Table 1.
Bacterial strains and plasmids utilized in this study.
Table 2.
Primers utilized in this study.
Fig 1.
SDS-PAGE and western blot of His-tagged recombinant proteins in L. casei MCJΔ1.
(A) SDS-PAGE analysis. Lane M, marker. Lanes 1–3, whole protein extracts of the pELX1, pELX1-ManB and pELX1-GusA transformants. (B) Western blot analysis, which suggested that both recombinant proteins were expressed. Lane M, marker. Lanes 1–3, whole protein extracts of the pELX1, pELX1-ManB and pELX1-GusA transformants. The protein concentrations of all samples were normalized to 3 mg·ml-1, and the sample volume loaded was15 μl in both the SDS-PAGE and western blot analysis.
Fig 2.
Expression of ManB and GusA in E. coli JM109.
The specific activity of ManB was high and remained proportional to cell mass throughout growth, whereas GusA activity decreased severely in the late exponential growth phase (from 450 U·mg-1 WCP to 180 U·mg-1 WCP in 1.5 h) and stabilized in the stationary phase (130–180 U·mg-1 WCP). In the growth curve, there were no significant difference between the negative control E. coli JM109 pELX1 transformant, the E. coli JM109 ManB transformant and the E. coli JM109 GusA transformant. Error bars represent standard errors from three replicate experiments.
Fig 3.
Expression of ManB and GusA in L. casei MCJΔ1.
In the growth curves, L. casei MCJΔ1 showed exponential growth until OD600 = 1.8, and the stationary phase was from OD600 = 1.8 to 6.8. In the growth curve, there were no significant difference between the negative control L. casei MCJΔ1 pELX1 transformant, the L. casei MCJΔ1 ManB transformant and the L. casei MCJΔ1 GusA transformant. the OD600 in the activity curve of L. casei MCJΔ1/pELX1-ManB transformant were 0.2, 0.5, 1.0, 1.8, 2.9, 4.3, 5.0 and 6.7, and those of L. casei MCJΔ1/pELX1-GusA transformant were 0.2, 0.5, 1.0, 2.2, 3.6, 4.6, 5.8 and 6.6, respectively. The specific activity of ManB increased in the exponential growth phase until the OD600 of the culture reached 1.8 (23 U·mg-1 WCP). Then, ManB activity decreased gradually to 15 U·mg-1 WCP in the late growth phase before remaining relatively constant. The specific activity of GusA peaked during the exponential growth phase (OD600 = 1.0, 75 U·mg-1 WCP) before decreasing rapidly in the late growth phase by more than 60% (OD600 = 1.0 versus 4.6). Error bars represent standard errors from three replicate experiments.
Fig 4.
Western blot detection of ManB and GusA in L. casei.
The final concentration of all the WCP samples were adjusted to 0.2 mg·ml-1 for ManB samples and 0.1 mg·ml-1 for GusA samples, and 10-μl aliquots were added in each lane of the electrophoresis gels. (A) Western blot of His-tagged ManB from the L. casei MCJΔ1 transformant. These results show a similar increase in the amount of ManB protein and ManB activity during early exponential growth (until OD600 = 1.8). Lane M, marker. Lanes 1–6, ManB from the L. casei MCJΔ1 transformant at different cell densities (OD600 = 1.0, 1.8, 2.9, 4.3, 5.0 and 6.7). (B) Western blot of His-tagged GusA from the L. casei MCJΔ1 transformant. These results show that although GusA activity decreased rapidly from the exponential growth phase to the stationary growth phase, the western blot analysis of GusA revealed large amounts of this protein in late growth-phase cell samples, when the specific activity of the enzyme dropped markedly. Lane M, marker. Lanes 1–6, GusA of the L. casei MCJΔ1 transformant at different cell densities (OD600 = 1.0, 2.2, 3.6, 4.6, 5.8 and 6.6).
Fig 5.
Expression cassette of related expression vectors.
Fig 6.
Expression and secretion of ManB in L. casei MCJΔ1/pELSH-ManB.
(A) ManB activity in the supernatant. ManB activity reached 8.85 U·ml-1 during the exponential growth phase of L. casei MCJΔ1/pELSH-ManB and remained relatively constant above 8 U·ml-1 throughout the late growth phase (between OD600 = 1.9 to 4.8) before decreasing. (B) Western blot of His-tagged ManB in the supernatant. Aliquots of 10 μl were added in each lane of the electrophoresis gels. These results clearly showed that secreted mannanase activity was correlated with the accumulation of ManB during cell growth. Lane M, marker. Lanes 1–8, ManB of the L. casei MCJΔ1 transformant at different cell densities (OD600 = 0.5, 0.9, 1.9, 2.8, 3.4, 4.8, 5.8 and 6.2). Error bars represent standard errors from three replicate experiments.
Fig 7.
Expression and secretion of the ManB-SlpA fusion protein in L. casei MCJΔ1/ pELWH-ManB.
(A) S-layer-bound mannanase activity from the functional fusion protein on the cell surface of L. casei MCJΔ1/pELWH-ManB. The mannanase activity increased during the early growth phase, peaking at OD600 = 3.7 with an activity of 28.4 mU·5×109 cfu-1, and then decreased. (B) SDS-PAGE and western blot analysis of the composition of the S-layer proteins. L. casei MCJΔ1 and L. casei MCJΔ1/pELWH were used as controls (the OD600 of all cell samples was 3.0). The fusion protein was barely detectable in L. casei MCJΔ1/pELWH-ManB (Lane 6), as reflected by a very low mannanase activity on the cell surface. Lane M, Marker. Lanes 1–3, SDS-PAGE-analyzed S-layer proteins from L. casei MCJΔ1, L. casei MCJΔ1/pELWH and L. casei MCJΔ1/pELWH-ManB. Lanes 4–6, western blot of His-tagged target proteins from the S-layer proteins of L. casei MCJΔ1, L. casei MCJΔ1/pELWH and L. casei MCJΔ1/pELWH-ManB. The red arrow represents the target proteins. Error bars represent standard errors from three replicate experiments.