Facile Multilayer Deposition of Polyethylenimine and κ-Carrageenan on a 3D-Printed Lattice for Cellulase Immobilization

Technological developments for cellulase immobilization aim to address the challenges of costly cellulose hydrolysis. Herein, dissimilarly charged polyethylenimine (PEI) and κ-carrageenan (κ-Ca) were deposited layer-by-layer on a modular 3D-printed lattice for cellulase immobilization, and an enzyme loading improvement of 54.9% was achieved after the deposition of 3 PEI/κ-Ca bilayers. Microscopy analyses revealed that the deposition of 3 PEI/κ-Ca layers increased the average surface roughness by 65.4%, implying improved availability of potential enzyme binding sites. Thermal stability and reusability of the cellulase-loaded lattice were determined. They demonstrated activity retention of 79.43% at 70 °C reaction temperature and 83.97% after ten enzymatic hydrolysis cycles with carboxymethylcellulose as the substrate. Moreover, the cellulase-loaded lattice exhibited a 64% higher turnover frequency than cellulase loaded on nanosupports. Utilizing the cellulase-loaded lattice in the hydrolysis of a macroalgae cellulosic residue yielded a saccharification yield of 44.91% in 48 h. The synergisms between PEI and κ-Ca improve enzyme stability, as depicted from the operational stability studies where E-GA-PkG showed ∼60% better activity retention than free cellulase at pH 7 using MCR as the substrate. Furthermore, the flexibility of prototyping complex geometries via 3D printing allows further intuition into more efficient reactor designs for integrated biorefineries.