てで振とう培 養した。 4 X 1 03菌 に達した時点で、 培養液に終濃度 1 mMになるようにEscherichia coli JM109 carrying plasmid pTrc-Ppk was inoculated into 2XYT medium 30 containing 10OigZ ^ ampicillin, and cultured with shaking. Once at the 4 X 1 0 3 bacteria to a final concentration of 1 mM to the culture solution
I PTGを添加し、 さらに 3 0でで 5時間振とう培養を続けた。 培養終了後、 遠 心分離 ( 9, 0 0 0 X g, 1 0分) により菌体を回収し、 6 0 の緩衝液 ( 5 0 mM トリス塩酸 (pH7. 5) 、 5 mM EDTA、 0. 1 %トライ トン X— 1 0 0、I PTG was added, and shaking culture was further continued at 30 for 5 hours. After completion of the culture, the cells were collected by centrifugation (9,000 X g, 10 minutes), and the cells were collected in 60 buffer (50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0. 1% Triton X—100,
0. 2mgZ7 ^リブチーム) に懸濁した。 3 7 °Cで 1時間保温した後、 超音波処理 を行い、 菌体を破砕し、 さらに遠心分離 ( 2 0, 0 0 0 x g、 1 0分) により菌 体残さを除去した。 このように得られた上清画分を 5mM 塩化マグネシウム及び0.2 mg Z7 ^ rib team). After incubating at 37 ° C for 1 hour, the cells were sonicated to disrupt the cells, and the cells were removed by centrifugation (20,000 xg, 10 minutes). The supernatant fraction thus obtained was treated with 5 mM magnesium chloride and
1 mM 2—メルカプトエタノールを含有する 5 OmMトリス塩酸 (pH7. 8 ) に対 して透析を行い、 粗酵素液とした。 It was dialyzed against 5 OmM Tris-HCl (pH 7.8) containing 1 mM 2-mercaptoethanol to obtain a crude enzyme solution.
粗酵素液におけるポリリン酸キナーゼ比活性は、 0. 1 9ユニッ ト/ mg蛋白質 であり、 対照菌 (p T r c 9 9 Aを保持する大腸菌 JM 1 0 9菌) の比活性 ( 0. 0 0 0 1 8ュニッ ト Zmg蛋白質) の約 1 0 0 0倍であった。 次に粗酵素液 を D E A E トヨパール 6 5 0 M (ト一ツー (株) ) を用いて 0〜 0. 5 M Na の濃度勾配にて分画し、 ポリ リン酸キナーゼ画分を得た。 この画分をポ リ リン酸キナーゼ酵素標品とした。 なお、 この酵素標品におけるポリ リン酸キナ 一ゼの比活性は、 0. 6ユニッ ト Zmg蛋白質であった。 The specific activity of the polyphosphate kinase in the crude enzyme solution was 0.19 unit / mg protein, which was the specific activity of the control bacterium (E. coli JM109 bacterium harboring pTrc99A). 0.18 units (Zmg protein). Next, the crude enzyme solution was fractionated using DEAE Toyopearl 6.5 M (Toy Corporation) with a concentration gradient of 0 to 0.5 M Na to obtain a polyphosphate kinase fraction. This fraction was used as a polyphosphate kinase enzyme preparation. The specific activity of polyphosphate kinase in this enzyme preparation was 0.6 unit Zmg protein.
なお、 本発明におけるポリ リン酸キナーゼ活性の単位 (ュニッ ト) は、 以下に 示す方法で測定、 算出したものである。 すなわち、 5mM塩化マグネシウム、 1 0 OmM硫安、 5mM A D P、 及びポリ リン酸 (無機リン酸として 1 0 0 ) を 含有する 2 5 mMトリス塩酸緩衝液 (pH 7. 8 ) に酵素標品を添加して、 3 7°Cで 保温することで反応を行い、 1 0 0°C、 1分間の熱処理により反応を停止させる c 高速液体ク αマトグラフィ一 (HP L C) を用いて反応液中の ΑΤΡを定量し、 3 7てで 1分間に 1 mo^e の ATPを生成する活性を 1単位 (ュニッ ト) とす
る o The unit (unit) of polyphosphate kinase activity in the present invention is measured and calculated by the following method. That is, the enzyme preparation was added to a 25 mM Tris-HCl buffer (pH 7.8) containing 5 mM magnesium chloride, 10 OmM ammonium sulfate, 5 mM ADP, and polyphosphoric acid (100 as inorganic phosphoric acid). Te, 3 7 ° to carry out the reaction by incubating at C, and ΑΤΡ in the reaction solution with 1 0 0 ° C, 1 minute annealing c high-performance liquid α Matogurafi one to stop the reaction by (HP LC) Quantify the activity to produce 1 mo ^ e of ATP per minute at 37 ° C. O
( 3 ) 大腸菌アデニレ一トキナ一ゼのクローニング (3) Cloning of E. coli adenylate kinase
大腸菌 1 2株 JM 1 0 9菌 (宝酒造 (株) より入手) の染色体 DNAを斉藤 と三浦の方法 (Biochim. Biophys. Acta. , 72, 619(1963)) で調製した。 この DNAをテンペレートとして、 以下に示す 2種類のプライマー DN Aを常法に従 つて合成し、 PCR法により大腸菌アデユレ—トキナーゼ (a d k) 遺伝子を増 幅した。 Chromosomal DNA of Escherichia coli 12 strain JM109 (obtained from Takara Shuzo Co., Ltd.) was prepared by the method of Saito and Miura (Biochim. Biophys. Acta., 72, 619 (1963)). Using this DNA as a template, the following two primers, DNA, were synthesized according to a conventional method, and the Escherichia coli adduct kinase (adk) gene was amplified by PCR.
