It is also plausible that the concentration of protease inhibitors in a given cocktail may be sufficient to prevent protein degradation, but insufficient to inhibit or kill bacteria in the samples. Accordingly, several investigators have reported that a concentration-dependent relationship exists between protease inhibitor and bacterial growth (Labbe et al., 2001). Grenier et al. (2001a) showed that there was no inhibition of bacteria with bestatin at 0.02 μg mL−1, but when the concentration of bestatin approached 10 μg mL−1, the bactericidal function Compound C in vivo reached a maximum. In the presence

of aprotinin, the growth of S. alboniger was also partially or completely inhibited, depending on the concentration of the protease inhibitor (Lopes et al., 1999). Clearly, then, a higher dose of protease inhibitor has the potential to interfere with the proliferation of bacteria, resulting in an alteration of bacterial composition. Because massive degradation of proteins caused by proteases has been

observed in proteomic studies, it was suggested that PI should be added in the preparation of samples. Here, we have presented results indicating that saliva samples with and without PI showed similar protein diversity in fractions both with high-molecular-weight proteins and low-molecular-weight species as judged by both 1D SDS-PAGE and LC-MS/MS analysis. Addition of protease inhibitors seemed to have no significant effect on the integrity of salivary samples. Alternatively keeping the samples on ice and processing them in <1 h may have been sufficient to preserve protein integrity. In summary, our study this website lends considerable

evidence that a protease cocktail containing AEBSF, aprotinin, bestatin, E64, leupeptin, and pepstatin A has no effect on oral bacterial growth or total bacterial composition. These findings suggest that the addition of protease inhibitors in the preparation of saliva samples for protein research will not interfere with microbial DNA analysis. The study was supported by the National Institute of Dental and Craniofacial Research (NIDCR) Grant no. U19 DE018385. Program Officer: Dr Isaac R. Rodriguez-Chavez. “
“A halophilic isolate Salimicrobium halophilum strain LY20 producing extracellular amylase OSBPL9 and protease was isolated from Yuncheng, China. Production of both enzymes was synchronized with bacterial growth and reached a maximum level during the early-stationary phase. The amylase and protease were purified to homogeneity with molecular weights of 81 and 30 kDa, respectively. Optimal amylase activity was observed at 70 °C, pH 10.0% and 10% NaCl. Complete inhibition by EDTA, diethyl pyrocarbonate (DEPC), and phenylarsine oxide (PAO) indicated that the amylase was a metalloenzyme with histidine and cysteine residues essential for its catalysis. Maltose was the main product of starch hydrolysis, indicating an β-amylase activity. The purified protease from LY20 showed highest activity at 80 °C, pH 10.0% and 12.5% NaCl.