Cloning, Expression and Characterization of aeruginosa EGYII L-Asparaginase from Pseudomonas aeruginosa strain EGYII DSM 101801 in E.coli BL21(DE3) pLysS

Publication date: Available online 21 June 2016 Source:Journal of Molecular Catalysis B: Enzymatic Author(s): Amany S. El-Sharkawy, Aida M. Farag, Amira M. Embaby, Hesham Saeed, Mohamed El-Shenawy Shortcomings encountered in commercial L-Asparaginases greatly restrict their therapeutic potential and hence, searching for novel L-Asparaginases has become a mandatory issue. Present work highlights cloning, expression and characterization of L-Asparaginase open reading frame (ORF) from Pseudomonas aeruginosa strain EGYII DSM 101801 in E.coli BL21 (DE3) pLysS. A DNA fragment of 984bp encoding L-Asparaginase ORF was cloned on pGEM-T easy vector via polymerase chain reaction. Successful expression of ORF fragment, harbored in frame on pET28-a(+) expression vector, was achieved in E.coli DE3 (BL21) pLysS as a Six His-Tag fusion protein after 18hrs of induction with 1mM IPTG at 37°C. The recombinant enzyme (aeruginosa EGYII L-Asparaginase) was purified to homogeneity using Ni2+ chelated Fast Flow Sepharose resin with 8.567 purification fold and 14.42% recovery. It exhibited an approximate molecular mass of 36kDa as deduced from SDS-PAGE. The recombinant enzyme exhibited its maximal activity at pH 8.5 and 45°C. It retained almost 80% and 60% of its activity at 37°C after 20min and 30min, respectively. Two fold enhancement in enzyme activity was retained in presence of 5mM MgCl2. EDTA, β-mercaptoethanol and ZnCl2 at a concentration of 1mM each slightly enhanced enzyme ...
Source: Journal of Molecular Catalysis B: Enzymatic - Category: Biochemistry Source Type: research