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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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Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, showing the relationships between strain NEAE-82 and related species of the genus Streptomyces.Only bootstrap values above 50%, expressed as percentages of 1000 replications, are shown at the branch points. GenBank sequence accession numbers are indicated in parentheses after the strain names. Phylogenetic analyses were conducted in the software package MEGA4. Bar, 0.2 substitution per nucleotide position.
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f4: Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, showing the relationships between strain NEAE-82 and related species of the genus Streptomyces.Only bootstrap values above 50%, expressed as percentages of 1000 replications, are shown at the branch points. GenBank sequence accession numbers are indicated in parentheses after the strain names. Phylogenetic analyses were conducted in the software package MEGA4. Bar, 0.2 substitution per nucleotide position.

Mentions: The 16S rRNA gene sequence (1508 bp) was determined for strain NEAE- 82. A BLAST search18 of the GenBank database using this sequence showed its similarity to that of many species of the genus Streptomyces. A phylogenetic tree (Fig. 4) based on 16S rRNA gene sequences of members of the genus Streptomyces was constructed according to the neighbour-joining method of Saitou and Nei19 with MEGA420. This tree shows the close phylogenetic association of strain NEAE-82 with certain other Streptomyces species. Phylogenetic analysis indicated that the strain NEAE-82 consistently falls into a clade together with Streptomyces somaliensis strain DSM 40738 (GenBank/EMBL/DDBJ accession No. NR_025292.1) and Streptomyces fradiae strain CBT BR13 (GenBank/EMBL/DDBJ accession No. KP230701.1).


Purification, characterization, cytotoxicity and anticancer activities of L-asparaginase, anti-colon cancer protein, from the newly isolated alkaliphilic Streptomyces fradiae NEAE-82
Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, showing the relationships between strain NEAE-82 and related species of the genus Streptomyces.Only bootstrap values above 50%, expressed as percentages of 1000 replications, are shown at the branch points. GenBank sequence accession numbers are indicated in parentheses after the strain names. Phylogenetic analyses were conducted in the software package MEGA4. Bar, 0.2 substitution per nucleotide position.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, showing the relationships between strain NEAE-82 and related species of the genus Streptomyces.Only bootstrap values above 50%, expressed as percentages of 1000 replications, are shown at the branch points. GenBank sequence accession numbers are indicated in parentheses after the strain names. Phylogenetic analyses were conducted in the software package MEGA4. Bar, 0.2 substitution per nucleotide position.
Mentions: The 16S rRNA gene sequence (1508 bp) was determined for strain NEAE- 82. A BLAST search18 of the GenBank database using this sequence showed its similarity to that of many species of the genus Streptomyces. A phylogenetic tree (Fig. 4) based on 16S rRNA gene sequences of members of the genus Streptomyces was constructed according to the neighbour-joining method of Saitou and Nei19 with MEGA420. This tree shows the close phylogenetic association of strain NEAE-82 with certain other Streptomyces species. Phylogenetic analysis indicated that the strain NEAE-82 consistently falls into a clade together with Streptomyces somaliensis strain DSM 40738 (GenBank/EMBL/DDBJ accession No. NR_025292.1) and Streptomyces fradiae strain CBT BR13 (GenBank/EMBL/DDBJ accession No. KP230701.1).

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