微嗜酸寡养单胞菌中的漆酶对黄曲霉毒素B1降解脱毒的生物活性

Characterization of the aflatoxin B1 degradation role of laccases in Stenotrophomonas acidaminiphila

摘要:

[目的] 探究微嗜酸寡养单胞菌中的漆酶对AFB1的降解活性，并确定漆酶在菌株CW117降解代谢AFB1过程中的贡献。[方法] 从微嗜酸寡养单胞菌基因组中，共筛选到两个漆酶基因lc1和lc2，并用大肠杆菌BL21外源表达蛋白rLC1和rLC2，在体外检测其对AFB1的降解活性。同时参考前人报道，研究了氧化性辅剂对漆酶AFB1降解的促进作用。在体外实验基础上，利用自杀质粒pK18mobsacB，以同源重组方法构建了两株漆酶缺失株CW117^△lc1和CW117^△lc1-lc2，验证了漆酶基因（lc1和lc2）对AFB1体内降解作用。[结果] 体外实验显示，重组酶rLC1具有AFB1降解活性，氧化性辅剂ABTS、AS或SA可显著地提高rLC1降解活性，但rLC2未显示降解活性。突变株CW117^△lc1和CW117^△lc1-lc2对AFB1仍显示了较高的降解活性，且在大多数降解时间点与野生株CW117无显著差异。[结论] 微嗜酸寡养单胞菌CW117菌株中，LC1在体外显示了AFB1的降解活性，且降解活性可以被氧化性辅助因子增强，LC2未显示体外降解活性；体内试验发现，漆酶基因lc1和lc2对菌株CW117降解AFB1的贡献较小，该菌株还存在其他降解途径。

Abstract:

[Objective] Characterization of the aflatoxin B1 (AFB1) degradation roles of the laccases screened from Stenotrophomonas acidaminiphila CW117. [Methods] Two laccase genes lc1 and lc2 from strain CW117 genome were screened, and their AFB1 degrading activity was examined in vitro by heterologous expressed proteins of rLC1 and rLC2 in E. coli BL21. On the basis of in vitro test, two laccase-deficient strains CW117^△lc1 and CW117^△lc1-lc2 were constructed by homologous recombination method by using suicide plasmid pK18mobsacB, and the laccases (lc1 and lc2) AFB1 degradation role on strain CW117 was validation in vivo. [Results] Laccase rLC1 showed the AFB1 degradation activity in vitro, but rLC2 did not show degradation activity. Degradation activity of rLC1 was improved by redox mediators of 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid), acetosyringone or syringaldehyde. The degrading activity of mutants CW117^△lc1 and CW117^△lc1-lc2 showed similar degradation activity to the wild-type strain CW117 in most evaluation time-points. [Conclusion] Laccase LC1 from S. acidaminiphila showed AFB1 degradation activity, and the degradation activity could be enhanced by redox mediators as previous study. However, the laccases’ contribution to AFB1 degradation in strain CW117 was minimal, and other degradation pathways existed in the strain.