プライマー (A) : 5 ' -ATGGATCCCGTTTCAGCCCCAGG Primer (A): 5'-ATGGATCCCGTTTCAGCCCCAGG
TGCC- 3 ' TGCC- 3 '
プライマー (B) : 5 ' — ATAAGCTTGGC CTGAGATTGCTG Primer (B): 5 '— ATAAGCTTGGC CTGAGATTGCTG
ATAAG- 3 ' ATAAG- 3 '
P CRによる a d k遺伝子の増幅は、 反応液 1 00 中 ( 50 mM 塩化力リウ ム、 1 0 mM ト リ ス塩酸 (pH 8. 3 ) 、 1. 5 raM 塩化マグネ シウム、 0. 0 0 1 %ゼラチン、 テンペレー ト DNA 0. 1 g、 プライマ一 DNA (A) (B) 各々 0. 1 M、 Amp 1 i T a q DNAポリ メラ一ゼ 2. 5ュ ニッ ト) を P e r k i n— E lme r C e t u s I n s t r ume n t社製 DNA Th e rma l C y c 1 e rを用いて、 熱変性 ( 94で、 1分) 、 ァ ニーリング ( 5 6 °C、 1. 0分) 、 ポリメライゼーシヨン ( 72て、 3. 0分) のステップを 25回繰り返すことにより行った。 Amplification of the adk gene by PCR was performed in a reaction mixture of 100 mM (50 mM chloride, 10 mM Tris-HCl (pH 8.3), 1.5 raM magnesium chloride, 0.001% 0.1 g of gelatin and temperate DNA, 0.1 M of each of primer DNA (A) and (B), 2.5 M of Amp1i Taq DNA polymerase (2.5 units) were added to Perkin-Elmer C Thermal denaturation (94 min., 1 min.), annealing (56 ° C, 1.0 min.), polymerization (72 min.) were performed using DNA Thermal Cyc 1 er manufactured by etus International. And 3.0 minutes) were repeated 25 times.
遺伝子増幅後、 反応液をフニノール Zクロ口ホルム ( 1 : 1) 混合液で処理し、 水溶性画分に 2倍容のェタノールを添加し DN Aを沈殿させた。 沈殿回収した DNAを文献 (Molecular cloning. 前述) の方法に従ってァガロースゲル電気 泳動により分離し、 1. Okb相当の DNA断片を精製した。 該 DNAを制限酵素 B a m H I及び H i n d I IIで切断 し、 同 じ く 制限酵素 B a m H I 及び H i n d III で消化したプラスミ ド p UC 1 8 (宝酒造 (株) より入手) と T 4 DNAリガーゼを用いて連結した。 連結反応液を用いて大腸菌 JM 1 09菌
を形質転換し、 得られたアンピシリ ン耐性形質転換体よりプラスミ ド p UC— A DKを単離した。 p UC— ADKは、 pUC 1 8の l a cプロモ一夕一下流の B amH I -H i n d ΠΙ切断部位に大腸菌 a d k遺伝子を含有する B amH I — H i n d UI DNA断片が挿入されたものである。 After gene amplification, the reaction solution was treated with a mixture of funinol Z-cloth form (1: 1), and DNA was precipitated by adding twice the volume of ethanol to the water-soluble fraction. The DNA collected by precipitation was separated by agarose gel electrophoresis according to the method described in the literature (Molecular cloning. Supra), and a DNA fragment equivalent to Okb was purified. The DNA was digested with the restriction enzymes BamHI and HindIII, and the plasmid pUC18 (obtained from Takara Shuzo) and T4 DNA digested with the restriction enzymes BamHI and HindIII. Ligation was performed using ligase. Escherichia coli JM109 using ligation reaction solution And the plasmid pUC-ADK was isolated from the resulting ampicillin-resistant transformant. pUC-ADK is obtained by inserting a BamHI-Hind UI DNA fragment containing the E. coli adk gene into the BamHI-HindII cleavage site downstream of the lac promoter overnight in pUC18.
