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

概要

論文の詳細を見る
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_5GleNAc_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_5GleNAc_2, 52% for Man_3GleNAc_2, 17% for Man_6GleNAc_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℃ , but the enzyme was active between 0-70℃. 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 0.483mM amd aV_<max> of 101nmol/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.
社団法人日本生物工学会の論文
2001-12-25