Improvement of phenolic profile and biological activities of wild mustard sprouts.

The current study aimed to assess the effect of the germination process of wild mustard seeds on the phenolic profile, antioxidant, antibacterial, and antidiabetic properties, and some relevant enzyme activities. The total phenolic and flavonoid contents increased 5- and 10-fold, respectively, and were maximized on 5-days sprouts. One new phenolic compound was identified on 5-days sprout extract using HPLC. The concentrations of the identified phenolic compounds increased 1.5-4.3 folds on 5-days sprouts compared with dry seeds. The total antioxidant activity multiplied 17- and 21-fold on 5-days sprouts using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) assays, respectively. The activity of carbohydrate-cleaving, phenolic-synthesizing and antioxidant enzymes also increased during germination. On 5-days sprouts, there was a substantial correlation between the highest ß-glucosidase and peroxidase activities with highest phenolic and flavonoid levels and maximum antioxidant activity. The phenolic extract of 5-days sprouts exhibited antimicrobial activities against Escherichia coli and Staphylococcus aureus and showed potent antidiabetic activity established by its inhibitory effect against α-amylase and α-glucosidase compared to dry seeds.

Exploration of antimicrobial and anticancer activities of L-amino acid oxidase from Egyptian Naja haje venom.

Hepatocellular carcinoma and bacterial resistance are major health burdens nowadays. Thus, providing new therapies that overcome that resistance is of great interest, particularly those derived from nature rather than chemotherapeutics to avoid cytotoxicity on normal cells. Venomous animals are among the natural sources that assisted in the discovery of novel therapeutic regimens. L-amino acid oxidase Nh-LAAO (140 kDa), purified from Egyptian Naja haje venom by a successive two-step chromatography protocol, has an optimal pH and temperature of 8 and 37 °C. Under standard assay conditions, Nh-LAAO exhibited the highest specificity toward L-Arg, L-Met and L-Leu, with Km and Vmax values of 3.5 mM and 10.4 µmol/min/ml, respectively. Among the metal ions, Ca+2, Na+, and K+ ions are activators, whereas Fe+2 inhibited LAAO activity. PMSF and EDTA slightly inhibited the Nh-LAAO activity. In addition, Nh-LAAO showed antibacterial and antifungal activities, particularly against Gentamicin-resistant P. aeruginosa and E. coli strains with MIC of 18 ± 2 µg/ml, as well as F. proliferatum and A. parasiticus among the selected human pathogenic strains. Furthermore, Nh-LAAO exhibited anti-proliferative activity against cancer HepG2 and Huh7 cells with IC50 of 79.37 and 60.11 µg/ml, respectively, with no detectable effect on normal WI-38 cells. Consequently, the apoptosis % of the HepG2 and Huh7 cells were 12 ± 1 and 34.5 ± 2.5 %, respectively, upon Nh-LAAO treatment. Further, the Nh-LAAO arrested the HepG2 and Huh7 cell cycles in the G0/G1 phase. Thus, the powerful selective cytotoxicity of L-amino acid oxidase opens up the possibility as a good candidate for clinical cancer therapy.

Development of chia gum/alginate-polymer support for horseradish peroxidase immobilization and its application in phenolic removal.

Chia gum's molecular structure with distinctive properties as well as the alginate-based hydrogel's three-dimensionally cross-linked structure can provide a potent matrix for immobilization of enzyme. Herein, chia gum (CG)/alginate (A)-polymeric complex was synthesized and employed as a support material for the immobilization of horseradish peroxidase (HRP). HRP was successfully immobilized on the developed ACG-polymeric support, and the highest immobilization recovery (75%) was observed at 1.0% CG and 2% A, pH 7.0, and 50 units of the enzyme. The structure, morphology, and thermal properties of the prepared ACG-HRP were demonstrated using Fourier Transform Infrared (FTIR), Scanning Electron Microscope, and Thermogravimetric (TGA) analyses. ACG-HRP showed a good reusability (60%) over ten reuses. The immobilized ACG-HRP displayed an acidic pH optimum (6.0), a higher temperature optimum (50 °C), and improved thermal stability (30-50 °C) compared to the soluble HRP at pH 7.0, 40 °C and (30-40 °C), respectively. ACG-HRP has a lower affinity for hydrogen peroxide (H2O2) and guaiacol and a higher oxidizing affinity for a number of phenolic substrates. The ACG-HRP demonstrated greater resistance to heavy metals, isopropanol, urea, Triton X-100, and urea, as well as improved efficiency for eliminating phenol and p-chlorophenol. The developed ACG-polymeric support provided improved enzyme properties, allowed the reuse of the immobilized HRP in 10 cycles, and made it promising for several biotechnological applications.

