Bionanofactory for green synthesis of collagen nanoparticles, characterization, optimization, in-vitro and in-vivo anticancer activities.

Collagen nanoparticles (collagen-NPs) are promising biological polymer nanoparticles due to their exceptional biodegradability and biocompatibility. Collagen-NPs were bio-fabricated from pure marine collagen using the cell-free supernatant of a newly isolated strain, Streptomyces sp. strain NEAA-3. Streptomyces sp. strain NEAA-3 was identified as Streptomyces plicatus strain NEAA-3 based on its cultural, morphological, physiological properties and 16S rRNA sequence analysis. The sequence data has been deposited under accession number OR501412.1 in the GenBank database. The face-centered central composite design (FCCD) was used to improve collagen-NPs biosynthesis. The maximum yield of collagen-NPs was 9.33 mg/mL with a collagen concentration of 10 mg/mL, an initial pH of 7, an incubation time of 72 h, and a temperature of 35 °C. Using the desirability function approach, the collagen-NPs biosynthesis obtained after FCCD optimization (9.53 mg/mL) was 3.92 times more than the collagen-NPs biosynthesis obtained before optimization process (2.43 mg/mL). The TEM analysis of collagen-NPs revealed hollow sphere nanoscale particles with an average diameter of 33.15 ± 10.02 nm. FTIR spectra confirmed the functional groups of the collagen, collagen-NPs and the cell-free supernatant that are essential for the efficient capping of collagen-NPs. The biosynthesized collagen-NPs exhibited antioxidant activity and anticancer activity against HeP-G2, MCF-7 and HCT116 cell lines. Collagen-NPs assessed as an effective drug loading carrier with methotrexate (MTX), a chemotherapeutic agent. The TEM analysis revealed that the average size of MTX-loaded collagen-NPs was 35.4 ± 8.9 nm. The percentages of drug loading (DL%) and encapsulation efficiency (EE%) were respectively 22.67 and 45.81%.

Green synthesis of collagen nanoparticles by Streptomyces xinghaiensis NEAA-1, statistical optimization, characterization, and evaluation of their anticancer potential.

Collagen nanoparticles (collagen-NPs) are promising biopolymeric nanoparticles due to their superior biodegradability and biocompatibility. The low immunogenicity and non-toxicity of collagen-NPs makes it preferable for a wide range of applications. A total of eight morphologically distinct actinomycetes strains were newly isolated from various soil samples in Egypt. The cell-free supernatants of these strains were tested for their ability. These strains' cell-free supernatants were tested for their ability to synthesize collagen-NPs. Five isolates had the ability to biosynthesize collagen-NPs. Among these, a potential culture, Streptomyces sp. NEAA-1, was chosen and identified as Streptomyces xinghaiensis NEAA-1 based on 16S rRNA sequence analysis as well as morphological, cultural and physiological properties. The sequence data has been deposited at the GenBank database under the accession No. OQ652077.1. Face-centered central composite design (FCCD) has been conducted to maximize collagen-NPs biosynthesis. Maximum collagen-NPs was 8.92 mg/mL under the condition of 10 mg/mL of collagen concentration, initial pH 7, incubation time of 48 h and temperature of 35 °C. The yield of collagen-NPs obtained via FCCD optimization (8.92 mg/mL) was 3.32-fold compared to the yield obtained under non-optimized conditions (2.5 mg/mL). TEM analysis of collagen-NPs showed hollow sphere nanoscale particles with mean of 32.63 ± 14.59 nm in diameter. FTIR spectra showed major peaks of amide I, amide II and amide III of collagen and also the cell-free supernatant involved in effective capping of collagen-NPs. The biosynthesized collagen-NPs exhibited anti-hemolytic, antioxidant and cytotoxic activities. The inhibitory concentrations (IC50) against MCF-7, HeP-G2 and HCT116 cell lines were 11.62 ± 0.8, 19.60 ± 1.2 and 41.67 ± 2.2 µg/mL; respectively. The in-vivo investigation showed that collagen-NPs can suppress Ehrlich ascites carcinoma (EAC) growth in mice. The collagen-NPs/DOX combination treatment showed considerable tumor growth suppression (95.58%). Collagen-NPs evaluated as nanocarrier with a chemotherapeutic agent, methotrexate (MTX). The average size of MTX loaded collagen-NPs was 42.73 ± 3.5 nm. Encapsulation efficiency percentage (EE %) was 48.91% and drug loading percentage (DL %) was 24.45%.

Optimization of Carotenoids Production from Rhodotorula sp. Strain ATL72 for Enhancing Its Biotechnological Applications.

