WO1998033894A1 - Procede pour la production de sucre de diphosphate nucleosidique (dpn) - Google Patents

Procede pour la production de sucre de diphosphate nucleosidique (dpn) Download PDF

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Publication number
WO1998033894A1
WO1998033894A1 PCT/DE1998/000185 DE9800185W WO9833894A1 WO 1998033894 A1 WO1998033894 A1 WO 1998033894A1 DE 9800185 W DE9800185 W DE 9800185W WO 9833894 A1 WO9833894 A1 WO 9833894A1
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WO
WIPO (PCT)
Prior art keywords
reaction
sugar
gtp
mannose
phosphate
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PCT/DE1998/000185
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German (de)
English (en)
Inventor
Sven Fey
Udo Kragl
Christian Wandrey
Lothar Elling
Maria-Regina Kula
Original Assignee
Forschungszentrum Jülich GmbH
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.)
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Publication date
Application filed by Forschungszentrum Jülich GmbH filed Critical Forschungszentrum Jülich GmbH
Priority to EP98906829A priority Critical patent/EP0958352A1/fr
Priority to JP53243498A priority patent/JP2001509671A/ja
Publication of WO1998033894A1 publication Critical patent/WO1998033894A1/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

Definitions

  • the invention relates to a method for producing NDP sugar (nucleoside diphosphate sugar), which are also referred to as sugar nucleotides.
  • NDP sugar nucleoside diphosphate sugar
  • These compounds are important intermediates in the biosynthesis of glycoconjugates. Their synthesis on a larger scale is therefore very important for the in vitro synthesis of oligosaccharides, especially with regard to the possible meanings of oligosaccharides in medical diagnostic and therapeutic applications.
  • NDP sugars can be synthesized by enzymatically reacting a sugar 1-phosphate with a nucleoside triphosphate (NTP).
  • NTP nucleoside triphosphate
  • they are usually insulated from natural material. Pyrophosphosrylases are used as enzymes, which catalyze the reaction with the help of divalent metal cations as cofactors.
  • a large number of such reactions are known in which different sugar 1-phosphates are reacted with different nucleoside triphosphates.
  • UDP-glucose, CDP-glucose, dTDP-glucose ( deoxy TDP-glucose), UDP-N-acetylglucosamine, UDP-galactose, UDP-N-
  • Acetylgalactosamine, GDP-fucose and GDP-mannose are listed.
  • By-products of these reactions are pyrophosphate anions PP i; which are converted into two phosphate anions (2 Pi) in a further enzymatic subsequent reaction.
  • the syntheses are usually carried out in stirred tanks which are operated in batch mode.
  • a flow-through membrane reactor is also known which is used in chemical process engineering.
  • the reactants to be reacted are passed through a reaction space, the cross section of which is separated by a membrane which is designed as an ultrafiltration membrane and retains molecules from a defined size.
  • the pyrophosphosrylases used in the reactions described above are subject to inhibitory influences, which reduce the enzyme activity to a large extent, so that the reaction rate decreases during the reaction.
  • the NDP sugar formed during the reaction inhibits the product.
  • This product inhibition competes with the reaction of the enzyme with the NTP.
  • the pyrophosphate anions PP i formed during the reaction also lower the activity of the pyrophosphorylases - however, these are removed from the equilibrium by a further degradation reaction, which reduces this effect.
  • nucleoside diphosphate sugars using pyrophosphorylases is to be made accessible for production on a larger scale.
  • Fig. 1 A general scheme for the enzymatic reaction of guanosine triphosphate (GTP) with mannose-1-phosphate to guanosine diphospho-mannose (GDP-mannose)
  • Fig. 2 The concentration profile of the reaction components Mg + and GTP during the reaction according to the prior art.
  • Fig. 4 The dependence of the pyrophosphorylase activity on the Mg 2+ / GTP ratio for the conversion of GTP and mannose-1-phosphate to GDP-mannose.
  • Fig. 5 The time course of the pyrophosphorylase activity for a process according to the prior art and according to the method according to the invention.
  • Fig. 6 A flow reactor suitable for carrying out the reaction.
  • Fig. 7 A graphic with the time course of the continuous production of GDP-mannose in the flow reactor under different operating conditions.
  • Fig. 8 A comparative illustration of the
  • Fig. 9 A comparative graphic between a reaction procedure according to the invention in a flow reactor and a cascade consisting of two flow reactors.
