Three-phase partitioning (TPP) purification, characterization and dye decolorization application of laccase from Pseudolagarobasidium acaciicola TDW-48

Abstract

This study focused on the efficient and economical purification of laccase from P. acaciicola TDW-48 and its application in dye decolorization. Firstly, the P. acaciicola TDW-48's laccase was purified by the three-phase partitioning (TPP) method, and then the artificial neural network-genetic algorithm (ANN-GA) optimization was used to improve the purification yield. The result indicated that the TPP method successfully purified P. acaciicola TDW-48's laccase with a high purification yield. After ANN-GA optimization, the strong interactive effects of the TPP parameters on purification yield were demonstrated by a high-accuracy ANN model (R-value of 0.99918 for all datasets). An optimum TPP system was developed, achieving 138.7 % activity recovery and 1.62-fold purity at a 57.82 % salt concentration, pH 5.75, and a t-butanol/enzyme ratio of 1.5. In the following, the enzyme characteristics and application potential of purified laccase were determined. The P. acaciicola TDW-48's laccase showed a molecular weight of 60.5 kDa and functioned optimally at pH 3 and 30 °C. The kinetic parameters implied high affinity and catalytic efficiency for the ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) substrate, with a low Km (37.9 μM) and a high Vmax (46.01 mM/min). Moreover, the purified laccase showed excellent potential for dye decolorization, with 44 % Congo red, 80 % bromophenol blue, and 58 % phenol red decolorized after 8 h of treatment.