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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

1Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences, Abdul Qadir Jillani Road, Lahore, Pakistan.
2Riphah International University, Raiwind Road Campus, Lahore 
3Department of Biotechnology, Virtual University of Pakistan
4Department of Chemistry, University of Agriculture Faisalabad, Faisalabad
 
* Corresponding author: muhammad.tayyab@uvas.edu.pk; tayyab_pakistan@yahoo.com

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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Pakistan Journal of Zoology

October

Vol. 54, Iss. 5, Pages 2003-2500

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