Chitosan-regulated biomimetic hybrid nanoflower for efficiently immobilizing enzymes to enhance stability and by-product tolerance

Abstract

Organic-inorganic hybrid nano-materials have been considered to be promising immobilization matrixes for enzymes due to their significantly enhanced reusability and stability of enzymes. Herein, we constructed a novel organic-inorganic hybrid nanoflower via biomacromolecule-regulated biomimetic mineralization to immobilize sucrose phosphorylase (SPase). It was found that chitosan (CS) effectively regulated the biomimetic mineralization of calcium phosphate (CaP), leading to the formation of flower-like hybrid materials for the entrapment of SPase via self-assembly to establish a nano-biocatalyst (CS-CaP@SPase). Upon immobilization, the obtained CS-CaP@SPase exhibited excellent pH, by-product and organic solvents tolerance, and storage stability. Specifically, at acidic condition (pH 4), CS-CaP@SPase performed over 80 % of initial activity, which was 2.42-folds higher than that of free SPase. The catalytic activity of free SPase was severely inhibited about 30 % in the presence of fructose (1.2 M), but CS-CaP@SPase only lost 5 % relative activity. The CS-CaP@SPase retained over 80 % of its relative activity, while the free SPase maintained <20 % of its relative activity in acetonitrile. The relative activity of CS-CaP@SPase was still retained about 80 % after 10 cycles and maintained 75 % after 15 days. Based on Raman spectra analysis, it was also found that the increased β-folding component of SPase in the secondary structure after immobilization was the main factor for its enhanced stability. It is reasonable to believe that biomacromolecule-regulated biomimetic mineralization could be potentially used as a promising method to immobilize enzymes with excellent stability and recyclability, thereby facilitating the preparation of highly efficient catalysts for industrial biocatalysts, biosensing, and biomedicine.