Laccases (benzenediol oxygen oxidoreductase; EC 1.10.3.2) have many biotechnological applications because of their oxidation ability towards a wide range of phenolic compounds. Within recent years, researchers have been highly interested in the identification and characterization of laccases from bacterial sources. In this study, we have isolated and cloned a gene encoding laccase (CotA) from Bacillus sp. HR03 and then expressed it under microaerobic conditions and decreased temperature in order to obtain high amounts of soluble protein. The laccase was purified and its biochemical properties were investigated using three common laccase substrates, 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), syringaldazine (SGZ) and 2,6-dimethoxyphenol (2,6-DMP). K(M) and k(cat) were calculated 535 microM and 127 s(-1) for ABTS, 53 microM and 3 s(-1) for 2, 6-DMP and 5 microM and 20 s(-1) for SGZ when the whole reactions were carried out at room temperature. Laccase activity was also studied when the enzyme was preincubated at 70 and 80 degrees C. With SGZ as the substrate, the activity was increased three-fold after 50 min preincubation at 70 degrees C and 2.4-fold after 10 min preincubation at 80 degrees C. Preincubation of the enzyme in 70 degrees C for 30 min raised the activity four-fold with ABTS as the substrate. Also, L-dopa was used as a substrate. The enzyme was able to oxidize L-dopa with the K(M) and k(cat) of 1,493 microM and 194 s(-1), respectively.
In our previous study, we reported an increase in the thermal stability of bacterial laccase from Bacillus sp. HR03 using site‐directed mutagenesis. Three‐dimensional model of this enzyme showed a negative patch in the connecting loop between domains 1 and 2. In the present study, the stability of laccase in organic solvents was improved by introducing nonpolar (E188 → A, I, L, and V) and positively charged (E188 → K and R) residues in this region by site‐directed mutagenesis. Irreversible thermoinactivation, C50 value (organic solvent concentration at which 50% of enzyme activity remains), change in transition‐state stabilization energy, and kinetic parameters of the wild type and its variants were calculated in the presence and absence of various organic solvents (ethanol, methanol, and 1‐propanol). All variants showed higher C50 values when compared to the wild type. Nonpolar amino acid substitutions were found to be the most efficient mutants for their remarkable increase in C50 value and a decrease in thermoinactivation rate in the presence of mentioned solvents. Data showed that replacing a negative residue with hydrophobic residues on the surface of a protein could enhance thermoresistance as well as solvent stability. The stability of the resulting enzymes was dependent on the length of the alkyl chain. Results demonstrated that solvent tolerance was positively correlated with thermal stability.
This work reports a novel nanobiosensor based on a thioglycolic acid (TGA)-capped CdTe quantum dot-laccase (Lac) enzyme system for sensitive detection of dopamine (DA). The enzyme used catalyzes the oxidation of DA to dopamine-o-quinone (DOQ), which can selectively quench the strong luminescence of CdTe nanocrystals at neutral pH. The relationship between luminescence intensity of CdTe nanocrystals and DA concentration is nicely described by the Stern-Volmer equation. At an optimum pH of 7.4, the proposed sensor gives a linear calibration over a DA concentration range of 0.3 to 100 μM, with a limit of detection of 0.16 μM and a response time of 2 min. The relative standard deviation for seven replicate determinations of 6.0 μM of DA was found to be 3.7%. The sensor was successfully applied to the determination of DA in a blood plasma sample and in a DA injection formulation.
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