Secretory expression of recombinant small laccase from Streptomyces coelicolor A3(2) in Pichia pastoris

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 alpha 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 degrees C for 30 min. The enzyme (similar to 38 kDa) had an optimum activity at 80 degrees C, but optimum pH varied with substrate used. K-m values for ABTS, SGZ and 2,6-DMP were 142.85 mu M, 10 mu M and 54.55 mu M and the corresponding k(cat) values were 60.6 s(-1), 25.36 s(-1) and 27.84 s(-1), respectively. The t(1/2) values of the rSLAC at 60 degrees C, 70 degrees C, 80 degrees C were 60 h, 32 h and 10 h, respectively. The enzyme deactivation energy (E-d) was 117.275 kJ/mol while Delta G, Delta H and Delta S 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. (C) 2017 Elsevier B.V. All rights reserved.