Effects of Glycerol and Phenolics on Myceliophthora heterothallica Endoxylanase Expressed in K. phaffii

Industrial applications of xylanases in high-temperature settings are limited by enzyme instability. This study evaluated glycerol and phenolic compounds as modulators of the catalytic and structural properties of a recombinant <i>Myceliophthora heterothallica</i> endoxylanase (rMhXyn) expressed in <i>Komagataella phaffii</i>. Glycerol (20% <i>v</i>/<i>v</i>) significantly improved thermostability (5-fold increase in half-life at 55 °C), decreased the activation energy for catalysis, and enhanced structural rigidity as evidenced by molecular dynamics simulations (reduced RMSD and Rg). In contrast, phenolic acids provided only short-term stabilization at moderate temperatures and did not confer structural benefits. Enzyme kinetics revealed that glycerol enhanced catalytic turnover (↑<i>V</i>_max), while phenolic compounds modified both <i>K</i>' and cooperativity (Hill coefficient). Thermodynamic analysis supported glycerol's stabilizing effect, with increased ∆<i>H</i>_(D) and a positive shift in ∆<i>S</i>_(D). These results suggest glycerol as a superior stabilizer for rMhXyn in high-temperature bioprocesses such as lignocellulosic biomass hydrolysis. These findings highlight the potential of targeted additives to improve enzyme performance for biotechnological applications.