State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, Jiangsu, China.
The Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu, China.
Molecules. 2019 Feb 7;24(3):592. doi: 10.3390/molecules24030592.
In this study, the K-3 glutaminase was successfully over-expressed in the GRAS (Generally Recognized as Safe) strain 168 by integration of the gene in the 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 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 and genes, which encode specific proteases, were disrupted by integration of the 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.
在这项研究中，通过将基因整合到 168 染色体的特定位点，成功地在 GRAS（一般认为安全）菌株 168 中过表达 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产生高浓度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评估了 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。为了进一步提高谷氨酰胺酶的产量，通过整合 基因破坏了编码特定蛋白酶的 和 基因。在不添加抗生素的情况下进行连续细胞培养后，整合的重组菌株表现出优异的遗传稳定性，显示出良好的工业化潜力。优化发酵温度后，在 24°C 下使用 5L 生物反应器进行重组谷氨酰胺酶生产菌株的分批补料发酵，获得的最高酶活约为 357.6 U/mL。
