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中国精品科技期刊2020
纪明华, 赵利超, 史吉平, 孙俊松. 一种芽孢杆菌中性蛋白酶启动子的克隆和验证[J]. 食品工业科技, 2016, (13): 192-197. DOI: 10.13386/j.issn1002-0306.2016.13.030
引用本文: 纪明华, 赵利超, 史吉平, 孙俊松. 一种芽孢杆菌中性蛋白酶启动子的克隆和验证[J]. 食品工业科技, 2016, (13): 192-197. DOI: 10.13386/j.issn1002-0306.2016.13.030
JI Ming- hua, ZHAO Li-chao, SHI Ji-ping, SUN Jun-song. Characterization of a neutral protease promoter derived from a Bacillus amyloliquefaciens strain[J]. Science and Technology of Food Industry, 2016, (13): 192-197. DOI: 10.13386/j.issn1002-0306.2016.13.030
Citation: JI Ming- hua, ZHAO Li-chao, SHI Ji-ping, SUN Jun-song. Characterization of a neutral protease promoter derived from a Bacillus amyloliquefaciens strain[J]. Science and Technology of Food Industry, 2016, (13): 192-197. DOI: 10.13386/j.issn1002-0306.2016.13.030

一种芽孢杆菌中性蛋白酶启动子的克隆和验证

Characterization of a neutral protease promoter derived from a Bacillus amyloliquefaciens strain

  • 摘要: 为研究1398中性蛋白酶高效表达的分子机制,对其生产菌株进行了基因组测序和比对,确认了表达该中性蛋白酶的操纵子序列及结构。通过构建1398中性蛋白酶表达质粒,将其转入枯草芽孢杆菌;以gfp为报告基因,将包含P1398在内的不同启动子分别构建到质粒p MK4,转入枯草芽孢杆菌。发酵研究结果表明:P1398是一种底物自发诱导的高效启动子,是菌株表达1398中性蛋白酶的关键因素。1398中性蛋白酶在枯草芽孢杆菌中可获得稳定的表达量,其中在164中活性最高(1210 U/m L);重组枯草芽孢杆菌在LB中培养时,P1398介导的GFP的表达量低于P43和Pxyl A,而在工业培养基PPB中,其表达量为Pxyl A介导的GFP的2.14倍。因此,P1398可用于食品工业酶重组生产菌株的构建。 

     

    Abstract: To obtain the DNA elements responsible for transcriptional regulation of the gene expressing 1398 neutral protease( npr E),the genome sequencing of the native bacillus strain was performed and analyzed,the complete operon including upstream region of the possible location of 1398 npr E cassette was introduced into B.subtilis for heterologous expression.gfp was then used as the reporter gene for full investigation of the promoter of 1398 npr E( P1398) and was compared with known promoter Pxyl Aand P43. It was found that,P1398 was an efficient and substrate- induced promoter as well as a key factor in the expression of 1398 Npr E.Stable expression of 1398 Npr E in B.subtilis was detected and the highest enzyme activity was 1210 U / m L in 164.Compared with PxylAand P43,the advantage of P1398 was its capability of bringing better yield of GFP protein in B. subtilis with medium used for industrial production of enzymes,the GFP level was 214% of that mediated by Pxyl A,therefore,P1398 was a good candidate of promoter choice for construction of recombinant enzymes' production using Bacillus.

     

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