Reversing lignin inhibition on enzymatic hydrolysis through regulating supramolecular assembly

The non-productive binding of enzymes to lignin is a well-documented barrier to efficient enzymatic hydrolysis. While significant attention has been paid to the influence of lignin's chemical structure on enzyme-lignin interactions, the role of its supramolecular assembly in this process has largely been overlooked. In this study, we regulated the supramolecular assembly of ethanol organosolv lignin (EOL) through solvent exchange dialysis and investigated its impact on the enzymatic hydrolysis of cellulose. Our findings reveal that tuning the supramolecular assembly of lignin significantly influenced its physicochemical properties, particularly particle size and hydrophobicity. Larger lignin particles, formed through self-assembly during dialysis, exhibited reduced hydrophobicity. Notably, the size of the lignin aggregates exercised very distinct effects on the performance of enzymatic hydrolysis. Certain large lignin assembled particles even reversed its inhibitory effects on the enzymatic hydrolysis of Avicel. EOL-M1 with the smallest particle size decreased 72 h glucose yields from 70.5 % to 62.9 %, whereas EOL-M100 with the largest particle size increased 72 h glucose yields to 82.3 %. Langmuir adsorption isotherms analysis, and X-ray photoelectron spectroscopy (XPS) analysis demonstrated that lignin with larger particle sizes and lower hydrophobicity reduced the enzyme-binding capacity. Furthermore, this phenomenon was consistently validated with three different lignin samples obtained from organosolv and kraft pulping processes. This study demonstrated the unusual functions of lignin particles in the enzymatic saccharification process. It also provides new insights into the mechanism underlying the influences of lignin on enzyme-lignin interactions and bioprocessing at large.