New strategies for enzyme stabilization involving molecular evolution and immobilization in mesoporous materials

Heme containing peroxidase, for example manganese peroxidase (MnP), is easily inactivated by the hydrogen peroxide (H2O2) presented in the reaction. Here we extremely increased the H2O2 stability of MnP by molecular evolution and immobilization in mesoporous materials. A mutant MnP library containing three randomized amino acid residues located in the entry site of H2O2-binding pocket of MnP was evolved on a 384-well plate using SIMPLEX (single-molecule PCR-linked in vitro expression). The screening of more than 104 samples independently expressed for improved H2O2 stability led to four positive mutants, the H2O2 stability of which was nine times higher than that of the wild-type. Immobilized MnP mutant in mesoporous material (FSM) showed the high H2O2 stability, more than 50 folds than wild type MnP. But the stability of immobilized wild type MnP was not improved so much as that for immobilized mutant MnP.