Carbohydrate microcapsules tailored and grafted for covalent immobilization of glucose isomerase for pharmaceutical and food industries.

Carrageenan is one of the most common carbohydrates utilised in the entrapment industry to immobilise cells and enzymes. However, it lacks functionality. Carrageenan has been grafted to produce fructose by covalently immobilising glucose isomerase (GI). Fructose is one of the most widely used sweeteners in beverages, food production, and the pharmaceutical business. Up to 91.1 U g-1 gel beads are immobilised by the grafted beads. Immobilized GI has a Vmax of 13.8 times that of the free enzyme. pH of immobilized GI was improved from 6.5-7 to 6-7.5 that means more stability in wide pH range. Also, optimum temperature was improved and become 65-75 °C while it was at 70 °C for free enzyme. The immovability and tolerance of the gel beads immobilised with GI over 15 consecutive cycles were demonstrated in a reusability test, with 88 percent of the enzyme's original activity retained, compared to 60 percent by other authors. These findings are encouraging for high-fructose corn syrup producers.

High expression level of antioxidants and pharmaceutical bioactivities of endophytic fungus Chaetomium globosum JN711454.

In order to maximize antioxidant activity of pharmaceutical bioactive endophytic fungus Chaetomium globosum JN711454 during fermentation process, designed fermentation experiments of culture media for three levels of eight culture factors were performed using a Taguchi orthogonal array (OA) design with layout L18 (2(1) × 3(7)). The agitation and the potato extract were the most significant affecting factors, and their interaction contributed significantly to fungus activity. The production of antioxidants was more favorable for static condition with 25 g potato extract/100 m. The remaining factors had no strong impact when considered individually. The validation of statistically optimized medium indicated the improvement of antioxidant activity to a level of twofold with approximately overall 40% enhancement in activity. The extract of optimized medium was investigated for various pharmaceutical bioactivities; it revealed a moderate antimicrobial activity, strong anticancer activity against HepG-2, UACC62 cell lines, an antiviral activity against HSV-2 virus, and strong inhibitory activity to butyrylcholinesterase enzyme, one of the neurohydrolase enzymes that play a major role in development of Alzheimer's disease. As a result of applying statistical fermentation designs, the optimized conditions of endophytic fungus C. globosum JN711454 developed a cost-effective production medium by using inexpensive commercial potato extracts statically, which can lower the energy requirement and could become an efficient, economic, and viable fermentation process for production of pharmaceutical secondary metabolites.

Isolation of Enterococcus faecium NM113, Enterococcus faecium NM213 and Lactobacillus casei NM512 as novel probiotics with immunomodulatory properties.

Probiotics, defined as living bacteria that are beneficial for human health, mainly function through their immunomodulatory abilities. Hence, these microorganisms have proven successful for treating diseases resulting from immune deregulation. The aim of this study was to find novel candidates to improve on and complement current probiotic treatment strategies. Of 60 lactic acid bacterial strains that were isolated from fecal samples of healthy, full-term, breast-fed infants, three were chosen because of their ability to activate human immune cells. These candidates were then tested with regard to immunomodulatory properties, antimicrobial effects on pathogens, required pharmacological properties and their safety profiles. To identify the immunomodulatory structures of the selected isolates, activation of specific innate immune receptors was studied. The three candidates for probiotic treatment were assigned Enterococcus faecium NM113, Enterococcus faecium NM213 and Lactobacillus casei NM512. Compared with the established allergy-protective strain Lactococcus lactis G121, these isolates induced release of similar amounts of IL-12, a potent inducer of T helper 1 cells. In addition, all three neonatal isolates had antimicrobial activity against pathogens. Analysis of pharmacological suitability showed high tolerance of low pH, bile salts and pancreatic enzymes. In terms of safe application in humans, the isolates were sensitive to three antibiotics (chloramphenicol, tetracycline and erythromycin). In addition, the Enterococcus isolates were free from the four major virulence genes (cylA, agg, efaAfs and ccf). Moreover, the isolates strongly activated Toll-like receptor 2, which suggests lipopeptides as their active immunomodulatory structure. Thus, three novel bacterial strains with great potential as probiotic candidates and promising immunomodulatory properties have here been identified and characterized.

