Fabrication of lignin-based sub-micro hybrid particle as a novel support for adenylate cyclase immobilization

Abstract

This study developed a surface functionalized lignin-based sub-microsphere as an innovative support for enzyme immobilization. Lignin was first modified with a silane reagent leading to lignin/SiO₂ (LS) organic/inorganic hybrid particles, displayed as regular sub-micro spheres with a SiO₂ shell as demonstrated in SEM images. The LS particles were further modified to introduce nickel ions, as evidenced in XPS spectra, facilitating affinity adsorption with a his-tagged enzyme. The immobilization of adenylate cyclase from Haloactinopolyspora alba (HaAC), expressed in Escherichia coli, was conducted on the surface functionalized LS (LS-G-NTA-Ni). The immobilization conditions were optimized to achieve the highest relative activity, which were determined to be using a Ni²⁺ concentration of 62.5 mM, at pH=9.5 and 25 °C, with an enzyme-to-support ratio of 4.0 for a duration of 2 h. The immobilized HaAC shows maximum relative activity at pH=9.5 and 40 °C, and exhibits significantly improved thermal stability compared to the free enzyme. After undergoing five reusing cycles, the immobilized HaAC maintains a satisfactory activity (54.15%), which is due to the surface chemistry and the structural stability of the functionalized LS. This work provides a valuable exploration for high-value application of industrial lignin.

Keywords: Catalytic activity; Enzyme immobilization; Lignin; Sub-microsphere; Surface functionalization.