( 4) 大腸菌アデ二レートキナーゼの調製 (4) Preparation of E. coli adenylate kinase
プラスミ ド pUC— ADKを保持する大腸菌 JM 1 0 9菌を、 1 0 O z g/ のアンピシリンを含有する 2 XYT培地 3 0 0 に植菌し、 3 7 °Cで振とう培養 した。 4 X 1 08 菌ノ に達した時点で、 培養液に終濃度 1 mMになるようにEscherichia coli JM109 carrying plasmid pUC—ADK was inoculated into 300 XYT medium containing 100 Ozg / ampicillin and cultured at 37 ° C with shaking. Once at the 4 X 1 0 8 bacteria Bruno, to a final concentration of 1 mM to the culture solution
I PTGを添加し、 さらに 3 0°Cで 5時間振とう培養を続けた。 培養終了後、 遠 心分離 ( 9, 0 0 0 X g. 1 0分) により菌体を回収し、 6 07^の緩衝液 ( 5 0 mMトリス塩酸 (pH7. 5) 、 5 mM EDTA、 0. 1 %トライ トン X— 1 0 0、 0. 2mgZ^リゾチ一厶) に懸濁した。 3 7 °Cで 1時間保温した後、 超音波処理 を行い、 菌体を破砕し、 さらに遠心分離 (2 0, 0 0 0 X g. 1 0分) により菌 体残さを除去した。 IPTG was added, and shaking culture was continued at 30 ° C. for 5 hours. After completion of the culture, the cells were collected by centrifugation (9,000 X g., 10 minutes), and the buffer of 60 ^^ (50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0 mM 1% Triton X—100, 0.2 mg Z ^ rhizodium). After incubating at 37 ° C for 1 hour, the cells were sonicated to disrupt the cells, and the cells were removed by centrifugation (20,000 X g., 10 minutes).
このように得られた上清画分を 5 mM 塩化マグネシウム及び 1 mill 2—メルカ ブトエタノールを含有する 5 OmMトリス塩酸 (pH 7. 8 ) に対して透析を行い、 粗酵素液とした。 粗酵素液におけるアデ二レートキナーゼの比活性は、 1 3 4ュ ニッ ト Zmg蛋白質であり、 対照菌 (pUC 1 8を保持する大腸菌 JM 1 0 9菌) の比活性 ( 1. 9ュニッ ト/ mg蛋白質) の約 8 5倍であった。 次に粗酵素液を D E A E ト ヨノ、0—ル 6 5 0 M ( ト一ソ一 (株) ) を用いて 0〜 0. 5 M Na C _βの濃度勾配にて分画し、 アデ二レートキナーゼ活性のある画分を回収し た。 この画分をアデ二レートキナーゼ酵素標品とした。 なお、 この酵素標品にお けるポリ リン酸キナーゼの比活性は、 3 4 4ュニッ ト Zmg蛋白質であった。 なお、 本発明におけるアデ二レートキナーゼ活性の単位 (ュニッ ト) も次の方 法で測定、 算出した。 すなわち、 5 mM塩化マグネシウム、 5mM ATP、 及び 5 mM AMPを含有する 5 0 mMトリス塩酸緩衝液 (pH7. 8 ) に酵素標品を添加し
て 3 7°Cで保温することで反応を行い、 1 0 0°C、 1分間の熱処理により反応を 停止させる。 HP L Cを用いて反応液中の ADPを定量し、 3 7°Cで 1分間に 2 /mo^eの AD Pを生成する活性を 1単位 (ュニッ ト) とする。 The supernatant fraction thus obtained was dialyzed against 5 OmM Tris-HCl (pH 7.8) containing 5 mM magnesium chloride and 1 mill 2-mercaptoethanol to obtain a crude enzyme solution. The specific activity of adenylate kinase in the crude enzyme solution was 134 units of Zmg protein, and the specific activity of the control bacterium (E. coli JM109 carrying pUC18) (1.9 units / unit). mg protein). Then the crude enzyme solution DEAE preparative Yono, 0 - fractionated by gradient 0~ 0. 5 M Na C _β with Le 6 5 0 M (DOO one source i (Inc.)), Ade two rate Fractions with kinase activity were collected. This fraction was used as an adenylate kinase enzyme preparation. The specific activity of polyphosphate kinase in this enzyme preparation was 344 unit Zmg protein. The unit (unit) of the adenylate kinase activity in the present invention was also measured and calculated by the following method. That is, an enzyme preparation was added to 50 mM Tris-HCl buffer (pH 7.8) containing 5 mM magnesium chloride, 5 mM ATP, and 5 mM AMP. The reaction is carried out by keeping the temperature at 37 ° C, and the reaction is stopped by heat treatment at 100 ° C for 1 minute. Quantify ADP in the reaction solution using HP LC, and define the activity to produce 2 / mo ^ e of ADP per minute at 37 ° C as 1 unit (unit).
