Abstract
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−1min−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.
Highlights
L-asparaginase is an important enzyme as therapeutic agents used in combination with other drugs in the treatment of acute lymphoblastic leukemia
The colonial morphology of a 14 day culture of strain NEAE-82 grown on yeast extract/malt extract agar (ISP 2 medium) revealed that strain NEAE-82 had the typical characteristics of the genus Streptomyces[17]
No pigment found in medium in yeast extract -malt extract agar, oatmeal agar inorganic saltstarch agar, glycerol–asparagine agar or tyrosine agar, faint brown pigments formed in peptone-yeast extract iron agar
Summary
L-asparaginase is an important enzyme as therapeutic agents used in combination with other drugs in the treatment of acute lymphoblastic leukemia. 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 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. Clinical employments of L-asparaginase are accompanied with fatal allergenic reactions to the patients[4]. These effects are mainly due to L-asparaginase associated L-glutaminase activity and bacterial endotoxins in enzyme preparations[5]. There are reports of L-asparaginase production from Streptomyces noursei MTCC 10469, isolated from marine sponge Callyspongia diffusa[15] and Streptomyces aurantiacus isolated from mangroves of Bhitarkanika[16]
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