  • Fig. 10 A graph in which the conversion of mannose-1-phosphate in a flow rate Actuator depending on the concentration ratio between GTP and mannose-1-phosohate is shown.
  • Fig.11 A graphic that shows itself during the
  • guanosine triphosphate (GTP) is reacted with mannose-1-phosphate in an enzyme-catalyzed reaction with GDP-mannose pyrophosphorylase EC 2.7.7.13 (recombinant enzyme from Escherichia coli. Source of the gene: Salmonella Enterica) converted to guanosine diphospho-mannose (GDP-mannose). Mg is used as the cofactor.
  • the by-product is the pyrophosphate anion PP ⁇ , which is degraded in a subsequent reaction to phosphate anions P i # . It is known that in such a reaction the pyrophosphorylase is inhibited both by the GDP mannose, the pyrophosphate and by an excess of the cofactor Mg + .
  • FIG. 2 shows the concentration profile of the GTP that occurs in the reaction shown in FIG. 1
  • FIG. 3 shows the concentration curve of the same components as in FIG. 2 using the method according to the invention.
  • the abscissa x and ordinate designations y are the same here as in FIG. 2. It can be seen from FIG. 2 that, according to the invention, both the Mg 2+ (straight line 1) and the GTP concentration (straight line 2) are kept constant.
  • FIG. 4 shows graphically at which concentration ratio between Mg + and GTP the activity of the pyrophosphorylase is greatest.
  • concentration ratio between Mg + and GTP the activity of the pyrophosphorylase is greatest.
  • Abscissa x concentration ratio between Mg and GTP (c Mg2 + / c GTP ), dimensionless.
  • Ordinate y: Volume-related activity of GDP-mannose pyrophosphoryslase Dimension: U / ml (1U 1
  • the curve was drawn through a group of three different types of points, which correspond to different values for the GTP concentration (c GTP ).
  • FIG. 5 shows schematically the time course of the pyrophosphorylase activity in the comparison between the procedure according to the prior art shown in curve 1 and the procedure according to the invention shown in straight line 2, in which the concentration ratio between GTP and Mg + is kept constant.
  • the 6 comprises a feed line 1 in which GTP, mannose-1-phosphate and the cofactor Mg are supplied as substrates S.
  • the feed line 1 opens into the reaction space 2, which contains the enzyme pyrophosphorylase.
  • An ultrafiltration membrane 3 is attached in the reaction space 2 in such a way that it forms a cross section through the flow volume of the introduced substrate solution.
  • An outlet line 4 connects to the reaction chamber 2 behind the ultrafiltration membrane 3, through which the products P, in this example GDP-mannose and P i7, and unreacted GTP, mannose-1-phosphate and the cofactor Mg leave the flow-through reactor.
  • the flat structures 5 in the reaction space represent the enzyme pyrophosphorylase.
  • the graphic shown in FIG. 7 shows the yields of GDP-mannose which were achieved with continuous operation in a flow-through reactor with different residence times.
  • the ordinate y the yield in relation to mannose-1-phosphate ⁇ GD p- Man , Man-i- P dimension%.
  • the vertical dashed lines each characterize new test sections with new parameters for the residence time in the flow reactor.
  • the information ⁇ x min denotes the dwell times for the new test sections.
  • the one with the arrow ⁇ - dated point means a dosage of the enzyme of 100 ul of pyrophosphorylase at a concentration of 87.6 mU / ml.
  • FIG. 8 shows the increase in the reaction rate, which is caused by the use of an excess of GTP in relation to the concentration of mannose-1-phosphate when carrying out the classic reaction in a batch reactor.
  • GTP concentration of mannose-1-phosphate
  • the ordinate y the GDP-mannose concentration
  • the graphic shown in FIG. 9 shows the increase in space-time yield (RZA or ⁇ RZ ) when using two flow reactors arranged in a cascade. compared to carrying out the reaction in only one flow reactor.
  • the total pyrohosphorylase concentration as a parameter is constant.
  • ⁇ residence time in the reaction solution in the flow reactor, which was varied by computer simulation.
  • Curve 1 shows the course for a flow reactor, curve 2 for two reactors connected in series, through which the reaction solution passes in succession.
  • Fig. 9 is the result of a computer simulation based on the mathematical model in Formula 1.
  • the graph shown in FIG. 10 shows the dependence of the conversion of mannose-1-phosphate on the ratio of the concentrations of the GTP to mannose-1-phosphate.
