Mohamed G. Farahat

Halophilic bacteria are promising reservoirs for halotolerant enzymes that have gained much attention in biotechnological applications due to their remarkable activity and stability. In this study, 62 halophilic bacterial strains isolated from a solar saltern were screened for the production of various extracellular enzymes. The results revealed that 31 strains (50%) were positive for amylase production while 26 strains (41.9%) were positive for protease. Further, 22 strains (35.48%) exhibited β-glucosidase activity and only 17 (27.41%) demonstrated lipase activity. Of the investigated halophiles, ten strains growing in the presence of ≥15% NaCl (w/v) were selected and identified based on their 16S rRNA gene sequences as Halomonas meridiana, Salinivibrio costicola, Virgibacillus oceani, Virgibacillus marismortui, Marinobacter lipolyticus, Halobacillus karajensis, Salicola salis, Pseudoalteromonas shioyasakiensis, Salinicoccus amylolyticus, and Paracoccus salipaludis. Therefore, the present study highlights the diversity of the culturable halophilic bacteria in a Mediterranean solar saltern, harboring various valuable halotolerant enzymes.

D-Tagatose is a low-calorie sugar substitute that has gained increased attention as a functional sweetener owing to its nutraceutical and prebiotic properties. Traditionally, D-tagatose is produced via the enzymatic conversion of L-galactose to D-tagatose by L-arabinose isomerase (L-AI). Nonetheless, the most reported L-AI enzymes are ion-dependent enzymes requiring Mn2+ and/or Co2+ as cofactors for their reactions, which limits their application due to safety and health concerns. Herein, we addressed the facile bioconversion of L-galactose to D-tagatose using a novel recombinant metallic-ions-independent L-AI derived from endophytic Bacillus amyloliquefaciens CAAI isolated from cantaloupe fruits. The ORF (1500 bp) of the L-arabinose isomerase gene (araA) was cloned and over-expressed in Escherichia coli. The recombinant enzyme (BAAI) was purified to homogeneity using Ni-NTA affinity chromatography, yielding a single distinct band with an apparent molecular mass of approximately 59 kDa as deduced from SDS-PAGE analysis. The purified enzyme showed optimum activity at pH and temperature of 7.5 and 45 °C, respectively, with obvious enzymatic activity in the presence of ethylenediaminetetraacetic acid (EDTA), indicating the metallic-ions independence from BAAI. The Km values of BAAI for D-galactose and L-arabinose were 251.6 mM and 92.8 mM, respectively. The catalytic efficiency (kcat/Km) values for D-galactose and L-arabinose were found to be 2.34 and 46.85 mM–1 min–1, respectively. The results revealed the production of 47.2 g/L D-tagatose from D-galactose (100 g/L) with 47.2% bioconversion efficiency in a metallic-ions-free reaction system that could be implemented in safe-production of food-grade low-calorie sweetener, D-tagatose.

Gardenia blue (GB) is a natural pigment widely used in textiles and food industries as an eco-friendly alternative to synthetic dyes owing to its safety, biocompatibility and chemical stability. Herein we demonstrated a recyclable, reusable and efficient catalysis system for the biosynthesis of GB from geniposide using β-glucosidase embedded in copper phosphate hybrid nanoflowers. In this study, a promising β-glucosidase-secreting actinomycete was isolated and identified as Streptomyces variabilis BGPA1. The secreted enzyme was successfully immobilized in nanoflowers as evidenced by scanning electron microscopy (SEM) and Fourier-transform infrared spectroscopy (FTIR) analysis. Results revealed the functionality of the prepared nanoflowers for the bioconversion of geniposide into genipin which interacts with glycine yielding the blue pigment. The optimum pH and temperature for the bioconversion were found to be 6.0 and 50∘C, respectively. Interestingly, the prepared β-glucosidase–copper phosphate hybrid nanoflowers retained up to 94% of their initial activity after 15 cycles of repeated usage, indicating the remarkable recyclability and reusability of the biocatalytic system. This study suggests that β-glucosidase–copper phosphate hybrid nanoflowers could be used as a potential candidate for the facile eco-friendly production of GB.

