title: Human N-acetylglucosaminyltransferase I. Expression in Escherichia coli as a soluble enzyme, and application as an immobilized enzyme for the chemoenzymatic synthesis of N-linked oligosaccharides.
abstract: N-Acetylglucosaminyltransferase I (GnT-I), which catalyzes the transfer of an N-acetylglucosamine residue from UDP-N-acetylglucosamine to the alpha1,3-linked mannose on Man5GlcNAc2 (M5), is a critical enzyme for the synthesis of high-mannose-type to complex-type glycan structures in N-linked glycan processing. We developed a large-scale preparation system for recombinant human GnT-I (hGnT-I) using the maltose binding protein (MBP) fusion system to facilitate the chemoenzymatic route for complex-type N-linked glycan synthesis. MBP-fused GnT-I was purified by affinity chromatography on an amylose resin column. The relative activity of MBP-fused GnT-I toward high-mannose-type N-linked oligosaccharides was 100% for Man5GlcNAc2, 52% for Man3GlcNAc2, 17% for Man6GlcNAc2. MBP-fused GnT-I exhibited optimal activity at pH 6.5-9.5 and was more active between pH 6.5-9.0. The optimum temperature for MBP-fused GnT-I activity was 40 degrees C, but the enzyme was active between 0-70 degrees C. Mn2+ and Co2+ were critical for the enzyme activity, while Zn2+ and Ca2+ inhibited the activity. Kinetic analysis of the purified enzyme showed an apparent K(m) value of 0.483 mM and a V(max) of 101 nmol/mg/min for M5. Immobilization of MBP-fused GnT-I on the amylose resin led to an 80% yield of the high mannose-type-of oligosaccharide.
authors.affiliation: The International Center for Biotechnology, Osaka University, 2-1 Yamada-oka, Suita, Osaka 565-0871, Japan. fujiyama@icb.osaka-u.ac.jp
authors.forename: K
authors.initials: K
authors.lastname: Fujiyama
authors.forename: Y
authors.initials: Y
authors.lastname: Ido
authors.forename: R
authors.initials: R
authors.lastname: Misaki
authors.forename: D G
authors.initials: DG
authors.lastname: Moran
authors.forename: I
authors.initials: I
authors.lastname: Yanagihara
authors.forename: T
authors.initials: T
authors.lastname: Honda
authors.forename: S
authors.initials: S
authors.lastname: Nishimura
authors.forename: T
authors.initials: T
authors.lastname: Yoshida
authors.forename: T
authors.initials: T
authors.lastname: Seki