Molecular cloning, heterologus expression, and structural modeling of L-asparaginase from Pseudopedobacter saltans in e.coli

L-asparaginase (L-ASP), or l-Asparagine aminohydrolase (EC 3.5.1.1), is an enzyme that belongs to the class of hydrolases that is capable of hydrolyzing L-Asn into aspartic acid and ammonia, which is used in the treatment of acute lymphoid leukemia, which promotes the cleavage of the amino acid asparagine through the use of water and cleaves non-peptide carbon-nitrogen bonds. In this current study, cloning of the novel L-asparaginase gene from Pseudopedobacter saltans DM12145 was performed in E. coli with accession number NC_015177.1. The full-length of Pseudopedobacter saltans L-asparaginase is 1019bp, a protein-encoding 339 amino acids; and the molecular weight was evaluated to be 37.8 kDa, with a theoretical (pI) of 6.13. It was cloned on the expression vector pET-28-His (+) by the EZ Clone method, synonymously called ligation independent cloning (LIC) with protein ID WP_013634621.1 by Genscript Co., USA. It was cloned onto the expression vector pET-28a (+). The construct was nominated as pET-28a (+)/asp_pseudopedo. The recombinant L-asparaginase I gene of Pseudopedobacter saltans was expressed in pET28-His and transformed into E. coli BL21 (DE3) as a 6 His-tag fusion protein. Induced by one mM of IPTG for 18 h at 30 oC, and purified by IMAC chromatography, then analyzed by SDS-PAGE to assess the solubility and molecular weight of the recombinant protein band, which was exactly as expected at 36.0 kDa. The findings of this study reveal that the L-asparaginase I enzyme maintained its enzymatic activity at a pH of 8.0, the temperature of 60 oC, with a variable kinetics Km value equal to 3 mM and a Vmax of 168.2 mol/min/mg. The Silico analysis reveals that it is quite similar to L-asparaginases I of E. coli, which are distinctly specific for L-asparagine and act as homodimer cytosolic proteins. Finally, the cloning and expression of a novel-bacterial Pseudopedobacter saltans L-asparaginase enzyme in soluble and active stats were successfully achieved.