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In this study, the <i>Micrococcus luteus</i> K-3 glutaminase was successfully over-expressed in the GRAS (Generally Recognized as Safe) <i>Bacillus subtilis</i> strain 168 by integration of the <i>Mglu</i> gene in the <i>16S rDNA</i> 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 <i>B. subtilis</i> 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 <i>nprB</i> and <i>nprE</i> genes, which encode specific proteases, were disrupted by integration of the <i>Mglu</i> 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 &#176;C, and the highest enzyme activity achieved was approximately 357.6 U/mL.