栓菌漆酶的异源表达及重组酶碳端和氮端组氨酸标签修饰对酶学特性的影响

以来源Trametes sp.C30 的LAC3漆酶基因为研究对象，通过引物设计进行突变，在其C端和N端进行延长，分别引入6 个组氨酸标签，并异源表达于酿酒酵母Saccharomyces cerevisiae中，将获得的重组酶进行比较发现漆酶末端氨基酸序列的改变对酶学性质的影响较大。C端引入组氨酸标签的重组酶的表达量不受影响，但是在N末端引入组氨酸标签的重组酶表达量只有LAC3的一半。添加组氨酸标签的融合蛋白对ABTS和SGZ两种底物的亲和力得到增强。而在酸碱稳定性耐受方面，与LAC3相比，N末端氨基酸的改变使其在酸碱稳定性方面得到了增强，中碱性条件下仍能保持较佳活性。C端和N端可塑性的研究对于漆酶新性状的获得具有重要意义。

Heterologous Expression of Trametes sp.Laccase in Saccharomyces cerevisiae and Characterization of Recombinant Enzyme by Fusing a Six-His Tag at N and C Termini

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.