Purification and characterization of cysteine protease of Sarcocystis fusiformis from infected Egyptian water buffaloes.

Sarcocystis spp. infects water buffaloes (Bubalus bubalis) causing sarcocystosis. In the present study, Sarcocystis fusiformis was recognized in Egyptian water buffaloes based on histological observation and molecular analysis of internal transcribed spacer 1 (ITS1), 18S ribosomal RNA (18S rRNA) and cytochrome c oxidase subunit I (COX-1) gene fragments. Chemotherapy and vaccines against Sarcocystis spp. could potentially target proteases because they may play a crucial role in the infection. Cysteine proteases are multifunctional enzymes involved in vital metabolic processes. However, the involvement of proteases in S. fusiform infection has not yet been characterized. Here, the purification and study on some biochemical properties of protease isolated from cysts of S. fusiform were carried out. Protease with a molecular weight of 100 kDa was purified. LC-MS/MS analyzed the protein sequence of purified protease and the data suggested that the enzyme might be related to the cysteine protease. The purified protease exhibited maximum activity at pH 6 and a temperature of 50 °C. The Michaelis-Menten constant (Km), the maximum velocity (Vmax), and the turnover number (Kcat) were determined. The complete inhibition effect of cysteine inhibitors indicated that the purified enzyme is a cysteine protease. The results suggested that S. fusiform proteolytic enzyme may be necessary for parasite survival in water buffaloes by digesting host tissues. Therefore, cysteine protease could be a suitable target for vaccinations.

Statistical optimization, characterization, antioxidant and antibacterial properties of silver nanoparticle biosynthesized by saw palmetto seed phenolic extract.

On the global market, silver nanoparticles (Ag-NPs) are in high demand for their various applications in biomedicine, material engineering, and consumer products. This study highlighted the biosynthesis of the Ag-NPs using saw palmetto seed phenolic extract (SPS-phenolic extract), which contained vital antioxidant-phenolic compounds. Herein, central composite statistical design, response surface methodology, and sixteen runs were conducted to optimize Ag-NPs biosynthesis conditions for maximizing the production of Ag-NPs and their phenolic content. The best-produced SPS-Ag-NPs showed a surface plasmon resonance peak at 460 nm and nano-spherical sizes ranging from 11.17 to 38.32 nm using the UV spectrum analysis and TEM images, respectively. The produced SPS-Ag-NPs displayed a high negative zeta-potential value (- 32.8 mV) demonstrating their high stability. The FTIR analysis demonstrated that SPS-phenolic compounds were involved in sliver bio-reduction and in stabilizing, capping, and preventing Ag-NP aggregation. The thermogravimetric investigation revealed that the produced SPS-Ag-NPs have remarkable thermal stability. The produced SPS-Ag-NP exceeded total antioxidant activity (13.8 µmol Trolox equivalent) more than the SPS-phenolic extract (12.0 µmol Trolox equivalent). The biosynthesized SPS-Ag-NPs exhibited noticeably better antibacterial activity against multidrug-resistant Gram-negative E. coli and Gram-positive S. aureus compared to SPS-phenolic extract. Hence, the bio-synthesized SPS-Ag-NPs demonstrated great potential for use in biomedical and antimicrobial applications.

Biotechnology approach using watermelon rind for optimization of α-amylase enzyme production from Trichoderma virens using response surface methodology under solid-state fermentation.

