外源木聚糖酶在枯草芽胞杆菌中信号肽筛选

Screening of optimal signal peptide for heterologous xylanase secretion by Bacillus Subtilis

摘要:

摘要:【目的】本试验旨在筛选引导表达外源木聚糖酶基因高效分泌的信号肽，为枯草芽胞杆菌木聚糖酶高效分泌表达系统提供元件。【方法】构建信号肽筛选载体，载体是以含壮观霉素抗性基因的大肠-枯草穿梭载体为基本骨架，目标蛋白为耐碱性木聚糖酶，可在麦芽糖启动子Pglv 诱导下表达。从枯草芽胞杆菌A1747基因组中扩增获得24 个Sec 途径信号肽，并将其全部链接到至筛选载体上，并在枯草芽胞杆菌WB700 中实现表达分泌。重组菌在3% 麦芽糖诱导下培养24h 后用DNS 法测定上清酶活。【结果】成功构建信号肽筛选载体pG

Abstract:

Abstract:［Objective］ We searched optimal signal peptide for heterologous and exogenous secretion of xylanase in Bacillus subtilis． ［Methods］We constructed a screening vector for signal peptides from B．subtilis． The Alkali resistance xylanase gene(xynA) from Bacillus pumilus was chosen as reporter gene and cloned into E． coli and B． subtilis shuttle vector pGJ148 which has maltose-inducible promoter Pglv and spectinomycin resistant gene． 24 Sec-type signal peptides (SPs) was amplified from B． Subtilis 1A747 and cloned into the screening vector for the expression of xynA in B．Subtilis WB700． The xylanase activity of the culture supernatant were detected after 24h incubation．Results］ The screening of these signal peptides revealed differences in xylanase activity of the culture supernatants， The recombinant strain containing YnfF signal peptide showed the highest xylanase acitivity (37.2IU/mL)．［Conclusion］ Experiment proved screening of signal peptides is ffective way for optimization of the export of heterologous protein in B．subtilis．