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Expression of CotA laccase in Pichia pastoris and its electrocatalytic sensing application for hydrogen peroxide

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Abstract

The CotA laccase from Bacillus subtilis WD23 was successfully overexpressed in Pichia pastoris, and the production level reached 891.2 U/L. The recombinant CotA laccase was purified to homogeneity. The optimal enzymatic activity was found at pH 4.6, 6.6, and 6.8 for 2, 2′-azino-bis (3-ethylbenzothiazoline-6-sulfonate) (ABTS), 4-hydroxy-3, 5-dimethoxybenzaldehyde azine (SGZ), and 2, 6-dimethoxyphenol (2, 6-DMP) oxidation, respectively. The maximal enzyme activity was observed at 80 °C with SGZ as a substrate. The kinetic constant K m values for ABTS, SGZ, and 2, 6-DMP were 162 ± 20, 24 ± 2, and 166 ± 18 μM, respectively, with corresponding k cat values of 15 ± 1.0, 7.6 ± 1.5, and 0.87 ± 0.1 s−1. Remarkably, the laccase activity increased to 561.9 % of its initial activity at pH 9.0 after 7 days of incubation and the half-life of laccase inactivation was approximately 3 h at 80 °C, which indicated that the recombinant CotA was a highly thermo-alkali-stable laccase. Bioelectrocatalytic reduction of H2O2 by the CotA laccase was detected when the recombinant CotA was adsorbed on pyrogenation graphite electrodes. Based on the bioelectrocatalytic reduction, a mediator-free amperometric biosensor for hydrogen peroxide was designed. The linear range of the H2O2 biosensor was from 0.05 to 4.75 mM, with a detection limit of 3.1 μM. The amperometric biosensor for H2O2 by CotA-modified electrode is a novel application for CotA laccase.

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Acknowledgments

This research was supported jointly by the “948” project of the National Forestry Bureau (no. 2012-4-03) and the National Natural Science Foundation of China (no. 31470489, 31170553). The authors thank Chunlei Wang for the kind gift of B. subtilis WD23.

Ethical statement

This article does not contain any studies with human participants or animals performed by any of the authors. Informed consent was obtained from all individual participants included in the study.

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The authors declare that they have no competing interests.

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  1. Academy of Fundamental and Interdisciplinary Sciences, Harbin Institute of Technology, No. 2, Yikuang Street, Harbin, 150001, China

    Lili Fan & Yan Wang

  2. College of Life Science, Northeast Forestry University, Harbin, 150040, China

    Min Zhao

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  1. Lili Fan
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  2. Min Zhao
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  3. Yan Wang
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Correspondence to Yan Wang.

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Fan, L., Zhao, M. & Wang, Y. Expression of CotA laccase in Pichia pastoris and its electrocatalytic sensing application for hydrogen peroxide. Appl Microbiol Biotechnol 99, 9483–9493 (2015). https://doi.org/10.1007/s00253-015-6720-0

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