Optimal immobilization of ß-galactosidase onto κ-carrageenan gel beads using response surface methodology and its applications.

ß-Galactosidase (ß-gal) was immobilized by covalent binding on novel κ-carrageenan gel beads activated by two-step method; the gel beads were soaked in polyethyleneimine followed by glutaraldehyde. 2(2) full-factorial central composite experiment designs were employed to optimize the conditions for the maximum enzyme loading efficiency. 11.443 U of enzyme/g gel beads was achieved by soaking 40 units of enzyme with the gel beads for eight hours. Immobilization process increased the pH from 4.5 to 5.5 and operational temperature from 50 to 55 °C compared to the free enzyme. The apparent K(m) after immobilization was 61.6 mM compared to 22.9 mM for free enzyme. Maximum velocity Vmax was 131.2 µ mol · min(-1) while it was 177.1 µ mol · min(-1) for free enzyme. The full conversion experiment showed that the immobilized enzyme form is active as that of the free enzyme as both of them reached their maximum 100% relative hydrolysis at 4 h. The reusability test proved the durability of the κ-carrageenan beads loaded with ß -galactosidase for 20 cycles with retention of 60% of the immobilized enzyme activity to be more convenient for industrial uses.

HPLC with fluorescence detection for the bioanalysis and pharmacokinetic study of Doxorubicin and Prodigiosin loaded on eco-friendly casein nanomicelles in rat plasma.

A rapid, efficient, and sensitive liquid chromatographic assay hyphenated to fluorometric detector (HPLC-FLD) was developed and validated for the determination of doxorubicin (DXR) and prodigiosin (PDG) in rat plasma. The sample pre-treatment involves a protein precipitation with acetonitrile with satisfying extraction efficiency (98% and 85% for DXR and PDG, respectively). The chromatographic separation was accomplished using stationary phase: Agilent Zorbax Eclipse plus-C18 analytical column (250 × 4.6 mm, 5 µm) and gradient eluting mobile phase of ammonium acetate (pH = 3), acetonitrile and methanol with programmed fluorescence detection. As the proposed method has been validated, it was subsequently implemented to evaluate DXR and PDG loaded on novel eco-friendly Casein nano drug delivery system after intravenous injection in healthy rats. A comparative pharmacokinetics' study was carried out in rats for DXR in free form, DXR alone entrapped in the nanomicelle and DXR with PDG entrapped in the nano micelle. After testing the differences in pharmacokinetic parameters of the different formulations using ANOVA, the results showed insignificant differences among the tested parameters. This indicates that the presented nanomicelle delivery system has succeeded to incorporate PDG and DXR in a hydrophilic, safe, and potent formulation. This novel nanomicelle has negligible effect on the distribution and elimination of DXR.

Biological activities and variation of symbiotic fungi isolated from Coral reefs collected from Red Sea in Egypt.

