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

N-Acetylglucosaminyltransferase I (GnT-I), which catalyzes the transfer of an N-acetylglucosamine residue from UDP- N-acetylglucosamine to the α1,3-linked mannose on Man 5GlcNAc 2 (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 Man 5GlcNAc 2, 52% for Man 3GlcNAc 2, 17% for Man 6GlcNAc 2. 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°C, but the enzyme was active between 0–70°C. Mn 2+ and Co 2+ were critical for the enzyme activity, while Zn 2+ and Ca 2+ 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.