Özet:

L-Asparaginase aminohydrolase (EC 3.5.1) or L-asparaginase (L-ASP) is an enzyme capable of hydrolyzing LAsn into aspartic acid and ammonia, which is used as a treatment for acute lymphoid leukemia. Cloning of Lasparaginase gene from A-novel Pseudopedobacter sltans DM12145 in E. coli. with accession number NC_015177.1. full-length of P. saltans L- asparaginase is 1019pb, protein encoding 339 amino acids; and molecular weight evaluated to be 37.8kDa, with theoretical (pI) is 6.13 kDa. It was cloned on the expression vector pET-28α-His (+) by EZ Clone method synonymously called ligation independent cloning (LIC) with protein ID WP_013634621.1". by Genscript Co., USA. Respectively. The recombinant of L-asparaginase I gene of P. saltans was expressed in pET28-αHis and transformed in E. coli BL21 (DE3); as a (6 his-tag fusion protein).and induced by one mM of (IPTG) for 18 hours at 30˚C, and purify by IMAC Chromatography, then analyzed by SDS-PAGE to assess the solubility and molecular weight of recombinant protein band was exactly as expected at 36.0 kDa. P. saltans L-asparaginase I enzyme maintained its enzymatic activity at a pH8.5, temperature 60˚C, with variable of kinetics Km value equal to 3 mM and a Vmax of 168.2 µmol/min/mg, finding of this study revel It is quite similar to L-asparaginases I of E. coli which is distinctly specific for LAsparagine and act as homodimer cytosolic protein. Finally, the cloning and expression of A Novel-bacterial of P. saltans L-asparaginase enzyme in soluble and active stats, was successfully achieved.

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