Green synthesis of Superparamagnetic Iron Oxide and Silver Nanoparticles in Satureja hortensis Leave Extract: Evaluation of Antifungal Effects on Botryosphaeriaceae Species.

In recent years, green synthesis methods of metallic nanoparticles (MNPs) have been attractive because of the more facile, cheaper, and appropriate features associated with biomolecules in MNPs biosynthesis. This research represented an easy, fast, and environmentally friendly method to biosynthesis of superparamagnetic iron oxide nanoparticles (SPIONPs) and silver nanoparticles (AgNPs) by the Satureja hortensis leaf extract as stabilizer and reducer. The SPIONPs synthesized in co-precipitation method. The biosynthesized SPIONPs and AgNPs were studied their antifungal effects against three Botryosphaeriaceae plant pathogens, Botryosphaeria dothidea, Diplodia seriata, and Neofusicoccum parvum. UV-visible spectra (UV-Vis), X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), field emission scanning electron microscopy (Fe-SEM), energy-dispersive X-ray spectroscopy (EDX), and vibrating-sample magnetometer (VSM) analyses were used to evaluate the physicochemical properties and verify the formation of green synthesized SPIONPs and AgNPs. UV-Vis spectra revealed absorption peaks at 243 and 448 nm for SPIONs and 436 nm for AgNPs, respectively. Microscopic and XRD analysis showed that SPIONPs and AgNPs was found spherical in shape with an average particle size of SPIONPs and AgNPs 10 and 12 nm, respectively. The antifungal test against Botryosphaeriaceae species showed that SPIONPs and AgNPs possess antifungal properties against B. dothidea, D. seriata, and N. parvum. However, AgNPs exhibits greater antifungal activity than SPIONPs. The results of the cytotoxicity tests of SPIONs and AgNPs on the MCF-7 cell line showed that AgNPs was significantly more cytotoxic towards the MCF-7 cell line, whereas no significant cytotoxic effect was recorded by SPIONs. Therefore, these biosynthesized MNPs could be substituted for toxic fungicides that are extensively applied in agriculture and contribute to environmental health and food safety.

Heterologous Expression and Functional Characterization of CAP18 from Oryctolagus cuniculus.

Background: Antimicrobial peptides belong to the innate defence system of creatures. These peptides attach to the bacterial membrane in order to die microorganisms by penetrating them. Hence, biotechnology researchers pay more attention to produce antimicrobial peptides for use in various fields. The studies showed that rabbit tissue with inflammation and skin ulcers would be producing CAP18 peptide, which belongs to the cathelicidin group. Methods: In this study, the optimized sequence of the cap18 gene was placed into the pPICZAα plasmid after the alpha-factor signal and transformed into Pichia pastoris (X-33 strain). Purification of the recombinant peptide was done based on its histidine tail at C-terminal, and western blotting method was used to demonstrate the purification of rCAP18. The antibacterial activity of the purified and desalted rCAP18 was investigated at different concentrations against pathogenic bacteria. Results: The maximum expression level of rCAP18 (17.5 kDa) was seen 90 h after induction of alcohol oxidase I (AOX1) promoter with methanol. The concentration of rCAP18 was 33 mg/L after purification with Ni-NTA Sepharose column. The function of rCAP18 (4.3, 5.7, 7 µg/ml) was investigated against Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. Results showed that %CFU/cm2 reached 28% after P. aeruginosa cells treatment with 7 µg/ml of rCAP18. Conclusion: This study presented the findings related to heterologous expression of cap18 gene, and evaluation of rCAP18 antibacterial effects. Our results showed that rCAP18 plays a significant role in inhibiting bacterial growth, especially Gram-negative bacteria.

Identification and in-silico characterization of taxadien-5α-ol-O-acetyltransferase (TDAT) gene in Corylus avellana L.

Paclitaxel® (PC) is one of the most effective and profitable anti-cancer drugs. The most promising sources of this compound are natural materials such as tissue cultures of Taxus species and, more recently, hazelnut (Corylus avellana L.). A large part of the PC biosynthetic pathway in the yew tree and a few steps in the hazelnut have been identified. Since understanding the biosynthetic pathway of plant-based medicinal metabolites is an effective step toward their development and engineering, this paper aimed to identify taxadiene-5α-ol-O-acetyltransferase (TDAT) in hazelnut. TDAT is one of the key genes involved in the third step of the PC biosynthetic pathway. In this study, the TDAT gene was isolated using the nested-PCR method and then characterized. The cotyledon-derived cell mass induced with 150 µM of methyl jasmonate (MeJA) was utilized to isolate RNA and synthesize the first-strand cDNA. The full-length cDNA of TDAT is 1423 bp long and contains a 1302 bp ORF encoding 433 amino acids. The phylogenetic analysis of this gene revealed high homology with its ortholog genes in Quercus suber and Juglans regia. Bioinformatics analyses were used to predict the secondary and tertiary structures of the protein. Due to the lack of signal peptide, protein structure prediction suggested that this protein may operate at the cytoplasm. The homologous superfamily of the T5AT protein, encoded by TDAT, has two domains. The highest and lowest hydrophobicity of amino acids were found in proline 142 and lysine 56, respectively. T5AT protein fragment had 24 hydrophobic regions. The tertiary structure of this protein was designed using Modeler software (V.9.20), and its structure was verified based on the results of the Verify3D (89.46%) and ERRAT (90.3061) programs. The T5AT enzyme belongs to the superfamily of the transferase, and the amino acids histidine 164, cysteine 165, leucine 166, histidine 167, and Aspartic acid 168 resided at its active site. More characteristics of TDAT, which would aid PC engineering programs and maximize its production in hazelnut, were discussed.

Improvement Thermal Stability of D-Lactate Dehydrogenase by Hydrophobin-1 and in Silico Prediction of Protein-Protein Interactions.

Hydrophobins are small surface-active proteins. They can connect to hydrophobic or hydrophilic regions and oligomerize in solution to form massive construction. In nature, these proteins are produced by filamentous fungi at different stages of growth. So far, researchers have used them in various fields of biotechnology. In this study, recombinant hydrophobin-1 (rHFB1, 7.5 kDa) was used to stabilize recombinant D-lactate dehydrogenase (rD-LDH, 35 kDa). rD-LDH is a sensitive enzyme deactivated and oxidized by external agents such as O2 and lights. So, its stabilization with rHFB1 can be the best index to demonstrate the positive effect of rHFB1 on preserving and improving enzyme's activity. The unique ability of rHFB1 for interacting with hydrophobic regions of rD-LDH was predicted by protein-protein docking study with ClusPro and PIC servers and confirmed by fluorescence experiments, and Colorless Native-PAGE. Measurement of thermodynamic parameters allows for authenticating the role of rHFB1 as a thermal stabilizer in the protein-protein complex (rD-LDH@rHFB1). Interaction between rHFB1 and rD-LDH improved half-life of enzyme 2.25-fold at 40 °C. Investigation of the kinetic parameters proved that the presence of rHFB1 along with the rD-LDH enhancement strongly the affinity of the enzyme for pyruvate. Furthermore, an increase of Kcat/Km for complex displayed the effect of rHFB1 for improving the enzyme's catalytic efficiency.