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Semi-rational design of cellobiose dehydrogenase for increased stability in the presence of peroxide

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Abstract

Cellobiose dehydrogenase (CDH, EC 1.1.99.18) from white rot fungi Phanerochaete chrysosporium can be used for constructing biosensors and biofuel cells, for bleaching cotton in textile industry, and recently, the enzyme has found an important application in biomedicine as an antimicrobial and antibiofilm agent. Stability and activity of the wild-type (wt) CDH and mutants at methionine residues in the presence of hydrogen peroxide were investigated. Saturation mutagenesis libraries were made at the only methionine in heme domain M65 and two methionines M685 and M738 in the flavin domain that were closest to the active site. After screening the libraries, three mutants with increased activity and stability in the presence of peroxide were found, M65F with 70% of residual activity after 6 h of incubation in 0.3 M hydrogen peroxide, M738S with 80% of residual activity and M685Y with over 90% of residual activity compared to wild-type CDH that retained 40% of original activity. Combined mutants showed no activity. The most stable mutant M685Y with 5.8 times increased half-life in the presence of peroxide showed also 2.5 times increased kcat for lactose compared to wtCDH and could be good candidate for applications in biofuel cells and biocatalysis for lactobionic acid production.

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Funding

This work was supported by funds from the Ministry of Education and Science, Republic of Serbia by the Project Nos. III46010, ON172049 and ON173017.

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Authors and Affiliations

  1. Institute of Chemistry, Technology and Metallurgy, University of Belgrade, Njegoseva 12, 11000, Belgrade, Serbia

    Ana Marija Balaž & Marija Blazić

  2. Department of Biochemistry, Faculty of Chemistry, University of Belgrade, Studentski trg 12 – 16, 11000, Belgrade, Serbia

    Jelena Stevanović, Karla Ilić Đurđić & Radivoje Prodanović

  3. Hall for Discovery and Learning Research, Purdue University, 207 S. Martin Jischke Dr., West Lafayette, IN, 47907, USA

    Raluca Ostafe

  4. Institute of Molecular Biotechnology, RWTH Aachen University, Worringerweg 1, 52074, Aachen, Germany

    Rainer Fischer

  5. Single Cell Analytics Center, Indiana Bioscience Research Institute, 1345 W. 16th St. Suite 300, Indianapolis, IN, 46202, USA

    Rainer Fischer

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  1. Ana Marija Balaž
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  7. Radivoje Prodanović
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Correspondence to Radivoje Prodanović.

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Balaž, A.M., Stevanović, J., Ostafe, R. et al. Semi-rational design of cellobiose dehydrogenase for increased stability in the presence of peroxide. Mol Divers 24, 593–601 (2020). https://doi.org/10.1007/s11030-019-09965-0

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  • DOI: https://doi.org/10.1007/s11030-019-09965-0

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