Characterization of a Novel Recombinant β-Lactamase from Bacillus subtilis R5
Characterization of a Novel Recombinant β-Lactamase from Bacillus subtilis R5
Amjed Ali1, Muhammad Tayyab1*, Abu Saeed Hashmi2, Asif Nadeem3, Shumaila Hanif4, Sehrish Firyal1, Shagufta Saeed1, Ali Raza Awan1 and Muhammad Wasim1
ABSTRACT
Current study deals with the characterization of recombinant β-lactamase from a locally isolated strain Bacillus subtilis R5. The study was an initial step for the fulfillment of commercial needs of β-lactamase in Pakistan. The 1 kb β-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 β-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 °C. The characterization studies demonstrated the maximal enzyme activity at 37 °C in 50 mM sodium phosphate buffer pH 7. The presence of EDTA in the activity assay mixture reduced the β-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 β-lactamase activity. The enzyme exhibited the Km and Vmax values of 2.27 mM and 45.45µmol/min, respectively when benzylpenicillin was utilized as substrate. The degradative ability of recombinant β-lactamase to hydrolyze a variety of β-lactam ring containing antibiotics makes it a suitable candidate for its utilization as positive control in diagnostics and in antibiotic susceptibility testing experiments.
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