A scalable overexpression of a thermostable recombinant poly-histidine tag carboxyl esterase under lambda promoter: purification, characterization, and protein modelling.

BACKGROUND: As a white biotechnological trend, esterases are thought to be among the most active enzymes' classes in biocatalysis and synthesis of industrially importance organic compounds. Esterases are used in many applications such as the manufacture of pharmaceuticals, cosmetics, leather, textile, paper, food, dairy products, detergents, and treatment of some environmental pollutants. RESULTS: A poly-histidine moiety was added to the C-terminal end of the Geobacillus sp. gene encoding carboxyl esterase (EstB, ac: KJ735452) to facilitate one-step purification. This recombinant protein was successfully expressed in Escherichia coli (E. coli) under control of Lambda promoter (λ). An open reading frame (ORF) of 1500 bps encoding a polypeptide of 499 amino acid residues and a calculated molecular weight (54.7 kD) was identified as carboxyl-esterase B due to its conserved glycine-X-serine-X-glycine motif (G-X-S-X-G) and its high similarity toward other carboxyl esterases, where the 3-D tertiary structure of EstB was determined based on high homology % (94.8) to Est55. The expression was scaled up using 7-L stirred tank bioreactor, where a maximum yield of enzyme was obtained after 3.5 h with SEA 51.76 U/mg protein. The expressed protein was purified until unity using immobilized metal affinity chromatography (IMAC) charged with cobalt and then characterized. The purified enzyme was most active at pH 8.0 and remarkably stable at pH (8-10). Temperature optimum was recorded at 65 °C, and it kept 70% of its activity after 1-h exposure to 60 °C. The active half-live of enzyme was 25 min at 70 °C and a calculated T melting (Tm) at 70 °C. The determined reaction kinetics Michaelis-Menten constant (Km), maximum velocity rate (Vmax), the turnover number (Kcat), and catalytic efficiency (Kcat/Km) of the pure enzyme were found 22.756 mM, 164.47 U/ml (59.6 min-1), and (2.619 mol/ min), respectively. CONCLUSION: Creation of a recombinant 6 × -His estB derived from a thermophile Geobacillus sp. was performed successfully and then overexpressed under λ-promoter. In a bench scale bioreactor, the overexpression was grown up, followed by one-step purification and biochemical characterization. The recorded promising pH and temperature stability properties suggest that this expressed carboxyl esterase could be used in many industrial sectors.

The dual activity of CaONPs as a cancer treatment substance and at the same time resistance to harmful microbes.

Nanotechnology holds significant promise for the development of novel and necessary products that enhance human health. Pharmacology and nanotechnology have contributed to developing advanced and highly effective drugs for cancer treatment and combating microbial infections. The microbiological effectiveness against the variety of examined microorganisms was assessed using the time killer curve, scanning electron microscopy (SEM), MIC techniques, and the agar well diffusion method. SEM was utilized to enhance the analysis of the mechanisms underlying the bio-interface interaction and intracellular localization of calcium oxide nanoparticles (CaONPs). The MTT test was used to examine the cytotoxicity of CaONP anticancer activity in various cancer cells, including colon, breast, and hepatic cells. The efficacy of CaONPs as an anticancer medication was elucidated by analyzing the gene expression of both treated and untreated cancer cells. MIC and MBC of CaONPs against Escherichia coli and Staphylococcus epidermidis were 150, 150, 150, and 200 µg/ml, respectively. The MIC and MFC of CaONPs against Candida albicans were 200 µg/ml and 250 µg/ml, respectively. The IC50 values of various CaONPs vary depending on the type of cancer cells. The gene expression analysis of breast cancer cells undergoing treatment revealed the identification of several cancer-controlling genes, namely BAX, BCL2, P53, TERT, KRAS1, KRAS2, and RB1. The study demonstrated the notable antibacterial efficacy of CaONPs, highlighting their potential as cancer therapies.

Nanocomposite fibers based on cellulose acetate loaded with fullerene for cancer therapy: preparation, characterization and in-vitro evaluation.

