Statistical factorial designs for optimum production of thermostable α-amylase by the degradative bacterium Parageobacillus thermoglucosidasius Pharon1 isolated from Sinai, Egypt.

BACKGROUND: This study aimed to isolate potent thermophilic and amylolytic bacteria from a hot spring of Pharaoh's bath, Sinai, Egypt, and screen its degradative activity. The amylolytic activity was further optimized using a statistical full factorial design followed by response surface methodology. RESULTS: A thermophilic bacterium was isolated from the hot spring of Pharaoh's Bath, Sinai, Egypt. The isolate produced amylase, cellulase, and caseinase and was identified by 16S rRNA gene sequencing as Parageobacillus thermoglucosidasius Pharon1 (MG965879). A growth medium containing 1% soluble starch was found to optimize the amylase production. Dinitrosalycalic acid method (DNS) was used to estimate the amount of reducing sugar produced. Statistical full factorial and response surface designs were employed to optimize physical variables affecting the α-amylase production and determine the significant interactions of the studied variables during the fermentation process. According to the results obtained by the response optimizer, the maximum amylase activity reached 76.07 U/mL/ min at 54.1°C, pH 5.6 after 98.5 h incubation under aerobic conditions. Moreover, the produced enzyme was thermostable and retained most of its activity when exposed to a high temperature of 100°C for 120 min. Maximum enzyme activity was attained when the enzyme was incubated at 70°C for 30 min. CONCLUSIONS: This is the first report of the production of thermostable α-amylase by the potent thermophilic Parageobacillus thermoglucosidasius. The enzyme endured extreme conditions of temperature and pH which are important criteria for commercial and industrial applications.

Assessment of exopolysaccharides, bacteriocins and in vitro and in vivo hypocholesterolemic potential of some Egyptian Lactobacillus spp.

Lactobacilli probiotics have been suggested to reduce cholesterol with low side effects to host. Bacteriocins and exopolysaccharides (EPSs) production are two meaningful examples of functional applications of lactobacilli in the food industry. Eight Lactobacillus strains were isolated from some Egyptian fermented food and tested for their probiotic properties. Analysis of the monosaccharide composition by thin layer chromatography showed the presence of glucose, galactose and unknown sugar. The main functional groups of EPSs were elucidated by Fourier-Transform Infrared Spectroscopy. Their fermentation cultures displayed powerful antioxidant activities extending from 97.5 to 99%, 40-75% for their EPSs and free cells, respectively, and exhibited in vitro cholesterol downgrading from 48 to 82% and 72 to 91% after 48 and 120 h, respectively. Their EPSs showed good anticancer activities against carcinoma cells with low IC50 values for HCT-116, PC-3 and HepG-2 cells. To the best of our knowledge, there have been no previous reports on the potential of Lactobacillus EPSs activity against PC-3. The selected strains, L. plantarum KU985433 and L. rhamnosus KU985436 produced two different bacteriocins as detected by gel permeation chromatography with good antimicrobial activities. In vivo study demonstrated that feeding Westar rats with fermented milk exhibited greater cholesterol, LDL and blood triglyceride reduction for both strains. Whereas, HDL was increased by about 43 and 38%, respectively, and the atherogenic indices decreased.

Response surface methodology-mediated improvement of the irradiated endophytic fungal strain, Alternaria brassicae AGF041 for Huperzine A-hyperproduction.

Huperzine A (HupA) is an anti-Alzheimer's therapeutic and a dietary supplement for memory boosting that is extracted mainly from Huperziacae plants. Endophytes represent the upcoming refuge to protect the plant resource from distinction but their HupA yield is still far from commercialization. In this context, UV and gamma radiation mutagenesis of the newly isolated HupA-producing Alternaria brassicae AGF041 would be applied in this study for improving the endophytic HupA yield. Compared to non-irradiated cultures, UV (30-40 min, exposure) and γ (0·5 KGy, dose) irradiated cultures, each separately, showed a significant higher HupA yield (17·2 and 30·3%, respectively). While, application of a statistically optimized compound irradiation (0·70 KGy of γ treatment and 42·49 min of UV exposure, sequentially) via Response Surface Methodology (RSM) resulted in 53·1% production increase. Moreover, a stable selected mutant strain CM003 underwent batch cultivation using a 6·6 l bioreactor for the first time and was successful for scaling up the HupA production to 261·6 µg l-1 . Findings of this research are demonstrated to be valuable as the employed batch fermentation represents a successful starting step towards the promising endophytic HupA production at an industrial scale.