Ten specimens of coral reefs were collected from the Red Sea in the Ein El-Sukhna region. Fungal isolation was done using two media, Dextrose Yeast Extract Agar (DYA) and Rose Bengal Agar (RBA). The morphological traits identified 18 fungal isolates belonging to the phyla Ascomycota, Mucoromycota and Deuteromycota. Five genera in three orders have been isolated: Eutrotiales (Aspergillus, Penicillium and Byssochlamys), Mucorales (Rhizopus) and Moniliales (Curvularia). The heat mapping clustering of the isolated fungi declared that Aspergillus and Penicillium were the most frequently isolate fungi in coral reefs. It was found that A. fumigatus colonised eight coral samples with 80% colonisation rate. Moreover, about 50% of the isolated fungal species were specific to one coral reef only such as A.candidus and A.carneus isolated from Isophyllastrea rigida only, A.japonicus and A.ochraceopetaliformis from Glaxaea fascicularis, A.niger van Tieghem from Porites astreoides, A.sydowii, A.terreus and P.waksmanii from Cladocora arbuscula, P.janthinellum from Pterogorgia guadalupensis and Curvularia tuberculata, Byssochlamys spectabilis and Rhizopus oryzae from Acropora humilis. Biological activities (antimicrobial, antioxidant antiradical and cytotoxicity) of the most predominant fungal species were investigated. The antimicrobial activity of coral fungal filtrates were investigated against six pathogenic bacteria including Escherichia coli ATCC11775, Neisseria gonorrhoeae ATCC19424, Pseudomonas aeruginosa ATCC10145, Streptococcus faecalis ATCC19433, Staphylococcus aureus subsp. aureus ATCC25923, Bacillus subtilis subsp. spizizenii ATCC6633 and two pathogenic yeast including Candida albicans ATCC7102 and Candida parapsilosis ATCC22019. Most of these fungal filtrates exhibited moderate to high antibacterial activities against both gram positive and gram negative bacteria, however it showed relatively low bioactivity towards the pathogenic Candida species. Investigating the free radical scavenging activity using DPPH reagent showed low to moderate bioactivities. The highest cytotoxic activity against liver cancer cell line Hep-G2 with an IC50 values of 18.8 µg/ml was exhibited by Aspergillus ochraceopetaliformis MN083316 and a metabolomics study was done on the ethyl acetate extract of this strain using LC-ESI-MS fingerprints leading to the isolation and purification of compound 1. Using 1D and 2D NMR techniques compound 1 was identified as ditryptophenaline. Compound 1 exhibited a strong antimicrobial, antioxidant activities as well as cytotoxic activities against MCF-7 and HEPG2 with IC50 values of 5.8 and 7.6 mmole, respectively. The objective of this study, isolation of Coral-reef associated fungi and studying their biological activities to produce the most active secondary metabolite which might possess a novel biological activity.

Biofiltration technology for the removal of toluene from polluted air using Streptomyces griseus.

Biofiltration technology has been recognized as a promising biotechnology for treating the volatile organic compounds (VOCs) present in polluted air. This study aims to investigate the performance of a biofiltration system of Streptomyces griseus sp. DSM-40759 immobilized on activated carbon (PICA S23) towards the adsorption and degradation of toluene vapour as well as to regenerate the activated carbon in situ. The batch studies were performed using nutrient agar medium and basal salt medium (BSM) for microbial growth. Initially the pre-cultures were incubated at a temperature of 28°C on a rotary shaker at 150 rpm. After two days, the strain S. griseus DSM-40759 was immobilized on a known weight of activated carbon (12 g). The results of biofilter performance showed three different stages with a quick adsorption phase with approximately 95% of toluene removal after 70 min, a slow biotransformation phase by immobilized cells. In the later, the removal efficiency decreased significantly with the extension of time and reached 60% during this stage. Moreover, a final quick removal phase by the immobilized cells had an average removal efficiency of toluene around 95% after 500 min. The toluene degradation was found to be more than 84% after the second cycle and the biofilter was still capable of removing additional toluene. Thus, the results demonstrated the feasibility and reusability of a new biofilter system for toluene removal as well as extending the activated carbon's capacity and this could be a potential solution to reuse the activated carbon in industrial application.

Immobilization of halophilic Aspergillus awamori EM66 exochitinase on grafted k-carrageenan-alginate beads.

A novel extreme halophilic exochitinase enzyme was produced by honey isolate Aspergillus awamori EM66. The enzyme was immobilized successfully on k-carrageenan-alginate gel carrier (CA) with 93 % immobilization yield. The immobilization process significantly improved the enzyme specific activity 2.6-fold compared to the free form. The significant factors influencing the immobilization process such as enzyme protein concentration and loading time were studied. Distinguishable characteristics of optimum pH and temperature, stability at different temperatures and NaCl tolerance for free and immobilized enzyme were studied. The immobilization process improved optimum temperature from 35 to 45 °C. The immobilized enzyme retained 76.70 % of its activity after 2 h at 75 °C compared to complete loss of activity for the free enzyme. The reusability test proved the durability of the CA gel beads for 28 cycles without losing its activity.

