Particulate and spherical cellulosic supports derived from sugarcane bagasse for oriented immobilization of recombinant CBD-tagged enzymes

This study developed and evaluated particulate and spherical cellulosic supports derived from sugarcane bagasse for the oriented immobilization of recombinant enzymes containing a cellulose-binding domain (CBD), using β-galactosidase-CBD (β-Gal-CBD) as a model. The bagasse was subjected to alkaline hydrolysis (L) and sequential acid and alkaline hydrolysis (LH), resulting in supports with progressively higher cellulose contents, as confirmed by chemical analysis, Fourier-transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), and scanning electron microscopy (SEM). The LH treatment yielded the support with the highest cellulosic purity, leading to superior performance in immobilization (LH-Gal). At a loading of 10 U/g, the particulate LH support achieved a recovered activity exceeding 100%, characterizing enzymatic hyperactivation, which is a highly promising result for supports derived from agro-industrial residues. Among the spherical supports (Sph) obtained by ionic gelation of sodium alginate, LH-Sph exhibited the highest yield and recovered activity values, with optimal conditions at 3 h and 1 U/sphere. The alginate control support presented the lowest immobilization parameters, demonstrating that the presence of cellulosic bagasse is determinant for the oriented immobilization. The economic viability of an industrial process for low-lactose milk production employing the LH-Gal biocatalyst was demonstrated in all evaluated scenarios (5-40 reuse cycles of the biocatalyst), with an internal rate of return (IRR) of 42.8-73.1%, a return on investment (ROI) of 340-634%, and a discounted payback period of 1.65-2.98 years. These results demonstrate that particulate and spherical cellulosic supports derived from sugarcane bagasse hold strong potential for the oriented immobilization of CBD-tagged enzymes, with promising applications in industrial bioprocesses.