WO1999049073A1 - Procede de production de cytidine 5'-diphosphate choline - Google Patents

Procede de production de cytidine 5'-diphosphate choline Download PDF

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Publication number
WO1999049073A1
WO1999049073A1 PCT/JP1999/001312 JP9901312W WO9949073A1 WO 1999049073 A1 WO1999049073 A1 WO 1999049073A1 JP 9901312 W JP9901312 W JP 9901312W WO 9949073 A1 WO9949073 A1 WO 9949073A1
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WO
WIPO (PCT)
Prior art keywords
choline
reaction
cdp
cytidine
cct
Prior art date
Application number
PCT/JP1999/001312
Other languages
English (en)
Japanese (ja)
Inventor
Toshitada Noguchi
Kiyoshi Okuyama
Tomoki Hamamoto
Kenji Takenouchi
Original Assignee
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
Publication of WO1999049073A1 publication Critical patent/WO1999049073A1/fr

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Classifications

    • 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
    • C12P19/305—Pyrimidine nucleotides

Definitions

  • the present invention head trauma, consciousness disorders associated with brain surgery, cytidine 5 1 are found using the improved treatment such as stroke - Ru der method of manufacturing a diphosphate choline (CDP-choline).
  • CDP-choline diphosphate choline
  • CCT choline kinase
  • CKI choline kinase
  • cytidine 5 Ichito only Urijin 5 1 coexist Li phosphate synthetase (PyrG) - monophosphate (5 1 - UMP) and choline
  • the present invention provides yeast cells, 5 1 - a UMP and choline, CCT, Ru der relates CDP- choline manufacturing method characterized by reacting in the presence of CKI and PyrG.
  • the present invention is a yeast cell, 5 1 - a UMP and phosphorylcholine
  • Ru der relates CDP- choline manufacturing method characterized by reacting in the presence of CCT and PyrG.
  • Figure 1 shows the effect of reaction temperature on CDP-choline productivity and the composition of the reaction mixture.
  • each substrate is preferably in the range of about 1 to 200 mM, particularly about 10 to 100 mM.
  • DNA preparation, restriction enzyme digestion, DNA ligation using T4 DNA ligase, and E. coli transformation were all performed by “Molecular cloning”.
  • Amplification of CCT gene by PCR is as follows: Reaction solution 10 ( ⁇ 1 [50 mM chloride, 10 mM Tris-HCl (pH 8.3), 1.5 mM magnesium chloride, 0.001% gelatin, 0.5 g of temperate DNA, primer DNA (A) and (B) in each 0.2 lambda M, AmpliTaq DNA polymerase La one peptidase 2.5 Yuni' preparative], using a Perkin-Elmer Cetus Instrument Co., Ltd.
  • the reaction solution was treated with a mixture of phenol nocroform-form (1: 1), and a two-fold solution of ethanol was added to the water-soluble fraction to precipitate DNA.
  • the DNA collected by precipitation was separated by agarose gel electrophoresis according to the method described in the literature (Molecular cloning, described above), and a DNA fragment corresponding to 2.0 kb was purified.
  • the DNA was cut with restriction enzymes Xbal and Pstl, and plasmid pTrc99A was digested with restriction enzymes Xbal and Pstl.
  • pTrc-CKI is obtained by inserting an Xbal-Pstl DNA fragment containing the yeast CKI gene into an Xbal-Pstl cleavage site downstream of the trc promoter of pTrc99A.
  • Plasmid pTrc-CCT was digested with restriction enzymes Ncol and O ⁇ Xbal, and the Ncol-Xbal fragment containing the CCT gene was separated and purified. The fragment was ligated with plasmid pTrc-CKI DNA cut with Ncol and al using T4 DNA ligase, and Escherichia coli JM109 was transformed using the ligation reaction solution. Plasmid pTrc-CCK was isolated from the obtained ampicillin-resistant transformant. pTrc-CCK is obtained by ligating and inserting yeast CCT and CKI genes downstream of the trc promoter of pTrc99A.
  • Escherichia coli JM109 harboring plasmid pTrc-CCK was inoculated into 300 ml of 2 XYT medium containing 100 g / ml ampicillin and cultured with shaking at 37 ° C. 4
  • IPTG isopropyl-/?-D-thiogalactovyranoside
  • the unit (unit) of each enzyme was measured and calculated by the following method.
  • the amount of CDP-choline in the reaction solution is analyzed by (HPLC).
  • the activity to produce 1 mole of CDP-choline per minute at 37 ° C is defined as 1 unit (unit).
  • the measurement of PyrG activity and the calculation of units were performed by the following methods. That is, an enzyme preparation was added to a 200 mM Tris-HCl buffer (pH 7.8) containing 5 mM UTP, 5 mM phosphocholine, 5 mM ATP, 10 mM magnesium chloride, 50 mM ammonium sulfate, and 1 unit / ml CCT. Perform the reaction at 37 ° C, and stop the reaction by heat treatment at 100 ° C for 1 minute. The amount of CDP-choline in the reaction solution is determined by the HPLC method. The activity to produce 1 ⁇ mol of CDP-choline per minute at 37 ° C is defined as 1 unit (unit).
  • the present invention provides a simple and efficient method for coexisting CCT, CKI (not required when phosphorylcholine is used) and PyrG in a reaction system of yeast cells, 5 "-UMP, and choline or phosphorylcholine.
  • the reaction temperature within the range of about 20-30 ° C, the synthesis yield of CDP-choline can be significantly increased, while the production of unintended products such as UDP-glucose can be kept low.
  • by dividing and adding the energy source it is possible to further improve the productivity of CDP-choline.

