通过信号肽筛选优化耐高温α-淀粉酶在枯草芽孢杆菌中的分泌

摘要

以大肠-枯草穿梭载体pMA5质粒为基本骨架,以来源于嗜热脂肪地芽孢杆菌Geobacillusstearothermophilus NUB3621的耐高温α-淀粉酶基因为目标基因,利用POE-PCR法,成功构建针对淀粉酶的信号肽筛选载体.从枯草芽孢杆菌168基因组中扩增得到46个信号肽,利用POE-PCR法,使46个信号肽分别与线性化的筛选载体形成对应的multimer产物,直接转化枯草芽孢杆菌1A751,得到含不同信号肽的重组菌株.发酵结果显示,除了5个与淀粉酶适配性很低的信号肽,其它信号肽均有不同的引导淀粉酶细胞外分泌的能力,其中bgls引导淀粉酶细胞外分泌的能力最强,上清酶活的峰值达1 393.3 U/mL.%A screening vector for signal peptides from B.subtilis was constructed using a POE-PCR cloning method.The thermostable a-amylase from Geobacillus stearothermophilus NUB3621 was chosen as the target gene and cloned into E.coli and B.subtilis shuttle vector pMA5.46 signal peptides was amplified from the genome of B.subtilis 168.The DNA multimer was generated based on each signal peptide and the linear vector backbone by prolonged overlap extension PCR was directly transformed into B.subtilis 1A751,then 46 strains,each amylase protein was driven by different signal peptide,were cultured.The fermentation results revealed that except 5 signal peptides showed inappropriate fit with desired secretion target protein,the others showed different ability of the export of α-amylase in B.subtilis.The highest enzyme activity of 1 393.3 U/mL was detected from the recombinant strain containing bgls signal peptide.