Novel n-acetylglucosamine-2-epimerase and method for producing cmp-neuraminic acid using the same

一种乙酰神经氨酸、乙酰葡糖胺的技术，应用在异构酶、生物化学设备和方法、酶等方向，能够解决N-乙酰甘露糖胺很贵、胞苷三磷酸产量低、纯化ManNAc过程复杂等问题

Active Publication Date: 2010-03-10
GENECHEM
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] However, method (1) has a problem in that raw material N-acetyl mannosamine (ManNAc) is very expensive, and method (2) uses cheap N-acetyl glucosamine (GlcNAc), but has a problem in that it is obtained from GlcNAc and N - Purification of ManNAc from a mixture of acetylmannosamine (ManNAc) is complicated
However, this method has a problem in that it requires multiple steps to produce CMP-N-acetylneuraminic acid, and the conversion of cytidylic acid (CMP) used as a substrate into cytidine triphosphate (CTP) is low in yield

Method used

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  • Novel n-acetylglucosamine-2-epimerase and method for producing cmp-neuraminic acid using the same
  • Novel n-acetylglucosamine-2-epimerase and method for producing cmp-neuraminic acid using the same
  • Novel n-acetylglucosamine-2-epimerase and method for producing cmp-neuraminic acid using the same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1: Cloning and Transformation of Genes

[0054] 1-1: nanE gene encoding N-acetylglucosamine-2-epimerase (SEQ ID NO: 1) cloning and transformation of

[0055] In order to eliminate the step of isomerizing N-acetyl-D-glucosamine into N-acetyl-D-mannosamine (which serves as the goal of increasing the production yield of CMP-N-acetylneuraminic acid and reducing production costs) Biggest problem pointed out), N-acetylglucosamine-2-epimerase (GlcNAc2 epimerase) was sought from various sources.

[0056] Three N-acetylglucosamine-2-epimerase-like genes were discovered from the anaerobic microorganism Bacteroides fragilis NCTC 9343 (NC_003228).

[0057] Candidate gene No.1 for N-acetylglucosamine-2-epimerase is located in the region of 947856-949034, has a size of 1179bp, and shows the same The corresponding genes of have 23.68-24.44% similarity.

[0058] Candidate gene No.2 for N-acetylglucosamine-2-epimerase is located in the scope of 1996625-1997809, has the ...

Embodiment 2

[0099] Example 2: Enzyme expression analysis

[0100] Inoculate 500 ml of seed culture obtained by culturing each transformant E.coli / pNANe, E.coli / pNANa, E.coli / pCMK, E.coli / pACKa and E.coli / pSYNb in LB medium to 5 liters of LB medium. When the cell concentration (OD 600 ) reached approximately 3-5, the cells were harvested. The culture conditions of the transformants containing various enzymes are shown in Table 1 below. Harvested cells were disrupted using a disruptor or French press, and the expression of each enzyme was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) ( Figure 7 ).

[0101] As a result, it was observed that the enzyme encoded by the gene inserted into each transformant was expressed in a large amount.

[0102] Table 1: Culture conditions of strains producing each enzyme

[0103]

[0104] (CMK)

Embodiment 3

[0105] Embodiment 3: the purification of enzyme

[0106] The N-acetylglucosamine-2-epimerase (GlcAc2-isomerase), N-acetylneuraminic acid aldolase (NeuAc aldolase), cytidine 5'-phosphate kinase, acetate kinase and CMP-N-acetylneuraminic acid synthetase (CMP-NeuAc synthetase) were precipitated using ammonium sulfate and purified using an ion exchange resin column (Protein purification techniques "Protein purification techniques", 2nd edition, Oxford University Press, 2001).

[0107] The concentration of ammonium sulfate used for each enzyme precipitation can be properly adjusted in the range of 30-80%. Each enzyme is precipitated using ammonium sulfate in a refrigerator below 10°C, preferably 3-5°C, and stirred for 1-5 hours.

[0108] The precipitated enzyme was dissolved in a small amount of 50 mM Tris HCl (pH 7.5) buffer and desalted through a dialysis membrane. The purified and concentrated enzyme solution was applied on a UNO sphereQ (Bio-RAD) column packed with strong i...

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Abstract

The present invention relates to a novel N-acetylglucosamine-2- epimerase and a method for preparing CMP-N-acetylneuraminic acid, more specifically, relates to a N-acetylglucosamine-2-epimerase derived from Bacteroides fragilis NCTC 9343, and a method for preparing CMP-N- acetylneuraminic acid using said N-acetylglucosamine-2-epimerase. According to the present invention, CMP-N-acetylneuraminic acid can be produced economically in a large amount through a one-step reaction using cytidine monophosphate and N-acetyl-D-glucosamine which are inexpensive substrates.

Description

technical field [0001] The present invention relates to novel N-acetylglucosamine-2-epimerases and methods for the production of CMP-neuraminic acid, more particularly to N-acetylglucosamine derived from Bacteroides fragilis NCTC9343 -2-Epimerase and method for producing CMP-N-acetylneuraminic acid using said N-acetylglucosamine-2-epimerase. Background technique [0002] Recently, due to the rapid development of studies on the structure and function of sugar chains, the development of physiological activities of oligosaccharides, glycolipids and glycoproteins and their analogs and their use as drugs or functional substances has attracted attention. Among them, sialic acid-containing sugar chains having N-acetylneuraminic acid (NeuAc) at the end have important functions, such as functioning as receptors at the time of cell adhesion or virus infection. [0003] Sialic acid-containing sugar chains are generally catalyzed by sialyltransferases. Sialyltransferase is an enzyme t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/00
CPCC12P17/16C12N9/90C12N9/00C12N9/88
Inventor 禹真锡宋在京金秉祺姜善烨金大熙张庆淳梁智英郑永洙徐元敏吉泰建沈相熙许仁康
Owner GENECHEM
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