Biological evaluation of endophytic fungus, Chaetomium globosum JN711454, as potential candidate for improving drug discovery.

The main objective of this research work focused on investigating the biological and chemical aspects of endophytic fungus Chaetomium globosum, for pharmaceutical purposes to improve the drug discovery process. The endophytic C. globosum was isolated from healthy leaves of Egyptian medicinal plant Adiantum capillus-veneris collected from Saint Katherine Protectorate, Sinai, Egypt. The identification of C. globosum was on the basis of classical and molecular taxonomy. Gene encoding for 18S rRNA was partially sequenced, submitted to the GenBank and got the accession number JN711454, to resolve the phylogenetic relations with fungal ancestor using phylogenetic tree. To explore the biosynthetic power of endophytic C. globosum JN711454, the fungus was cultivated over five different media, oatmeal, rice, yeast malt glucose, potato dextrose agar (PDA) and Czapek's dox media, for 3 weeks at 30 °C, followed by extraction with different solvents, ethyl acetate (EA), and methanol. The ethyl acetate extract of C. globosum cultivated on PDA medium was the most potent extract. It showed strong antioxidant activity with EC50 11.5 µg/ml, potent anticancer activity with 55 % toxicity toward HepG-2 cells at 100 µg/ml and 66 % cytotoxicity to FGC4 cells at 250 µg/ml, promising butyrylcholinesterase inhibitory activities (>85 %), and moderate antimicrobial and stopped the attachment of HSV-2 virus to VERO cells. The metabolomic profiling of PDA-EA extract using LC-MS revealed the presence of several metabolites to which the observed bioactivities could be attributed. Here we report for the first time inhibitory activity of endophytic C. globosum JN711454 secondary metabolites to butyrylcholinesterase, one of neuro hydrolase enzymes that play a major role in development of Alzheimer's disease.

Lipase production from a novel thermophilic Bacillus sp.: application of Plackett-Burman design for evaluating culture conditions affecting enzyme formation.

A novel thermophilic Bacillus sp. capable of producing lipase was locally isolated. Phylogenetic analysis based on 16SrDNA sequence revealed its close relationship to Bacillus thermoleovorans. Plackett-Burman experimental design was used to evaluate cultural conditions affecting lipase production process. Fifteen variables and four dummy variables were examined in the experimental design. Tween 80, temperature, olive oil, aeration, beef extract and inoculum age were found to be the highest positive significant variables affecting lipase activity, whereas pH and calcium chloride were the highest negative significant variables. Moreover, Tween 80, temperature and olive oil positively affected lipase specific activity. On the other hand, gum arabic, inoculum size and calcium chloride had the highest negative effect on lipase specific activity. This study would improve the further optimization steps on the bioprocess development track.

Improvement of rifemycins production by Amycolatopsis mediterranei in batch and fed-batch cultures.

The production of rifamycins B and SV using glucose as main C-source by Amycolatopsis mediterranei in batch and fed-batch culture was investigated. Fed-batch culture using glucose as mono feeding substrate either in the form of pulse addition, in case of shake flask, or with constant feeding rate, in bioreactor level, proved to be an alternative production system with a significant increase in both volumetric and specific antibiotic production. The maximal concentrations of about 1146 mg/l and 2500 mg/l of rifamycins B and SV, respectively, was obtained in fed-batch culture in bioreactor level under non-oxygen limitation. On the other hand, the rate of rifamycins production was increased from 6.58 to 12.13 mg/l x h for rifamycin B and from 9.47 to 31.83 mg/l x h for rifamycin SV on the bioprocess transfer and improvement from the conventional batch cultivation in shake flask to fed-batch cultivation in stirred tank bioreactor.