( 5) ポリ リ ン酸キナーゼとアデニレ一トキナーゼによる AT Pの合成 (その 1 ) 1 OmM 塩化マグネシウム、 1 0 OmM 硫酸アンモニゥム、 ポリ リ ン酸 (無機 リ ン酸として 7 5mM) 、 4mM AMPを含有する 5 OmM トリス塩酸緩衝液 (pH 7. 8 ) に酵素標品あるいは菌体抽出液を添加し、 3 7°Cで 1 5 0分保温した。 反応終了後反応液中のヌクレオチドを HP L Cを用いて定量した。 その結果を表 (5) Synthesis of ATP by polyphosphate kinase and adenylate kinase (Part 1) Contains 1 OmM magnesium chloride, 10 OmM ammonium sulfate, polyphosphate (75 mM as inorganic phosphate), and 4 mM AMP The enzyme preparation or the cell extract was added to 5 OmM Tris-HCl buffer (pH 7.8), and the mixture was kept at 37 ° C for 150 minutes. After completion of the reaction, nucleotides in the reaction solution were quantified using HPLC. The result is displayed
1に示す o
O shown in 1
表 1 添加酵素標品 ATP(mM) ADP(raM)Table 1 Added enzyme preparations ATP (mM) ADP (raM)
PPK画分(0.02ュニッ ト Ζτηβ) < 0.01 < 0.01 ADK画分(0.2ュニッ ト /m < 0.01 < 0.01PPK fraction (0.02 units Ζτηβ) <0.01 <0.01 ADK fraction (0.2 units / m <0.01 <0.01
PPK画分(0.02ュニッ ト Z ) + ADK画分(0.2ュニッ ト τηβ) 2.58 1.82PPK fraction (0.02 unit Z) + ADK fraction (0.2 unit τηβ) 2.58 1.82
ΡΡΚ画分(0.02ュニッ ト Z ) + ADK画分(0.02ュニッ ト Ζτηβ) 0.78 1. 11ΡΡΚ fraction (0.02 unit Z) + ADK fraction (0.02 unit Ζτηβ) 0.78 1.11
PPK画分(0.02ュニッ ト Z ) + JM109[pUC18]粗抽出液(2// g/m6) < 0.01 < 0.01PPK fraction (0.02 unit Z) + JM109 [pUC18] crude extract (2 // g / m6) <0.01 <0.01
PPK画分(0.02ュニッ ト Z ) + JM109[pliC- AM]粗抽出液(2 g/τηβ) 0.20 1.37 PPK fraction (0.02 unit Z) + JM109 [pliC-AM] crude extract (2 g / τηβ) 0.20 1.37
PPK: ポリ リ ン酸キナーゼ、 ADK: アデ二レー トキナ一ゼ
PPK: Polyphosphate kinase, ADK: Adenylate kinase
表 1に示すように、 ポリ リ ン酸キナーゼ並びにアデニレ一トキナーゼそれぞれ 単独では AMPをリン酸化し、 ADP及び ATPを生成する活性は存在せず、 両 者が同時に存在することで初めて AMPのリ ン酸化が生じた。 また、 通常の大腸 菌 ( JM 1 0 9 CpUC 1 8) ) より調製した粗酵素液とポリ リ ン酸キナーゼを 混合しても AMPのリ ン酸化は生じないが、 アデニレ一 トキナーゼ高生産株 ( JM 1 0 9 Cp UC -ADK) ) より調製した粗酵素液をポリ リ ン酸キナーゼ と混合すると顕著な AMPのリ ン酸化反応が生じた。 以上のことからポリ リ ン酸 キナーゼとアデ二レートキナ一ゼが共存することで AMPのリ ン酸化が生じるこ とは明らかである。 As shown in Table 1, polyphosphate kinase and adenylate kinase alone do not phosphorylate AMP and have no activity to produce ADP and ATP, and only when both are present at the same time do AMP phosphorylation. Oxidation occurred. AMP phosphorylation does not occur when a crude enzyme solution prepared from normal E. coli (JM109 CpUC18)) is mixed with polyphosphoric acid kinase, but adenylate kinase-producing strain ( When the crude enzyme solution prepared from JM109CpUC-ADK)) was mixed with polyphosphate kinase, a marked AMP phosphorylation reaction occurred. From the above, it is clear that co-presence of polyphosphate kinase and adenylate kinase causes AMP phosphorylation.
