Cloning And Expression Of Staphylococcus Simulans Lysostaphin Enzyme Gene In Bacillus Subtilis Wb600

Lysostaphin is a glycylglycine endopeptidase, secreted by Staphylococcus simulans, capable of specifically hydrolyzing pentaglycine crosslinks present in the peptidoglycan of the Staphylococcus aureus cell wall. In this paper, we describe the cloning and expression of the lysostaphin enzyme gene in Bacillus subtilis WB600 host using pWB980 expression system. Plasmid pACK1 of S. simulans was extracted using the alkaline lysis method. Lysostaphin gene was isolated by PCR and cloned into pTZ57R/T-Vector, then transformed into Escherichia coli DH5 alpha. The amplified gene fragment and uncloned pWB980 vector were digested using PstI and Xba. enzymes and purified. The restricted gene fragment was ligated into the pWB980 expression vector by the standard protocols, then the recombinant plasmid was transformed into B. subtilis WB600 using electroporation method. The recombinant protein was evaluated by the SDS-PAGE method and confirmed by western immunoblot. Analysis of the target protein showed a band corresponding to 27kDa r-lysostaphin. Protein content was estimated 91 mg/L by Bradford assay. The recombinant lysostaphin represented 90% of its maximum activity at 40 degrees C and displayed good thermostability by keeping about 80% of its maximum activity at 45 degrees C. Heat residual activity assay of recombinant lysostaphin demonstrated that the enzyme stability was up to 40 degrees C and showed good stability at 40 degrees C for 16 h incubation.