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 high ly reactive substrate for lignin peroxidase (LIP), exhibiting a k(cat) of 4 1.7 s(-1). Despite the high reactivity, two modes of inactivation prevailed during the oxidation of FA. The first, H2O2-dependent inactivation, was ev idenced by incomplete substrate oxidation and accumulation of LIP compound III (LIPIII), even at relatively low H2O2 concentrations. This was attribut ed to the high turnover rate along with the inability of FA to revert LIPII I to the native state, as evidenced by pre-steady-state kinetics. H2O2-depe ndent inactivation could be avoided by inclusion of veratryl alcohol (VA), which efficiently reverts LIPIII to the native state. However, VA also medi ated 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 oxidat ion products to the enzyme and its extent directly correlated with the amou nt of FA consumed. This inactivation could be considerably suppressed by in clusion of gelatin. Therefore, during the oxidation of highly reactive phen olics, different kinds of protectors are required for efficient oxidation a nd maintaining LIP activity over time. This is of importance when consideri ng emerging biotechnological applications for LIP. (C) 2001 Elsevier Scienc e Inc. All rights reserved.