Bacillus sonorensis L. Asparaginase: Cloning, Expression in E. coli and Characterization.

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.

• Authors: Nihal Aly, Amani El-Ahwany, Farid Shokry Ataya, Hesham Saeed
• Journal: The protein journal (Protein J) Vol. 39 Issue 6 Pg. 717-729 (12 2020) ISSN: 1875-8355 [Electronic] Netherlands
• PMID: 33106988 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Bacterial Proteins
• Recombinant Proteins
• Asparaginase

Topics

• Asparaginase (biosynthesis, chemistry, genetics)
• Bacillus (enzymology, genetics)
• Bacterial Proteins (biosynthesis, chemistry, genetics)
• Cloning, Molecular
• Enzyme Stability
• Escherichia coli (genetics, metabolism)
• Gene Expression
• Recombinant Proteins (biosynthesis, chemistry, genetics)