Background: Bacterial resistance to antibiotics is an overwhelming solemn challenge worldwide. This calls for exploratory for novel origins of antibacterial drugs. Tetracycline resistance is mediated via mutations within the ribosomal binding site and/or the attainment of mobile genetic elements bearing tetracycline-specific resistance genes. The aim of the study: Design of novel antimicrobial tetracycline analogs and screening of their in vitro antibacterial activity. Methodology: Our study type was a screening experimental study. In this work, in vitro antimicrobial novel tetracycline, analog activity semi-synthetically produced from Streptomyces species was evaluated by standard agar dilution technique determining their minimum inhibitory concentrations (MICs) of growth of different pathogenic bacteria in Egypt. Tetracycline was purified by aqueous two-phase systems consisting of cholinium-based salts and polyethylene glycol, then modified by chemo-informatics. The addition of electron-with drawing Iodide anion at carbon 7 position to tetracycline originated tetracycline analog B (iodocycline). Results and discussion iodocycline(tetracycline analog B) antibiotic was a more active bacteriostatic antibacterial agent than tetracycline but demonstrated less bacterial resistance. Tetracycline analog B showed MICs of less than 10 micrograms/ml for bacterial growth which reflected its powerful antimicrobial activity in a comparison with the chloramphenicol prototype antibiotic.

Background: Hydrolysis of L-arginine to L-ornithine and urea is catalyzed by the crucial L-arginase enzyme. L-arginase nowadays is necessary for medicine due to its significance as an anticancer agent against auxotrophic cancers to arginine. It exists and is widely distributed in plants, fungi, chordates, and non chordates, however, there are few available reports for L-arginase from bacteria. Auxotrophic cancer cells for L-arginine can not synthesize their L-arginine as a sole metabolic source of carbon and nitrogen, while normal cells can synthesize their L-arginine for normal growth. Deprivation of auxotrophic cancer cells from L-arginine causes their killing. The aim of the study: Our study was aimed at isolation, screening, and molecular detection of L-arginase expressing bacteria from different soil environments in Egypt and characterization of the enzyme of activity as an anticancer mediator against auxotrophic cancer cells for arginine.Materials and methods: 50Grassland soil samples were collected from different locations in Egypt. The activity of L-arginase was confirmed by rapid plate assay screening using mineral arginine agar (MAA) media and estimating the optical density of Urea level spectrophotometrically. DNA Northen blotting hybridization technique, Gram stain, and biochemical reactions were used to identify the predominant soil bacterial isolates producing L-arginase. Tissue culturing techniques were applied for screening of anticancer activity of bacterial L-arginase against auxotrophic cancers for arginine.Results: Only the bacterial isolates which were able to utilize L-arginine as the sole metabolic source for carbon and nitrogen were grown on MAA media. Molecular detection and the biochemical reactions revealed that Bacillus subtilis, strain W23 (74%), and Bacillus anthracis, strain 34F2 (26%) were the predominant bacterial isolates producing L-arginase as an anticancer mediator for auxotrophic cancers for arginine. Bacterial L-arginase showed excellent anticancer activity against auxotrophic cancers for L-arginine in presence of Co, Ni, and Mn metal ions as co-factors. The molecular weight of bacterial L-arginase was found to be 39KDa as determined by the mass spectrometer.IC50 of bacterial L-arginase was 2.31 U/ml against hepatic cancer cell line,2.19 U/ml against melanoma cancer cell line and 4.51 U/ml against colorectal cancer cell line,4.64 U/ml against lung adenocarcinoma cell line (Calu-3) and 4.28 U/ml against cardiac cancer line(HL1). The Km and Vmax values of L-arginase were 8.63 mmol/l and 7.41 μmol /min. Conclusion: Our study was a promising approach study due to discovering a new bacterial L-arginase enzyme as an anticancer agent from different soil environments in Egypt.

