The activity of the integrated vgb was demonstrated using spectrophotometry
since a peak at 420 nm results from absorbance due
to CO binding to VHb (Liu and Webster, 1974). This peak was present
in B. subtilis S18-3-vgb+, but absent in B. subtilis S18-3-vgb
(Fig. 1).
3.3. Comparison of cell densities and c-PGA yield among B. subtilis
S18-3, B. subtilis S18-3-vgb and B. subtilis S18-3-vgb+ by flask
culture
Cell densities, c-PGA yield, and sucrose and glutamic acid concentrations
are shown in Fig. 2. The logarithmic phase of B. subtilis
S18-3-vgb+ was 2 h longer than that of B. subtilis S18-3 and B. subtilis
S18-3-vgb, and in the stationary phase, the cell density of B.
subtilis S18-3-vgb+ was significantly greater than that of B. subtilis
S18-3 and B. subtilis S18-3-vgb. This result indicated that the
expression of VHb in B. subtilis S18-3-vgb+ improved growth. A
similar observation has been reported for Bacillus thuringiensis,
where the cell density and the yield of insecticidal crystal proteins
were improved under low aeration/agitation conditions (Feng
et al., 2007).
Fig. 1. CO-difference spectra analysis of B. subtilis S18-3-vgb+ and B. subtilis S18-3-
vgb.
Table 1
Primers used in this study.
Primer Primer sequence 50–30
amyE-up1 ATTGCTCGAGATGTTTGCAAAACGATTCAAA
amyE-up2 GGATAAGCTTTGTGTGTTTCCATGTGTCCAGT
amyE-down1 ATTGTCTAGAGCTGTGCTTTATCCTGATGATA
amyE-down2 ATTACCGCGGTCAATGGGGAAGAGAACCGCT
C1 AATGAAGCTTACGGCAATAGTTACCCTTATT
C2 ACTGGAATTCTGTAATATAAAAACCTTCTTC
P1 ATTAGAATTCTGTCGACGTGCATGCAGGC
P2 GTAGGATCCGCTTCTGTTATTAATTCTTG
V1 ATTGGATCCGGAAGACCCTCATGTTAGA
V2 ATTATCTAGATTATTCAACCGCTTGAGCGTA
The underline is the restriction enzyme cleavage site.