( 6) ポリ リ ン酸キナーゼとアデ二レートキナ一ゼによる AT Pの合成 (その 2) 1 OmM 塩化マグネシウム、 1 0 OmM 硫酸アンモニゥ厶、 ポリ リ ン酸 (無機 リ ン酸としてそれぞれ OmM、 3 OmM, 7 5 mM、 1 5 0 mM) 、 4 mM AMPを含有 する 5 OmM トリス塩酸緩衝液 (pH 7. 8) に 0. 1ユニッ ト Ζτ^ポリ リン酸キ ナ一ゼ及び 0. 5ユニッ ト / τηβ アデ二レー トキナーゼを添加し、 3 7 で 1 2 0分保温した。 反応終了後反応液中のヌクレオチドを H P L Cを用いて定量 した。 その結果を表 2に示す。 (6) Synthesis of ATP by polyphosphate kinase and adenylate kinase (Part 2) 1 OmM magnesium chloride, 10 OmM ammonium sulfate, polyphosphate (OmM and 3 OmM as inorganic phosphate, respectively) , 5τ ^ polyphosphate kinase and 0.5 unit in 5 OmM Tris-HCl buffer (pH 7.8) containing 4 mM AMP / τηβ adenylate kinase was added, and the mixture was kept at 37 for 120 minutes. After completion of the reaction, nucleotides in the reaction solution were quantified using HPLC. The results are shown in Table 2.
表 2 ポリ リ ン酸濃度 (リン酸として) ATP(mM) ADP(mM) Table 2 Polyphosphate concentration (as phosphoric acid) ATP (mM) ADP (mM)
OmM <0.01 ぐ 0.01 OmM <0.01 over 0.01
30 0.37 0.76 30 0.37 0.76
75 0.67 1.52 75 0.67 1.52
150 <0.01 0.09 表 2に示すように、 ポリ リ ン酸キナーゼとアデ二レートキナ一ゼによるリ ン酸 化活性はポリ リ ン酸濃度に依存しているが、 高濃度ポリ リ ン酸存在下ではその活 性も阻害された。
( 7) ポリ リ ン酸キナーゼとアデニレ一トキナ一ゼによる AT Pの合成 (その 3 ) 1 OmM 塩化マグネシウム、 1 0 OmM 硫酸アンモニゥ厶、 ポリ リ ン酸 (無機 リ ン酸として 7 5mM) 、 4mM AMPを含有する 5 OmM トリス塩酸緩衝液 (pH 7. 8) に 0. 1ユニッ ト ポリ リ ン酸キナ一ゼ及び様々な濃度のアデニレ一 トキナーゼ酵素標品を添加し、 3 7てで 7 0分保温した。 反応終了後反応液中の ヌクレオチドを H P L Cを用いて定量した。 その結果を表 3に示す。 150 <0.01 0.09 As shown in Table 2, the phosphorylation activity of polyphosphate kinase and adenylate kinase depends on the concentration of polyphosphate, but in the presence of high concentrations of polyphosphate, Its activity was also impaired. (7) Synthesis of ATP by polyphosphate kinase and adenylate kinase (Part 3) 1 OmM magnesium chloride, 10 OmM ammonium sulfate, polyphosphate (75 mM as inorganic phosphate), 4 mM Add 0.1 unit of polyphosphate kinase and various concentrations of adenylate kinase enzyme to 5 OmM Tris-HCl buffer (pH 7.8) containing AMP. Keep warm for a minute. After completion of the reaction, nucleotides in the reaction solution were quantified using HPLC. The results are shown in Table 3.
表 3 ポリ リ ン酸キナーゼ アデ二レートキナーゼ ATP(mM) ADP(mM) Table 3 Polyphosphate kinase adenylate kinase ATP (mM) ADP (mM)
0.10ュニッ mk 0.05ュニッ m. 0.18 0.14 0.10 unit mk 0.05 unit m. 0.18 0.14
0.10 0.25 0.85 1.16 0.10 0.25 0.85 1.16
0.10 0.50 0.89 1.26 0.10 0.50 0.89 1.26
0.10 2.50 1.04 1.20 0.10 2.50 1.04 1.20
( 8 ) ポリ リ ン酸キナーゼとアデニレ一トキナーゼによる AT Pの合成 (その 4 ) 1 0 mM 塩化マグネシゥム、 1 0 0 mM 硫酸ァンモニゥ厶、 ポリ リ ン酸 (無機 リン酸として 7 5 ) 、 4mM AMPを含有する 5 OmM トリス塩酸緩衝液 (pH 7. 8 ) に 0. 1ユニッ ト ポリ リン酸キナーゼ及び 2. 5ユニッ ト アデ 二レートキナーゼ酵素標品を添加し、 3 7°Cで 4 0 0分保温した。 反応終了後反 応液中のヌクレオチドを HPL Cを用いて定量した。 その結果を図 1に示す。 反 応終了時の反応液中のヌクレオチド濃度は、 ATP 2. 3mM、 ADP 1. 3 mM、 AMP 0. 4mMであった。 (8) Synthesis of ATP by polyphosphate kinase and adenylate kinase (Part 4) 100 mM magnesium chloride, 100 mM ammonium sulfate, polyphosphate (75 as inorganic phosphate), 4 mM AMP Add 0.1 units of polyphosphate kinase and 2.5 units of adenylate kinase enzyme to 5 OmM Tris-HCl buffer (pH 7.8) containing Keep warm for a minute. After the reaction was completed, nucleotides in the reaction solution were quantified using HPLC. Figure 1 shows the results. At the end of the reaction, the nucleotide concentrations in the reaction solution were ATP 2.3 mM, ADP 1.3 mM, and AMP 0.4 mM.