  • Abzzissa x the time (dimensionless) and the ordinate y: the quantitative ratio between Mg 2+ and GTP (Mg 2+ / GTP dimensionless). This course arises in one Stir kettle when working in batch mode according to the state of the art.
  • the quantitative ratio of Mg + and guanosine triphosphate (GTP), ie Mg 2+ / GTP, is kept constant during the production of GDP mannose. Reaction control in which this prerequisite is fulfilled can be ensured by operating the flow reactor shown in FIG. 6.
  • the GDP-mannose pyrophosphorylase EC 2.7.7.13 required for carrying out the reaction is located in reaction chamber 2 in aqueous solution, which is separated by a tris
  • Buffer is kept constant at pH 8.
  • Mg + , mannose-1-phosphate and GTP are fed in through feed line 1 - the GTP concentration is kept at a constant value.
  • the products resulting from the reaction, GDP-mannose and phosphate, which are produced by the breakdown of pyrophosphate, and excess Mg leave reaction chamber 2 via the ultrafiltration membrane, which retains the pyrophosphorylase as a macromolecule.
  • the inorganic pyrophosphatase that breaks down the pyrophosphate PP ⁇ is also retained by the ultrafiltration membrane.
  • the Mg + / GTP ratio is kept constant, which surprisingly leads to a consistently high enzyme activity of the pyrophosphorylase and thus a higher space-time yield compared to a reaction in a stirred tank with batch operation.
  • this concentration ratio constant is very beneficial for the space-time yield, even if the substrate GTP (guanosine triphosphate) is in the contraction saturation range with regard to the enzyme reaction, in which, according to the previous conception, a decrease in the GTP concentration within the saturation range has no influence had the reaction speed.
  • a constant, maximum, reaction speed is set in continuous operation (Fig. 5).
  • Varying the residence time of the reaction mixture leads to different conversions and space-time yields.
  • the sections delimited by the vertical dashed lines in FIG. 7 represent test sections in which the flow rate of the reaction solution, which is inversely proportional to the residence time ⁇ , was varied as a parameter. The figure shows that when using the same Because segments characterized by the same period can achieve the same sales. The sales that decrease with increasing operating time of the flow reactor are due to natural denaturation of the pyrophosphorylase. The increase in sales in segment 2 of FIG. 7 is only due to an increase in temperature during operation.
  • the invention is not limited to the reaction of guanosine triphosphate with mannose-1-phosphate under the catalytic action of GDP-mannose pyrophosphorylase in combination with Mg + as a cofactor.
  • the surprising effect is that the enzyme activity in the reaction of nucleoside triphosphates with sugar-1-phosphates with the participation of cofactors in the form of divalent metal cations can be kept constant at a high level for long periods of time if the ratio between the nucleoside triphosphate and the concentration of the divalent metal cation cofactor is kept constant, has generally been found.
  • nucleotides uridine (U), guanosine (G), cytidine (C), thymidine (T) and d-thymidine (dT) with the sugar-1-phosphates of mannose, galactose, or Glucose, fucose, N-acetylglucosamine or N-acetylglucosamine are implemented.
  • the cofactors Mg + , Mn or, for example, Zn + can be used for each reaction.
  • the pyrophosphosylases which convert the corresponding sugar 1-phosphate serve as pyrophoyphorylases.
  • the effect according to the invention that the STA is increased or the enzyme activity remains costly over long periods apart from natural denaturation can also be achieved if a stirred tank, ie in batch mode, is used with a decreasing NTP concentration and a complexing agent is added, which complexes the cofactor, such as Mg + , in accordance with the decrease in the NTP concentration and thus removes the reaction equilibrium.
  • a stirred tank ie in batch mode
  • a complexing agent is added, which complexes the cofactor, such as Mg + , in accordance with the decrease in the NTP concentration and thus removes the reaction equilibrium.
  • the effect according to the invention also occurs if the NTP concentration is kept constant and the concentration of the cofactor fluctuates slightly.
  • the limits for good results are given in this case by the fact that the cofactor concentration must not drop to such an extent that the cofactor does not act as a limiting factor for the reaction rate and that on the other hand no excess cofactor is allowed to arise so that none inhibitory effect occurs. However, these limits are different for each reaction and can be determined by simple experiments.