The current prevalence of cancerous diseases necessitates the exploration of materials that can effectively treat these conditions while minimizing the occurrence of adverse side effects. This study aims to identify materials with the potential to inhibit the metastasis of cancerous diseases within the human body while concurrently serving as therapeutic agents for their treatment. A novel approach was employed to enhance the anti-cancer properties of electrospun cellulose fibers by incorporating fullerene nanoparticles (NPs) into cellulose acetate (CA) fibers, resulting in a composite material called Fullerene@CA. This development aimed at utilizing the anti-cancer properties of fullerenes for potential therapeutic applications. This process has been demonstrated in vitro against various types of cancer, and it was found that Fullerene@CA nanocomposite fibers displayed robust anticancer activity. Cancer cells (Caco-2, MDA-MB 231, and HepG-2 cells) were inhibited by 0.3 and 0.5 mg.g-1 fullerene doses by 58.62-62.87%, 47.86-56.43%, and 48.60-57.73%, respectively. The tested cancer cells shrink and lose their spindle shape due to morphological changes. The investigation of the prepared nanocomposite reveals its impact on various genes, such as BCL2, NF-KB, p53, Bax, and p21, highlighting the therapeutic compounds' effectiveness. The experimental results demonstrated that the incorporation of NPs into CA fibers resulted in a significant improvement in their anti-cancer efficacy. Therefore, it is suggested that these modified fibers could be utilized as a novel therapeutic approach for the treatment and prevention of cancer metastasis.

Characterization of an isolated lactase enzyme produced by Bacillus licheniformis ALSZ2 as a potential pharmaceutical supplement for lactose intolerance.

Introduction: Lactose intolerance is a widespread problem that affects people of many different races all over the world. The following pharmacological supplements can improve the lives of those who suffer from this issue. Methods: This work focused on lactase producer isolation and statistical design (Plackett-Burman, and BOX-Behnken) to maximize the effectiveness of environmental factors. A lactase-producing bacterium was chosen from a discovery of 100 strains in soil that had previously been polluted with dairy products. Plackett-Burman investigated fifteen variables. Results: The most critical variables that lead to increased lactase synthesis are glucose, peptone, and magnesium sulfate (MgSO4). The ideal process conditions for the creation of lactase yield among the stated variables were then determined using a BOX-Benken design. To establish a polynomial quadratic relationship between the three variables and lactase activity, the Box-Behnken design level was used. The EXCEL-solver nonlinear optimization technique was used to predict the best form for lactase production. The ideal temperature and pH levels have been determined, both before and after the lactase purification process, to achieve the highest performance of isolated lactase. Conclusion: According to this study, Bacillus licheniformis is a perfect supply of the lactase enzyme (ß -Galactosidase), It can be used as a product to assist people who have health issues due to lactose intolerance.

The role of Algerian Ephedra alata ethanolic extract in inhibiting the growth of breast cancer cells by inducing apoptosis in a p53- dependent pathway.

Background: Ephedra alata, a member of the Ephedraceae family, was used to treat different diseases and it might be shown a strong efficacy to inhibit cancer cell lines. Methods: Due to the limited research available about this plant, the objective of this research was to evaluate the antioxidant, cytotoxic and apoptotic effects of Ephedra alata ethanolic extract (EAEE), against different human cancer cell lines. Results: EAEE inhibited the growth of the liver (HepG2), breast (MCF-7), and colon cancer cells (Caco-2). MCF-7 cells with an IC50 of 153 µg/ml, were the most sensitive to the extract. Furthermore, exploration using flow cytometry using Annexin V-FITC/PI assay demonstrated that EAEE caused death for all human cancer cells mainly through apoptosis. Very interestingly, qRT-PCR analysis using the ΔΔCt method revealed that four genes, Bax, p21, RB1, and TP53 were up-regulated in MCF-7 cells treated either with EAEE or S-FU drug. These findings let us believe that the mechanism by which EAEE kills breast cancer cells seems to be apoptosis via a P53-dependent manner, which involved intrinsic pathways through the induction of Bax, p21, and RB1. Conclusions: EAEE exhibits good biological properties in contradiction of HepG-2, MCF-7, and Caco-2 cell lines. This study appoints for the first time that EAEE increases the expression in MCF-7 cells of p53 and three more genetic traits that control cellular proliferation and apoptosis. Therefore, this plant could serve as a potential source to find new pro-apoptotic drugs for cancer treatment.

Bioprocess development for extraction and purification of cellulases from Aspergillus niger 3ASZ using statistical experimental design techniques.

The amount of cellulosic materials is large and may lead to environmental pollution, so they can be converted into useful materials for use in food or energy. Statistical design (Plackett-Burman and Box-Behnken) was the main topic of this study and was used to optimize the effect of environmental factors on cellulase production by Aspergillus niger. Cellulase production using Plackett-Burman was 6.86-fold higher than the production of cellulase using the basal medium. B0X-Benken showed an increase in the cellulase production equal to 18 times compared to the basal medium, where the cellulase produced had an activity equal to 79.4 U/mL/min. Ammonium sulfate precipitation was applied to the crude enzyme, followed by sequential fractionation with an Amicon system. The Amicon was used to demonstrate the final volume, total enzyme activity, specific activity, purification fold, and yield of cellulase (partially purified enzyme). Numerous cellulolytic enzymes are abundant in Aspergillus species. All of the data showed that Aspergillus sp. might be a reliable source of industrially and economically useful cellulases. By statistically calculating the relevance of a large number of elements in one experiment using a multifactorial statistical design, time may be saved while still maintaining the validity of each component.

