|Budget Amount *help
¥12,600,000 (Direct Cost : ¥12,600,000)
Fiscal Year 2001 : ¥3,800,000 (Direct Cost : ¥3,800,000)
Fiscal Year 2000 : ¥3,800,000 (Direct Cost : ¥3,800,000)
Fiscal Year 1999 : ¥5,000,000 (Direct Cost : ¥5,000,000)
An 'enzyme-based automated glycosynthesizer' which can be applied for efficient and practical synthesis of glycoconjugates has been developed. The first version of the synthesizer named 'Golgi' has been accomplished in this project. Immobilized glycosyltransferases catalyze transfer reactions of sugar molecules from sugar nucleotides to water-soluble primer polymers in which sugars bound on the flexible polymers. Recently, a variety of recombinant glycosyltransferases are becoming practicable owing to advances in cloning studies of glycosyltransferases. Five of these recombinant glycosyltransferases, b-1,4-GalT, a-2,3-SiaT, a-2,6-SiaT, b-1,3-GlcNacT and a-1,3-FucT, have been immobilized successfully and is implemented in our synthesizer. A fusion protein composed of b-1,3-N-acetyl-D- glucosaminyltransferase (b-1,3-GlcNAcT) from Streptococcus agalactiae type Ia and maltose-binding protein (MBP) have been produced in Escherichia coli as a soluble and highly active form. Water-soluble primer polymers, which are polyacrylamide-based materials with simple sugar side-chains as glycosyl acceptors, drastically enhance the affinity between recombinant glycosyltransferases and the primer polymers. In addition, the primer polymers can be easily isolated from the reaction mixture after the sequential sugar-elongation reactions by employing simple gel- permeation chromatography. The sugar-elongation processes by enzyme can be monitored by both NMR spectroscopy and fluorescence resonance energy transfer (FRET)-based analysis.
Although sugar-elongation reactions by GalT proceed quantitatively, the efficiency of glycosylation by SiaT and FucT are low at the present time. We will examine the condition of glycosylation by these glycosyltransferases to improve the efficiency of the transfer reactions for practical usage of the automated synthesizer.