枯草芽孢杆菌麦芽糖诱导型启动子Pglv-M1的改造

通过定点突变对麦芽糖诱导型启动子Pglv-M1进行改造,提高启动子启动能力。分析Pglv-M1的Sextama-35区与Pri-bmow-10区,通过对这些区域模拟随机突变,软件打分后获得最高分的新片段。将新启动子与载体p HCMC04-sva连接,并在枯草芽孢杆菌Bacillus subtilis 1A857中诱导表达,每12 h取粗酶液进行酶活测定。研究结果表明:除突变启动子Pglv-35以外,其余突变体启动效果均有所下降,有突变体构成的表达载体Pglv-35-pHCMC04-sva诱导表达后,得到的单位粗酶活力最高点出现在36 h处,较突变前提高1.31倍。PglvM1作为诱导型启动子Pglv的改造产物,其各个区域的相互影响较为紧密。通过对启动子关键区域单独或同时突变,仅获得一组启动能力有所提高的启动子Pglv-35,这为今后针对启动子的思考、研究方向提供参考。

Modification of Bacillus subtilis Maltose Inducible Promoter Pglv-M1

The maltose-inducible promoter Pglv-M1 was modified by site-directed mutagenesis to improve the start-up ability of the promoter. The Sextama-35 and Pri-bmow-10 areas of Pglv-M1 were analyzed, and by simulating random mutations in these areas, the software scored the new fragments with the highest score. The new promoter was ligated to the vector pHCMC04-sva,and express in Bacillus subtilis 1A857, and the enzyme activity was measured every 12 h. The results showed that except for the mutant promoter Pglv-35, the start-up effect of the other mutants reduced. After the expression vector Pglv-35-pHCMC04-sva was expressed by the mutant, the highest activity of the crude enzyme per unit appeared at 36 h, which was 1.31 times higher than that of the mutant before the mutation. As a modified product of the inducible promoter Pglv,Pglv-M1 has a relatively close interaction with each other. By separately or simultaneously mutating the key regions of the promoter, only one promoter Pglv-35 with improved starting ability is obtained, which provides a reference for thinking and research directions of the promoter in the future.