Preparation and application of cross-linked aggregates of chloroperoxidase with enhanced hydrogen peroxide tolerance

A cross-linked enzyme aggregate (CLEA®) of chloroperoxidase (CPO) was created that exhibited greatly improved stability in the presence of hydrogen peroxide concentrations as high as 1.2 M. The CPO-CLEA was generated by oxidizing the protein with sodium periodate and precipitating and cross-linking with ammonium sulfate and sodium borohydride. CLEA® production parameters, including the concentrations of these three reagents, were optimized to maximize the activity of the biocatalyst in oxidizing 7-azaindole to 7-azaoxindole. Additionally, the in situ production of the CLEA® was demonstrated, resulting in a process for converting >90% of 5 g/l 7-azaindole in <1 h while requiring neither gradual peroxide addition nor immobilized enzyme isolation.