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Extension of Polyphenolics by CWPO-C Peroxidase Mutant Containing Radical-Robust Surface Active Site

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Abstract

Expressed as insoluble forms in Escherichia coli, native cationic cell wall peroxidase (CWPO-C) from the poplar tree and mutant variants were successfully reactivated via refolding experiments and used to elucidate the previously presumed existence of an electron transfer (ET) pathway in the CWPO-C structure. Their catalytic properties were fully characterized through various analyses including steady-state kinetic, direct oxidation of lignin macromolecules and their respective stabilities during the polymerization reactions. The analysis results proved that the 74th residue on the CWPO-C surface plays an important role in catalyzing the macromolecules via supposed ET mechanism. By comparing the residual activities of wild-type CWPO-C and mutant 74W CWPO-C after 3 min, mutation of tyrosine 74 residue to tryptophan increased the radical resistance of peroxidase up to ten times dramatically while maintaining its capability to oxidize lignin macromolecules. Furthermore, extension of poly(catechin) as well as lignin macromolecules with CWPO-C Y74W mutant clearly showed that this radical-resistant peroxidase mutant can increase the molecular weight of various kinds of polyphenolics by using surface-located active site. The anti-oxidation activity of the synthesized poly(catechin) was confirmed by xanthine oxidase assay. The elucidation of a uniquely catalytic mechanism in CWPO-C may improve the applicability of the peroxidase/H2O2 catalyst to green polymer chemistry.

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Acknowledgments

This work was supported by the Korean Government (NRF-2009-0093281, 2013K000361) and Kwangwoon University 2013; we, the authors, are thankful for this support.

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Authors and Affiliations

  1. Department of Chemical Engineering, Kwangwoon University, 447-1, Wolgye-Dong, Nowon-Gu, Seoul, 139-701, Republic of Korea

    L. T. Mai Pham & Y. Hwan Kim

  2. Korea Research Institute of Chemical Technology, Sinseong-no 19, Yuseong-gu, Daejeon, 305-600, Republic of Korea

    S. Jin Kim, U. Suk Ahn & B. Keun Song

  3. Department of Forest Sciences, College of Agriculture and Life Science, Seoul National University, 599 Gwanak-ro, Gwanak-gu, Seoul, 151-921, Republic of Korea

    J. Weon Choi

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  1. L. T. Mai Pham
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Correspondence to Y. Hwan Kim.

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Pham, L.T.M., Kim, S.J., Ahn, U.S. et al. Extension of Polyphenolics by CWPO-C Peroxidase Mutant Containing Radical-Robust Surface Active Site. Appl Biochem Biotechnol 172, 792–805 (2014). https://doi.org/10.1007/s12010-013-0534-2

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