Background: Laccases are copper-containing enzymes which have been used
as green biocatalysts for many industrial processes. Although bacterial
laccases have high stabilities which facilitate their application under
harsh conditions, their activities and production yields are usually
very low. In this work, we attempt to use a combinatorial strategy,
including site-directed mutagenesis, codon and cultivation
optimization, for improving the productivity of a thermo-alkali stable
bacterial laccase in Pichia pastoris. Results: A D500G mutant of
Bacillus licheniformis LS04 laccase, which was constructed by
site-directed mutagenesis, demonstrated 2.1-fold higher activity when
expressed in P. pastoris. The D500G variant retained similar catalytic
characteristics to the wild-type laccase, and could efficiently
decolorize synthetic dyes at alkaline conditions. Various cultivation
factors such as medium components, pH and temperature were investigated
for their effects on laccase expression. After cultivation
optimization, a laccase activity of 347 \ub1 7 U/L was finally
achieved for D500G after 3 d of induction, which was about 9.3 times
higher than that of wild-type enzyme. The protein yield under the
optimized conditions was about 59 mg/L for D500G. Conclusions: The
productivity of the thermo-alkali stable laccase from B. licheniformis
expressed in P. pastoris was significantly improved through the
combination of site-directed mutagenesis and optimization of the
cultivation process. The mutant enzyme retains good stability under
high temperature and alkaline conditions, and is a good candidate for
industrial application in dye decolorization