Method of producing uridine 5'-diphospho-N-acetylgalactosamine

The technology of acetylgalactosamine and acetylgalactose is applied in the field of preparation of uridine 5'-diphosphate-N-acetylgalactosamine, which can solve the problems of unpractical method, difficult separation, difficult preparation of enzymes, etc. Easy effect of enzyme preparation

Active Publication Date: 2007-10-10
YAMASA SHOYU CO LTD
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Problems solved by technology

[0004] However, due to the following problems: (A) is only a small-scale reaction in the laboratory, (B) the preparation of the enzyme is difficult, (C) the ATP regeneration system must be supplemented during phosphorylation of galactosamine, and the yield of the whole reaction process is also low. (D) In ​​the case where UDP-galactosamine remains in the reaction solution, there is a problem in separating the target UDP-GalNAc from it, so it is not necessarily a practical method (Methods Enzymol., 28, 271-277, (1972), J. Org. Chem., 57, 152-157. (1992))
[0005] In addition, conversion of uridine 5'-diphosphate-N-acetylglucosamine 4-epimerase (UDP-GlcNAc 4-epimerase) to The method (WO2002 / 050267) of sugar amine (UDP-GlcNAc) synthesizing UDP-GalNAc, because there are following problems: (i) the existence amount of UDP-GlcNAc 4-epimerase in animal tissue or thalline is very little, ( ii) Although UDP-GlcNAc 4-epimerase can be prepared by the recombinant DNA method, the enzyme reaction is an equilibrium reaction so the conversion rate is low, and the UDP-GlcNAc as the substrate is not completely consumed, and a large amount remains in the reaction solution, Therefore the separation of UDP-GalNAc and UDP-GlcNAc is very difficult and thus this method is not practical

Method used

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  • Method of producing uridine 5'-diphospho-N-acetylgalactosamine

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Embodiment 1

[0075] (1) Cloning of the GALK 2 gene encoding N-acetylgalactosamine kinase derived from human kidney

[0076] Using the cDNA library derived from human kidney (obtainable from Crontec Company) as a template, the two primer DNAs shown below were synthesized according to conventional methods, and the human kidney GALK 2 gene (submitted to the National Center for Biotechnology Information ( NCBI), the accession number is NM_002044, (Submitted to NCBI, Accession No.NM_002044)) amplification.

[0077] Primer (A): 5'-CGGGGATCCATGGCTACAGAGAGCCCTGCT-3'

[0078] Primer (B): 5'-TACGTCGACTTAGGCCTCAAGCAAAACCAA-3'

[0079] When the GALK 2 gene was amplified by the PCR method, 100 μl of the reaction solution (50 mM potassium chloride, 10 mM Tris hydrochloric acid (pH 8.3), 1.5 mM magnesium chloride, 0.001% gelatin, 0.2 mM dATP, 0.2mM dGTP, 0.2mM dCTP, 0.2mM dTTP, template DNA 0.1ng, primer DNA (A) and (B) each 0.2μM, ExTaq DNA polymerase 2.5U) were heat denatured (94°C, 1 minute), The s...

Embodiment 2

[0096] (1) Synthesis of UDP-GalNAc with human N-acetylgalactosamine kinase and Escherichia coli UDP-GlcNAc pyrophosphorylase

[0097] In 0.2ml solution containing 100mM Tris hydrochloric acid buffer solution (pH7.5), 10mM magnesium chloride, 5mM 5'-UTP·3Na, 5mM GalNAc, 5mM 5'-ATP·3Na, 5mM sodium fluoride, add The obtained N-acetylgalactosamine kinase enzyme solution (0.094 U), UDP-GlcNAc pyrophosphorylase enzyme solution (4.4 U) and inorganic pyrophosphatase (manufactured by Sigma, 0.5 U) at a specified activity amount were kept at 37° C. react.

[0098] During the reaction, an appropriate amount was taken from the reaction liquid, and after boiling for 5 minutes, centrifugation was carried out, and the supernatant was subjected to HPLC analysis. Analysis of the reaction solution 4 hours after the start of the reaction confirmed that 3.2 mM of UDP-GalNAc was synthesized in the presence of N-acetylgalactosamine kinase, UDP-GlcNAc pyrophosphorylase, and inorganic pyrophosphoryl...

Embodiment 3

[0100] (1) Synthesis of UDP-GalNAc using dry yeast and human N-acetylgalactosamine kinase and Escherichia coli UDP-GlcNAc pyrophosphorylase

[0101]In 1ml of solution containing 200mM glucose, 50mM GalNAc, 50mM UMP, 200mM potassium phosphate (pH8.0), 20mM magnesium chloride, 3% (w / v) dry baker's yeast (Oriental Yeast Industry), add the mixture prepared in Example 1. Recombinant enzyme (N-acetylgalactosamine kinase; 0.32 U, UDP-GlcNAc pyrophosphorylase; 10.4 U) was reacted at 26° C. while stirring at a stirring speed of 300 rpm. Then, 0.1 ml of a 2M glucose solution was added to the reaction solution at 16, 24, 40, and 48 hours after the start of the reaction. The results of analyzing the reaction solution at different times are shown in FIG. 2 . The accumulated amount of UDP-GalNAc reached 32mM after 49 hours of reaction.

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Abstract

A method of producing uridine 5'-diphospho-N-acetylgalactosamine (UDP-GalNAc), which is an important substrate in synthesizing an oligosaccharide, characterized in that in enzymatically producing uridine 5'-diphospho-N-acetylgalactosamine from uridine 5'-triphosphate (UTP) and N-acetylgalactosamine-1-phosphate (GalNAc1-P), uridine 5'-diphospho-N-acetylgalactosamine pyrophosphorylase (UDP-GlcNAc pyrophosphorylase) originating in a microorganism (excluding pathogenic ones) is used as an enzyme. In this method, it is also possible to use, as GalNAc-1P, a product prepared from N-acetylgalactosamine and a phosphate donor in a reaction system with the use of N-acetylgalactosamine kinase. According to this method, uridine 5'-diphospho-N-acetylgalactosamine can be efficiently produced by using a relatively inexpensive substrate.

Description

technical field [0001] The invention relates to a preparation method of uridine 5'-diphosphate-N-acetylgalactosamine (UDP-GalNAc), an important substrate for oligosaccharide synthesis. Background technique [0002] The method for synthesizing oligosaccharides with an enzyme includes two methods, a method using the reverse reaction of an oligosaccharide hydrolase and a method using a transglycosylase. The synthesis method using transglycosylase is better than the reverse reaction method using hydrolase in terms of synthesis yield and use in the synthesis of oligosaccharides with complex structures. In addition, with the advancement of recombinant DNA technology in recent years, Large-scale production of various transglycosylases has also been developed along with this technology. [0003] However, except for a part of the sugar donor sugar nucleotides used in the synthesis of transglycosylases, the price of others is still relatively high, and the current supply is only at t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/30C12N15/62C12N15/54C12N9/12
CPCC12N9/1241C12P19/305C12N9/12C12N15/52C12N15/62C12P19/04
Inventor 奥山洁野口利忠
Owner YAMASA SHOYU CO LTD
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