title: Expression of CotA laccase in Pichia pastoris and its electrocatalytic sensing application for hydrogen peroxide.
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.
authors.affiliation: Academy of Fundamental and Interdisciplinary Sciences, Harbin Institute of Technology, No. 2, Yikuang Street, Harbin, 150001, China.
authors.forename: Lili
authors.initials: L
authors.lastname: Fan
authors.affiliation: College of Life Science, Northeast Forestry University, Harbin, 150040, China.
authors.forename: Min
authors.initials: M
authors.lastname: Zhao
authors.affiliation: Academy of Fundamental and Interdisciplinary Sciences, Harbin Institute of Technology, No. 2, Yikuang Street, Harbin, 150001, China. wangy_msn@hotmail.com.
authors.forename: Yan
authors.initials: Y
authors.lastname: Wang
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015374:Biosensing Techniques; D003001:Cloning, Molecular; D006207:Half-Life; D006861:Hydrogen Peroxide / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D042845:Laccase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010843:Pichia / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D006861:Hydrogen Peroxide; D042845:Laccase
keywords: Bioelectrocatalytic; CotA; H2O2 biosensor; Laccase; Secretory expression