The biosynthesis of nanoparticles using green technology is emerging as a cost-efficient, eco-friendly and risk-free strategy in nanotechnology. Recently, tellurium nanoparticles (TeNPs) have attracted growing attention due to their unique properties in biomedicine, electronics, and other industrial applications. The current investigation addresses the green synthesis of TeNPs using a newly isolated mangrove-associated bacterium, Gayadomonas sp. TNPM15, and their impact on the phytopathogenic fungi Fusarium oxysporum and Alternaria alternata. The biogenic TeNPs were characterized using transmission electron microscopy (TEM), scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), X-ray diffraction (XRD), Raman spectroscopy and Fourier transform infrared (FTIR). The results of TEM revealed the intracellular biosynthesis of rod-shaped nanostructures with a diameter range from 15 to 23 nm and different lengths reaching up to 243 nm. Furthermore, the successful formation of tellurium nanorods was verified by SEM-EDX, and the XRD pattern revealed their crystallinity. In addition, the FTIR spectrum provided evidence for the presence of proteinaceous capping agents. The bioinspired TeNPs exhibited obvious inhibitory effect on the spores of both investigated phytopathogens accomplished with prominent ultrastructure alternations, as evidenced by TEM observations. The biogenic TeNPs impeded spore germination of F. oxysporum and A. alternata completely at 48.1 and 27.6 µg/mL, respectively. Furthermore, an increase in DNA and protein leakage was observed upon exposure of fungal spores to the biogenic TeNPs, indicating the disruption of membrane permeability and integrity. Besides their potent influence on fungal spores, the biogenic TeNPs demonstrated remarkable inhibitory effects on the production of various plant cell wall-degrading enzymes. Moreover, the cytotoxicity investigations revealed the biocompatibility of the as-prepared biogenic TeNPs and their low toxicity against the human skin fibroblast (HSF) cell line. The biogenic TeNPs showed no significant cytotoxic effect towards HSF cells at concentrations up to 80 μg/mL, with a half-maximal inhibitory concentration (IC50) value of 125 μg/mL. The present work spotlights the antifungal potential of the biogenic TeNPs produced by marine bacterium against phytopathogenic fungi as a promising candidate to combat fungal infections.

The current investigation addressed the green synthesis of silver nanoparticles (AgNPs) using newly isolated silver-resistant rare actinomycetes, Glutamicibacter nicotianae SNPRA1 and Leucobacter aridicollis SNPRA2, and investigated their impact on the mycotoxigenic fungi Aspergillus flavus ATCC 11498 and Aspergillus ochraceus ATCC 60532. The formation of AgNPs was evidenced by the reaction’s color change to brownish and the appearance of the characteristic surface plasmon resonance. The transmission electron microscopy of biogenic AgNPs produced by G. nicotianae SNPRA1 and L. aridicollis SNPRA2 (designated Gn-AgNPs and La-AgNPs, respectively) revealed the generation of monodispersed spherical nanoparticles with average sizes of 8.48 ± 1.72 nm and 9.67 ± 2.64 nm, respectively. Furthermore, the XRD patterns reflected their crystallinity and the FTIR spectra demonstrated the presence of proteins as capping agents. Both bioinspired AgNPs exhibited a remarkable inhibitory effect on the conidial germination of the investigated mycotoxigenic fungi. The bioinspired AgNPs caused an increase in DNA and protein leakage, suggesting the disruption of membrane permeability and integrity. Interestingly, the biogenic AgNPs completely inhibited the production of total aflatoxins and ochratoxin A at concentrations less than 8 μg/mL. At the same time, cytotoxicity investigations revealed the low toxicity of the biogenic AgNPs against the human skin fibroblast (HSF) cell line. Both biogenic AgNPs exhibited feasible biocompatibility with HSF cells at concentrations up to 10 μg/mL and their IC50 values were 31.78 and 25.83 μg/mL for Gn-AgNPs and La-AgNPs, respectively. The present work sheds light on the antifungal prospect of the biogenic AgNPs produced by rare actinomycetes against mycotoxigenic fungi as promising candidates to combat mycotoxin formation in food chains at nontoxic doses.