Production of amylases by fungi under solid-state fermentation is considered the best methodology for commercial scaling that addresses the ever-escalating needs of the worldwide enzyme market. Here response surface methodology (RSM) was used for the optimization of process variables for α-amylase enzyme production from Trichoderma virens using watermelon rinds (WMR) under solid-state fermentation (SSF). The statistical model included four variables, each detected at two levels, followed by model development with partial purification and characterization of α-amylase. The partially purified α-amylase was characterized with regard to optimum pH, temperature, kinetic constant, and substrate specificity. The results indicated that both pH and moisture content had a significant effect (P < 0.05) on α-amylase production (880 U/g) under optimized process conditions at a 3-day incubation time, moisture content of 50%, 30 °C, and pH 6.98. Statistical optimization using RSM showed R2 values of 0.9934, demonstrating the validity of the model. Five α-amylases were separated by using DEAE-Sepharose and characterized with a wide range of optimized pH values (pH 4.5-9.0), temperature optima (40-60 °C), low Km values (2.27-3.3 mg/mL), and high substrate specificity toward large substrates. In conclusion, this study presents an efficient and green approach for utilization of agro-waste for production of the valuable α-amylase enzyme using RSM under SSF. RSM was particularly beneficial for the optimization and analysis of the effective process parameters.

Egyptian chia seeds (Salvia hispanica L.) during germination: Upgrading of phenolic profile, antioxidant, antibacterial properties and relevant enzymes activities.

Little studies on chia sprouts were not deeply address the polyphenols profiles and their functional properties during long period of germination. This study aims to evaluate the impact of germination process on the phenolic profile, antioxidant and antibacterial properties and relevant enzymes activities of Egyptian chia seeds. The total phenolic and flavonoid contents of chia sprouts increased several times during ten days of germination and maximized on 7-day sprouts (6.4 and 11.5 folds, respectively). In HPLC analysis, seventeen phenolic compounds were detected on 7-day sprouts compared to fifteen in dry seeds, where two new phenolic compounds (p-coumaric acid and kaempferol) were detected. The concentrations of all the identified phenolic compounds increased several folds (1.8-27) on 7-day sprouts. The total antioxidant activity increased 10, 17, and 29 folds on 7-day sprouts using DPPH, ABTS and PMC antioxidant methods, respectively compared to the dry seeds. Both antioxidant and carbohydrate-cleaving enzymes increased in chia sprouts and correlated with their phenolic content and antioxidant activity. The phenolic content of 7-day sprouts showed a potent antibacterial activity against some human enteric pathogenic bacteria including Escherichia coli O157-H7, Salmonella typhi, Pseudomonas aeruginosa and Staphylococcus aureus with lower MIC values compared to the raw seeds.

Diabetic complications and oxidative stress: The role of phenolic-rich extracts of saw palmetto and date palm seeds.

Recently, we reported that the date palm seed (DP) and saw palmetto seed (SP) extracts possessed a great amount of phenolic contents with potent antioxidant, antimicrobial, and anti-inflammatory activities. Therefore, this study aimed to assess the role of DP and SP phenolic-rich extracts in modulating diabetic complications and oxidative stress in the STZ- diabetic rat. DP and SP extracts significantly inhibited both microbial and pancreatic α-amylases. The STZ-induced diabetic rat groups treated with DP and SP extracts exhibited reversed hyperglycemia (40% and 54%, p < .001-.01) and body weight (70%, p < .001) alteration close to normal. Moreover, DP and SP extracts modulated serious damages in the structures of the pancreas, kidney, and liver of diabetic rats. DP and SP extracts improved the decline of the activities of antioxidant enzymes: Catalase, glutathione-S-transferase, glutathione reductase, and glutathione peroxidase in liver, kidney, and pancreas of the diabetic rats. PRACTICAL APPLICATIONS: Generally, date seed is a rich source of dietary fibers, polyphenols, and antioxidants and used in foods and pharmaceuticals. Our study reported that date palm seed (DP) and saw palmetto seed (SP) phenolic-rich extracts attenuated diabetes and its complications, probably tissue regeneration and normalizing the oxidative stress in the STZ-induced diabetic rat.

Improving the catalytic efficiency of thermostable Geobacillus stearothermophilus xylanase XT6 by single-amino acid substitution.

