Layered Polymeric Carbon Nitride as a Green Support for Cellulase Immobilization: Improved Stability, Activity, and Reusability

Polymeric carbon nitride (PCN) has attracted considerable attention in recent years due to its layered structure and donor groups such as terminal amines and triazine units. In this study, PCN was synthesized from melamine and employed for the first time as a support material for the immobilization of cellulase, a key enzyme used in numerous industrial applications. Transmission electron microscopy (TEM) analysis revealed that ranged from 219 to 375 nm in size, thereby confirming their nanoscale layered morphology. Cellulase from Aspergillus sp. was immobilized onto PCN using three methods. In the first approach, cellulase was adsorbed onto PCN to form PCN@cellulase. In the second method, PCN@cellulase was cross-linked with glutaraldehyde (PCN@cellulase/Glu). Finally, the primary amino group of PCN was modified with glutaraldehyde, and the cellulase was immobilized onto this support via covalent attachment (PCN/Glu@cellulase). Each cellulase preparation was assessed for its optimal pH, temperature conditions, thermal stability, and enzyme kinetics. The optimal pH was 5.5 for all cellulase preparations, while the optimal temperature was 45 °C for free cellulase and 55 °C for all immobilized cellulase preparations. Thermal stability of PCN@cellulase, PCN@cellulase/Glu, and PCN/Glu@cellulase increased by 2.1-, 2.7-, and 3.7-fold, respectively, compared to the free cellulase. PCN/Glu@cellulase showed a 1.4-fold higher catalytic efficiency than the free cellulase and retained 80% of its initial activity after 10 reuses. This work highlights PCN as a green, metal-free, and easy-to-prepare support for enzyme immobilization, thereby opening new possibilities for sustainable bioprocessing applications.