Characterization and antimicrobial activity of lectins purified from three Egyptian leguminous seeds.

Lectins are carbohydrate-binding proteins that play vital roles in many biological processes. In this study, lectins from three Egyptian cultivars (fava bean, lentil, and pea) were isolated by precipitation with different concentrations of ammonium sulfate. The purification process was performed by affinity chromatography using mannose agarose. The highest concentration of purified lectins (1.48 mg/g) was recorded in pea at 90% saturation. SDS-PAGE of the purified lectins revealed bands of low molecular weights (14 to 18 kDa). The complete amino acid sequences of purified lectins were assessed using mass spectrometry (MS), which indicated the presence of the peptides favin, p54, and psl in fava bean, lentil, and pea, respectively. The lectins showed antimicrobial activity. The highest inhibition zone (35 mm) was measured with lectin purified from lentil against Staphylococcus aureus ATCC 6538, followed by pea lectin (33.4 mm) against Pseudomonas aeruginosa ATCC 10145. To the best of our knowledge, the legume lectins in this study are the first lectins to exhibit antifungal activity against Candida albicans, with the maximum inhibition zone (25.1 mm) observed with purified lectins of fava bean. Additionally, the first scanning electron microscope (SEM) images showing agglutination and clumping of microbial cells exposed to tested lectins are provided. These findings proved that Egyptian legume lectins are distinct from other lectins reported in previous studies and demonstrated their potential as antimicrobial agents against human pathogenic microorganisms.

Optimizing the Reduction of Molybdate by Two Novel Thermophilic Bacilli Isolated from Sinai, Egypt.

The aim of this study was to isolate thermophilic bacteria to be used in in situ bioremediation of molybdate at elevated temperatures. Two molybdate reducing bacteria (Pharon2 and Pharon3) were isolated from the hot Spring at Pharaoh's Bath, Sinai, Egypt. The isolates were identified by 16S rRNA genes sequencing and were submitted to GenBank as Bacillus tequilensis strain Pharon2 (MK078034) and Bacillus sonorensis strain Pharon3 (MK078035). The molybdenum blue production was optimized using multifactorial statistical approaches, Plackett-Burman and central composite designs. According to the results obtained by response optimizer, the maximum molybdenum blue production achieved was 1.04 and 1.12 represented as absorbance at 865 nm, with the optimum salt concentration of 1.1 and 2.5%; at pH 7.02 and 7.07; incubation temperature of 46.1 and 52.2 °C; sucrose and glucose as an electron donor for Bacillus tequilensis strain Pharon2 and Bacillus sonorensis strain Pharon3, respectively. In conclusion, the thermophilic bacterial isolates belonging to the genus Bacillus could be used in in situ bioremediation under elevated temperatures. To the best of our knowledge, this is the first record of molybdenum blue production by thermophilic Bacillus tequilensis and Bacillus sonorensis.

Production and enhancement of the acetylcholinesterase inhibitor, huperzine A, from an endophytic Alternaria brassicae AGF041.

Huperzine A (HupA) is a potent acetylcholinesterase (AChE) inhibitor of a great consideration as a prospective drug candidate for Alzheimer's disease treatment. Production of HupA by endophytes offers an alternative challenge to reduce the massive plant harvest needed to meet the increasing demand of HupA. In the current study, some endophytic fungal and actinobacterial isolates from the Chinese herb, Huperzia serrata, underwent liquid fermentation, alkaloid extraction, and screening for AChE inhibition and HupA production. Among these isolates, Alternaria brassicae AGF041 strain was the only positive strain for HupA production with the maximum AChE inhibition of 75.5%. Chromatographic analyses verified the identity of the produced HupA. The HupA production was efficiently maximized up to 42.89 µg/g of dry mycelia, after optimization of thirteen process parameters using multifactorial statistical approaches, Plackett-Burman and central composite designs. The statistical optimization resulted in a 40.8% increase in HupA production. This is the first report to isolate endophytic actinobacteria with anti-AChE activity from H. serrata, and to identify an endophytic fungus A. brassicae as a new promising start strain for a higher HupA yield.

