Formation of inclusion bodies is an important obstacle to the production of active recombinant protein in Escherichia coli. Thus, soluble expression of penicillin G acylase from Kluyvera citrophila was investigated in BL21(DE3). In this study, the yield of active enzyme was significantly enhanced by the composition of the medium and induction opportunity. When 0.5 mmol/L IPTG was added to complex medium at 15 h after incubation, the volumetric and specific activities of penicillin G acylase both achieved the highest values, respectively. However, aggravation of intracellular proteolysis and decline of enzyme expression were also observed if induction occurred too much later. Ca2+ ion was another critical factor in cell growth and protein expression. When 24 mmol/L Ca2+ ion was adding to the medium at the beginning of fermentation, a greater than 2-fold increase in cell density and a 7-fold increase in volumetric activity of penicillin G acylase were reached. Nevertheless, no significant benefit for recombination protein expression was found when excess Ca2+ was added after induction time. This study demonstrates that the induction starting time and Ca2+ ion are two critical factors for the expression of active penicillin G acylase.

Inhalational anthrax is caused by B. anthracis, a virulent sporeforming bacterium which secretes anthrax toxins consisting of protective antigen (PA), lethal factor (LF) and edema factor (EF). LF is a Zn-dependent metalloprotease and is the main determinant in the pathogenesis of anthrax. Here we report the identification of a lead small-molecule inhibitor of anthrax lethal factor by screening an available synthetic small-molecule inhibitor library using fluorescence-based high-throughput screening (HTS) approach. Seven small molecules were found to have inhibitory effect against LF activity, among which SM157 had the highest inhibitory activity. All theses small molecule inhibitors inhibited LF in a noncompetitive inhibition mode. SM157 and SM167 are from the same family, both having an identical group complex, which is predicted to insert into S1' pocket of LF. More potent small-molecule inhibitors could be developed by modifying SM157 based on this identical group complex. [BMB reports 2011; 44(12): 811-815]