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WO1998048031A1 - Procede de production d'adenosine 5'-triphosphate et utilisation de ladite substance - Google Patents

Procede de production d'adenosine 5'-triphosphate et utilisation de ladite substance Download PDF

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Publication number
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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Prior art keywords
kinase
polyphosphate
adenosine
atp
reaction
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Application number
PCT/JP1998/001711
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English (en)
Japanese (ja)
Inventor
Kazuya Ishige
Toshitada Noguchi
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Yamasa Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Yamasa Corporation filed Critical Yamasa Corporation
Priority to JP54545098A priority Critical patent/JP3764755B2/ja
Publication of WO1998048031A1 publication Critical patent/WO1998048031A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • 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

Procédé de production d'adénosine 5'-triphosphate (ATP) par mise en réaction d'adénosine 5'-monophosphate (AMP) avec une polyphosphate kinase, une adénylate kinase et un acide phosphorique. Ledit procédé permet de produire de l'ATP facilement et de manière peu onéreuse.
PCT/JP1998/001711 1997-04-18 1998-04-15 Procede de production d'adenosine 5'-triphosphate et utilisation de ladite substance WO1998048031A1 (fr)

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Application Number Priority Date Filing Date Title
JP54545098A JP3764755B2 (ja) 1997-04-18 1998-04-15 「アデノシン５’−三リン酸の製造法及びその応用」

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JP11627297 1997-04-18
JP9/116272 1997-04-18

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001053513A1 (fr) * 2000-01-17 2001-07-26 Satake Corporation Systemes et procede de reaction de regeneration atp permettant d'examiner le nucleotide d'adenine, procede de detection d'arn et procede d'amplification d'atp
EP1652934A1 (fr) * 2003-07-29 2006-05-03 Japan Science and Technology Agency Procede ameliore d'amplification de l'atp et son utilisation
WO2006095696A1 (fr) * 2005-03-10 2006-09-14 Bussan Nanotech Research Institute, Inc. Methode de detection de l’adenosine triphosphate et reactif de detection utilise
JP2009050264A (ja) * 2002-05-29 2009-03-12 Yamasa Shoyu Co Ltd 新規なポリリン酸：ａｍｐリン酸転移酵素
EP2100956A1 (fr) 2005-01-25 2009-09-16 Yamasa Corporation Procédé de synthèse enzymatique de 3'-phosphoadenosine-5'-phophosulfate
JP2020504615A (ja) * 2016-12-30 2020-02-13 エヌティーエックスバイオ，エルエルシー 新規無機リン酸ベースエネルギー再生機能を備えた無細胞発現系
US12227778B2 (en) 2016-12-30 2025-02-18 Nature's Toolbox, Inc. Cell-free expression system having novel inorganic polyphosphate-based energy regeneration

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP5800218B2 (ja) * 2011-07-20 2015-10-28 国立大学法人広島大学 Ａｔｐの製造方法およびその利用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS4882087A (fr) * 1972-02-07 1973-11-02
JPS57166992A (en) * 1981-04-08 1982-10-14 Seitetsu Kagaku Co Ltd Preparation of adenosine-5'-triphosphate
JPS59106296A (ja) * 1982-12-09 1984-06-19 Kazutomo Imahori 複合酵素法による生理活性物質の製造法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS4882087A (fr) * 1972-02-07 1973-11-02
JPS57166992A (en) * 1981-04-08 1982-10-14 Seitetsu Kagaku Co Ltd Preparation of adenosine-5'-triphosphate
JPS59106296A (ja) * 1982-12-09 1984-06-19 Kazutomo Imahori 複合酵素法による生理活性物質の製造法

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001053513A1 (fr) * 2000-01-17 2001-07-26 Satake Corporation Systemes et procede de reaction de regeneration atp permettant d'examiner le nucleotide d'adenine, procede de detection d'arn et procede d'amplification d'atp
JP2009050264A (ja) * 2002-05-29 2009-03-12 Yamasa Shoyu Co Ltd 新規なポリリン酸：ａｍｐリン酸転移酵素
US7745160B2 (en) 2003-07-29 2010-06-29 Japan Science And Technology Agency Method of amplifying ATP and use thereof
EP1652934A4 (fr) * 2003-07-29 2008-03-12 Japan Science & Tech Agency Procede ameliore d'amplification de l'atp et son utilisation
EP1652934A1 (fr) * 2003-07-29 2006-05-03 Japan Science and Technology Agency Procede ameliore d'amplification de l'atp et son utilisation
US8003341B2 (en) 2003-07-29 2011-08-23 Japan Science And Technology Agency Method of amplifying ATP and use thereof
EP2100956A1 (fr) 2005-01-25 2009-09-16 Yamasa Corporation Procédé de synthèse enzymatique de 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 (fr) * 2005-03-10 2006-09-14 Bussan Nanotech Research Institute, Inc. Methode de detection de l’adenosine triphosphate et reactif de detection utilise
JP2020504615A (ja) * 2016-12-30 2020-02-13 エヌティーエックスバイオ，エルエルシー 新規無機リン酸ベースエネルギー再生機能を備えた無細胞発現系
US11136586B2 (en) 2016-12-30 2021-10-05 Ntxbio, Llc Cell-free expression system having novel inorganic polyphosphate-based energy regeneration
US12227778B2 (en) 2016-12-30 2025-02-18 Nature's Toolbox, Inc. Cell-free expression system having novel inorganic polyphosphate-based energy regeneration

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