Tri-Enzyme immobilized on magnetized multiwall carbon nanotubes for single pot Lignocellulosic biomass hydrolysis

Single pot is an innovative idea by combining multi-enzyme in single process for hydrolysis of lignocellulosic biomass. Native enzymes have several drawbacks in the industrial setting, including low enzyme activity, poor stability, uneconomical nature, and challenging separation from the final product. This recent study addresses the co-immobilization of xylanase, laccase, and cellulase on magnetized multiwall carbon nanotubes that have been synthesized and functionalized using an environmentally friendly method that combines water-based synthesis of functionalized multiwall carbon nanotubes with mild acid oxidation of pristine multiwall carbon nanotubes. The immobilized enzymes were characterized in terms of stability and reusability prior to the establishment of a single-pot system for the hydrolysis of paddy straw into reducing sugars (glucose and xylose). Through mild acid treatments, 8 M of acid, 8 h, and 80 °C of reflux time and temperature were examined as ideal working conditions. The establishment of a water-based system in the synthesis of iron oxides on p-MWCNTs was confirmed by energy-dispersive X-ray spectroscopy (EDX), with 10.49% iron detected on the surface of MWCNTs. Immobilization of enzymes on m-MWCNTs was successfully achieved via the adsorption method with > 95% binding efficiency for all enzymes, which was further confirmed with the aid of Fourier-transform infrared spectroscopy (FTIR) peaks, scanning electron microscopy (SEM) images, and EDX analysis. The optimum enzyme concentration for immobilization was recorded at 5 mg/mL for both cellulase and xylanase and 7 mg/mL for laccase. Based on the stability study, the optimum temperature of immobilized cellulase, laccase, and xylanase corresponds to 50, 60, and 70 °C, respectively. The optimum pH of 5 was recorded with immobilized cellulase and laccase, while pH 6 was recorded with immobilized xylanase. A reusability study on model substrate showed all immobilized enzymes retained more than 50% of relative activity after five cycles of analysis, while up to 30% of relative activity was still retained with immobilized cellulase and xylanase that were subjected to the hydrolysis of paddy straw. The detection of a FTIR peak at 1657 cm-1 using paddy straw treated with immobilized laccase on m-MWCNTs indicates successful delignification. As for the single-pot system, the operation was conducted at 50 °C, pH 5, and 100 rpm for 16 h using untreated and acid-pretreated paddy straw. Untreated biomass exhibited higher glucose and xylose content during each cycle of analysis. From high-performance liquid chromatography (HPLC) analysis, 1.03 g/L and 1.04 g/L of glucose and xylose were secreted from untreated biomass through a single-pot system after the third cycle of analysis, highlight the potential of immobilized enzymes on m-MWCNTs synthesized using a water-based for lignocellulosic biomass hydrolysis.