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Molecules 2019, 24(3), 592; https://doi.org/10.3390/molecules24030592

Improving the Production of Salt-Tolerant Glutaminase by Integrating Multiple Copies of Mglu into the Protease and 16S rDNA Genes of Bacillus subtilis 168

1
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, Jiangsu, China
2
The Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu, China
3
School of Medicine, Yichun University, Yichun 336000, Jiangxi, China
*
Authors to whom correspondence should be addressed.
Received: 6 January 2019 / Revised: 28 January 2019 / Accepted: 4 February 2019 / Published: 7 February 2019
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Abstract

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. View Full-Text
Keywords: Bacillus subtilis; glutaminase; integrated expression; 16S rDNA Bacillus subtilis; glutaminase; integrated expression; 16S rDNA
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Zhang, X.; Xu, Z.; Liu, S.; Qian, K.; Xu, M.; Yang, T.; Xu, J.; Rao, Z. Improving the Production of Salt-Tolerant Glutaminase by Integrating Multiple Copies of Mglu into the Protease and 16S rDNA Genes of Bacillus subtilis 168. Molecules 2019, 24, 592.

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