Astaxanthin is a xanthophyll carotenoid possessing impressive nutraceutical, antioxidant, and bioactive merits. Traditionally, astaxanthin is extracted from crustacean wastes via solvent extraction methods. However, the rigid structure of shells that comprise complex proteins and chitin challenges the extraction process. This investigation addressed an efficient microbial-assisted method to facilitate astaxanthin recovery from crab exoskeleton waste utilizing chitinolytic and proteolytic microorganisms. Herein, we evaluated the effect of pretreatment of the exoskeleton waste with a newly isolated probiotic strain, Bacillus amyloliquefaciens CPFD8, showing remarkable protease and chitinase activity and a proteolytic Saccharomyces cerevisiae 006-001 before solvent extraction, using acetone/hexane, on astaxanthin recovery. Furthermore, the antioxidant and anti-inflammatory activities of the recovered astaxanthin were inspected. Results revealed that both strains boosted the astaxanthin yield from the crab (Callinectes sapidus) exoskeleton compared with solvent extraction using acetone/hexane. Under optimum conditions, astaxanthin yield was 217 and 91 µg/g crab exoskeleton in samples treated with B. amyloliquefaciens CPFD8 and S. cerevisiae 006-001, respectively. Interestingly, pretreatment of crab exoskeleton waste with B. amyloliquefaciens CPFD8 yielded more than 6-fold astaxanthin compared with the solvent extraction method that yielded just 35 µg/g. This increase could be attributed to the proteolytic activity of B. amyloliquefaciens CPFD8 that rendered deproteinized shell chitin accessible to chitinase, facilitating the penetration of solvents and the recovery of astaxanthin. The recovered astaxanthin exhibited excellent antioxidant activity in scavenging DPPH or ABTS free radicals with IC50 values of 50.93 and 17.56 µg/mL, respectively. In addition, the recovered astaxanthin showed a remarkable anti-inflammatory impact on LPS-induced murine macrophage RAW264.7 cells and significantly inhibited the production of nitric oxide, TNF-α, and IL-6 compared with the untreated control. These findings suggest the potential use of the developed microbial-assisted method utilizing chitinolytic and proteolytic B. amyloliquefaciens CPFD8 to maximize the recovery of bioactive astaxanthin from crab (C. sapidus) exoskeleton waste.

Natural environments contain a variety of microorganisms, of which, a limited number are able to develop bioremediation process. The main goal of the present paper was to perform microbiological investigations to culturable bacteria isolated from uranium ore samples from Aborshid Egypt, and characterize their response to 15 antibiotics and 10 heavy metals beside uranium. Among the potential environmental isolates, there was no detection of any acquired antibiotic resistance, which supports the idea that their resistance mechanisms are mainly intrinsic. On the contrary, the potentially pathogenic isolates presented a broad diversity of acquired antibiotic resistance toward different antibiotic classes. Furthermore, results revealed a varying response of the ore bacteria to the tested heavy metals. All isolates showed multiple metal resistances toward 10 heavy metals, with MIC ranging from 50 to 1000 ppm. We found that optimum temperature for growth of bacteria in general ranges from 30 to 50 °C and bio-sorption of metal increased with increasing pH from 1.0 to 5.0, then decreased upon further increase to 7.0. The decrease in the sorption efficiency at pH higher than 5.0 may be due to the precipitation as hydroxide. We found possible correlation between antibiotic resistance and heavy-metal resistance patterns of Escherichia coli strains isolated from uranium ore; the most potent of the strains in both groups were resistant to Pb, Ni, Cu and Zn. It was concluded that highly metal-resistant bacteria could be used with potential application for treatment of wastewaters.

Levan is an industrially important, functional biopolymer with considerable applications in the food and pharmaceutical fields owing to its safety and biocompatibility. Here, levan-type exopolysaccharide produced by Pantoea agglomerans ZMR7 was purified by cold ethanol precipitation and characterized using TLC, FTIR, 1H, and 13C NMR spectroscopy. The maximum production of levan (28.4 g/l) was achieved when sucrose and ammonium chloride were used as carbon and nitrogen sources, respectively, at 35°C and an initial pH of 8.0. Some biomedical applications of levan like antitumor, antiparasitic, and antioxidant activities were investigated in vitro. The results revealed the ability of levan at different concentrations to decrease the viability of rhabdomyosarcoma and breast cancer cells compared with untreated cancer cells. Levan appeared also to have high antiparasitic activity against the promastigote of Leishmania tropica. Furthermore, levan had strong DPPH radical scavenging (antioxidant) activity. These findings suggest that levan produced by P. agglomerans ZMR7 can serve as a natural biopolymer candidate for the pharmaceutical and medical fields.

