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.

Enhanced catalytic efficiency of CotA-laccase by DNA shuffling

“…Laccases activity was determined using ABTS and SGZ as substrates as previously described [16]. One unit is defined as the amount of enzyme that oxidizes 1 μmol of substrate per min at 30°C.…”

An Inferred Ancestral CotA Laccase with Improved Expression and Kinetic Efficiency

“…The laccase BsCotA from Bacillus subtilis displays exceptional heat tolerance, with a half-life of inactivation at 80 °C for 2 h [ 10 ]. However, its practical application is hindered by folding difficulties that limit protein yield in Escherichia coli and Pichia pastoris [ 11 , 12 , 13 ]. These limitations restrict the broad application prospects of BsCotA.…”

Hydrogen Peroxide-Resistant CotA and YjqC of Bacillus altitudinis Spores Are a Promising Biocatalyst for Catalyzing Reduction of Sinapic Acid and Sinapine in Rapeseed Meal

“…The CotA protein is located in the outer spore coat and contributes to protecting spores from UV light, H 2 O 2 and other chemical agents. Thus, the CotA laccase is supposed to be more resistant to H 2 O 2 or high salinity than fungal laccases [ 10 , 12 ]. Compared with the extensive use of heat-, alkali- and halide-resistant CotA laccases, little attention has been focused on Bacillus CotA laccases with high resistance to H 2 O 2 [ 12 ].…”