Carboxylated nanocellulose from quinoa husk for enhanced protease immobilization and stability of protease in biotechnological applications.

Herein, an efficient and feasible approach was developed to oxidize low-cost agricultural waste (quinoa husk, QS) for the synthesis of carboxylated nanocellulose (CNC). The as-prepared rod-like CNCs (average diameter of 10 nm and length of 103 nm) with a high specific surface area (173 m/g) were utilized for the immobilization of a model protease enzyme (PersiProtease1) either physically or via covalent attachment. For chemical immobilization, CNCs were firstly functionalized with N, N'-dicyclohexylcarbodiimide (DCC) to provide DCNCs nanocarrier which could covalently bond to enzyme trough nucleophilic substitution reaction and formation of the amide bond between DCNCs and enzyme. The immobilization efficiency, activity, stability, kinetic parameters, and reusability of covalently attached and physically immobilized PersiProtease1 were similar to those of the free enzyme. Enzyme immobilization resulted in higher thermal stability of the enzyme at elevated temperatures (> 80 °C), and the covalently immobilized enzyme displayed higher reusability than its physically immobilized form (56% vs. 37% activity, after 15 consecutive cycles), which would be rooted in a more tightly attached and less leached enzyme in the case of PersiProtease1/DCNCs. This study demonstrates the significance of using agricultural by-products and the enhanced performance and stability of immobilized proteases.