Purification, characterization, and anticancer and antioxidant activities of L-glutaminase from Aspergillus versicolor Faesay4.

L-Glutaminase is an amidohydrolase which can act as a vital chemotherapeutic agent against various malignancies. In the present work, L-glutaminase productivity from Aspergillus versicolor Faesay4 was significantly increased by 7.72-fold (from 12.33 ± 0.47 to 95.15 ± 0.89 U/mL) by optimizing submerged fermentation parameters in Czapek's Dox (CZD) medium including an incubation period from 3 (12.33 ± 0.47 U/mL) to 6 days (23.36 ± 0.58 U/mL), an incubation temperature from 30 °C (23.36 ± 0.49 U/mL) to 25 °C (31.08 ± 0.60 U/mL), initial pH from pH 5.0 (8.49 ± 0.21 U/mL)  to pH 7.0 (32.18 ± 0.57 U/mL), replacement of glucose (30.19 ± 0.52 U/mL) by sucrose (48.97 ± 0.67 U/mL) as the carbon source at a concentration of 2.0% (w/v), increasing glutamine concentration as the nitrogen source from 1.0% (w/v, 48.54 ± 0.48 U/mL) to 1.5% (w/v, 63.01 ± 0.60 U/mL), and addition of a mixture of KH2PO4 and NaCl (0.5% w/v for both) to SZD as the metal supplementation (95.15 ± 0.89 U/mL). Faesay4 L-glutaminase was purified to yield total activity 13,160 ± 22.76 (U), specific activity 398.79 ± 9.81 (U/mg of protein), and purification fold 2.1 ± 3.18 with final enzyme recovery 57.22 ± 2.17%. The pure enzyme showed a molecular weight of 61.80 kDa, and it was stable and retained 100.0% of its activity at a temperature ranged from 10 to 40 °C and pH 7.0. In our trials, to increase the enzyme activity by optimizing the assay conditions (which were temperature 60 °C, pH 7.0, substrate glutamine, substrate concentration 1.0%, and reaction time 60 min), the enzyme activity increased by 358.8% after changing the assay temperature from 60 to 30 °C and then increased by 138% after decreasing the reaction time from 60 to 40 min. However, both pH 7.0 and glutamine as the substrate remain the best assay parameters for the L-glutaminase activity. When the glutamine in the assay as the reaction substrate was replaced by asparagine, lysine, proline, methionine, cysteine, glycine, valine, phenylalanine, L-alanine, aspartic acid, tyrosine, and serine, the enzyme lost 23.86%, 29.0%, 31.0%, 48.3%, 50.0%, 73.6%, 74.51%, 80.42%, 82.5%, 83.43%, 88.36%, and 89.78% of its activity with glutamine, respectively. Furthermore, Mn2+, K+, Na+, and Fe3+ were enzymatic activators that increased the L-glutaminase activity by 25.0%, 18.05%, 10.97%, and 8.0%, respectively. Faesay4 L-glutaminase was characterized as a serine protease enzyme as a result of complete inhibition by all serine protease inhibitors (PMSF, benzamidine, and TLCK). Purified L-glutaminase isolated from Aspergillus versicolor Faesay4 showed potent DPPH scavenging activities with IC50 = 50 µg/mL and anticancer activities against human liver (HepG-2), colon (HCT-116), breast (MCF-7), lung (A-549), and cervical (Hela) cancer cell lines with IC50 39.61, 12.8, 6.18, 11.48, and 7.25 µg/mL, respectively.

Evaluation of carcinogenic activities and sperm abnormalities of Gram-negative bacterial metabolites isolated from cancer patients after subcutaneous injection in albino rats.

Microbial pathogens drive tumorigenesis in 20% of cancer cases, so the present study is aimed to evaluate the carcinogenic activities, sperm abnormalities and other dangerous effects of the subcutaneous injection of extracts obtained from various clinical Gram-negative bacteria derived from cancer patients using albino rats. We isolated, identified and extracted of their secondary metabolites of carbapenem resistant Gram-negative bacteria derived from cancer patients. Various methods have been used to determine hepatotoxicity, nephrotoxicity, tumorigenesis, inflammatory and sperm abnormalities in the albino rats injected with extracts. In comparison with the normal animals group, all extracts induced hepatotoxicity which was evidenced by the significant elevation in the activity of the serum alanine aminotransferase, aspartate aminotransferase, gamma-glutamyltransferase and alkaline phosphatase; also, nephrotoxicity that was indicated through the marked increase in the serum urea and creatinine levels; tumorigenesis was achieved from the sharp elevation in serum levels of alpha fetoprotein, carcinoembryonic antigen and lactate dehydrogenase values as tumor markers; as well as severe inflammatory characteristics were monitored from the marked raise of tumor necrosis factor alpha and interleukin-1beta. Furthermore, the proportion of micronuclei in polychromatic erythrocytes and sperm abnormalities were statistically significant in all groups compared to control group. Various kinds of head abnormalities and coiled tail were noted. Histopathological examination of hepatic tissue came in line with the biochemical and cytological findings. It could conclude that the extracts of Serratia sp. Esraa 1, Stenotrophomonas sp. Esraa 2, Acinetobacter sp. Esraa 3, Escherichia sp. Esraa 4 and Pseudomonas sp. Esraa 5 were able to initiate cytotoxicity and tumorigenesis in rats.

