Deactivation mechanisms of chloroperoxidase during biotransformations.

Inactivation mechanisms of chloroperoxidase (CPO) from Caldariomyces fumago have been investigated with the aim of improving the practical utility of CPO for hydrocarbon oxidation. Deactivation studies in the presence of oxidants (i.e., hydrogen peroxide and t-butyl hydroperoxide) indicated that CPO lost oxidation activity toward hydrocarbon substrates during dismutation of hydrogen peroxide. The loss of enzyme activity was accompanied by the apparent destruction of the heme rather than aggregation or denaturation of the apo-protein. The decrease of enzyme activity was significantly retarded by adding the radical scavenger t-butyl alcohol at pH 4.1, or by optimizing the reaction pH. CPO retained greatest oxidation activity at pH 5-6, which may produce a more favorable ionization state of the key amino acid (Glu-183) and thus reduce radical formation. As a result of higher activity at pH 5-6, the total turnover numbers (TTN, defined as the amount of product produced over the catalytic lifetime of the enzyme) for the oxidation of toluene and o-, m-, p-xylenes in substrate/aqueous emulsion systems ranged from ca. 10% to 110% higher at pH 5.5 (20,000 to 45,000 mol product/mol enzyme) compared to pH 4.1. Furthermore, TTNs of CPO increased with increasing turnover frequencies, indicating that higher activity toward reducing substrates reduces radical formation and stabilizes CPO toward inactivation by H(2)O(2). These findings demonstrate the important relationship between CPO stability and activity, and illustrate that large improvements in CPO activity and stability can be achieved through solvent engineering.