Development of alanine aminotransferase reactor based on polymer@Fe3O4 nanoparticles for enzyme inhibitors screening by chiral ligand exchange capillary electrophoresis

Abstract

Alanine aminotransferase (ALT) plays significant role in biological and clinical research. In this study, a unique ALT enzyme reactor based on multifunctional polymer@magnetic nanoparticles has been constructed for the first time and the enzymolysis efficiency has been evaluated by chiral ligand exchange capillary electrophoresis technique. Poly(N-acryloxysuccinimide) has been synthesized by reversible addition-fragmentation chain transfer polymerization method and immobilized on the magnetic nanoparticles via the succinimide group in the polymer. Interestingly, the enzyme also could easily react with the succinimide group, which enables of ALT covalent bonding onto the polymer. The enzyme amount immobilized and the immobilization time have been investigated. Comparing with free ALT in solution (V_max of free enzyme = 0.6 mM min^-1), the resultant enzyme reactor has exhibited good reusability and stability, and displayed about five times enhanced enzymolysis efficiency with L-alanine as the substrate (V_max of enzyme reactor = 3.4 mM min^-1). Furthermore, the prepared enzyme reactor has been applied in ALT inhibitors screening. The enzyme reactors based on the multifunctional polymer@magnetic nanoparticles have depicted great potential in anti-liver drugs development, liver diseases study and ALT related biological process inspect.

Keywords: Alanine aminotransferase; Chiral ligand exchange capillary electrophoresis; Enzyme inhibitors screening; Multifunctional polymer@magnetic nanoparticles based enzyme reactor.