Evaluation of a new antimicrobial agent production (RSMM C3) by using metagenomics approaches from Egyptian marine biota.

Diseases and epidemics in the current days need new types of antibiotics in order to be able to eliminate them. The goal of this research is to use metagenomics to identify isolated utilitarian gene (s) as antimicrobial specialists. Collection of diverse locations from sea sediment samples from Alexandria and extraction of total DNA, restriction enzyme fragmentation, cloning into pUC19 vector, and expression of the isolated gene(s) in E. coli DH5α were all part of the process. Characterization of Antimicrobial agent was done for the best clone for antimicrobial agent's production to detect efficiency, optimum pH, thermal stability, pH stability, effect of different compounds on antimicrobial activity, and residual activity of product after preservation in room temperature. Amino acid sequence of RSMM C3 gene (1250 bp) was 72% identity with Herbaspirillum sp. The ideal temperature level of the RSMM C3 antimicrobial agent production was 36 °C. The antimicrobial agent RSMM C3's stability was stable at -20 °Celsius for up to two months without thawing. The antibacterial agent RSMM C3 was stable at 4 °C for 14 days without loss in activity. The ideal pH level of the RSMM C3 antimicrobial agent was 6. Remain activity was gradually decreased at pH 5, 6, 6.5 and 7 (86.1, 96.9, 97.2 and 94.9%, respectively). On the other hand, residual activity was (92 and 84%) at (pH 7.5 and 8) for 8 days. The tested antimicrobial RSMM C3 was stable against 1 mM of different compounds (DMSO, Glycerol, NaCl, CaCl2, MgCl2, ZnCl2, FeSO4, MnSO4 and CuSO4). The research provides for the Metagenomics technique that has the ability for the production of novel antimicrobial agents produced by clone RSMM C3 which has a wide spectrum activity towards different microorganisms comparing to other antibiotics as Ampicillin and Tetracycline.

Sustainable Microbial and Heavy Metal Reduction in Water Purification Systems Based on PVA/IC Nanofiber Membrane Doped with PANI/GO.

Effective and efficient removal of both heavy metal pollutants and bacterial contamination from fresh water is an open issue, especially in developing countries. In this work, a novel eco-friendly functional composite for water treatment application was investigated. The composite consisted of electrospun nanofiber membrane from blended polyvinyl alcohol (PVA)/iota carrageenan (IC) polymers doped with equal concentrations of graphene oxide (GO) nanoparticles and polyaniline (PANI). The effectiveness of this composite as a water purification fixed-bed filter was optimized in a batch system for the removal of cadmium (Cd+2) and lead (Pb+2) ions, and additionally characterized for its antimicrobial and antifungal properties and cytotoxicity effect. The fiber nanocomposite exhibited efficient antibacterial activity, with maximum adsorption capacity of about 459 mg g-1 after 120 min for Cd+2 and of about 486 mg g-1 after 90 min for Pb+2. The optimized conditions for removal of both metals were assessed by using a response surface methodology model. The resulting scores at 25 °C were 91.4% (Cd+2) removal at 117 min contact time for 89.5 mg L-1 of initial concentration and 29.6 cm2 membrane area, and 97.19% (Pb+2) removal at contact time 105 min for 83.2 mg L-1 of initial concentration and 30.9 cm2 nanofiber composite membrane. Adsorption kinetics and isotherm followed a pseudo-second-order model and Langmuir and Freundlich isotherm model, respectively. The prepared membrane appears to be promising for possible use in domestic water purification systems.

Cloning of cellulase gene using metagenomic approach of soils collected from Wadi El Natrun, an extremophilic desert valley in Egypt.

