ABSTRACT
Herein, we report the purification and characterization of an alkaline protease from the alkaliphilic Salipaludibacillus agaradhaerens (formerly Bacillus agaradhaerens) strain AK-R, which was previously isolated from Egyptian soda lakes. The purification procedures resulted in enzyme purification up to 13.3-fold, with a recovery yield of 16.3% and a specific activity of 3488 U/mg protein. AK-R protease was a monomeric protein with an estimated molecular weight of 33.0 kDa. The optimum pH and temperature for AK-R protease were pH 10 and 60 °C, respectively. The enzyme thermostability was significantly enhanced in the presence of CaCl2 by approximately 1.3-fold. Moreover, under optimal conditions, the K m and V max values of the enzyme were 2.63 mg/ml and 4166.7 U/mg, respectively. PMSF caused complete inhibition of the enzyme activity, suggesting that AK-R belongs to the serine protease family. In addition, the enzyme was completely inhibited by EDTA, revealing the requirement of metal ions for AK-R protease activity; hence, it can be classified as a metalloprotease. AK-R protease is a mostly thiol-independent enzyme, since thiol reductants such as ß-mercaptoethanol and dithiothreitol had no effect on the enzyme activity. AK-R protease exhibited high stability in several organic solvents, including butanol, amyl alcohol, dimethyl ether, toluene, diethyl ether and methanol. Moreover, AK-R protease showed significant stability to a variety of surfactants and commercial detergents. The features and properties of AK-R alkaline protease are favourable and suggest its potential applications in various industries, particularly in the laundry detergent industry.
ABSTRACT
Background Enterococcus faecalis is considered to be one of most prevalent species in the oral cavity, particularly in endodontic infections. The aim of the present study was to investigate the prevalence of E. faecalis in dental root canals, clonal diversity by restriction fragment length polymorphism (RFLP) and randomly amplified polymorphic DNA (RAPD-PCR) analysis, and the antibiotic susceptibility of E. faecalis isolates. Results Among the bacterial strains isolated from dental root canal specimens (n = 82), E. faecalis was determined to have the highest prevalence followed by Streptococcus viridians, Leuconostoc mesenteroides, Staphylococcus aureus, Streptococcus mitis, and Pediococcus pentosaceus. Cluster analysis of RAPD-PCR and RFLP patterns of the E. faecalis isolates discriminated five and six different genotypes, respectively. Among the tested strains, 43%, 52% and 5% were susceptible, intermediate resistant, and resistant to erythromycin, respectively. In addition, one strain (E-12) was intermediate resistant to linezolid, and one isolate (E-16) was resistant to tetracycline. Interestingly, many of the intermediate resistant/resistant strains were grouped in clusters 5 and 6, according RAPD and to RFLP, respectively. Conclusions E. faecalis demonstrated the highest prevalence in the tested dental root canal specimens collected from Saudi patients and were grouped into five to six different genotypes. Different levels of antimicrobial susceptibility were observed in the tested E. faecalis strains, which clearly indicated that although bacterial strains may be similar, point mutations can result in extreme susceptibility or resistance to various antibiotics. This phenomenon is a cause for concern for clinicians in the treatment of dental infections caused by E. faecalis.
