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

• P. aeruginosa strain EGYII DSM 101801 was used as source for L-Asparaginase.
• ORF of L-Asparaginase was cloned and expressed in E.coli BL21(DE3) pLysS.
• Optimal activity of recombinant L-Asparaginase is at pH 8.5 and 45 °C.
• Two fold enhancements in recombinant enzyme activity was retained with 5 mM MgCl2.
• Recombinant L-Asparaginase has Km of 66 mM and Vmax of 1121 μM min−1 mg−1.

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 984 bp 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 18 h of induction with 1 mM 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 36 kDa 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 20 min and 30 min, respectively. Two fold enhancement in enzyme activity was retained in presence of 5 mM MgCl2. EDTA, β-mercaptoethanol and ZnCl2 at a concentration of 1 mM each slightly enhanced enzyme activity. Kinetics parameters of the recombinant enzyme were Km (63.11 mM) and Vmax (1121 μmol min−1 mg−1) for L-Asparagine as calculated from Lineweaver–Burk plot. Present data would encourage testing the therapeutic potential of the recombinant aeruginosa EGYII L-Asparaginase.

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