Directed evolution using error-prone polymerase chain reaction was employed in the current study to enhance the catalytic efficiency of a thermostable Geobacillus stearothermophilus xylanase XT6 parent. High-throughput screening identified two variants with enhanced activity. Sequencing analysis revealed the presence of a single-amino acid substitution (P209L or V161L) in each variant. The maximum activity of mutant V161L and P209L was at 85°C and 70°C, respectively. Both mutants exhibited maximum activity at pH 7. The thermal and alkaline tolerance of mutant V161L only were markedly improved. The two mutants were more resistant to ethanol inhibition than the parent. Substrate specificity of the two mutants was shifted from beechwood xylan to birchwood xylan. The potential of the two mutants to hydrolyze rice straw and sugarcane bagasse increased. Both turnover number (kcat) and catalytic efficiency (kcat/kM) increased 12.2- and 5.7-folds for variant P209L and 13- and 6.5-folds for variant V161L, respectively, towards birchwood xylan. Based on the previously published crystal structure of extracellular G. stearothermophilus xylanase XT6, V161L and P209L mutation locate on ßα-loops. Conformational changes of the respective loops could potentiate the loop swinging, product release and consequently result in enhancement of the catalytic performance.

l-Amino acid oxidase from Cerastes vipera snake venom: Isolation, characterization and biological effects on bacteria and tumor cell lines.

A homodimeric l-amino acid oxidase enzyme (Cv-LAAOI) was isolated from the venom of Cerastes vipera (Egyptian Sand viper) using gel filtration followed by anion exchange chromatography. The molecular mass of Cv-LAAO is 120 kDa in its native form and 60 kDa in its monomeric form. The optimum enzyme activity was achieved on l-Leucine as a substrate in 50 mM buffer pH 7.5 at 50 °C. The Cv-LAAOI activity was significantly reduced by increasing the temperature over 40 °C, lost 75% of its activity at 60 °C and inhibited completely at 80 °C. The Cv-LAAOI attains the highest substrate specificity towards L-Met. The results have also indicated that Mn2+ enhances the enzyme activity by 10%, while Cu2+, Hg2+, Ni2+, Co2+ have suppressive effects on the Cv-LAAOI activity. On the other hand, EDTA has no significant effect on the enzyme activity. The kinetic parameters of Cv-LAAOI activity (Km, Kcat and Vmax) estimated on l-Leucine at pH 8 and 37 °C were found to be 2 mM, 12 S-1 and 16.7 µmol/min/ml, respectively. In addition, the results have shown that Cv-LAAOI exhibits a significant bactericidal activity against gram-positive and gram-negative bacteria, particularly Staphylococcus aureus and Escherichia coli with MIC values of 20 µg/ml. Moreover, Cv-LAAOI has exhibited a considerable cytotoxic activity against breast cancer cell line (MCF-7) with IC50 value 2.75 ±â€¯0.38 µg/ml compared with different tumor cell lines (liver HepG2, lung A549, colon HCT116 and prostate PC3). Furthermore, Cv-LAAOI has triggered antiproliferative activity via extensive H2O2 generation as indicated by the increase in H2O2 and TBARS levels accompanied by the depletion in the catalase activity (CAT) in MCF-7 treated cells compared to the untreated ones. Thus, these findings clearly indicate that Cv-LAAOI has a selective cytotoxic effect on breast cancer cell line, demonstrating a great prospective for future use in cancer therapy.

Biochemical characterization of an extracellular polygalacturonase from Trichoderma harzianum.

An extracellular polygalacturonase (PGII) from Trichoderma harzianum was purified to homogeneity by two chromatography steps using DEAE-Sepharose and Sephacryl S-200. The molecular weight of T. harzianum PGII was 31,000 Da by gel filtration and SDS-PAGE. PGII had isoelectric point of 4.5 and optimum pH of 5.0. PGII was very stable at the pH 5.0. The extent of hydrolysis of different pectins by enzyme was decreased with increasing of degree of esterification (DE). PGII had very low activity toward non-pectic polysaccharides. The apparent K(m) value and K(cat) value for hydrolyzing polygalacturonic acid (PGA) were 3.4 mg/ml and 592 s(-1), respectively. PGII was found to have temperature optimum at 40 degrees C and was approximately stable up to 30 degrees C for 60 min of incubation. All the examined metal cations showed inhibitory effects on the enzyme activity. A 1,10-phenanthroline, Tween 20, Tween 80, Triton X-100 and SDS had no effect on the enzyme activity. The rate of enzyme catalyzed reduction of viscosity of solutions of PGA or pectin was higher three times than the rate of release of reducing sugars indicating that the enzyme had an endo-action. The storage stability of the enzyme in liquid and powder forms was studied, where the activity of the powder form was stable up to 1 year. These properties of T. harzianum PGII with appreciable activity would be potentially novel source of enzyme for food processing.