Abstract:

Although some amino acid components and sequence of the active center are strongly related with laccase
activity, the impact of C- and N-terminal modifications cannot be neglected. The effects of C- and N- terminal plasticity on
the activity, structure and properties of Basidiomycetes laccase are still unclear so far. In this study, the laccase gene LAC3
was cloned from Trametes sp. C30 and two mutants were obtained by fusing an additional 6-histidine tag at both C and
N termini. All these stains were successfully expressed in the yeast Saccharomyces cerevisiae. The recombinant enzymes
obtained were compared and the results showed that the modification of terminal amino acid sequence of the enzyme
considerably affected its characteristics. Compared with LAC3, the production of C-terminal histidine-tagged recombinant enzyme
was not affected, but the production of N-terminal histidine-tagged recombinant enzyme was only half of the amount of LAC3.
The affinity to the substrate ABTS and SGZ was enhanced by both histidine-tagged fusion proteins. Compared with LAC3, the
pH stability was enhanced and the activity was maintained better under alkaline or neutral conditions with the modification on the
N-terminal. Studies of the plasticity of the C and N termini have great significance to obtain new laccases.

Key words: laccase, heterologous expression, his-tag, C terminus, N terminus