The potential of a novel enzyme-based surface plasmon resonance biosensor for direct detection of dopamine.

Dopamine is one of the significant neurotransmitters and its monitoring in biological fluids is a critical issue in healthcare and modern biomedical technology. Here, we have developed a dopamine biosensor based on surface plasmon resonance (SPR). For this purpose, the carboxymethyl dextran SPR chip was used as a surface to immobilize laccase as a bioaffinity recognition element. Data analysis exhibited that the acidic pH value is the optimal condition for dopamine interaction. Calculated kinetic affinity (KD) (48,545 nM), obtained from a molecular docking study, showed strong association of dopamine with the active site of laccase. The biosensor exhibited a linearity from 0.01 to 189 µg/ml and a lower detection limit of 0.1 ng/ml (signal-to-noise ratio (S/N) = 3) that is significantly higher than the most direct dopamine detecting sensors reported so far. Experiments for specificity in the presence of compounds that can co-exist with dopamine detection such as ascorbic acid, urea and L-dopa showed no significant interference. The current dopamine biosensor with high sensitivity and specificity, represent a novel detection tool that offers a label-free, simple procedure and cost effective monitoring system.

Biosynthesis Optimization of Antibacterial-Magnetic Iron Oxide Nanoparticles from Bacillus megaterium.

The occurrence of antibiotic resistance on common bacterial agents and the need to use new generations of antibiotics have led to the use of various strategies for production. Taking inspiration from nature, using bio-imitation patterns, in addition to the low cost of production, is advantageous and highly accurate. In this research, we were able to control the temperature, shake, and synthesis time of the synthesis conditions of Bacillus megaterium bacteria as a model for the synthesis of magnetic iron nanoparticles and optimize the ratio of reducing salt to bacterial regenerating agents as well as the concentration of salt to create iron oxide nanoparticles with more favorable properties and produced with more antibacterial properties. Bacterial growth was investigated by changing the incubation times of pre-culture and overnight culture in the range of the logarithmic phase. The synthesis time, salt ratio, and concentration were optimized to achieve the size, charge, colloidal stability, and magnetic and antibacterial properties of nanoparticles. The amount of the effective substance produced by the bacteria was selected by measuring the amount of the active substance synthesized using the free radical reduction (DPPH) method. With the help of DPPH, the duration of the synthesis was determined to be one week. Characterizations such as UV-vis spectroscopy, FTIR, FESEM, X-ray, and scattering optical dynamics were performed and showed that the nanoparticles synthesized with a salt concentration of 80 mM and a bacterial suspension to salt ratio of 2:1 are smaller in size and have a light scattering index, a PDI index close to 0.1, and a greater amount of reducing salt used in the reaction during one week compared to other samples. Moreover, they had more antibacterial properties than the concentration of 100 mM. As a result, better characteristics and more antibacterial properties than common antibiotics were created on E. coli and Bacillus cereus.

Synthesis and evaluation of nanosystem containing chondroitinase ABCI based on hydroxyapatite.

The bacterial enzyme chondroitinase ABCI (chABCI), which has been isolated from Proteus Vulgaris, is crucial in the treatment of spinal cord injuries. However, due to its short lifespan, the maintenance and clinical application of this enzyme are very constrained. In this study, the immobilization of this enzyme on hydroxyapatite has been carried out and assessed with the aim of enhancing the characteristics and efficiency of chABCI. Hydroxyapatite particles (HAPs) are a potential candidate for drug-delivery carriers because of their excellent biocompatibility, shape controllability, and high adsorption. The use of the nanometer scale allows efficient access to the enzyme's substrate. It demonstrates important biological application capabilities in this way. Field emission gun-scanning electron microscopy (FEG-SEM), X-ray diffraction (XRD), infrared spectroscopy (FT-IR), in vitro release study, and cytotoxicity test were used to characterize the drug nanosystem's properties. According to the findings, electrostatic bindings was formed between charged groups of the enzyme and hydroxyapatite nanoparticles. The results also demonstrated that immobilized chABCI on hydroxyapatite has beneficial properties, such as more manageable drug release, minimal toxicity and side effects, and a high potential to enhance the efficacy of drug delivery and decrease the need for repeated injections.

Bacterial nano-factories as a tool for the biosynthesis of TiO2 nanoparticles: characterization and potential application in wastewater treatment.

