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.

Keywords: 16S rDNA; Bacillus subtilis; glutaminase; integrated expression.

Figure 1

Comparison of the Mglu gene transcriptional levels of the transformants with Mglu integrated into the 16S rDNA gene of B. subtilis. Transcriptional levels were measured using the $2^{-\Delta \mathrm{tt}}$ assay. The transcriptional level of strain No. 8 was defined as 100%. The standard errors are calculated from three independent biological experiments.

Figure 2

Shake flask fermentation analysis of recombinant strains and B. subtilis 168. (A) Growth curves of the recombinant and B. subtilis 168 strains. The values shown are the average of three independent measurements and are presented as the error bars; (B) the maximum glutaminase activity of the recombinant strains and B. subtilis 168 produced by fermentation. The wild-type B. subtilis 168 strain was used as a negative control. The standard errors are calculated from three independent biological experiments.

Figure 3

SDS-PAGE analysis of the expression of glutaminase in BSM4. Lane M: protein marker; lane l: cell extract of B. subtilis 168 (negative control); lane 2: cell extract of recombinant BSM4; and lane 3: purified glutaminase.

Figure 4

Analysis of the genetic stability of the recombinant strains BSM4 and BSM. The standard errors are calculated from three independent biological experiments.

Figure 5

The effect of temperature on the fermentation of glutaminase. (A) Glutaminase fermentation at 37 °C; (B) glutaminase fermentation at 30 °C; (C) glutaminase fermentation at 24 °C; and (D) glutaminase fermentation at 20 °C. The standard errors are calculated from three independent biological experiments.

Figure 6

Fed-batch fermentation of the recombinant strain BSM4. The standard errors are calculated from three independent biological experiments.

Figure 7

Strategy for integrating the Mglu gene in the B. subtilis 168 chromosome. An integrative fragment contained the lox71-zeo-lox66 cassette with two regions having homology to the B. subtilis 16 chromosome and the HpaII-Mglu cassette. Due to the homologous regions that flank the lox71-zeo-lox66 cassette and the HpaII-Mglu cassette, the corresponding gene in the B. subtilis 168 chromosome DNA can be disrupted, and the zeo gene and HpaII-Mglu cassette is introduced. Next, the zeo gene was deleted through recombination between the lox71 and lox66 sites using the Cre/lox system, yielding the lox72 site.