Improving the Production of Salt-Tolerant Glutaminase by Integrating Multiple Copies of into the Protease and Genes of 168.

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.