Cloning and Purification of L-Asparaginase from Enterobacter carcerogenus

Abstract

In this study, the gene coding for EcL-ASP from Enterobacter carcerogenus was identified in full sequence and cloned into a mesophilic organism. The gene encoding L-asparaginase was transferred to the pET-28a (+) vector to ensure its expression in Escherichia coli BL21 (DE3) pLysS. The recombinant protein containing the N-terminal histidine tail (6xHis tag) was purified by Nickel affinity chromatography. As a result of SDS-PAGE, it was determined that the purified protein consisted of a single type of polypeptide. In the theoretical calculation, the subunit molecular weight of the recombinant protein containing the histidine tail was found to be 37 kDa. It was observed that the cloned enzyme had low L-glutaminase activity. The pH and temperature at which the recombinant enzyme showed the best activity were determined as 7.0 and 37 °C, respectively. From the drawn Lineweaver-Burk graph, it is estimated that the Km value is 0.06 mM and the Vmax value is 666.7 U mg-1 protein.

Keywords

• Enterobacter carcerogenus
• L-asparaginase
• recombinant DNA
• protein Purification

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