実施例 2 ; ATP再生系を利用したガラク トース— 1 一リ ン酸の製造 Example 2 Production of Galactose-1 Monophosphate Using ATP Regeneration System
( 1 ) 大腸菌ガラク トキナーゼの調製 (1) Preparation of E. coli galactokinase
大腸菌ガラク トキナーゼ遺伝子を含有するプラスミ ド P DR 5 4 0 (Gene, 20, 231(1982) 、 フアルマシア社より入手) を保持する大腸菌 JM 1 0 9菌を、 1 0 0 g/r のマンピシリ ンを含有する 2 X YT培地 3 0 0 に植菌し、
3 7 °Cで振とう培養した。 4 x 1 08菌 に達した時点で、 培養液に終濃度 1 mMになるように I PTGを添加し、 さらに 3 0 °Cで 5時間振とう培養を続けた。 培養終了後、 遠心分離 ( 9, 0 0 0 X g、 1 0分) により菌体を回収し、 6 Ο τηβ の緩衝液 ( 5 0 mM トリス塩酸 (pH 7. 5 ) 、 5 mM E D T A、 0. 1 %トライ トン X— 1 0 0、 0. 2mgZ7 ^リブチーム) に懸濁した。 3 7 °Cで 1時間保温し た後、 超音波処理を行い、 菌体を破砕し、 さらに遠心分離 ( 2 0, 0 0 0 X g,Escherichia coli JM109 containing plasmid PDR540 (Gene, 20, 231 (1982), obtained from Pharmacia) containing the Escherichia coli galactokinase gene was isolated from 100 g / r of manpicillin. Inoculate 2X YT medium 300 containing The cells were cultured at 37 ° C with shaking. Once at the 4 x 1 0 8 bacteria, was added I PTG to a final concentration of 1 mM to the culture solution was continued for an additional 3 0 ° C for 5 hours with shaking culture. After completion of the culture, the cells were collected by centrifugation (9,000 X g, 10 minutes), and a buffer of 6ητηβ (50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0 mM 1% Triton X—100, 0.2 mg Z7 ^ ribzyme). After incubation at 37 ° C for 1 hour, sonication was performed to disrupt the cells, and centrifugation (20,000 X g,
1 0分) により菌体残さを除去した。 このように得られた上清画分を 5mM 塩化 マグネシウム及び 1 mM 2—メルカプトエタノールを含有する 5 OmMトリス塩酸(10 minutes) to remove bacterial cell residues. The supernatant fraction obtained in this manner is treated with 5 OmM Tris-HCl containing 5 mM magnesium chloride and 1 mM 2-mercaptoethanol.
(pH7. 8 ) に対して透析を行い、 ガラク トキナ一ゼ酵素液とした。 酵素液にお けるガラク トキナ一ゼの比活性は、 1. 3 9ュニッ ト /mg蛋白質であった。 (pH 7.8) was dialyzed to obtain a galactokinase enzyme solution. The specific activity of galactokinase in the enzyme solution was 1.39 units / mg protein.
なお、 ガラク トキナーゼ活性の単位 (ュニッ ト) は、 以下に示す方法で測定、 算出したものである。 5 mM塩化マグネシウム、 1 OmM ATP、 1 0 mM ガラク ト一スを含有する 1 0 OmMトリス塩酸緩衝液 (pH7. 8 ) に酵素標品を添加して、 The unit (unit) of galactokinase activity was measured and calculated by the following method. Add enzyme preparation to 10 OmM Tris-HCl buffer (pH 7.8) containing 5 mM magnesium chloride, 1 OmM ATP, and 10 mM galactose.
3 7 °Cで保温することで反応を行い、 1 0 0 T:、 1分間の熱処理により反応を停 止させる。 糖分析装置 (ダイォネックス社) を用いて反応液中のガラク ト一ス一The reaction is carried out by keeping the temperature at 37 ° C, and the reaction is stopped by heat treatment at 100 T: 1 minute. Galactose in the reaction solution using a sugar analyzer (Dionex)
1 -リ ン酸を定量し、 3 7 °Cで 1分間に 1 mo^eのガラク ト一ス— 1 一リ ン酸 を生成する活性を 1単位 (ュニッ ト) とする。 The 1-phosphoric acid is quantified, and the activity to produce 1 mo ^ e of galactose- 1-phosphoric acid per minute at 37 ° C is defined as 1 unit (unit).
