The importance and applicability of ionic liquids (ILs) in biocatalysis have been well recognized. ILs have growing interest as new and highly efficient reaction mediums for biocatalytic reactions, but as a reaction milieu for firefly luciferase has not been tested. In this report, the effects of two tetramethylguanidine-based ionic liquids on the activity and stability of Photinus pyralis luciferase were investigated. In spite of a common cationic part, luciferase activity increased up to 0.25 M of [TMG][Lac] but decreased in the presence of similar concentrations of [TMG][Pro]. Optimum temperature and thermal stability studies show more stability of luciferase only in the presence of [TMG][Lac]. The change in light intensity of firefly luciferase in the presence of both ILs was brought about without effect on bioluminescence emission spectra. The rate of light decay in the presence of both ILs was slower than native luciferase.
Firefly luciferase catalyzes production of light from luciferin in the presence of Mg(2+)-ATP and oxygen. This enzyme has wide range of applications in biotechnology and development of biosensors. The low thermal stability of wild-type firefly luciferase is a limiting factor in most applications. Improvements in activity and stability of few enzymes in the presence of ionic liquids were shown in many reports. In this study, kinetic and thermal stability of firefly luciferase from Photinus pyralis in the presence of three tetramethylguanidine-based ionic liquids was investigated. The enzyme has shown improved activity in the presence of [1, 1, 3, 3-tetramethylguanidine][acetate], but in the presence of [TMG][trichloroacetate] and [TMG][triflouroacetate] activity, it decreased or unchanged significantly. Among these ionic liquids, only [TMG][Ac] has increased the thermal stability of luciferase. Incubation of [TMG][Ac] with firefly luciferase brought about with decrease of K(m) for ATP.
A new series of benzo[5,6]chromeno[3,2-c]quinoline derivatives were successfully synthesized using various arylglyoxal monohydrates, quinoline-2,4-dione, and β-naphthol in H2O:EtOH (2:1) as a green solvent in the presence of catalytic amounts p-toluenesulfonic acid as a mild catalyst under reflux conditions with high yields (83–92%). The reaction conditions were optimized in different solvents at variable thermal conditions, and the optimized reaction condition for this synthesis has been reported. The structures of all new products were defined by 1H-NMR, 13C-NMR, FT-IR, mass spectral data, and HRMS.
Objectivesα-amylases hydrolyze α-1,4 glycosidic bonds in starch. ILs used as co-solvent in different enzymatic reactions to improve activity, selectivity and stability of enzymes. In this study, fluorescence spectroscopy method was used to explain the effect of [emim][lactate] on kinetic and thermal stability of Aspergillus oryzae α-amylase.MethodsEffect of different concentrations of [emim][lactate] on activity of α-amylases was determined. Kinetic parameters, optimum pH and temperature and thermal stability were determined and compared with absence of [emim][lactate]. Intrinsic fluorescence spectroscopy for Trp residues was performed for both presence and absence of [emim][lactate].ResultsActivity of α-amylase decreases in presence of [emim][Lac]. Moreover, Km of α-amylase in the presence of [emim][lactate] increases while Vm decreased. Optimum temperature in presence of [emim][lactate] increases from 45 to 50 °C while optimum pH decreases from 9 to 7. Thermal stability of α-amylase in the presence of [emim][lactate] is similar to that in the absence of [emim][lactate] at 40 and 50 °C but decreases at 60 °C. Intrinsic fluorescence spectroscopy shows unfolding of native structure of α-amylase is dependent on [emim][lactate] concentration.ConclusionsPresence of [emim][lactate] ionic liquid as co-solvent leads to structural unfolding of α-amylase and loss of its activity and thermal stability.
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