caa a22 4500 605500843 CHVBK 20210128100558.0 cr unu---uuuuu 210128e20151101xx s 000 0 eng 10.1007/s00253-015-6720-0 doi (NATIONALLICENCE)springer-10.1007/s00253-015-6720-0 Expression of CotA laccase in Pichia pastoris and its electrocatalytic sensing application for hydrogen peroxide [Elektronische Daten] [Lili Fan, Min Zhao, Yan Wang] The CotA laccase from Bacillus subtilis WD23 was successfully overexpressed in Pichia pastoris, and the production level reached 891.2U/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±18μM, respectively, with corresponding k cat values of 15±1.0, 7.6±1.5, and 0.87±0.1s−1. Remarkably, the laccase activity increased to 561.9% of its initial activity at pH 9.0 after 7days of incubation and the half-life of laccase inactivation was approximately 3h 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 4.75mM, with a detection limit of 3.1μM. The amperometric biosensor for H2O2 by CotA-modified electrode is a novel application for CotA laccase. Springer-Verlag Berlin Heidelberg, 2015 CotA nationallicence Laccase nationallicence Secretory expression nationallicence Bioelectrocatalytic nationallicence H2O2 biosensor nationallicence Fan Lili Academy of Fundamental and Interdisciplinary Sciences, Harbin Institute of Technology, No. 2, Yikuang Street, 150001, Harbin, China aut Zhao Min College of Life Science, Northeast Forestry University, 150040, Harbin, China aut Wang Yan Academy of Fundamental and Interdisciplinary Sciences, Harbin Institute of Technology, No. 2, Yikuang Street, 150001, Harbin, China aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/22(2015-11-01), 9483-9493 0175-7598 99:22<9483 2015 99 253 https://doi.org/10.1007/s00253-015-6720-0 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-015-6720-0 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Fan Lili Academy of Fundamental and Interdisciplinary Sciences, Harbin Institute of Technology, No. 2, Yikuang Street, 150001, Harbin, China aut NATIONALLICENCE 700 1- Zhao Min College of Life Science, Northeast Forestry University, 150040, Harbin, China aut NATIONALLICENCE 700 1- Wang Yan Academy of Fundamental and Interdisciplinary Sciences, Harbin Institute of Technology, No. 2, Yikuang Street, 150001, Harbin, China aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/22(2015-11-01), 9483-9493 0175-7598 99:22<9483 2015 99 253