Covalent immobilization of thermotolerant recombinant nano-coupled xylanase for improved stability and reusability in the saccharification process

Xylanase, a key enzyme in the saccharification of lignocellulosic biomass (LCB) for bioethanol production, often faces limitations due to its limited reusability and poor stability. For this purpose, β-1,4-xylanase gene (Clocl_0045) of 1239 bp from Clostridium clariflavum (also known as Acetivibrio clariflavus) was cloned and expressed into expression system i.e., E.coli BL21. A rational approach was developed to covalently immobilize cloned xylanase on iron oxide (Fe₃O₄) magnetic nanoparticles (MNPs) coated with silica (SiO₂), enhancing stability, reusability, and recovery from the reaction system. Multiple alignment analysis and structural modeling studies revealed that recombinant xylanase contained a conserved GH (glycosyl hydrolase) domain. The enzyme's catalytic site included Glu-166 and Glu-271 residues for substrate binding. The successful immobilization of nano-coupled xylanase was analyzed using Fourier-transform infrared spectroscopy (FTIR) to observe changes in the shift, from CO to CN. Additionally, crystalline nature of iron oxide nanoparticles was confirmed by X-ray diffraction (XRD) analysis. The results indicated that the the immobilized xylanase exhibited activity of 6.45 ± 0.21 U/mL,metal ion stability with calcium ions, thermal stability at 90 °C after 4 h with residual activity of 38.5 % and pH stability with residual activity of 93.7 % at pH 7.0. Furthermore, the immobilized enzyme demonstrated enhanced residual activity in the reusability assay for upto 15 cycles on xylan (substrate) and 5 cycles on pretreated sugarcane bagasse. Saccharification time for pretreated biomasses was found to be 72 h. In conclusion, all these findings highlight the effectiveness and competitiveness of the immobilized xylanase with high reusability and stability for better industrial implementation.