Purification, characterization, cytotoxicity and anticancer activities of L-asparaginase, anti-colon cancer protein, from the newly isolated alkaliphilic Streptomyces fradiae NEAE-82.
L-asparaginase is an important enzyme as therapeutic agents used in combination with other drugs in the treatment of acute lymphoblastic leukemia. A newly isolated actinomycetes strain, Streptomyces sp. NEAE-82, was potentially producing extracellular L-asparaginase, it was identified as Streptomyces fradiae NEAE-82, sequencing product was deposited in the GenBank database under accession number KJ467538. L-asparaginase was purified from the crude enzyme using ammonium sulfate precipitation, dialysis and ion exchange chromatography using DEAE Sepharose CL-6B. Further the kinetic studies of purified enzyme were carried out. The optimum pH, temperature and incubation time for maximum L-asparaginase activity were found to be 8.5, 40 °C and 30 min, respectively. The optimum substrate concentration was found to be 0.06 M. The Km and Vmax of the enzyme were 0.01007 M and 95.08 Uml(-1)min(-1), respectively. The half-life time (T1/2) was 184.91 min at 50 °С, while being 179.53 min at 60 °С. The molecular weight of the subunits of L-asparaginase was found to be approximately 53 kDa by SDS-PAGE analysis. The purified L-asparaginase showed a final specific activity of 30.636 U/mg protein and was purified 3.338-fold. The present work for the first time reported more information in the production, purification and characterization of L-asparaginase produced by newly isolated actinomycetes Streptomyces fradiae NEAE-82.
Department of Bioprocess Development, Genetic Engineering and Biotechnology Research Institute, City of Scientific Research and Technological Applications, Alexandria, Egypt.
Noura El-Ahmady
NE
El-Naggar
Department of Protein Research, Genetic Engineering and Biotechnology Research Institute, City of Scientific Research &Technological Applications, Alexandria, Egypt.
Sahar F
SF
Deraz
Department of Botany, Faculty of Science, Mansoura University, Mansoura, Egypt.
Hoda M
HM
Soliman
Biopharmacetical Product Research Department, Genetic Engineering and Biotechnology Research Institute, City of Scientific Research and Technological Applications, Alexandria, Egypt.
Nehal M
NM
El-Deeb
Department of Bioprocess Development, Genetic Engineering and Biotechnology Research Institute, City of Scientific Research and Technological Applications, Alexandria, Egypt.
Sara M
SM
El-Ewasy
D039903:Actinobacteria / Q000201:enzymology / Q000648:ultrastructure; D000890:Anti-Infective Agents / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000302:isolation & purification* / Q000494:pharmacology* / Q000633:toxicity*; D001215:Asparaginase / Q000302:isolation & purification* / Q000494:pharmacology* / Q000633:toxicity*; D003110:Colonic Neoplasms / Q000188:drug therapy*; D030561:Databases, Nucleic Acid; D004789:Enzyme Activation; D057075:Enzyme Assays; D004795:Enzyme Stability; D012335:RNA, Ribosomal / Q000235:genetics; D013302:Streptomyces / Q000201:enzymology* / Q000648:ultrastructure; D013379:Substrate Specificity
D000890:Anti-Infective Agents; D000970:Antineoplastic Agents; D012335:RNA, Ribosomal; D001215:Asparaginase