Abstract
Chemical modification is a valuable strategy for tuning enzyme functionality by introducing new reactive groups without disrupting the overall fold. Conventional amination using ethylenediamine (EDA) is effective, but the resulting modified proteins show limited reactivity for conjugation at neutral pH, and the modifier itself poses safety concerns due to its volatility and corrosive nature. Dihydrazides, in contrast, offer a safer and more versatile alternative: they operate through the same carboxyl-activation mechanism while enabling systematic investigation of chain-length effects. In this study, Thermomyces lanuginosus lipase (TLL) and Myceliophthora thermophila laccase (MTL) were modified using dihydrazides with different alkyl chain lengths (carbonyl (CZ), oxalyl (OX), succinyl (SC), and adipic (AA)), and compared to EDA-modified and unmodified enzymes to evaluate their effects on catalytic performance. Hydrazide-modified variants exhibited enhanced catalytic performance, reaching up to 2.5-fold (TLL-CZ) and 4.2-fold (MTL-AA and MTL-OX) higher efficiencies than unmodified and EDA-modified enzymes. Notably, AA provided the most consistent improvement across both enzymes (1.3-fold in TLL and the best in MTL). Molecular dynamics and docking analyses supported these findings, linking increased flexibility (higher RoG and RMSF) with higher k(cat), and changes in substrate binding with lower k(m). Overall, hydrazide-based modification broadens the spectrum of enzyme variants attainable through amination, while offering safer procedures, thus representing an alternative that overcomes the limitations of using EDA as a conventional aminating agent.