Abstract

L'invention concerne un procédé pour la production de sucre de diphosphate-nucléosidique (DPN), dans lequel un sucre-1-phosphate réagit avec un triphosphate nucléosidique en présence d'un cation métallique bivalent comme co-facteur par l'intermédiaire d'une pyrophosphorylase transformant le sucre-1-phosphate. Selon l'invention, la concentration en TPN ou bien de préférence le rapport de concentration entre le sucre de DPN et le cation métallique bivalent sont maintenus constant. Etonnamment, lors d'une telle réaction, l'inhibition de la pyrophosphorylase est largement empêchée, la réaction présentant ainsi, en raison d'une activité enzymatique constante, un rendement-espace-temps élevé.
PCT/DE1998/000185 1997-01-29 1998-01-20 Procede pour la production de sucre de diphosphate nucleosidique (dpn) WO1998033894A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP98906829A EP0958352A1 (fr) 1997-01-29 1998-01-20 Procede pour la production de sucre de diphosphate nucleosidique (dpn)
JP53243498A JP2001509671A (ja) 1997-01-29 1998-01-20 Ｎｄｐ−糖類の製造方法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE1997103031 DE19703031C2 (de) 1997-01-29 1997-01-29 Verfahren zur Herstellung von NDP-Zucker
DE19703031.9 1997-01-29

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1034797A2 (fr) * 1999-03-01 2000-09-13 Forschungszentrum Jülich Gmbh Procédé d'obtention d'un réactif de glycosylation marqué au fluor-18 ainsi qu'un réactif de glycosylation marqué au fluor-18 et son utilisation
EP1816206A1 (fr) * 2004-10-21 2007-08-08 Yamasa Corporation Procede de production d'uridine 5'-diphospho-n-acetylgalactosamine

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3937892A1 (de) * 1989-11-15 1991-05-16 Forschungszentrum Juelich Gmbh Enzymmembranreaktor
WO1996027670A2 (fr) * 1995-03-03 1996-09-12 Forschungszentrum Jülich GmbH PROCEDE ENZYMATIQUE DE FABRICATION DE GDP-α-D-MANNOSE, GDP-MANNOSE PYROPHOSPHORYLASE ET PHOSPHOMANNOMUTASE APPROPRIEES ET LEUR OBTENTION, EPREUVE ENZYMATIQUE

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0202094B1 (fr) * 1985-05-13 1988-09-21 Unitika Ltd. Procédé de production d'une substance physiologiquement active

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3937892A1 (de) * 1989-11-15 1991-05-16 Forschungszentrum Juelich Gmbh Enzymmembranreaktor
WO1996027670A2 (fr) * 1995-03-03 1996-09-12 Forschungszentrum Jülich GmbH PROCEDE ENZYMATIQUE DE FABRICATION DE GDP-α-D-MANNOSE, GDP-MANNOSE PYROPHOSPHORYLASE ET PHOSPHOMANNOMUTASE APPROPRIEES ET LEUR OBTENTION, EPREUVE ENZYMATIQUE

Non-Patent Citations (2)

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Title
C.J. GRAY: "Enzyme-catalized reactions", 1971, VAN NOSTRAND REINHOLD PUBL., LONDON, XP002065264 *
PREISS J ET AL: "SUGAR NUCLEOTIDE REACTIONS IN ARTHOBACTER I. GUANOSINE DIPHOSPHATE MANNOSE PYROPHOSPHORYLASE: PURIFICATION AND PROPERTIES", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 239, no. 10, 1 October 1964 (1964-10-01), pages 3119 - 3126, XP000574872 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1034797A2 (fr) * 1999-03-01 2000-09-13 Forschungszentrum Jülich Gmbh Procédé d'obtention d'un réactif de glycosylation marqué au fluor-18 ainsi qu'un réactif de glycosylation marqué au fluor-18 et son utilisation
EP1034797A3 (fr) * 1999-03-01 2000-09-20 Forschungszentrum Jülich Gmbh Procédé d'obtention d'un réactif de glycosylation marqué au fluor-18 ainsi qu'un réactif de glycosylation marqué au fluor-18 et son utilisation
EP1816206A1 (fr) * 2004-10-21 2007-08-08 Yamasa Corporation Procede de production d'uridine 5'-diphospho-n-acetylgalactosamine
EP1816206A4 (fr) * 2004-10-21 2011-10-26 Yamasa Corp Procede de production d'uridine 5'-diphospho-n-acetylgalactosamine

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EP0958352A1 (fr) 1999-11-24
DE19703031C2 (de) 1998-10-29
JP2001509671A (ja) 2001-07-24
DE19703031A1 (de) 1998-07-30

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