蜡样芽胞杆菌蛋白酶的克隆及在枯草芽胞杆菌系统中的高效表达

Cloning and over-expression of protease gene from Bacillus cereus WQ9-2 in Bacillus subtilis

基于来源于Bacillus cereus WQ9-2的耐有机溶剂蛋白酶WQ的液相色谱-双质谱(LC-MS-MS)分析结果,设计引物,克隆耐有机溶剂蛋白酶WQ的基因,测序分析表明该蛋白酶的开放阅读框(ORF)大小为1 701 bp,编码566个氨基酸,其中含有信号肽(28个氨基酸)、前肽(220个氨基酸)及成熟肽序列(含954 bp编码318个氨基酸),相对分子质量约为3.7×104。将不带自身信号肽的蛋白酶基因apr WQ插入穿梭质粒p MA5中,构建了表达载体p MA5/apr WQ。该表达载体导入枯草芽胞杆菌WB600中获得阳性重组子WB600-p MA5/apr WQ,通过优化培养基成分以及培养条件,重组子发酵产蛋白酶体积酶活达到17 400 U/m L,约为高产野生菌产酶量的5倍。重组蛋白酶在多种有机溶剂(体积分数为50%)中表现出了良好的耐受性,验证了重组菌表达的蛋白酶与来源于B.cereus WQ9-2的耐有机溶剂蛋白酶性质一致,该耐有机溶剂蛋白酶的高效表达为进一步发挥其高效生物催化作用等实际应用奠定了基础。

Based on the liquid chromatography-mass-mass spectrometry （ LC-MS-MS） analysis of trypsin-digested protein fragments of the solvent-stable protease WQ from a solvent-stable strain Bacillus cereus WQ9-2,solvent-stable protease gene was successfully cloned. The gene contains an open reading frame of 1 701 bp,encoding a pre-pro-protein enzyme of 566 amino acids（28-aa pre-signal peptide,220-aa pro-peptide and 318-aa mature protein with the molecular mass of 3.7×10^4）. The expression plasmid pMA5/aprWQ was constructed by inserting the aprWQ gene into the vector pMA5 without native signal peptide sequence. The maximum concentration of the recombinant protease was 17 400 U/mL,5 fold higher than the natural production level. Molecular weight, tolerance in organic solvent of recombinant solvent-stable protease was identical to the native protease. The findings provides the basis for further expansion of the catalytic applications of organic solvent-stable protease.