( 2 ) ポリ リ ン酸キナーゼとアデニレ一トキナ一ゼによる ATP再生とガラク ト キナーゼによるガラク トース— 1 ―リ ン酸の合成 (2) ATP regeneration by polyphosphate kinase and adenylate kinase and synthesis of galactose-1-phosphate by galactokinase
1 OmM 塩化マグネシウム、 1 0 OmM 硫安、 ポリ リ ン酸 (無機リン酸として 7 5m ) 及び 4mM AMPを含有する 1 0 0 mM トリス塩酸緩衝液 (ρΗ7 · 8 ) に 0. 1ユニッ ト/ / ポリ リ ン酸キナーゼ及び 2. 5ユニッ ト/ アデ二レート キナーゼ酵素標品を添加し、 3 7°Cで 1 2 0分保温した。 該 AMPリ ン酸化反応 終了時の反応液中のヌクレオチド濃度は、 ATP 1. 4mM、 AD P 1. 7 mM, AMP 0. 9mMであった。 この反応液に終濃度 4 0 mMとなるように D (十) ガ ラク トースを添加し、 さらに 1. 0ユニッ ト/ となるようにガラク トキナーゼ
を添加し、 3 7°Cで 4. 5時間保温した。 反応終了液を糖分析装置 (ダイォネッ クス社) を用いて分析したところ、 2 8. 4 mMのガラク トースー 1 —リ ン酸の生 成が確認された。 産業上の利用可能性 1 Omm magnesium chloride, 1 0 Omm ammonium sulfate, 0.1 poly-phosphate 1 0 0 mM Tris-HCl buffer containing and 4 mM AMP (7 5 m as inorganic phosphate) (ρΗ7 · 8) 1 unit / / poly Phosphate kinase and 2.5 units / adenylate kinase enzyme preparation were added, and the mixture was incubated at 37 ° C for 120 minutes. The nucleotide concentrations in the reaction solution at the end of the AMP phosphorylation reaction were ATP 1.4 mM, ADP 1.7 mM, and AMP 0.9 mM. D (10) galactose was added to this reaction solution to a final concentration of 40 mM, and galactokinase was further added to 1.0 unit /. Was added and kept at 37 ° C. for 4.5 hours. Analysis of the reaction solution using a sugar analyzer (Dionex) confirmed the production of 28.4 mM galactose-1-phosphate. Industrial applicability
本発明により、 簡便かつ安価に AMPから ATPを製造することが可能となつ た。 この AT P合成系を AT Pを消費する酵素反応系と組み合わせることにより、 効率的に目的とする化合物を製造することが可能となった。
According to the present invention, it has become possible to easily and inexpensively produce ATP from AMP. By combining this ATP synthesis system with an ATP-consuming enzyme reaction system, it has become possible to efficiently produce the target compound.
Claims
1 . アデノシン 5 ' —一リ ン酸にポリ リン酸キナーゼ、 アデ二レートキナーゼ 及びポリ リ ン酸を作用せしめることを特徴とするアデノシン 5 ' —三リ ン酸の製 造法。 1. Adenosine 5'-a method for producing adenosine 5'-triphosphate, which comprises reacting monophosphate with polyphosphate kinase, adenylate kinase and polyphosphate.
2 . アデノシン 5 ' —三リ ン酸を消費する酵素反応を利用した化合物の製造法 において、 アデノシン 5 ' —一リ ン酸にポリ リン酸キナーゼ、 アデ二レートキナ ーゼ及びポリ リン酸を作用せしめてアデノシン 5 ' —三リ ン酸を生成させ、 当該 酵素反応に供給することを特徴とする当該化合物の製造法。 2. In a method for producing a compound using an enzymatic reaction that consumes adenosine 5'-triphosphate, adenosine 5'-monophosphate is reacted with polyphosphate kinase, adenylate kinase and polyphosphate. Adenosine 5′-triphosphate is produced by the reaction and supplied to the enzymatic reaction.
3 . アデノシン 5 ' —三リン酸を消費する酵素反応を利用した化合物の製造法 において、 生成したアデノシン 5 ' ——リン酸及び Zまたはアデノシン 5 ' —二 リン酸にポリ リ ン酸キナーゼ、 アデニレ一トキナーゼ及びポリ リ ン酸を作用せし めてアデノシン 5 ' —三リ ン酸を再生しながら当該酵素反応を行うことを特徴と する当該化合物の製造法。 3. Adenosine 5 '-In the production of compounds using an enzymatic reaction that consumes triphosphate, adenosine 5'--phosphate and Z or adenosine 5 '-diphosphate are converted to polyphosphate kinase, adenylate. A process for producing the compound, characterized in that the enzyme reaction is carried out while adenosine 5'-triphosphate is regenerated by reacting monokinase and polyphosphate.
