Characterization of a Recombinant Glutaminase-Free L-Asparaginase (ansA3) Enzyme with High Catalytic Activity from Bacillus licheniformis

• Published in: Applied biochemistry and biotechnology 2014-12, Vol.174 (7), p.2504-2515
• Main Authors: Sudhir, Ankit P, Dave, Bhaumik R, Prajapati, Anil S, Panchal, Ketankumar, Patel, Darshan, Subramanian, R. B
• Format: Article
• Language: English
• Subjects: Asparaginase - biosynthesis; Asparaginase - chemistry; Asparaginase - genetics; Asparaginase - isolation & purification; Asparagine; Bacillus - enzymology; Bacillus - genetics; Bacillus licheniformis; Bacteria; Biochemistry; Biological and medical sciences; Biotechnology; Catalysis; catalytic activity; Catalytic polymerization; Chemistry; Chemistry and Materials Science; E coli; Enzymes; Escherichia coli; Escherichia coli - chemistry; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; gene overexpression; genes; glutaminase; Leukemia; lymphocytic leukemia; polyacrylamide gel electrophoresis; purification methods; recombinant proteins; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; sodium dodecyl sulfate; Substrate Specificity
• ISSN: 0273-2289; 1559-0291
• DOI: 10.1007/s12010-014-1200-z

L-Asparaginase (3.5.1.1) is an enzyme widely used to treat the acute lymphoblastic leukemia. Two genes coding for L-asparaginase (ansA1 and ansA3) from Bacillus licheniformis MTCC 429 were cloned and overexpressed in Escherichia coli BL21 (DE3) cells. The recombinant proteins were purified to homogeneity by one-step purification process and further characterized for various biochemical parameters. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that both the enzymes are monomers of ∼37 kDa. Recombinant ansA1 was found to be highly unstable, and recombinant ansA3 was catalytically active and stable, which showed an optimum activity of 407.65 IU/mg at 37 °C and pH 8. Recombinant ansA3 showed higher substrate specificity for L-asparagine with negligible glutaminase activity. Kinetic parameters like K ₘ , Vₘₐₓ, kcₐₜ, and kcₐₜ/K ₘ were calculated for recombinant ansA3.