الفهرس 
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Abstract
In the current work, we studied Microbiological and Biochemical studies on methioninase enzyme produced from Streptomycetes as Anticancer Agent. Thirty-four random Streptomycetes isolates were found in soil, marine water, sediment. The maximum number of Streptomycetes isolates were found in Dakahlia, followed by Gharbia and Giza, while the lowest number was found in Alexandria and South Sinai (Sharm El-Sheikh).Streptomycetes isolates were chosen based on the distinctive colony morphology, which is often spherical, convex, and has deeply rooted development into the medium. Typically, dry, powdery spore masses cover the surface of the colonies.
The release of methioninase by thirty-four Streptomyces isolates is tested using a rapid plate assay test technique revealed that only nine Streptomyces isolates were distinguished by the presence of a pink hue around their colonial growth as evidence and have the greatest ability for the formation of extracellular methioninase.
The most effective degrading isolate (D10) was found by further investigation, and it was identified and described by morphological, cultural, physiological, biochemical, and 16S rRNA sequencing studies. Isolates (D10) had smooth spore surface ornamentation along with a straight flexuous spore chain morphology. Yellow-colored spore masses contrast with the pale yellow-white coloration of the substrate mycelium. The genus Streptomyces, as listed in Bergey’s handbook, is strongly implied by all of the isolate’s (D10) characteristics. However, isolate (D10) appears to be similar to S. albidoflavus based on biochemical and morphological evidence.
Methioninase production by Streptomyces albidoflavus strain 4MR was affected by an incubation time test. reveal that methioninase synthesis gradually increases until 7 days, at which point it reaches its maximum (4.65 U/ml), and subsequently enzyme activity declines. And it also affected by PH. Thus, at pH 7.0, the maximum enzyme yield was observed to be (5.21 U/ml). Reduced enzyme synthesis was brought on by either an increase or a DROP in the medium’s Ph.
Maximal enzyme yield was noticed at pH 7.0 was (5.21 U/ml). either excess or lowering in the pH of the medium produced in reduced enzyme production.
The results also validated a substantial correlation between Met-Nase synthesis and incubation temperature up to 30 °C, where Met-Nase output of (5.42 U/ml) was recorded. Compared to the optimal temperature value, there was a significant DROP in enzyme synthesis at 35 and 40 °C, respectively. The carbon source employed significantly affected the synthesis of Met-Nase, with glucose being the favored carbon source and yielding enzymes with an activity of activity of (6.35U/ml). also, peptone was found to be the optimum nitrogen source, increasing the maximum enzyme output (6.81 U/ml).
Various concentrations of Methionine were used in the enzyme production medium. The 0.4% concentration gave maximal enzyme production (5.50 U/ml). But, by increase in methioninase concentration, there was a reduced ion in enzyme production.
Using a Sephacryl S-300 column, Met-Nase isolated from Streptomyces albidoflavus was purified by ammonium sulfate precipitation. One fraction of the twelve recovered from the cell free extract showed Met-Nase activity in contrast to the total protein, and total activity, which all declined correspondingly, the particular enzyme activity increased with each purification step. With a specific enzyme activity of roughly 5.01 U/mg, 359.3 mg of protein and 1800.0 U of Met-Nase activity were produced from the crude extract. 291 mg of protein and 1740.0 U of enzyme activity, with an enhanced specific activity of 5.98 U/mg, were obtained using the purification process of Lyophilization.
Electrophoresis on sodium dodecyl sulfate-polyacrylamide gel was used to gauge the homogeneity and molecular weight of the purified Met-Nase (SDS-PAGE). When compared to the common molecular weight markers, the results showed that the Met-Nase preparation was pure since just one unique protein band with an apparent molecular weight of 45.4 kDa was found. The results of the Met-Nase protein’s SDS-PAGE and Met-Nase activity assay demonstrated that the enzyme purification procedure was effective and that the purified enzyme lacked methioninase activity.
Kinetic characterization of the purified methioninase showed that the ideal pH range for Streptomyces 4MR Met-Nase activity was shown to be ranging from 3.0 to 11.0 and the enzyme displayed stability in the pH range of 6.0 to 8.0. The enzyme activity evaluated at 37 ° C was found to be at its highest and was therefore considered to be the ideal temperature for activity. However, the enzyme activity dropped off significantly beyond 70 ° C, losing 60% of its initial activity at 80 °C. According to the results of the temperature stability, the enzyme was stable at temperatures between 25.0-45.0 ° C (for 30 minutes) and 25.0-45.0 °C (for 60 minutes). In the presence of various metal ions, MET-Nase activity was also measured. Only Hg2+, Cu2+, Cd2+, Ba2+, Ni2+, and Cd2+ among the ions examined showed a significant reduction of activity, with Na+, Mg2+, Ni2+, and Li2+ serving as an inducer at 5.0 mM EDTA. Hg2+, Cu2+, and Cd2+-induced inhibition of enzyme activity.
Steady-state kinetic analysis was performed on the purified Met-Nase The isolated Met-Nase from Streptomyces sp. Strain 4MR had Km and Vmax values of 14.1 mM and 30.3 U/ml, respectively. The apparent Km and Vmax of purified enzyme were calculated from a Lineweaver-Burk plot. Anticancer activity of Met-Nase against cancer cell lines the outcomes of the MTT experiment used to gauge the cytotoxicity of Met-Nase to Hep-G2, MDA-MB-231and A375 cells were evaluated. A robust dose-response association between the cytotoxic ability of Met-Nase from Streptomyces sp. strain 4MR and the data obtained from this test was apparent. Cancerous cells gradually lost viability with increasing dosages of Met-Nase, The IC50 of Met-Nase was determined to be 10.82 µg/ml and 9.35 µg/ml against HepG-2 and A375 cells, respectively. Accordingly, the IC50 of Met-Nase showed greater toxicity on MDA-MB-231 cells, with an IC50 of 69.70 µg/ml, which was supported by phase-contrast images that showed Met-Nase concentration-dependent alterations in cell shape. Met-Nase treatment resulted in cell fragmentation, apoptotic cell shrinkage, and membrane blebbing in Hep-G2, MDA-MB-231, and A375 cells