BACKGROUND: Wadi El Natrun microorganisms have been considered as a new resource for natural products due to its extreme condition of salinity and alkalinity. Therefore, this study was devoted to generate metagemic library from soils collected from such an extreme environment in order to clone a novel cellulase for physique industrial applications. RESULTS: Total soil-DNA was successfully extracted, and then digested by different restriction enzymes. Purified fragments ranged ~ 200-6500 bp were ligated and were cloned into plasmid cloning vector (pUC19) by using Escherichia coli DH5α (E. coli) host cells. A constructed metagenomic library composed of 270 clones was screened on carboxymethylcellulose (CMC) agar plate where the active clones had been characterized by the formation of the yellowish halo zone. Thereafter, clone 1 was selected as the most active as being based on cellulase activity quantification (19 µ/ml). Plasmid related to clone 1 encoded cellSNSY gene of approximately 1.5 kb was subjected to molecular characterization; the obtained partial sequence of 861 bps encoded 287 amino acids showing 76% similarity to the endoglucanase gene of Bacillus amyloliquefaciens. The recombinant cellSNSY was expressed under lacz promoter at 1 mM of isopropyl ß-d-1-thiogalactopyranoside (IPTG), giving 21 µ/ml cellulase after ~ 27 h. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and an activity staining of the recombinant cellSNSY which revealed an active band with a molecular mass ~ 59 kDa appeared in the induced sample. The maximum enzyme activity of crude cellSNSY was observed at 45 °C and for a pH of 8.5. Interestingly, the enzyme activity was slightly inhibited by ethylenediamine tetraacetic acid (EDTA) and methanol. It showed high resistance to the tested heavy metals and the surfactant which ordered Zn> (SDS,Fe)>Mn>Cu. CONCLUSIONS: This study established an easy and a skillful way to clone/express a new found cellulase gene(s) under lacZ promoter. The isolated recombinant cellSNSY showed 76% similarity to endoglucanase gene, and the enzyme showed tolerance to the mostly tested agents including heavy metals, surfactant, solvents, and EDTA. Additionally, the studied recombinant showed a high stability up to 55 °C and for alkaline pH 8.5. These features make it an ample and viable for many applications.

Novel scaffold based graphene oxide doped electrospun iota carrageenan/polyvinyl alcohol for wound healing and pathogen reduction: in-vitro and in-vivo study.

Wound healing is a complicated multicellular process that involves several kinds of cells including macrophages, fibroblasts, endothelial cells, keratinocytes and platelets that are leading to their differentiation towards an anti-inflammatory response for producing several chemokines, cytokine and growth factors. In this study, electrospun nanofiber scaffold named (MNS) is composed of polyvinyl alcohol (PVA)/iota carrageenan (IC) and doped with partially reduced graphene oxide (prGO) that is successfully synthesized for wound healing and skin repair. The fabricated MNS was tested in case of infection and un-infection with E. coli and Staphylococcus and in both of the presence and in the absence of yeast as a natural nutritional supplement. Numerous biochemical parameters including total protein, albumin, urea and LDH, and hematological parameters were evaluated. Results revealed that the MNS was proved to be effective on most of the measured parameters and had exhibited efficient antibacterial inhibition activity. Whereas it can be used as an effective antimicrobial agent in wound healing, however, histopathological findings confirmed that the MNS caused re-epithelialization and the presence of yeast induced hair follicles growth and subsequently it may be used to hide formed head wound scar.

Morphological Formation, Fatty Acid Profile, and Molecular Identification of Some Landraces of Ethiopian Brassica as a Promising Crop to Support Breeding Programs.

There has been an increased interest in oilseed crops for agro-industry research and development breeding programs to secure sustainable food and agriculture. The introgression of exotic genotypes of oilseed Brassica into cultivated relatives is inevitable in the genetic improvement of oilseed crops. This experimental attempt aimed to characterize the morphological and molecular basis for the identification and characterization of some Brassica genotypes. Fatty acid profile, yield, and morphology are under genetic control and can be used to identify genotypes. Characterization and identification were fulfilled for five accessions from Brassica spp. Plant height, height of first branch, number of branches and pods per plant, seed yield per plant, average pod length, number of seeds per pod, protein and oil contents (%), and fatty acid profile were examined. Besides, the relationship between seed yield and seed yield-contributing characteristics was estimated, as well as the phylogenetic relationship of the internal transcribed spacer (ITS). The genotypes varied significantly for all examined traits, taking into account the most important traits: seed yield per plant and oil content. For example, oil content in the samples ranged between 41.1 and 49.3%. Path analysis results showed a high and positive direct effect between each number of primary branches and the number of pods per plant with seed yield per plant (0.48). The morphological and molecular observations suggest that the Fay1, Fay3, Fay4, and Fay6 accessions belong to Brassica rapa, while Fay2 belongs to Brassica carinata. It can be concluded based on the present findings that the Fay3 genotype with the highest oil content and the lowest erucic acid content compared to the other genotypes can be proposed as a potential donor for future breeding programs for oil production and quality, while Fay1 can be utilized as donor to increase the seed yield per plant.

Stress-Based Production, and Characterization of Glutathione Peroxidase and Glutathione S-Transferase Enzymes From Lactobacillus plantarum.

