Dataset Information

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

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.

SUBMITTER: Zhang X

PROVIDER: S-EPMC6384544 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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Publications

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

Zhang Xian X Xu Zhaoyang Z Liu Song S Qian Kai K Xu Meijuan M Yang Taowei T Xu Jianzhong J Rao Zhiming Z

Molecules (Basel, Switzerland) 20190207 3

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 ...[more]

PMID: 30736411

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Project description:The real-time PCR (qPCR) and digital PCR (dPCR) to amplify a single-copy of house-keeping genes (i.e., hsp60, pheS or tuf) are used for the assay of limited microbial species. In general, with a single-copy gene, there are obviously varied DNA sequences for even the same microbial species, which could cause difficulties with design of primers and probes for PCR when targeting various single copy genes. In general, for identification by dPCR (as a representative case: Lactobacillus paracasei), accumulated DNA sequence information of 16S rDNA, which is much more frequently used, should be targeted. In contrast, next-generation sequencing revealed that there are five copies of 16S rDNA in a live L. paracasei MCC1849. Therefore, we aimed to reveal, if heat-killed L. paracasei supplemented in nutritional foods that aid the host immune system have the relevant five copies per chromosomal DNA, and if the relevant copies remain unchanged on the same chromosomal DNA or remain to be different chromosomal DNA fragments. So, we revealed the actual distribution of the potential original five copies of 16S rDNA using our innovative dPCR, in which both 16S rDNA and hsp60 genes were simultaneously elongated. The molecular ratios of 16S rDNA/hsp60 dispersed in the dPCR chip were then estimated. The 16S rDNA/hsp60 molecular ratios of the heat-killed L. paracasei in foods, resultantly ranged from 5.0 to 7.2, being the same or higher than that of the five copies determined by next-generation sequencing. The 16S rDNA copy number/ratio indicated the chromosomal DNA molecular number and the associated cell number. As significance, different nutritional foods could potentially cause the loss of chromosomal DNA of supplemented beneficial microbes to a much greater degree. Our absolute dPCR does not require standard correlative samples for the estimation of final products. The estimation principle of the ratio of 16S rDNA/a house-keeping single-copy gene by our absolute dPCR could lead to a useful and accurate assay for various nutritional foods.

Dataset Information

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

Publications

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

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