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.