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Purification, characterization, cytotoxicity and anticancer activities of L-asparaginase, anti-colon cancer protein, from the newly isolated alkaliphilic Streptomyces fradiae NEAE-82

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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.

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Scanning electron micrograph showing the spore-chain morphology and spore-surface ornamentation of strain NEAE-82 grown on starch nitrate agar medium for 14 days at 30 °C at magnification of 4000X (A) and 10000X (B).
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f2: Scanning electron micrograph showing the spore-chain morphology and spore-surface ornamentation of strain NEAE-82 grown on starch nitrate agar medium for 14 days at 30 °C at magnification of 4000X (A) and 10000X (B).

Mentions: 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 Streptomyces17. It is aerobic, mesophilic, Gram-positive actinomycete that develops abundant and well-developed substrate and aerial mycelium. Strain NEAE-82 produced reddish brown aerial mycelium (Fig. 1) on yeast extract-malt extract agar, oatmeal agar, inorganic salts-starch agar, and peptone-yeast extract iron agar. Not-distinctive aerial mycelium on glycerol-asparagine agar and tyrosine agar. Substrate mycelium with no distinctive pigments on most tested medium and brown substrate mycelium was produced on peptone-yeast extract iron agar (Table 1). Substrate mycelium pigment is not a pH indicator. No pigment found in medium in yeast extract -malt extract agar, oatmeal agar inorganic salt-starch agar, glycerol–asparagine agar or tyrosine agar, faint brown pigments formed in peptone-yeast extract iron agar. Melanoid pigments not formed in peptone-yeast extract iron agar and tyrosine agar. Strain NEAE-82 grew well on yeast extract -malt extract agar (ISP medium 2), oatmeal agar (ISP medium 3), inorganic salt-starch agar (ISP medium 4), peptone-yeast extract iron agar (ISP medium 6) but poor growth on glycerol–asparagine agar (ISP medium 5) and tyrosine agar (ISP medium 7). Strain NEAE-82 formed an extensively branched substrate mycelium and aerial hyphae which differentiated into long straight spore chains. Spore chains in section Retinaculiaperti including open spiral spore chains (Fig. 2). Flexuous or spiral spore chains are seen on starch-nitrate agar. Mature spore chains generally have 10–50 spores per chain; longer chains are sometimes observed. Spore surface is smooth. Verticils are not present. The mycelium does not fragment.


Purification, characterization, cytotoxicity and anticancer activities of L-asparaginase, anti-colon cancer protein, from the newly isolated alkaliphilic Streptomyces fradiae NEAE-82
Scanning electron micrograph showing the spore-chain morphology and spore-surface ornamentation of strain NEAE-82 grown on starch nitrate agar medium for 14 days at 30 °C at magnification of 4000X (A) and 10000X (B).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5015098&req=5

f2: Scanning electron micrograph showing the spore-chain morphology and spore-surface ornamentation of strain NEAE-82 grown on starch nitrate agar medium for 14 days at 30 °C at magnification of 4000X (A) and 10000X (B).
Mentions: 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 Streptomyces17. It is aerobic, mesophilic, Gram-positive actinomycete that develops abundant and well-developed substrate and aerial mycelium. Strain NEAE-82 produced reddish brown aerial mycelium (Fig. 1) on yeast extract-malt extract agar, oatmeal agar, inorganic salts-starch agar, and peptone-yeast extract iron agar. Not-distinctive aerial mycelium on glycerol-asparagine agar and tyrosine agar. Substrate mycelium with no distinctive pigments on most tested medium and brown substrate mycelium was produced on peptone-yeast extract iron agar (Table 1). Substrate mycelium pigment is not a pH indicator. No pigment found in medium in yeast extract -malt extract agar, oatmeal agar inorganic salt-starch agar, glycerol–asparagine agar or tyrosine agar, faint brown pigments formed in peptone-yeast extract iron agar. Melanoid pigments not formed in peptone-yeast extract iron agar and tyrosine agar. Strain NEAE-82 grew well on yeast extract -malt extract agar (ISP medium 2), oatmeal agar (ISP medium 3), inorganic salt-starch agar (ISP medium 4), peptone-yeast extract iron agar (ISP medium 6) but poor growth on glycerol–asparagine agar (ISP medium 5) and tyrosine agar (ISP medium 7). Strain NEAE-82 formed an extensively branched substrate mycelium and aerial hyphae which differentiated into long straight spore chains. Spore chains in section Retinaculiaperti including open spiral spore chains (Fig. 2). Flexuous or spiral spore chains are seen on starch-nitrate agar. Mature spore chains generally have 10–50 spores per chain; longer chains are sometimes observed. Spore surface is smooth. Verticils are not present. The mycelium does not fragment.

View Article: PubMed Central - PubMed

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.

No MeSH data available.


Related in: MedlinePlus