Characterization of a Novel Recombinant ß-Lactamase from Bacillus subtilis R5

Current study deals with the characterization of recombinant ss-lactamase from a locally isolated strain Bacillus subtilis R5. The study was an initial step for the fulfillment of commercial needs of ss-lactamase in Pakistan. The 1 kb ss-lactamase gene was amplified by PCR using the genomic DNA of B. subtilis R5 as template. The purified PCR product was cloned in pTZ57R/T and sub-cloned in pET21a. Expression of recombinant protein was examined in BL21 CodonPlus (DE3) cells. SDS-PAGE confirmed the size of recombinant protein as 34 kDa. Recombinant ss-lactamase was produced optimally when the BL21 CodonPlus (DE3) cells were induced with 0.6 mM IPTG with a post induction time of 5h at 37 degrees C. The characterization studies demonstrated the maximal enzyme activity at 37 degrees C in 50 mM sodium phosphate buffer pH 7. The presence of EDTA in the activity assay mixture reduced the ss-lactamase activity to 91% while Zn2+, Co2+, Mn2+ enhanced the enzymatic activity to 144, 121 and 108% when used at a final concentration of 1 mM. The ionic and non-ionic detergents showed slight inhibitory impact on the recombinant ss-lactamase activity. The enzyme exhibited the K (m) and V (max) values of 2.27 mM and 45.45 mu mol/min, respectively when benzylpenicillin was utilized as substrate. The degradative ability of recombinant ss-lactamase to hydrolyze a variety of ss-lactam ring containing antibiotics makes it a suitable candidate for its utilization as positive control in diagnostics and in antibiotic susceptibility testing experiments.