الفهرس 
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Abstract
The aim of our study is to isolate Actinomycetes bacteria from different agricultural soils, screening for their biological activities, isolation and identification of the bioactive compound from the most potent Actinomycetes crude extract.
In the present study, a number of 52 Actinomycete isolates were collected from 13 agricultural soil samples, from Beni-Suef governorate, Egypt,
At which 20 isolates out of 52 Actinomycetes isolates showed marked antimicrobial activities to at least one tested organism.
Four isolates out of the total active twenty Actinomycetes isolates showing broad spectrum activity, eleven isolates showing activity against gram positive bacteria and five isolates showing activity against fungus, gram–positive and gram–negative bacteria.
To facilitate the screening for antitumor activity, we Fermenting and extracting the secondary metabolites and total proteins of three Actinomycetes isolates in small scales, in which after centrifugation of the cell free supernatant, it divided into two portions; one was used for extraction of total secondary metabolites and the other used for extraction of total proteins. After testing the extracted secondary metabolites and proteins against the indicated test organisms, we found the activity as secondary metabolites from isolates AGM13-1, AGM2-1 and AGM12-1 plus the protein activity from AGM13-1.
The all four tested substances showed anti-tumor toxicity against both cell lines (HepG2 hepatocellular carcinoma cell line and HCT 116 human colon carcinoma) where the survival fraction showed significant decrease as the concentration of these compounds increased, and The anti-tumor activity of these compounds against HepG2 cell line was arranged ascendingly as follow AGM12-1, AGM2-1, AGM13-1 (protein) and AGM13-1 while this arrangement for HCT116 was AGM12-1, AGM13-1 (protein), AGM13-1 and AGM2-1.
The most potent isolate Actinomycete AGM12-1 exhibited a wide spectrum of antibacterial and antifungal activities plus showing the highest cytotoxicity against HepG2 and HCT 116 cell lines. Our results revealed that isolate AGM12-1 produced its inhibitory compound in both broth and agar media. So, the Actinomycetes isolate AGM12-1 was selected due to its high productivity for further studies.
The identification of the most potent Actinomycete isolate AGM12-1
was attempted using two methods, Phenotypic and Genotypic characterizations, The morphological observation of the 7 days old culture using light microscopic showed a straight chain section with no fragmentation of the aerial mycelium and the using SEM showed crimpy spore surface with aerial and vegetative hyphae.
The growth capability, pigment production and color of both aerial and substrate mycelium of AGM12-1 strain was determined using different growth media; ISP-3, ISP-4, ISP-5, Czapek Dox, Sato, nutrient agar and starch nitrate agar
The physiological and biochemical characterization of the isolate such as Production of Catalase, Lecithinase, Protease, Lipolytic, Pectinolytic, Amylolytic, Hydrogen sulphide, Nitrate reductase, Urease, Gelatinase and Melanin besides screening for utilization of different nitrogen sources (peptone, protease peptone, potassium nitrate, yeast extract, ammonium sulfate) and carbon sources (starch, glucose, sucrose, fructose, mannitol) and ability to grow at wide range of pH from 5-9 were performed and it exhibited that the Actinomycetes isolate related to a group of Streptomyces according to the traditional methods of Actinomycetes identification.
The 16S rRNA gene amplification and sequencing revealed that, this isolate has a 99% similarity with Streptomyces vinaceusdrappus according to NCBI BLAST. The resulted sequence was aligned to 39 of the closely related Streptomyces species by retrieving their sequences from the NCBI GenBank database and assembled in MEGA7 software for phylogenetic analysis using the UPGMA method and the evolutionary distances were computed using the Maximum Composite Likelihood method.
Detection for the presence of antibiotic biosynthetic genes involved in the production of glycopeptide antibiotics (OXY B), non-ribosomal peptide synthase (NRPS), type I polyketide synthase (PKS I) and type II polyketide synthase (ARO-PKS II) was conducted using PCR specific primers targeting these specific genes that involved in the synthesis of the core antibiotic structures. The result revealed presence of type II of polyketide synthase system which is mostly responsible for the synthesis of aromatic antibiotic.
The optimum conditions for production of antimicrobial and antitumor agents from Streptomyces vinaceusdrappus AGM12-1 was screened and observed according to zone of inhibitions. It was found that the maximum productivity was achieved after using mannitol and ammonium sulphate at concentrations of 2.5% and 0.2% respectively. Other factors like pH 7, 2% starting inoculums and incubation for 11 days at 30 °c were found to produce high yield of antimicrobial and antitumor substance from Streptomyces vinaceusdrappus.
The active metabolites were extracted by ethyl acetate at the level of (1:1 v/v) at which the crude extract was purified by HPLC and all collected fractions were examined against Sarcina lutea to determine the active purified fraction.
Based on 1HNMR and MS as well as comparing the results with previously published one, the isolated compound was identified as Cyclo (S-Pro-S-Val).
The identification of antimicrobial and antitumor compound revealed its relatedness to diketopiprazine family that have important biological activities like inhibition of plasminogen activator inhibitor-1 (PAI-1) and alteration of cardiovascular and blood-clotting functions They also have activities as antitumour, antiviral, antifungal, antibacterial, and antihyperglycaemic agents.
We concluded that the Actinomycetes especially Streptomycetes still an important sources of bioactive compounds that are used for treating infections, cancer circulation and many other diseases. We characterized a derivative of di-ketopiprazine produced by Streptomyces vinaceusdrappus AGM12-1, isolated from Beni Suef governorate, Egypt, demonstrated obvious inhibitory affects against Gram positive, Gram negative, fungi, and an anti-tumor toxicity against human liver and colon cell lines (HepG 2 and HCT 116). To our knowledge, this is the first time to characterize diketopiprazine derivative as a secondary metabolite recovered from Streptomyces vinaceusdrappus in Egypt.