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

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(-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.

Figure 1. Color of the aerial mycelium of Streptomyces sp. NEAE-82 grown on starch -nitrate agar medium for 7–14 days of incubation at 30 °C.

Figure 2

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).

Figure 3

(A) Plate assay showing zone of hydrolysis of starch by strain NEAE 82. All the starch in the medium near the microbe has been hydrolyzed by extracellular amylases; (B) Coagulation and peptonization of milk by strain NEAE 82.

Figure 4. 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.

Figure 5. Purification of L-asparaginase produced by Streptomyces fradiae NEAE-82 using ion exchange on DEAE Sepharose CL-6B.

(▲) refer to protein, (●) refer to L-asparaginase activity.

Figure 6. Activity of L-asparaginase as a function of the pH of the reaction.

Figure 7. Effect of the temperature on L-asparaginase activity.

Figure 8. Effect of the substrate concentration of the reaction on L-asparaginase activity.

Figure 9. Michaelis-Menten plot for L-asparaginase produced by Streptomyces fradiae NEAE-82.

Figure 10. Effect of different incubation periods on L-asparaginase activity.

Figure 11. Thermal stability of L-asparaginase as a function of the time of the reaction.

Figure 12. SDS-polyacrylamide gel electrophoresis of the purified L-asparaginase from Streptomyces fradiae NEAE-82.

Lane 1: Protein marker; Lane 2–4: Ammonium sulphate fractions (50, 60, 70% respectively); Lane 5, 6: Purified protein.

Figure 13. Cytotoxicity and safety assay of the purified L-asparaginase on non-cancerous human fibroblast cells.

Figure 14. The anticancer effects of the purified L-asparaginase on Caco2, Hep2 and HepG2 cells.

Figure 15. The anticancer effect of the purified L-asparaginase on Caco2 cells after 48 of treatment, cells undergoing apoptosis are characterized by cellular rounding up, shrinkage, membrane blebbing and loss of cell adhesion.