Heme containing peroxidase, for example manganese peroxidase (MnP), is easily inactivated by the hydrogen peroxide (H
2O
2) presented in the reaction. Here we extremely increased the H
2O
2 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 H
2O
2-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 10
4 samples independently expressed for improved H
2O
2 stability led to four positive mutants, the H
2O
2 stability of which was nine times higher than that of the wild-type. Immobilized MnP mutant in mesoporous material (FSM) showed the high H
2O
2 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. [DOI: 10.1380/ejssnt.2005.207]
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