Laccase CotA from Bacillus subtilis 168 was successfully displayed on the membrane of Escherichia coli cells using poly-γ-glutamate synthetase A protein (PgsA) from B. subtilis as an anchoring matrix. Further analyses demonstrated that the fusion protein PgsA/CotA efficiently translocates to the cell surface of E. coli with an enzymatic activity of 65 U/10 cells. Surface-displayed CotA was shown to possess improved enzymatic properties compared with those of the wild-type CotA, including higher thermal stability (above 90% activity at 70 °C and nearly 40% activity at 90 °C after 5-h incubation) and stronger inhibitor tolerance (approximately 80 and 65% activity when incubated with 200 and 400 mM NaCl, respectively). Furthermore, the whole-cell system was demonstrated to have high enzymatic activity against anthraquinone dye, Acid Blue 62, triphenylmethane dye, Malachite Green, and azo dye, Methyl Orange with the decolorization percentages of 91, 45, and 75%, after 5-h incubation, respectively.
In this study, stable CotA laccase from Bacillus subtilis 168 was adsorbed on electrode modified with a thiol graphene‐gold nanoparticle (thGP‐AuNPs) nanocomposite film. The novel bacterial laccase biosensor was employed for quantitative detection of hydroquinone (HQ) and the electrochemical properties of this laccase biosensor were investigated. The results indicate that the immobilized CotA shows great oxidation activity towards HQ in the presence of oxygen and the biosensor shows linear electrocatalytic activity in the concentration range from 1.6 to 409.6 μM, with a detection limit of 0.3 μM. Further, the CotA modified electrode, when compared to fungal laccase‐modified biosensors, shows better alkaline stability (retaining approximately 80 % and 70 % of response current at pH 8 and 9, respectively) and reusability (retaining ∼87 % of response current after 100 days). The development of this new kind of laccase on a biosensor will offer a novel tool for substance detection applications in hostile environments, especially for industrial pollutants.
Photoisomerization of azobenzene spatially confined in silver nanoparticle (AgNP) aggregates and in ethanol solution was monitored by Raman spectroscopy inside optically clear colorimetric dishes. After ultraviolet irradiation, overall Raman intensity assigned to trans-azobenzene of these two samples decreased, and notably, a cis peak appeared at 599 cm −1 , assigned to C─N═N─C torsional mode. The Raman intensity at 1,007 cm −1 , assigned to trans-azobenzene, was recorded versus irradiation time for analyzing the trans-to-cis photoisomerization quantum yield (PQY). By combining our theoretical derivation and standard curve of Raman spectra, the trans-to-cis PQY of azobenzene in AgNP aggregates was roughly calculated to be 79.0% higher than that in ethanol solution. The enhancement mechanism was discussed in terms of the competition between enhanced local optical field from surface plasmon resonance and the accelerated nonradiative decay of excited azobenzene molecules together.
K E Y W O R D Sabsorption spectrum, azobenzene, photoisomerization quantum yield, Raman spectroscopy, silver nanoparticles
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