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Cloning, Heterologous Expression, Purification and Characterization of M12 Mutant of Aspergillus niger Glucose Oxidase in Yeast Pichia pastoris KM71H

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Abstract

Aspergillus niger glucose oxidase (GOx) genes for wild-type (GenBank accession no. X16061, swiss-Prot; P13006) and M12 mutant (N2Y, K13E, T30 V, I94 V, K152R) were cloned into pPICZαA vector for expression in Pichia pastoris KM71H strain. The highest expression level of 17.5 U/mL of fermentation media was obtained in 0.5 % (v/v) methanol after 9 days of fermentation. The recombinant GOx was purified by cross-flow ultrafiltration using membranes of 30 kDa molecular cutoff and DEAE ion-exchange chromatography at pH 6.0. Purified wt GOx had k cat of 189.4 s−1 and K m of 28.26 mM while M12 GOx had k cat of 352.0 s−1 and K m of 13.33 mM for glucose at pH 5.5. Specificity constants k cat/K m of wt (6.70 mM−1 s−1) and M12 GOx (26.7 mM−1 s−1) expressed in P. pastoris KM71H were around three times higher than for the same enzymes previously expressed in Saccharomyces cerevisiae InvSc1 strain. The pH optimum and sugar specificity of M12 mutant of GOx remained similar to the wild-type form of the enzyme, while thermostability was slightly decreased. M12 GOx expressed in P. pastoris showed three times higher activity compared to the wt GOx toward redox mediators like N,N-dimethyl-nitroso-aniline used for glucose strips manufacturing. M12 mutant of GOx produced in P. pastoris KM71H could be useful for manufacturing of glucose biosensors and biofuel cells.

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Acknowledgments

This work was supported by Grant No. ON172049 and ON173017 sponsored by the Ministry of Education and Science, Republic of Serbia.

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Authors and Affiliations

  1. Faculty of Chemistry, University of Belgrade, Studentski trg 12, 11000, Belgrade, Serbia

    Gordana Kovačević, Bojana Draganić, Marija Gavrović-Jankulović & Radivoje Prodanović

  2. Center for Chemistry, IHTM, University of Belgrade, Njegoševa 12, 11000, Belgrade, Serbia

    Marija Blažić

  3. Molecular Biotechnology, Faculty of Biology, RWTH Aachen University, Worringerweg 1, 52078, Aachen, Germany

    Raluca Ostafe & Rainer Fischer

Authors
  1. Gordana Kovačević
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  2. Marija Blažić
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  3. Bojana Draganić
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  4. Raluca Ostafe
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  5. Marija Gavrović-Jankulović
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  6. Rainer Fischer
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  7. Radivoje Prodanović
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Correspondence to Radivoje Prodanović.

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Kovačević, G., Blažić, M., Draganić, B. et al. Cloning, Heterologous Expression, Purification and Characterization of M12 Mutant of Aspergillus niger Glucose Oxidase in Yeast Pichia pastoris KM71H. Mol Biotechnol 56, 305–311 (2014). https://doi.org/10.1007/s12033-013-9709-x

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  • DOI: https://doi.org/10.1007/s12033-013-9709-x

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