Inactivation of lignin peroxidase during oxidation of the highly reactive substrate ferulic acid

Ferulic acid (4-hydroxy-3-methoxycinnamic acid) (FA) was found to be a highly reactive substrate for lignin peroxidase (LIP), exhibiting a k_cat of 41.7 s^-1. Despite the high reactivity, two modes of inactivation prevailed during the oxidation of FA. The first, H₂O₂-dependent inactivation, was evidenced by incomplete substrate oxidation and accumulation of LIP compound III (LIPIII), even at relatively low H₂O₂ concentrations. This was attributed to the high turnover rate along with the inability of FA to revert LIPIII to the native state, as evidenced by pre-steady-state kinetics. H₂O₂-dependent inactivation could be avoided by inclusion of veratryl alcohol (VA), which efficiently reverts LIPIII to the native state. However, VA also mediated FA oxidation, and significantly decreased the reaction rate, which is unlike for previously reported VA-mediated reactions. The second mechanism of LIP inactivation was attributed to binding of phenoxy radicals or oxidation products to the enzyme and its extent directly correlated with the amount of FA consumed. This inactivation could be considerably suppressed by inclusion of gelatin. Therefore, during the oxidation of highly reactive phenolics, different kinds of protectors are required for efficient oxidation and maintaining LIP activity over time. This is of importance when considering emerging biotechnological applications for LIP.