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  • Title: Bacillus sonorensis L. Asparaginase: Cloning, Expression in E. coli and Characterization.
    Author: Aly N, El-Ahwany A, Ataya FS, Saeed H.
    Journal: Protein J; 2020 Dec; 39(6):717-729. PubMed ID: 33106988.
    Abstract:
    L-asparaginases (L-ASNases; EC 3.5.1.1) are aminohydrolases that catalyze the hydrolysis of L-asparagine (L-Asn) to L-aspartic acid and ammonia, resulting in the death of acute lymphoblastic leukemic cells and other blood cancer cells. In this study, Bacillus sonorensis (accession number MK523484) uncharacterized L-ASNase gene (accession number MN562875) was isolated by polymerase chain reaction (PCR), cloned into pET28a (+) vector, and expressed in Escherichia coli as a cytosolic protein. The recombinant enzyme was purified by affinity chromatography at 23.79-fold and 49.37% recovery. Denaturing polyacrylamide gel (10%) analysis of the purified enzyme resulted in a single protein band at 36 kDa that immunoreacted strongly with 6His-tag monoclonal antibody. The purified enzyme exhibited optimal activity at 45 °C and pH 7.0 and retained 92% and 85% of its initial activity after incubation for 60 min at 37 °C and 45 °C, respectively. The purified enzyme exhibited substrate specificity toward L-asparagine and low glutaminase activity (15.72%) toward L-glutamine at a concentration of 10 mM. The Km and Vmax values were 2.004 mM and 3723 µmol min1-, respectively.
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