Biogenic production of silver nanoparticles by Enterobacter cloacae Ism26.

A bacterial isolate capable of tolerating 30 mM silver nitrate (AgNO3) was recovered from soil contaminated with industrial waste. The isolate was identified by 16S rRNA as Enterobacter cloacae Ism26 (KP988024) and its capability to synthesize silver nanoparticles (AgNPs) was investigated. AgNPs were produced by mixing 1 mM AgNO3 solution with bacterial cell lysate under light conditions. The UV-Vis spectrum of the aqueous medium containing AgNPs exhibited a peak at 440 nm corresponding to the surface plasmon resonance of the AgNPs. The crystalline nature of the particles was confirmed by X-ray difractometer. High-resolution transmission electron microscopy revealed that the AgNPs were spherical and well dispersed and ranged in size from 7 to 25 nm. The average size range of the produced AgNPs was confirmed by dynamic light scattering. Fourier transform infrared spectroscopy revealed possible involvement of reductive groups on the surface of the nanoparticles. The biosynthesized AgNPs were stable for 6 months and inhibited both gram-positive and gram-negative bacteria. This work describes the exploitation of a low-cost biomaterial and an easy method for the synthesis of AgNPs with desirable and advantageous characteristics.

A new low molecular mass alkaline cyclodextrin glucanotransferase from Amphibacillus sp. NRC-WN isolated from an Egyptian soda lake

Background: Cyclodextrin glucanotransferase (CGTase) is one of the most industrially important enzymes used in the commercial production of cyclodextrins (CDs). Alkaliphilic bacteria have attracted much interest in the last few decades because of their ability to produce extracellular enzymes that are active and stable at high pH values. Here, we report the isolation of a new CGTase from alkaliphilic bacteria collected from Egyptian soda lakes and describe the purification and biochemical characterization of this CGTase. Results: Screening for CGTase-producing alkaliphilic bacteria from sediment and water samples collected from Egyptian soda lakes located in the Wadi Natrun valley resulted in the isolation of a potent CGTase-producing alkaliphilic bacterial strain, designated NRC-WN. Strain NRC-WN was belonging to genus Amplibacullus by 16S rDNA sequence analysis (similarity: ca. 98%). Among the tested nitrogen and carbon sources, peptone (0.15%, w/v) and soluble starch (0.4%, w/v) allowed maximal CGTase production by Amphibacillus sp. NRC-WN. CGTase was successfully purified from Amphibacillus sp. NRC-WN up to 159.7-fold through a combination of starch adsorption and anion exchange chromatography, resulting in a yield of 84.7%. SDS-PAGE analysis indicated that the enzyme was purified to homogeneity and revealed an estimated molecular mass of 36 kDa, which makes it one of the smallest CGTases reported in the literature. The purified enzyme exhibited maximum activity at 50ºC and was stable up to 70ºC, retaining 93% of its initial activity after treatment for 1 hr. Furthermore, Ca2+ ions (10 mM) significantly enhanced the thermal stability of the CGTase. The purified enzyme was active and stable over a wide pH range, showing maximal activity at pH 9.5. The enzyme was significantly stimulated by Zn2+, Ca2+ and Co2+ but was completely inhibited in the presence of Fe3+ and mercaptoethanol. The Km and Vmax values of the purified CGTase were estimated to be 0.0434 mg/ml and 3,333.3 mg β-CD/ml/min, respectively. β-CD was the predominant product of starch degradation by the Amphibacillus sp. NRC-WN CGTase, followed by α-and γ-CDs. Conclusions: A new low molecular mass alkaline CGTase was purified from a newly identified alkaliphilic Amphibacillus sp. NRC-WN isolate from the Egyptian soda lakes. The enzyme showed promising thermal and pH stability and a high affinity toward starch as a natural substrate.