More attention has been recently directed toward glutathione peroxidase and s-transferase enzymes because of the great importance they hold with respect to their applications in the pharmaceutical field. This work was conducted to optimize the production and characterize glutathione peroxidase and glutathione s-transferase produced by Lactobacillus plantarum KU720558 using Plackett-Burman and Box-Behnken statistical designs. To assess the impact of the culture conditions on the microbial production of the enzymes, colorimetric methods were used. Following data analysis, the optimum conditions that enhanced the s-transferase yield were the De Man-Rogosa-Sharp (MRS) broth as a basal medium supplemented with 0.1% urea, 0.075% H2O2, 0.5% 1-butanol, 0.0125% amino acids, and 0.05% SDS at pH 6.0 and anaerobically incubated for 24 h at 40°C. The optimum s-transferase specific activity was 1789.5 U/mg of protein, which was ~12 times the activity of the basal medium. For peroxidase, the best medium composition was 0.17% urea, 0.025% bile salt, 7.5% Na Cl, 0.05% H2O2, 0.05% SDS, and 2% ethanol added to the MRS broth at pH 6.0 and anaerobically incubated for 24 h at 40°C. Furthermore, the optimum peroxidase specific activity was 612.5 U/mg of protein, indicating that its activity was 22 times higher than the activity recorded in the basal medium. After SDS-PAGE analysis, GST and GPx showed a single protein band of 25 and 18 kDa, respectively. They were able to retain their activities at an optimal temperature of 40°C for an hour and pH range 4-7. The 3D model of both enzymes was constructed showing helical structures, sheet and loops. Protein cavities were also detected to define druggable sites. GST model had two large pockets; 185Å3 and 71 Å3 with druggability score 0.5-0.8. For GPx, the pockets were relatively smaller, 71 Å3 and 32 Å3 with druggability score (0.65-0.66). Therefore, the present study showed that the consortium components as well as the stress-based conditions used could express both enzymes with enhanced productivity, recommending their application based on the obtained results.

Production of Cold-Active Lipase by Free and Immobilized Marine Bacillus cereus HSS: Application in Wastewater Treatment.

Lipases are enzymes that have the potential to hydrolyze triacylglycerol to free fatty acids and glycerol and have various applications. The aim of the present study was to isolate and screen marine bacteria for lipase production, optimize the production, and treat wastewater. A total of 20 marine bacterial isolates were obtained from the Mediterranean Sea and were screened for lipase production. All isolates were found to have lipolytic ability. The differences between the isolates were studied using RAPD-PCR. The most promising lipase producer (isolate 3) that exhibited the highest lipolytic hydrolysis (20 mm) was identified as Bacillus cereus HSS using 16S rDNA analysis and had the accession number MF581790. Optimization of lipase production was carried out using the Plackett-Burman experimental design with cotton seed oil as the inducer under shaking conditions at 10°C. The most significant factors that affected lipase production were FeSO4, KCl, and oil concentrations. By using the optimized culture conditions, the lipase activity increased by 1.8-fold compared with basal conditions. Immobilization by adsorption of cells on sponge and recycling raised lipase activity by 2.8-fold compared with free cells. The repeated reuse of the immobilized B. cereus HSS maintained reasonable lipase activity. A trial for the economic treatment of oily wastewater was carried out. Removal efficiencies of biological oxygen demand, total suspended solids, and oil and grease were 87.63, 90, and 94.7%, respectively, which is promising for future applications.

Optimization of Reduced Glutathione Production by a Lactobacillus plantarum Isolate Using Plackett-Burman and Box-Behnken Designs.

In this work, we aim to optimize the production of reduced glutathione (GSH) synthesized intracellularly by a food-grade microorganism through a statistical approach. Using a colorimetric method, 25 Lactobacillus plantarum isolates were screened in an attempt to find a GSH-producing strain. It was found that 36% of the tested isolates showed positive result. Isolate (L7) was found to produce 152.61 µM glutathione per gram which was the highest amount produced intracellularly. Accordingly, the later isolate was selected for the optimization process using Plackett-Burman and Box-Behnken designs. Temperature, amino acids, and urea were found to be the most significant independent variables. Following data analysis, the composition of the optimized medium was De Man-Sharp-Rogosa broth as a basal medium supplemented with NaCl (5%), H2O2 (0.05%), sodium dodecyl sulfate (0.05%), amino acids (0.0281%), and urea (0.192%). The pH of the medium was adjusted to 8 and incubated for 24 h at 40°C. The GSH amount was increased by 10-fold (851%) using the optimized medium. Hence, our optimization design estimated the biotechnological potential of L. plantarum (L7) for the production of GSH in the industry.