Improvement the expression and purification of Loa22: a lipoprotein with OmpA domain from pathogenic Leptospira serovars.

Background and Objectives: One of the highly conserved outer membrane proteins expressed only by pathogenic Leptospires is Loa22. The study aims is to achieve the optimum conditions for high expression of recombinant Loa22 (rLoa22) protein. Materials and Methods: Complete coding sequence of loa22 gene sub-cloned into a pET32a (+) expression vector. BL21 competent E. coli (pLysS) used as expression host for transformation. The recombinant clones selected on ampicillin plates and subjected to PCR by using pET T7 primers. Then expression conditions optimized by adjusting parameters such as culture media, induction time, temperature, and IPTG concentration. Results: SDS-PAGE analysis showed that high production of rLoa22 protein obtained when post induction incubation, IPTG concentration, and duration of induction were 37°C, 0.1 M and 5 h in 2×TY medium respectively. The purification of rLoa22 protein under native conditions using Ni-NTA pull-down was optimum in one hour binding at 37°C, five times washing process and elution buffer with a pH 7.4 and a 0.3 M imidazole concentration. Conclusion: The findings of the study led to high production of pure Loa22 protein, which can form the basis for future investigation on the design of rapid diagnostic tests and more effective vaccine candidates for leptospirosis.

Development of a newly designed biosensor using multi-walled carbon nanotubes (MWCNTs) with gold nanoparticles (AuNPs) in the presence of acetaminophen for detection of Escherichia coli.

Escherichia coli is one of the main indicators in the quality control of water, pharmaceuticals, and other samples. Compared with the time-consuming and high prices of the classical methods, with their high risks in the case of insensitive, contamination and offline detections, biosensors have long been a fast and accurate approach for identifying different bacteria. The present study reports the development of a newly electrochemical biosensor using a glassy carbon electrode (GCE) modified by multi-walled carbon nanotubes/AuNPs/E. coli polyclonal antibody/Bovine Serum Albumin in 0.1 M phosphate-buffered saline ( pH 7) in the presence of acetaminophen, for the detection of E. coli in pharmaceuticals and some real samples. Acetaminophen was added as an indicator for the detection of E. coli by changes in conductivity and current. To identify E. coli, square-wave voltammetry (SWV), differential pulse voltammetry (DPV), and cyclic voltammetry (CV) techniques were used. According to the obtained results of square-wave voltammetry, a limit of 3.02 CFU/ml E. coli detection in 3 min with desirable sensitivity, repeatability, and reproducibility was found with the designed biosensor. This biosensor could be a powerful tool for the detection of indicator bacteria in the food industry, drug safety, quality control, clinical diagnostics, and environmental monitoring.

Optimization of zinc bioleaching from paint sludge using Acidithiobacillus thiooxidans based on response surface methodology.

The reduction of zinc metal in the paint sludge, a hazardous waste, was investigated using Acidithiobacillus thiooxidans by a two-stage bioleaching process. This process was performed using the response surface methodology (RSM) method based on the central composite design (CCD). Four variables, a temperature range of 32-34.5-37 °C, rotation speed of shaker 120-150-180 rpm, pH of 4.2-3.2-2.2, and particle sizes of 1-2-3 mm, were used to optimize the experiments. The results showed that with a constant pulp density of 10 g/L at 32 °C, shaker speed of 120 rpm, a particle size of 1 mm and pH of 4.2, the highest removal predicted by the software (Design Expert version 11) was 22.89%. Repeating the experiments confirmed a decrease in zinc to the nearest predicted point. According to the ANOVA result, the rotation speed of the shaker has the greatest effect on the bioleaching process, followed by the two variables of the rotation speed of shaker and pH together affects. After the bioleaching process, energy dispersive X-ray (EDX) and mapping analysis showed quantitative changes in the chemical composition of the paint sludge, and morphological changes of texture were confirmed by scanning electron microscopy (SEM).

Inhibitory Potential of Acroptilon repens against Key Enzymes involved in Alzheimer and Diabetes, Phytochemical Profile, Radical Scavenging, and Antibacterial Activity.