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

Procédé de production efficace de cytidine 5'-diphosphate choline à utiliser pour l'amélioration de troubles de la conscience associés, entre autres, aux coups de chaleur ou à une opération du cerveau, à une attaque cérébrale, etc. Ledit procédé consiste à ajouter de la choline-phosphate citidyltransférase, de la choline kinase (inutile lorsque de la phosphorylcholine est utilisée) et de la cytidine 5'-triphosphate synthétase à un système réactionnel contenant des cellules de levure, de l'uridine 5'-monophosphate et de la choline ou de la phosphorylcholine. En régulant la température de réaction, de sorte qu'elle soit de 20 à 30 °C environ, et en ajoutant une source d'énergie dans certaines parties, en particulier, on peut encore accroître la productivité de la cytidine 5'-diphosphate choline.
PCT/JP1999/001312 1998-03-20 1999-03-17 Procede de production de cytidine 5'-diphosphate choline WO1999049073A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP10/92626 1998-03-20
JP9262698 1998-03-20

Publications (1)

Publication Number Publication Date
WO1999049073A1 true WO1999049073A1 (fr) 1999-09-30

Family

ID=14059664

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP1999/001312 WO1999049073A1 (fr) 1998-03-20 1999-03-17 Procede de production de cytidine 5'-diphosphate choline

Country Status (1)

Country Link
WO (1) WO1999049073A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003095660A1 (fr) 2002-05-08 2003-11-20 Kyowa Hakko Kogyo Co., Ltd. Procede de production de cytidine 5'-diphosphate choline
WO2007018259A1 (fr) 2005-08-10 2007-02-15 Kyowa Hakko Kogyo Co., Ltd. Procédé de purification de la cytidine diphosphocholine
CN111808899A (zh) * 2020-08-31 2020-10-23 宁波酶赛生物工程有限公司 一种胞磷胆碱钠的合成方法
CN116790466A (zh) * 2023-07-19 2023-09-22 山东理工大学 一种工程改造枯草芽孢杆菌发酵生产胞磷胆碱的方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5127754B1 (fr) * 1969-12-19 1976-08-14
JPH05276974A (ja) * 1992-01-30 1993-10-26 Kyowa Hakko Kogyo Co Ltd シチジンジリン酸コリンの製造法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5127754B1 (fr) * 1969-12-19 1976-08-14
JPH05276974A (ja) * 1992-01-30 1993-10-26 Kyowa Hakko Kogyo Co Ltd シチジンジリン酸コリンの製造法

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003095660A1 (fr) 2002-05-08 2003-11-20 Kyowa Hakko Kogyo Co., Ltd. Procede de production de cytidine 5'-diphosphate choline
WO2007018259A1 (fr) 2005-08-10 2007-02-15 Kyowa Hakko Kogyo Co., Ltd. Procédé de purification de la cytidine diphosphocholine
US8303820B2 (en) 2005-08-10 2012-11-06 Kyowa Hakko Bio Co., Ltd. Method of purifying cytidine diphosphate choline
CN111808899A (zh) * 2020-08-31 2020-10-23 宁波酶赛生物工程有限公司 一种胞磷胆碱钠的合成方法
CN116790466A (zh) * 2023-07-19 2023-09-22 山东理工大学 一种工程改造枯草芽孢杆菌发酵生产胞磷胆碱的方法
CN116790466B (zh) * 2023-07-19 2023-11-03 山东理工大学 一种工程改造枯草芽孢杆菌发酵生产胞磷胆碱的方法

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