The exploration of new sources of L-asparaginase with low glutaminase activity is of great interest in both medical and food applications. In the current study, a novel L-asparaginase gene (CobAsnase) from halotolerant Cobetia amphilecti AMI6 was cloned and over-expressed in Escherichia coli. The enzyme had a molecular mass of 37 kDa on SDS-PAGE and dynamic light scattering (DLS) analysis revealed that CobAsnase is a homotetramer in solution. The purified enzyme showed optimum activity at pH and temperature of 7 and 60 °C, respectively, with obvious thermal stability. It exhibited strict substrate specificity towards L-asparagine with no detectable activity on L-glutamine. Pre-treatment of potato slices by CobAsnase prior to frying reduced the acrylamide contents in the processed chips up to 81% compared with untreated control. These results suggest that CobAsnase is a potential candidate for applications in the food industry for mitigation of acrylamide formation in fried potato and baked foods.

Salinity is one of the most crucial stressors that restrain crop productivity. Endophytes with 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity possess the potential to ameliorate salt stress in plants by degrading the precursor molecule of ethylene, ACC, whose concentration is elevated in salt-stressed plants. The present investigation reports the efficiency of salt-tolerant endophyte Bacillus aryabhattai EWR29 exhibiting ACC deaminase activity as bioinoculant to improve the growth of the wheat plant under salinity stress conditions. The strain EWR29 manifested multifarious plant growth-promoting traits including, phosphate solubilization, and production of indole acetic acid (IAA), siderophore, HCN, and exopolysaccharides (EPS). Besides, it endures salinity (12%) and, drought stresses (−0.49 MPa) and showed a strong biofilm-formation potential. Moreover, acdS gene coding for ACC deaminase was sequenced and characterized at the molecular level. Inoculation of wheat plants with B. aryabhattai EWR29 alleviated the negative effects of salinity stress and promoted significant increases in growth criteria compared with un-inoculated plants grown at the same salinity levels. The bioinoculant induced up-regulation of the antioxidant enzymes peroxidase (POD) and superoxide dismutase (SOD), and decreased proline content in treated plants indicating the mitigation of salt effect. The results indicated the potential used of B. aryabhattai EWR29 as bioinoculant to enhance the growth of wheat plants cultivated in salt-affected agriculture fields and alleviate salt stress.

D-Tagatose is a promising low-calorie sucrose substitute having prebiotic properties. In this study, biosynthesis of D-tagatose was achieved by constructing and expressing a polycistronic plasmid encoding codon-optimized thermostable β-galactosidase from Marinomonas sp. BSi20414 and L-arabinose isomerase from Clostridium hylemonae, simultaneously, in Lactococcus lactis NZ3900. Both enzymes were expressed as soluble and functional proteins under control of the nisinA inducible promoter and the optimum concentration of nisin was found to be 1 ng/ml. The induced cells were immobilized in chitosan beads and used for D-tagatose production from lactose as a whole-cell biocatalyst. Results revealed an efficient bioconversion of lactose to D-tagatose and maximum production was accomplished at 60°C after 12 h, starting with 20% (w/ v) lactose. The recombinant cells entrapped in chitosan beads converted up to 34% of lactose into D-tagatose under optimum conditions in a single step that could be implemented in safe-production of food-grade low-calorie sweetener.

Edible films containing antimicrobial agents can be used as safe alternatives to preserve food products. Essential oils are well-recognized antimicrobials. However, their low water solubility, volatility and high sensitivity to oxygen and light limit their application in food preservation. These limitations could be overcome by embedding these essential oils in complexed product matrices exploiting the encapsulation efficiency of β-cyclodextrin. This study focused on the maximization of β-cyclodextrin production using cyclodextrin glucanotransferase (CGTase) and the evaluation of its encapsulation efficacy to fabricate edible antimicrobial films. Response surface methodology (RSM) was used to optimize CGTase production by Brevibacillus brevis AMI-2 isolated from mangrove sediments. This enzyme was partially purified using a starch adsorption method and entrapped in calcium alginate. Cyclodextrin produced by the immobilized enzyme was then confirmed using high performance thin layer chromatography, and its encapsulation efficiency was investigated. The clove oil/β-cyclodextrin inclusion complexes were prepared using the coprecipitation method, and incorporated into chitosan films, and subjected to antimicrobial testing. Results revealed that β-cyclodextrin was produced as a major product of the enzymatic reaction. In addition, the incorporation of clove oil/β-cyclodextrin inclusion complexes significantly increased the antimicrobial activity of chitosan films against Staphylococcus aureus, Staphylococcus epidermidis, Salmonella Typhimurium, Escherichia coli, and Candida albicans. In conclusion, B. brevis AMI-2 is a promising source for CGTase to synthesize β-cyclodextrin with considerable encapsulation efficiency. Further, the obtained results suggest that chitosan films containing clove oils encapsulated in β-cyclodextrin could serve as edible antimicrobial food-packaging materials to combat microbial contamination.

