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Application of galactokinase in synthesizing N-acetylgalactose-1-phosphoric acid and derivatives thereof

A technology of acetylgalactose and galactokinase, applied in the direction of transferase, fermentation, etc., can solve weak problems and achieve the effect of broad application prospects

Inactive Publication Date: 2011-07-20
SHANDONG UNIV
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  • Summary
  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

However, GalK discovered so far cannot utilize N-acetylgalactose (GalNAc) to generate GalNAc-1-P and its derivatives
[0008] At the same time, only Galk derived from lactic acid bacteria has been found to have a very weak ability to synthesize Glc-1-P

Method used

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  • Application of galactokinase in synthesizing N-acetylgalactose-1-phosphoric acid and derivatives thereof
  • Application of galactokinase in synthesizing N-acetylgalactose-1-phosphoric acid and derivatives thereof
  • Application of galactokinase in synthesizing N-acetylgalactose-1-phosphoric acid and derivatives thereof

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Experimental program
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Embodiment

[0049] 1 Materials and methods

[0050] 1.1 Strains and methods

[0051] E.coli DH5α was purchased from Gibco-BRL (Gaithersburg, MD), protein expression strain E.coli BL21(DE3)F-ompT hsdS B (r - B m - B ) gal dcm (DE3) was purchased from Novagen (Carlsbad, CA). The pMCSG7 plasmid was from Novagen (Madison, WI). Reagents and various restriction enzymes used for PCR were from Invitrogen (Carlsbad, CA). Qiagen (Valencia, CA). Plasmid extraction kit and gel recovery kit were purchased from Qiagen (Valencia, CA). Nickel ion affinity chromatography column (5ml) and HiLoad_16 / 60_superdex 200 column were purchased from Amersham Pharmacia Biotech (Piscataway, NJ).

[0052] 1.2 Clone the galK gene from S.pneumonia TIGR4.

[0053] The DNA sequence of the gene galK (GenBank accession No.AAK75925) was extracted from the S.pneumoniae TIGR4 genome, and the PCR primers were as follows:

[0054] GalKS: 5'-TACTTCCAATCCAATGCGATGGCACAACATCTTACT-3' (SEQ ID NO.3)

[0055] GalKA: 5'-TTAT...

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Abstract

The invention relates to application of galactokinase in synthesizing N-acetylgalactose-1-phosphoric acid and derivatives thereof, belonging to the technical field of biotechnology. The application method comprises the following steps: (1) preparing a galactokinase solution from galactokinase of which the amino acid sequence is disclosed as SEQ ID No.1; (2) preparing an N-acetylgalactose solution or N-acetylgalactose derivative solution from N-acetylgalactose or N-acetylgalactose derivatives; and (3) proportionally mixing the galactokinase solution with the N-acetylgalactose solution or N-acetylgalactose derivative solution, adding MgCl2 and ATP (adenosine triphosphate), reacting, and purifying after the reaction, thereby obtaining the N-acetylgalactose-1-phosphoric acid or derivatives thereof. The invention overcomes the technical defect that the galactokinase can not be applied to actual production since the galactokinase can not be synthesized into N-acetylgalactose-1-phosphoric acid or the synthesis efficiency of N-acetylgalactose-1-phosphoric acid is very low in the prior art.

Description

technical field [0001] The invention relates to the application of galactokinase in synthesizing N-acetylgalactose-1-phosphate and derivatives thereof, and belongs to the technical field of biotechnology. Background technique [0002] At present, many natural products and drugs have sugar groups, and changes in sugar groups will affect the biological functions and properties of these glycoconjugates, such as affecting their stability and half-life. To change the glycosyl groups of glycoconjugates, it is first necessary to obtain glycosyl donors. Therefore, how to obtain glycosyl donors has become a key step in research. [0003] N-acetylgalactose (GalNAc) is an important sugar group that exists widely in organisms and participates in many biological functions. The synthesis of glycoconjugates containing N-acetylgalactose (GalNAc) requires the nucleoside acetylgalactose (UDP-GalNAc) as a glycosyl donor. UDP-GalNAc can be obtained by converting nucleoside acetylglucose (UDP-...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P13/02C12N9/12
Inventor 陈敏陈蕾蕾沈杰王鹏
Owner SHANDONG UNIV
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