Sequential enzymatic glycosyltransfer reactions on a microfluidic device: Synthesis of a glycosaminoglycan linkage region tetrasaccharide.
A microfluidic chip carrying three reaction chambers was designed and constructed to examine sequential multiple enzymatic reactions. The synthesis of oligosaccharides in living cells is carried out in the Golgi apparatus where multiple enzymes such as glycosidase and glycosyltransferases act on a variety of substrates to generate glycoconjugates that include glycolipids and glycoproteins. The regulatory mechanism of the process however remains unknown. A microchip-based analysis platform may provide a valuable tool with which to address the issue by mimicking the Golgi function. We thus examined 3 sequential glycosyltransfer reactions on a chip, and succeeded in the synthesis of a tetrasaccharide using immobilized enzymes. Also, the kinetic parameters for a recently identified glycosyltransferase, proteoglycan GalT-I, were obtained for the first time.
Ono
Yasunari
Y
Mitsubishi Kagaku Institute of Life Sciences (MITILS), 11 Minamiooya, Machida-shi, Tokyo, 194-8511, Japan.
Kitajima
Motohiro
M
Daikoku
Shusaku
S
Shiroya
Toshifumi
T
Nishihara
Shoko
S
Kanie
Yoshimi
Y
Suzuki
Katsuhiko
K
Goto
Satoshi
S
Kanie
Osamu
O
D002240:Carbohydrate Sequence; D004800:Enzymes, Immobilized; D008401:Gas Chromatography-Mass Spectrometry; D006025:Glycosaminoglycans / Q000737:chemistry* / Q000378:metabolism; D006031:Glycosylation; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation* / Q000706:statistics & numerical data*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000138:chemical synthesis* / Q000737:chemistry; D012685:Sepharose / Q000737:chemistry
D004800:Enzymes, Immobilized; D006025:Glycosaminoglycans; D009844:Oligosaccharides; D012685:Sepharose