Background: This study was devoted to assessing the inhibitory potential of acetone, methanol, and ethanol extracts of Acroptilon repens against disease-associated enzymes, as well as their antioxidant/antibacterial activity and phytochemical composition. Methods: Comparative assessment using various antioxidant evaluation methods, including ferric reducing antioxidant power, scavenging ability on 2,2-diphenyl-1-picrylhydrazyl radical and hydrogen peroxide, and reducing power, indicated that the acetone extract presented the highest antioxidant activity, due to its highest total antioxidant content. Results: The total phenolic content and total flavonoids content of these extracts were 3.44 ± 0.32 mg GAE/g DW and 2.09 ± 0.2 mg QE/g DW, respectively. The hydrodistillation essential oil from A. repens was analyzed by gas chromatography-mass spectroscopy, and 17 compounds were identified. All extracts showed good inhibitory activities against disease-related enzyme acetylcholinesterase and α-amylase, with the lowest IC50 for acetonic extract. Extracts of A. repens exhibited inhibiting activities against the Gram-positive bacteria, with the most effect of acetone extract. Conclusion: Our findings suggest A. repens as a promising source of natural antioxidant, antimicrobial, anti-cholinesterase and anti-amylase agents for the management of oxidative damage, and pharmaceutical, food, and cosmeceutical purposes.

Rapid Detection of Escherichia coli by ß-Galactosidase Biosensor Based on ZnO NPs and MWCNTs: A Comparative Study.

The need for alternative approaches for identifying pathogens has led researchers to focus on nanobiotechnology. In this study, zinc oxide nanoparticles (ZnO NPs) and multi-wall carbon nanotubes (MWCNTs) were used as marker molecules. After measuring the best concentration of these nanomaterials to inhibit the lactase activity of the beta-galactosidase enzymes by binding to them, different concentrations of Escherichia coli were added to the medium and their detection ability was finally compared with each other. Due to small size and high reactivity, these compounds are able to detect very low amount of bacteria in the ambient. In fact, the bacteria are attached to the nanoparticles and detach them from the enzyme and lead to substrate decomposition by the enzyme. MWCNTs exhibited better performance than ZnO NPs in detection of bacteria at very low concentration of 101 CFU/ml in 15 min. As a result, they are very appropriate to be utilized especially in the food industry.

Effects of zinc chelating nutrients on Aflatoxin production in Aspergillus flavus.

Aflatoxins are carcinogenic metabolites produced by Aspergillus and Penicillium spp. Aflatoxin contamination of food is a serious health hazard. Some metal ions (such as Zn2+) affect Aspergillus growth and aflatoxin biosynthesis. Presence of zinc in the growth medium incites aflatoxin production. This study investigates the effect of zinc binding amino acids and peptides on aflatoxin synthesis in indigenous toxigenic Aspergillus species isolated from agro-ecological zones in Northern Iran. Zinc (II) chelating nutrients (such as Histidine (His), Cysteine (Cys), Histidine-Cysteine (His-Cys), and triple peptide (Asn-Cys-Ser) were added to the growth medium of toxigenic Aspergillus isolates and incubated at temperature range of 25-40 °C. Aflatoxin production on different culture media was tested using ELISA. Addition of cysteine to Sabouraud dextrose broth (SDB) medium significantly reduced aflatoxin production, which could be related to its zinc chelating property. Aflatoxin production was drastically restrained at high concentration of His, especially in combination with Cys, at high pH values and incubation temperature (pH = 7.5, temperature = 40 °C). Aflatoxin production was low in presence of triple peptide (Asn-Cys-Ser) at concentration of 500 mg/L. From the application perspective, natural zinc chelators can be used as harmless aflatoxin-production inhibitors.

ß-galactosidase Production by Aspergillus niger ATCC 9142 Using Inexpensive Substrates in Solid-State Fermentation: Optimization by Orthogonal Arrays Design.

BACKGROUND: Enzymatic hydrolysis of lactose is one of the most important biotechnological processes in the food industry, which is accomplished by enzyme ß-galactosidase (ß-gal, ß-D-galactoside galactohydrolase, EC 3.2.1.23), trivial called lactase. Orthogonal arrays design is an appropriate option for the optimization of biotechnological processes for the production of microbial enzymes. METHODS: Design of experimental (DOE) methodology using Taguchi orthogonal array (OA) was employed to screen the most significant levels of parameters, including the solid substrates (wheat straw, rice straw, and peanut pod), the carbon/nitrogen (C/N) ratios, the incubation time, and the inducer. The level of ß-gal production was measured by a photometric enzyme activity assay using the artificial substrate ortho-Nitrophenyl-ß-D-galactopyranoside. RESULTS: The results showed that C/N ratio (0.2% [w/v], incubation time (144 hour), and solid substrate (wheat straw) were the best conditions determined by the design of experiments using the Taguchi approach. CONCLUSION: Our finding showed that the use of rice straw and peanut pod, as solid-state substrates, led to 2.041-folds increase in the production of the enzyme, as compared to rice straw. In addition, the presence of an inducer did not have any significant impact on the enzyme production levels.