Improving the Production of Salt-Tolerant Glutaminase by Integrating Multiple Copies of Mglu into the Protease and 16S rDNA Genes of Bacillus subtilis 168
Improving the Production of Salt-Tolerant Glutaminase by Integrating Multiple Copies of Mglu into the Protease and 16S rDNA Genes of Bacillus subtilis 168
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DOI：
10.3390/molecules24030592
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发表时间：
2019-02
期刊：
影响因子：
4.6
通讯作者：
Xian Zhang;Zhaoyang Xu;Song Liu;K. Qian;Meijuan Xu;Tao-wei Yang;Jian-zhong Xu;Z. Rao
中科院分区：
文献类型：
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作者：
Xian Zhang;Zhaoyang Xu;Song Liu;K. Qian;Meijuan Xu;Tao-wei Yang;Jian-zhong Xu;Z. Rao
在本研究中，通过将Mglu基因整合到16S rDNA基因座上，成功地在GRAS(公认为安全的)枯草芽孢杆菌168菌株中过表达了黄色微球菌K-3谷氨酰胺酶。这样做是为了从选定的候选菌株中筛选出一株产生高水平重组谷氨酰胺酶的菌株。通过qPCR、SDS-PAGE分析和酶活性测定，进一步研究了枯草杆菌168染色体上谷氨酰胺酶基因的转录和谷氨酰胺酶蛋白的表达。为了进一步增加谷氨酰胺酶的产量，编码特定蛋白酶的nprB和nprE基因被整合到mglu基因中打乱。在不添加抗生素的情况下进行连续培养，整合后的重组菌株具有良好的遗传稳定性，具有良好的产业化潜力。在优化发酵温度后，利用5-L生物反应器在24℃下对重组谷氨酰胺酶产生菌进行补料分批发酵，最高酶活可达357.6 U/mL左右。
In this study, the Micrococcus luteus K-3 glutaminase was successfully over-expressed in the GRAS (Generally Recognized as Safe) Bacillus subtilis strain 168 by integration of the Mglu gene in the 16S rDNA locus. This was done in order to screen a strain producing high levels of recombinant glutaminase from the selected candidates. The transcription of the glutaminase genes in the B. subtilis 168 chromosome and the expression of glutaminase protein was further assessed by qPCR, SDS-PAGE analysis and an enzyme activity assay. To further increase the production of glutaminase, the nprB and nprE genes, which encode specific proteases, were disrupted by integration of the Mglu gene. After continuous cell culturing without the addition of antibiotics, the integrated recombinant strains showed excellent genetic stability, demonstrating favorable industrialization potential. After the fermentation temperature was optimized, a 5-L bioreactor was used for fed-batch fermentation of the recombinant glutaminase producing strain at 24 °C, and the highest enzyme activity achieved was approximately 357.6 U/mL.