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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.

No MeSH data available.


Thermal stability of L-asparaginase as a function of the time of the reaction.
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f11: Thermal stability of L-asparaginase as a function of the time of the reaction.

Mentions: The effect of temperature on the stability of L-asparaginase showed maximum enzyme activity at 50 °C (Fig. 11). Around 86.126% of the initial activity was retained by the enzyme after 20 min of incubation at 50 °C. About 72.072% of L-asparaginase activity was lost after incubation at 50 °C for 90 min, while a rapid decrease in the enzyme activity (16.874%) was observed after incubation at 80 °C for 90 min. Thermal inactivation process of the enzyme follows the theoretical curve of a sample first order reaction. However, linear regression of the obtained data was assayed to determine half life time (T1/2) as shown in Table 4. The half-life time (T1/2) was 184.91 min at 50 °С, while being 179.53 min at 60 °С. On the other hand, destruction of enzyme activity was observed at 80 °C with low half-life time (75.36 min). It can be concluded from the previous results that the higher thermal stability behavior of L-asparaginase was at 50 °C. Results presented in Table 4 were normalized to activity of un-incubated enzyme (time 0 = 100%) for the percentage of activity remaining. Heat inactivation half-life (T1/2) and heat deactivation constant (k) were determined by fitting the data to a first-order decay curve using Graph-Pad Prism software. An earlier study reported no significant loss of L-asparaginase activity purified from Streptomyces radiopugnans MS1, when the enzyme was pre-incubated at 40 °C for 60 min33. Similar results were recorded with Streptomyces noursei15, E. carotovora34, Pseudomonas stutzeri MB 40528.


Purification, characterization, cytotoxicity and anticancer activities of L-asparaginase, anti-colon cancer protein, from the newly isolated alkaliphilic Streptomyces fradiae NEAE-82
Thermal stability of L-asparaginase as a function of the time of the reaction.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f11: Thermal stability of L-asparaginase as a function of the time of the reaction.
Mentions: The effect of temperature on the stability of L-asparaginase showed maximum enzyme activity at 50 °C (Fig. 11). Around 86.126% of the initial activity was retained by the enzyme after 20 min of incubation at 50 °C. About 72.072% of L-asparaginase activity was lost after incubation at 50 °C for 90 min, while a rapid decrease in the enzyme activity (16.874%) was observed after incubation at 80 °C for 90 min. Thermal inactivation process of the enzyme follows the theoretical curve of a sample first order reaction. However, linear regression of the obtained data was assayed to determine half life time (T1/2) as shown in Table 4. The half-life time (T1/2) was 184.91 min at 50 °С, while being 179.53 min at 60 °С. On the other hand, destruction of enzyme activity was observed at 80 °C with low half-life time (75.36 min). It can be concluded from the previous results that the higher thermal stability behavior of L-asparaginase was at 50 °C. Results presented in Table 4 were normalized to activity of un-incubated enzyme (time 0 = 100%) for the percentage of activity remaining. Heat inactivation half-life (T1/2) and heat deactivation constant (k) were determined by fitting the data to a first-order decay curve using Graph-Pad Prism software. An earlier study reported no significant loss of L-asparaginase activity purified from Streptomyces radiopugnans MS1, when the enzyme was pre-incubated at 40 °C for 60 min33. Similar results were recorded with Streptomyces noursei15, E. carotovora34, Pseudomonas stutzeri MB 40528.

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.