The aim of the work: Finding and evaluating fungal arginine-degrading enzymes from various Egyptian soil habitats as anticancer mediators against auxotrophic cancer cells was the goal. Methods: Fungal cultures that produced enzymes degrading arginine were subcultured on malt agar medium. The enzymes were then readily isolated after expression by salting out with ammonium sulfate and purified using anion exchange resin chromatography. Additionally, the evaluation of their auxotrophic anticancer properties in male rabbit animal models weighing close to 2 kg and several cancer cell lines. In this work, fungal arginine degrading enzymes were generated at pH 6.5, 25 °C, and incubation for three days using mineral arginine agar [MAM] selective medium [developed to select only fungi capable of using arginine as a single metabolic source of carbon and nitrogen].Results: DNA blotting hybridization analyses identified Aspergillus as the primary fungal genus isolating these enzymes in the current investigation. Aspergillus fumigatus produced arginine decarboxylase and arginine deiminase, while Aspergillus niger was the producer of the L-arginase enzyme. They proved to be powerful anticancer drugs when used against these auxotrophic cancer cells. L-arginine amino acid is converted by arginine deiminase into Citrulline and ammonia. Ornithine and urea are produced from arginine by L-arginase. Extracellular proteins were identified as the enzymes degrading arginine. Based on spectrophotometric measurement of the Citrulline concentration and the ammonia concentration resulting from the hydrolysis of L-arginine by arginine deiminase, arginine deiminase synthesis and activity were examined. Using mass spectrometry and western blot-gel electrophoresis, the molecular masses of fungal L-arginase, arginine deiminase and arginine decarboxylase were determined to be 51 kDa, 53 kDa, and 37 kDa, respectively. L-arginase's Km and Vmax values were 8.63 mmol/l and 7.41 μmol/min, respectively.Conclusion: The study presented a unique fungal source of arginine-degrading enzymes from different soil conditions as anticancer mediators.

Background: Auxotrophic cancers are a leading cause of cancer-related deaths in the world. Because certain cancers are arginine amino acid auxotrophs, the Depletion of extracellular arginine using arginine deiminase (ADI) could be exploited to target such cancers. ADI is a potential anticancer agent against arginine auxotrophic cancers. It catalyzed the hydrolysis of L-arginine to L-citrulline and ammonia.Purpose of the research: Our study aimed to find new sources of bacterial arginine deiminases from different soil environments in Egypt and their production as anticancer agents by recombinant DNA technology.Methodology: This study was designed to screen for ADI-producing bacterial species using a simple and convenient method using metallo-arginine agar selective medium (MAA) and the Nesslarization assay. The isolated samples were characterized based on morphological features, biochemical reactions, and DNA probe hybridization. The antitumor activity of ADI was analyzed by MTT cell viability assay using various cancer cell lines. Results: Bacillus cereus strain AH173 was the major ADI-producing bacterial isolate from various soil environments in Egypt. The enzyme of interest showed excellent anticancer activity against L-arginine amino acid auxotrophic cancers. The molecular weight of ADI was 48 kDa. The Km and Vmax values of ADI were 8.75 mmol/L and 8.78 µmol/min. The IC50 for bacterial ADI was 2.42 U/ml for liver cancer cell lines, 2.17 U/ml for melanoma cancer cell lines, 4.63 U/ml for colon cancer cell lines, and 4.53 U/ml for lung adenocarcinoma cell line (Calu-3), 3.12 vs PANC-1 pancreatic cancer cell line, 3.77 vs RCC4 renal cancer cell line and 4.37 U/ml against a heart cancer line (HL1). The selectivity index of bacterial ADI against cancer cell lines was as the following: 3.93 vs (JHH4) liver cancer cell lines, 3.24 vs (IM1215) colon cancer cell lines, 3.61 vs (UPMM3) melanoma cancer cell lines, 3.18 vs (Calu-3) lung adenocarcinoma cell lines, 3.07 vs PANC-1 pancreatic cancer cell line, 3.81 vs RCC4 renal cancer cell line and 3.75 vs (HL1) heart cancer cell line. Conclusion: In this study, a new bacterial source for the production of ADI as an anticancer agent from various soil environments in Egypt was characterized.

