This work reports for the first time the secretory expression of the small laccase (SLAC) from Streptomyces coelicolor A3(2) in Pichia pastoris. Using an AOX1 promoter and α factor as a secretion signal, the recombinant P. pastoris harbouring the laccase gene (rSLAC) produced high titres of extracellular laccase (500 ± 10 U/l), which were further increased seven fold by pre-incubation at 80 °C for 30 min. The enzyme (∼38 kDa) had an optimum activity at 80 °C, but optimum pH varied with substrate used. Km values for ABTS, SGZ and 2,6-DMP were 142.85 μM, 10 μM and 54.55 μM and the corresponding kcat values were 60.6 s-1, 25.36 s-1 and 27.84 s-1, respectively. The t1/2 values of the rSLAC at 60 °C, 70 °C, 80 °C were 60 h, 32 h and 10 h, respectively. The enzyme deactivation energy (Ed) was 117.275 kJ/mol while ΔG, ΔH and ΔS for thermal inactivation of the rSLAC were all positive. The rSLAC decolourised more than 90% of Brilliant Blue G and Trypan Blue dye in 6 h without the addition of a mediator. High titres of SLAC expressed in P. pastoris enhance its potential for various industrial applications.
Keywords: Dye decolourization; Pichia secretory expression; Small laccase.
Copyright © 2017 Elsevier B.V. All rights reserved.