Sugar-coated sensor chip and nanoparticle surfaces for the in vitro enzymatic synthesis of starch-like materials

Abstract

The insoluble glucan polymer starch is a major player in the human diet; it is also an important bulk commodity. Nonetheless, our understanding of its biochemistry remains poor, not least because of the challenge of analysing enzymes that operate across the solid–liquid interface. In the present study, the enzymatic polymerisation of glucans immobilised on gold sensor chip and nanoparticle surfaces was achieved with Arabidopsis phosphorylase AtPHS2. The basis of the action of AtPHS2 on surface glucans could be rationalised through consideration of the X-ray crystal structure of this enzyme, which identified a previously unreported enzyme surface binding site for glucans. Extension of the glucan-coated sensor chip surfaces could be monitored in real time by SPR, enabling kinetic analysis of AtPHS2-mediated glucan synthesis, which showed similar efficiency to in solution analyses. Extension of both sensor and nanoparticles surfaces coated with glucan was analysed by TEM, which confirmed glucan polymerisation. The arrangement of newly formed glucan chains into ordered helical arrangements was evident from iodine staining, as well as from enzyme response characteristics that proved typical of starch-like material. As such, the glucan-modified sensor chip and nanoparticle surfaces represent novel tools with which to analyse starch-active enzymes.