5), the number of cycles at which fluorescence was reached the threshold line was 31.09 on the lipase gene and 5.09 on 16S rRNA, respectively.

In contrast, when we used RNA sample from the cells cultured in NB (3.0) the number of cycles was 28.00 on the lipase gene and 4.98 on 16S rRNA. Consequently, we estimated by the ΔΔCt method that the relative transcriptional level of lipase gene in 3% NaCl is 7.8 times higher SRT1720 datasheet than that in 0.5% NaCl. Moreover, as shown in Figure 8, the densities of the samples recovered at 12 and 24  hrs from the culture in NB (3.0) were certainly higher than those from the culture in NB (0.5), showing that the gene for the lipase is well transcribed in A. sobria under the condition of 3.0% NaCl. Transcription of the lipase gene by A. sobria in NB (3.0) at 12 and 24  hrs was more active than in NB (0.5). As shown, the amount of lipase in the culture supernatant from culturing in NB (3.0) was low compared with that in NB (0.5) (Fig. 1); however, transcription of the lipase gene by A. sobria was not suppressed in 3.0% NaCl and the mRNA of the lipase gene was produced well (Fig.  8). We therefore considered that the posttranscriptional process to become mature lipase had been disrupted in NB

(3.0). As shown in Figure 4, lipase expresses esterolytic Ferroptosis targets activity; we therefore examined the esterolytic activity of the culture supernatant, using pNpp Oxalosuccinic acid as the substrate. The supernatant

from the 24  hr culture in NB (0.5) expressed esterolytic activity, but that from the culture in NB (3.0) did not (Fig. 9). These findings suggest that the three-dimensional structure of the lipase differs from that of the active form when A. sobria is cultured in NB (3.0), and that the lipase produced in NB (3.0) is degraded by bacterial intracellular proteases. This explains why the amount of lipase in the supernatant from culture in NB (3.0) was low compared with that in NB (0.5). In this study, we found a protein of A. sobria whose production in the milieu was suppressed by NaCl in the medium. Analysis revealed that this protein is lipase; it degraded tributyrin, and expressed esterase activity against pNp-fatty acyl esters. We then cloned the lipase gene and determined the nucleotide sequence. The lipase substrate binding signature sequence (GLKVHFLGHSLGA) was contained in the sequence (28), supporting the contention that the protein is a lipase. The amino acid sequence deduced from the nucleotide sequence had 78.6% identity with the lipase of A. hydrophila AH-3 (11). Merino et al. have examined the substrate specificity of the lipase from A. hydrophila AH-3 (11). They found that E.