Over-activity and stability of laccase using ionic liquids: screening and application in dye decolorization

Abstract

The use of a wide range of water miscible and immiscible ionic liquids (ILs) as reaction media for ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) oxidation by Trametes versicolor laccase was studied. Thirteen ILs were shown to be suitable media for the laccase oxidation reaction, increasing the activity with respect to conventional media. Among them, the water-miscible IL choline dihydrogen phosphate [Chol][H₂PO₄] allowed over-laccase activity with an enhancement rate of 451% at 25 °C and pH 7.0. This ionic liquid improved the stability of the enzyme in the face of high temperature and high pH, while storage at room temperature in aqueous medium was increased up to 4.5 times. Moreover, it was found that in the reaction medium for decolourizing dyes (antraquinonic and azoic) using laccase increased the decolourization rate by up to 216% and 137% for the azoic dyes Acid Black 1 and Remazol Brilliant Blue R, respectively. A high decolorization rate was also obtained for a mixture of dyes (80% within 8 h). To understand the effect of [Chol][H₂PO₄] on the secondary protein structure of the laccase, several spectroscopic techniques were used such as Circular Dichroism (CD), Fourier transform infrared (FT-IR) and Fluorescence, all of which demonstrated that the β sheet structure was affected. A shift to an α-helix structure [Chol][H₂PO₄] could be responsible for the enhancement of the enzyme activity observed at 300 mM.