4 . ポリ リ ン酸キナーゼ、 アデ二レートキナ一ゼ及びポリ リ ン酸を組み合わせ てなるアデノシン 5 ' —一リ ン酸からアデノシン 5 ' —三リ ン酸の合成系。
4. Synthetic system of adenosine 5'-adenosine 5'-triphosphate by combining polyphosphate kinase, adenylate kinase and polyphosphate.
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JP54545098A JP3764755B2 (en) | 1997-04-18 | 1998-04-15 | "Production Method of Adenosine 5'-Triphosphate and Its Application" |
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Cited By (6)
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WO2001053513A1 (en) * | 2000-01-17 | 2001-07-26 | Satake Corporation | Atp regeneration reaction systems and method of examining adenine nucleotide, method of detecting rna and method of amplifying atp by using the |
EP1652934A1 (en) * | 2003-07-29 | 2006-05-03 | Japan Science and Technology Agency | Improved method of amplifying atp and use thereof |
WO2006095696A1 (en) * | 2005-03-10 | 2006-09-14 | Bussan Nanotech Research Institute, Inc. | Method of detecting adenosine triphosphate and detection reagent therefor |
JP2009050264A (en) * | 2002-05-29 | 2009-03-12 | Yamasa Shoyu Co Ltd | New polyphosphate: amp phosphotransferase |
EP2100956A1 (en) | 2005-01-25 | 2009-09-16 | Yamasa Corporation | Method of enzymatically synthesizing 3'-phosphoadenosine-5'-phophosulfate |
JP2020504615A (en) * | 2016-12-30 | 2020-02-13 | エヌティーエックスバイオ,エルエルシー | Cell-free expression system with novel inorganic phosphate-based energy regeneration function |
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JP5800218B2 (en) * | 2011-07-20 | 2015-10-28 | 国立大学法人広島大学 | ATP production method and use thereof |
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JPS4882087A (en) * | 1972-02-07 | 1973-11-02 | ||
JPS57166992A (en) * | 1981-04-08 | 1982-10-14 | Seitetsu Kagaku Co Ltd | Preparation of adenosine-5'-triphosphate |
JPS59106296A (en) * | 1982-12-09 | 1984-06-19 | Kazutomo Imahori | Preparation of physiologically active substance by composite enzyme process |
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JPS4882087A (en) * | 1972-02-07 | 1973-11-02 | ||
JPS57166992A (en) * | 1981-04-08 | 1982-10-14 | Seitetsu Kagaku Co Ltd | Preparation of adenosine-5'-triphosphate |
JPS59106296A (en) * | 1982-12-09 | 1984-06-19 | Kazutomo Imahori | Preparation of physiologically active substance by composite enzyme process |
Cited By (12)
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WO2001053513A1 (en) * | 2000-01-17 | 2001-07-26 | Satake Corporation | Atp regeneration reaction systems and method of examining adenine nucleotide, method of detecting rna and method of amplifying atp by using the |
JP2009050264A (en) * | 2002-05-29 | 2009-03-12 | Yamasa Shoyu Co Ltd | New polyphosphate: amp phosphotransferase |
EP1652934A1 (en) * | 2003-07-29 | 2006-05-03 | Japan Science and Technology Agency | Improved method of amplifying atp and use thereof |
EP1652934A4 (en) * | 2003-07-29 | 2008-03-12 | Japan Science & Tech Agency | Improved method of amplifying atp and use thereof |
US7745160B2 (en) | 2003-07-29 | 2010-06-29 | Japan Science And Technology Agency | Method of amplifying ATP and use thereof |
US8003341B2 (en) | 2003-07-29 | 2011-08-23 | Japan Science And Technology Agency | Method of amplifying ATP and use thereof |
EP2100956A1 (en) | 2005-01-25 | 2009-09-16 | Yamasa Corporation | Method of enzymatically synthesizing 3'-phosphoadenosine-5'-phophosulfate |
US8728789B2 (en) | 2005-01-25 | 2014-05-20 | Yamasa Corporation | DNA fragment encoding a polyphosphate-driven nucleoside 5′-diphosphate kinase polypeptide |
US9193958B2 (en) | 2005-01-25 | 2015-11-24 | Yamasa Corporation | Method of enzymatically synthesizing 3′-phosphoadenosine-5′-phosphosulfate |
WO2006095696A1 (en) * | 2005-03-10 | 2006-09-14 | Bussan Nanotech Research Institute, Inc. | Method of detecting adenosine triphosphate and detection reagent therefor |
JP2020504615A (en) * | 2016-12-30 | 2020-02-13 | エヌティーエックスバイオ,エルエルシー | Cell-free expression system with novel inorganic phosphate-based energy regeneration function |
US11136586B2 (en) | 2016-12-30 | 2021-10-05 | Ntxbio, Llc | Cell-free expression system having novel inorganic polyphosphate-based energy regeneration |
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