Rhodotorula yeasts which are known as carotenogenic yeasts have a great industrial value due to their ability to produce carotenoids. In particular, the isolated yeast Rhodotorula sp. (strain ATL72) has been reported to be a promising producer of high concentrations of carotenoids. A combination of central composite design (CCD) and Plackett-Burman (PB) design was used to optimize carotenoids produced by this yeast. The optimum production of carotenoids was completed when the yeast was grown in a production medium composed of 3.7 g/L malt extract, 7.7 g/L fructose, 9 g/L urea, 35 g/L NaCl, and 1 g/L yeast extract at 27.5 °C, pH 6.7, and 180 rpm. Two batch runs in 1 L and 7 L bioreactors were conducted which increased the productivity of carotenoid concentration from 21.5 mg/L after 98 h of incubation at the level of the shake flask to 229.9 mg/L after 47 h of incubation at the level of 7 L bioreactor. The carotenoid pigment was extracted in dimethylsulfoxide (DMSO), acetone, petroleum ether, and sodium chloride, and subsequently identified and characterized using UV-visible scanning, thin layer chromatography, and gas chromatography/mass spectrometry.

Extracellular cholesterol oxidase production by Streptomyces aegyptia, in vitro anticancer activities against rhabdomyosarcoma, breast cancer cell-lines and in vivo apoptosis.

In recent years, microbial cholesterol oxidases have gained great attention due to its widespread use in medical applications for serum cholesterol determination. Streptomyces aegyptia strain NEAE-102 exhibited high level of extracellular cholesterol oxidase production using a minimum medium containing cholesterol as the sole source of carbon. Fifteen variables were screened using Plackett-Burman design for the enhanced cholesterol oxidase production. The most significant variables affecting enzyme production were further optimized by using the face-centered central composite design. The statistical optimization resulted in an overall 4.97-fold increase (15.631 UmL-1) in cholesterol oxidase production in the optimized medium as compared with the unoptimized medium before applying Plackett Burman design (3.1 UmL-1). The purified cholesterol oxidase was evaluated for its in vitro anticancer activities against five human cancer cell lines. The selectivity index values on rhabdomyosarcoma and breast cancer cell lines were 3.26 and 2.56; respectively. The in vivo anticancer activity of cholesterol oxidase was evaluated against Ehrlich solid tumor model. Compared with control mice, tumors growth was significantly inhibited in the mice injected with cholesterol oxidase alone, doxorubicin alone and cholesterol oxidase/doxorubicin combination by 60.97%, 72.99% and 97.04%; respectively. These results demonstrated that cholesterol oxidase can be used as a promising natural anticancer drug.

Purification, characterization and amino acid content of cholesterol oxidase produced by Streptomyces aegyptia NEAE 102.

BACKGROUND: There is an increasing demand on cholesterol oxidase for its various industrial and clinical applications. The current research was focused on extracellular cholesterol oxidase production under submerged fermentation by a local isolate previously identified as Streptomyces aegyptia NEAE 102. The crude enzyme extract was purified by two purification steps, protein precipitation using ammonium sulfate followed by ion exchange chromatography using DEAE Sepharose CL-6B. The kinetic parameters of purified cholesterol oxidase from Streptomyces aegyptia NEAE 102 were studied. RESULTS: The best conditions for maximum cholesterol oxidase activity were found to be 105 min of incubation time, an initial pH of 7 and temperature of 37 °C. The optimum substrate concentration was found to be 0.4 mM. The higher thermal stability behavior of cholesterol oxidase was at 50 °C. Around 63.86% of the initial activity was retained by the enzyme after 20 min of incubation at 50 °C. The apparent molecular weight of the purified enzyme as sized by sodium dodecyl sulphate-polyacryalamide gel electrophoresis was approximately 46 KDa. On DEAE Sepharose CL-6B column cholesterol oxidase was purified to homogeneity with final specific activity of 16.08 U/mg protein and 3.14-fold enhancement. The amino acid analysis of the purified enzyme produced by Streptomyces aegyptia NEAE 102 illustrated that, cholesterol oxidase is composed of 361 residues with glutamic acid as the most represented amino acid with concentration of 11.49 µg/mL. CONCLUSIONS: Taking into account the extracellular production, wide pH tolerance, thermal stability and shelf life, cholesterol oxidase produced by Streptomyces aegyptia NEAE 102 suggested that the enzyme could be industrially useful.

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

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 °Ð¡, while being 179.53 min at 60 °Ð¡. 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.