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

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Abstract

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

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

Journal

  • e-Journal of Surface Science and Nanotechnology

    e-Journal of Surface Science and Nanotechnology (3), 207-212, 2005

    The Surface Science Society of Japan

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