Insights into histidine-assisted mitigation of pretreatment-derived inhibitors for sustainable sugar platform biorefinery

Abstract

Inhibitory compounds generated during the acidic pretreatment of lignocellulose (carbohydrate- and lignin-derived byproducts) hinder enzymatic hydrolysis by forming pseudo-lignin and restricting enzyme access to substrate. Enhancing (hemi)cellulase tolerance to these inhibitors could improve hydrolysis efficiency and reduce the need for costly detoxification steps. This study investigates the role of amino acids with diverse physicochemical properties in mitigating these effects, with a particular focus on histidine. Our findings show that charged amino acids—histidine (His), arginine (Arg), aspartic acid (Asp), and glutamic acid (Glu)—were most effective in improving glucose yields, which increased to over 60 % after 48 h of hydrolysis, compared to 46.7 % in the control. These amino acids also reduced pseudo-lignin formation, decreasing lignin content from 35.7 % to 21–25 %. His exhibited the highest inhibitor removal efficiency (91.2 %), followed by Asp (88.4 %), Glu (86.9 %), and Arg (86.5 %). In-depth in vitro and in silico analyses revealed that His’s imidazole side chain facilitated multiple synergistic interactions, including electrostatic interactions, hydrogen bonding, π-π stacking, and van der Waals forces, contributing to its superior binding characteristics. His demonstrated the highest binding constant (Ka: 69.18) and the most negative free energy ( ΔG : −11.4), enhancing the stability of inhibitor-enzyme complexes. These findings position histidine as a promising candidate for engineering next-generation (hemi)cellulases with enhanced inhibitor resistance, thereby improving the efficiency of lignocellulose conversion in sustainable biorefineries.