Background: Auxotrophic cancers for Arginine are a leading cause of death worldwide. The manufacture of novel arginine degrading enzymes such as Arginine deiminase enzyme is mandatory due to this crisis. Aim of the study: Since certain tumor cells are auxotrophic for Arginine, the depletion of the extracellular Arginine by means of Arginine deiminase enzyme was exploited in the present study to target such tumors. Methodology: Selective recovery of some bacterial isolates from different environmental sites in Egypt and assessment their capabilities for Arginine deiminase production. Studying environmental and physiological factors affecting Arginine deiminase production by some selected isolates. Characterization of of activity Arginine deiminase produced by certain selected isolates as well as its production through bacterial recombinant DNA technology. Results: The major bacterial isolates grown on mineral Arginine agar( MAA) plates producing ADI were further identified as Bacillus subtilis DE 111 using 16S rRNA sequencing technique. The Arginine deiminase production and activity were optimal at 40℃ and alkaline pH. Mn⁺²,Ni⁺² and Co⁺² metal ions as cofactors were optimum activators for production and activity of ADI. The results showed that the potent cytotoxic consequences of ADI were exerted on the renal and leukemic cancer cell lines. ADI produced via bacterial recombinant DNA technology showed efficacious IC₅₀ 10.31± 0.2 and 16.08± 0.3 µg/ml against renal( Caki-1) and leukemic( K-562) cancer cell lines, respectively. The purified monomeric ADI was 36.18 KDa molecular mass as determined using SDS-PAGE, the specific activity reached 36.07 U/mg. Km,Vmax and Kcat were 0.05871 M, 40.36 µmol/ml/min and 5.014min^-1respectively. Optimum pH and temperature for productivity and activity ranged from 6-10 and 37-70℃,respectively. Total protein estimation using Bar-ford assay was determined to be 5.68 mg during the initial culture. ADI purification was achieved using 70% Ammonium Sulfate followed by Ni⁺²-immobilized affinity column chromatography with a final purification fold of 15.03.In vitro determination of biological half life of ADI using nesslerization assay was observed to be nearly 300 min. Conclusion: ADI produced from Bacillus subtilis DE111 demonstrated efficacious anticancer activities against leukemic( K-562) and renal( Caki-1) auxotrophic cancers for Arginine due to the depletion of L-arginine from the external surrounding environments.

Background: Oral and non-invasive insulin delivery remains limited by rapid gastrointestinal degradation, poor epithelial permeability, and intrinsic instability of native insulin under acidic, enzymatic, and thermal conditions. Objective: To design a mechanism-engineered insulin analog (InsuRFPK) with intrinsic stability and evaluate dual delivery via oral enteric-coated and sublingual tablets. Methods: InsuRFPK was rationally engineered using HisB5→Arg substitution, D-PheB24 and D-LysB29 incorporation, N-methylation at ProB28, and lipidation. The analog was synthesized using in vitro transcription–translation (IVTT) and purified via RP-HPLC. Structural validation was performed using molecular docking and molecular dynamics simulations (RMSD, RMSF, SASA). The analog was formulated into enteric-coated micronized tablets and fast-dissolving sublingual tablets. In vitro studies included stability in SGF (pH 1.2), SIF (pH 6.8), enzymatic degradation (trypsin, chymotrypsin), and dissolution testing. In vivo pharmacokinetics (Cmax, Tmax, t1/2, bioavailability) and pharmacodynamics (glucose reduction) were evaluated in diabetic models. Results: InsuRFPK retained native folding with enhanced stability (lower RMSD and SASA). Binding affinity improved to −12.8 kcal/mol vs −11.2 kcal/mol for native insulin. The analog showed>90% stability in SGF (2 h), SIF (4 h), and enzymatic conditions, while native insulin retained<20%. Thermal stability exceeded 70°C. Enteric tablets exhibited90% at pH 6.8 within 8–10 h. Sublingual tablets disintegrated within 2 min enabling rapid absorption. Oral bioavailability reached ~21.6% with Cmax 48.7 µIU/mL, Tmax 2.3 h,and half-life 3.8 h. Sublingual delivery showed faster onset (Tmax 0.5–1 h). Sustained glucose reduction of 50–55% was observed over 8–10 h, outperforming native insulin. Conclusion: Intrinsic molecular stabilization combined with dual oral and sublingual delivery enables effective non-invasive insulin therapy with improved stability, bioavailability, and sustained pharmacological activity.