Phytochemical Screening, Antifungal, and Anticancer Activities of Medicinal Plants Thymelaea Hirsuta, Urginea Maritima, and Plantago Albicans.

Ethyl acetate, ethanol, and acetone extracts of the medicinal plants Thymelaea hirsuta L., Urginea maritima L., and Plantago albicans L. (aerial parts) were evaluated for their phytochemical compositions, antimycotic activity against dermatophytes, and antiproliferative activity against different human cancer cell lines. Among them, the ethanolic extracts showed the highest phytochemical contents along with hyperactivities and were then selected for gas chromatography-mass spectrometry and Fourier-transform infrared spectroscopy analysis. The Fourier-transform infrared spectroscopy analysis confirmed the presence of different characteristic peak values with various functional chemical groups of the active components. However, U. maritima extracts through Fourier-transform infrared spectroscopy analysis showed distinctive peaks related to phenolic, amines, amides, aromatic, alkanes, alkyne, cyclopentanone, conjugated aldehyde, nitro, methoxy, uronic acids, aromatic esters, tertiary alcohol or ester, secondary and primary alcohols, aliphatic ether, sulfoxide, vinylidene, and halo compounds. Many bioactive main compounds with reported biological activities were detected by GC/MS (%) in the ethanolic extract of T. hirsuta, U. maritima, and P. albicans. All studied dermatophytes included a diverse set of virulence factors, including phospholipase, protease, keratinase, hemolysis, and melanoid production, all of which play vital roles in dermatophytic infection. Ethanolic extract of P. albicans inhibited the growth of Trichophyton soudanense totally and Trichophyton erinacei in addition to all Microsporum species. In contrast, the ethanolic extract of Trichophyton hirsuta at concentrations of 25 g/mL totally prevented the growth of all Trichophyton species. EtOH extract of U. maritima completely prevented the growth (100% inhibition) of all dermatophytic strains under study at the lowest concentration of 12.5 µg/mL. Scanning electron microscope analysis revealed considerable morphological modifications and structural alterations in dermatophyte species exposed to ethanolic extract of these plants. The viability of HCT-116, HepG-2, MCF-7, and HeLa cell lines was reduced after treatment with the ethanolic extracts of T. hirsuta, U. maritima, and P. albicans individually with IC50 values (10.0, 9.97, 48.5, and 56.24 µg/mL), (26.98, 25.0, 17.11, and 9.52 µg/mL), and (9.32, 7.46, 12.50, and 16.32 µg/mL), respectively. Our work revealed the significance of these traditional ethnomedical plants as potent sources for biologically active pharmaceuticals with potential applicability for the treatment of fungal and cancer diseases.

Marine endophytic fungal metabolites: A whole new world of pharmaceutical therapy exploration.

The growing threat arises due to diseases such as cancer and the infections around the world leading to a critical requirement for novel and constructive compounds with unique ways of action capable of combating these deadly diseases. At present, it is evident that endophytic fungi constitute an enormous as well as comparatively untapped source of great biodiversity that can be considered as a wellspring of effective novel natural products for medical, agricultural and industrial use. Marine endophytic fungi have been found in every marine plants (algae, seagrass, driftwood, mangrove plants), marine vertebrates (mainly, fish) or marine invertebrates (mainly, sponge and coral) inter- and intra-cellular without causing any palpable symptoms of illness. Since evolution of microbes and eukaryotes to a higher level, coevolution has resulted in specific interaction mechanisms. Endophytic fungi are known to influence the life cycle and are necessary for the homeostasis of their eukaryotic hosts and the chemical signals of their host have been shown to activate gene expression in endophytes to induce expression of endophytic secondary metabolites. Marine endophytic fungi are receiving increasing attention by chemists because of their varied and structurally unmatched compounds that have strong biological roles in life as lead pharmaceutical compounds, including anticancer, antiviral, insulin mimetic, antineurodegenerative, antimicrobial, antioxidant and immuno-suppressant compounds. Moreover, fungal endophytes proved to have different biological activities for exploitation in the environmental and agricultural sustainability.