BACKGROUND AND STUDY AIMS: Colonized patients with carbapenamase producing Enterobacteriaceae (CPE) are vulnerable to invasive infections from their endogenous flora. We aimed to assess faecal colonization with (CPE) among children admitted to Cairo University paediatric intensive care units (ICUs). The phenotypic and genotypic characterizations of carbapenemase-producing Enterobacteriaceae were also studied. PATIENTS AND METHODS: A total of 413 Enterobacteriaceae isolates have been isolated from cultured rectal swabs of 100 children. All swabs were inoculated on ChromID™ CARBA agar to screen for carbapenem resistant Enterobacteriaceae (CRE). Disk diffusion method, Modified Hodge test (MHT) and further genotypic detection of carbapenemases genes (bla(OXA-48), bla(KPC) and bla(NDM-1), bla(VIM) and bla(IMP)) by multiplex PCR were done. RESULTS: Out of 413 Enterobacteriaceae isolates; 100 isolates were defined as CRE. Bla(OXA-48) was detected in (33%); Escherichia coli (n = 11), Klebsiella oxytoca (n = 3) and Klebsiella pneumoniae (n = 19), while (27%) carried bla(NDM-1)Escherichia coli (n = 7), and Klebsiella pneumoniae (n = 20). CONCLUSION: Prevalence of carbapenem resistant Enterobacteriaceae was 24%, various genes of carbapenemases were detected in 80% of carbapenem resistant Enterobacteriaceae with dominance of bla(OXA-48). Understanding the colonization status of our patients with strict infection control measures can reduce the risk of horizontal gene transfer of carbapenemases.

The neoagarooligosaccharides have received growing attention owing to their physiological activities. The aim of this study was the isolation of agarase-producing bacteria for production of agar hydrolysates with special emphasis on their anti-oxidant potential. An agarolytic strain NI125 was isolated from Nelson's Island, Alexandria, Egypt. Based on 16S rRNA analysis and extensive phenotypic characterization, it was identified as Aquimarina agarilytica. Maximum enzyme production was achieved after 24 h incubation at 20°C, and tryptone was recorded to be the best nitrogen source for agarase production. Extracellular agarase was partially purified by ammonium sulfate precipitation. The substrate specificity assay using p-nitrophenyl-α/β-D-galactopyranoside revealed the cleavage of the β-linkage rather than the α-linkage. Neoagarooligosaccharides produced by the partially purified β-agarase expressed promising anti-oxidant properties, with 23% free radical scavenging potential. Notable enhancement of the anti-oxidant potency of the oligosaccharides was achieved (up to 87% scavenging ability) by sulfation of the agar prior to hydrolysis for 12 h with β-agarase. Results obtained suggest the potential application of the produced neoagarooligosaccharides anti-oxidants as promising additives in food and feed products.

β-galactosidase (E.C.3.2.1.23) has been widely used in dairy and pharmaceutical industries. In this study, Bacillus megaterium NM56 showing maximum production of β-galactosidase was isolated from raw milk. Effects of various culture conditions, namely, incubation period, temperature, pH, carbon source and nitrogen source were investigated. Results revealed that the highest level of β-galactosidase was produced after 48 h of incubation. Optimal temperature and pH for production was observed at 40°C and 7.5, respectively. Galactose, lactose, tryptone and yeast extract were the best carbon and nitrogen sources for enzyme production. Permeabilization efficiency of different solvents was assessed and results indicated that iso-propanol was the most potent permeabilizing agent.