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 ±
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.