The development of reliable and eco-conscious processes for nanoparticle synthesis constitutes a significant element in nanotechnology. TiO2 nanoparticles (NPs) are becoming essential due to their potential uses in dentistry, surgery, agriculture, and pharmacy. This leads to the development of various procedures for producing TiO2 NPs using various physicochemical methods. Still, the drawbacks of these conventional methods are associated with the emission of toxic chemicals into the atmosphere and high energy demands in production, hence endangering the health and the environment. Problems issued are solved by green nanotechnology, which offers tools as nano-factories by utilizing biological sources to subside the improper effects of conventional methods and produces nanoparticles through synthesis methods that are clean, safe, energy-efficient, and cost-effective. Among the biogenic sources, microbial cells such as bacteria possess intrinsic pathways of converting metallic salt to nanoparticles due to their ability to produce reductase enzymes. Also, they can offer features to products such as high dispersity and produce sustainable nanoparticles at a large scale. Biosynthesized TiO2 NPs have high oxidizing potential and a wide range of applications, specifically as photosensitizers and antimicrobial agents. This review will address bacterial nano-factories that can be utilized for the biosynthesis of TiO2 NPs, the characterization of biosynthesized nanoparticles, and their potential application in wastewater treatment.

Improvement of thermal-stability of chondroitinase ABCI immobilized on graphene oxide for the repair of spinal cord injury.

Spinal cord injury healing has been shown to be aided by chondroitinase ABC I (cABCI) treatment. The transport of cABCI to target tissues is complicated by the enzyme's thermal instability; however, cABCI may be immobilized on nanosheets to boost stability and improve delivery efficiency. This investigation's goal was to assess the immobilization of cABC I on graphene oxide (GO). for this purpose, GO was produced from graphene using a modified version of Hummer's process. the immobilization of cABC I on GO was examined using SEM, XRD, and FTIR. The enzymatic activity of cABC I was evaluated in relation to substrate concentration. The enzyme was then surface-adsorption immobilized on GO, and its thermal stability was examined. As compared to the free enzyme, the results showed that the immobilized enzyme had a greater Km and a lower Vmax value. The stability of the enzyme was greatly improved by immobilization at 20, 4, 25, and 37 °C. For example, at 37 °C, the free enzyme retained 5% of its activity after 100 min, while the immobilized one retained 30% of its initial activity. The results showed, As a suitable surface for immobilizing cABC I, GO nano sheets boost the enzyme's stability, improving its capability to support axonal regeneration after CNC damage and guard against fast degradation.

Biomimetic synthesis of iron oxide nanoparticles from Bacillus megaterium to be used in hyperthermia therapy.

The suitable structural characteristics of magnetic nanoparticles have resulted in their widespread use in magnetic hyperthermia therapy. Moreover, they are considered a proper and operational choice for pharmaceutical nanocarriers. Using the biomimetic method, we were able to produce iron oxide magnetic nanoparticles from the bacterial source of PTCC1250, Bacillus megaterium, for therangostic diagnosis systems and targeted drug delivery. Some of the benefits of this method include mitigated environmental and biological dangers, low toxicity, high biocompatibility, cheap and short-term mass production possibilities in each synthesis round compared to other biological sources, simple equipment required for the synthesis; and the possibility of industrial-scale production. Bacillus megaterium is a magnetotactic bacteria (MTB) that has a magnetosome organelle capable of orienting based on external magnetic fields, caused by the mineralization of magnetic nanocrystals. Utilizing this capability and adding an iron nitrate solution to the bacterial suspension, we synthesized iron oxide nanoparticles. The extent of synthesis was measured using UV-visible spectrophotometry. The morphology was evaluated using FESEM. The crystallized structure was characterized using RAMAN and XRD. The size and distribution of the nanoparticles were assessed using DLS. The surface charge of the nanoparticles was measured using zeta potential. The synthesis of iron oxide nanoparticles was confirmed using FT-IR, and the magnetic property was measured using VSM. This study is continued to identify industrial and clinical applications.

Catalytic parameters and thermal stability of chondroitinase ABCI on red porous silicon nanoparticles.

The bacterial enzyme chondroitinase ABC, which digests extracellular chondroitin sulfate proteoglycans, has been shown to enhance axonal regeneration. However, the utilization of this enzyme as therapeutics is notably restricted due to its thermal instability. Therefore, red luminescent porous silicon that hold promise for potential applications in biological/medical imaging was used as a carrying matrix for chondroitinase with the aim of enhancing its stability. Porous Si nanoparticles were prepared by electrochemical etching of silicon wafers in ethanolic HF solution. The size of nanoparticles (210 nm) and the mean pore diameter (8 -20 nm) were determined using dynamic light scattering and scanning electron microscopy. Purified chondroitinase was then incorporated into the silicon pores. Results revealed similar Km and lower Vmax value for the immobilized enzyme when compared with the free enzyme. The immobilized chondroitinase exhibited about a 4 fold increase in stability at 37 °C after 50 min. It is likely possible that, the enzyme was protected inside the pores resulted in higher stability. Moreover, porous silicon was seen to be capable of holding the chondroitinase for repeated cyclic tests for three times. The cell viability assay exhibited no significant cytotoxicity for Psi-chondroitinase up to 24 h.

Porous silicon nanoparticle as a stabilizing support for chondroitinase.

Chondroitinase ABCI (cABCI) from Proteus vulgaris is a drug enzyme that can be used to treat spinal cord injuries. One of the main problems of chondroitinase ABC1 is its low thermal stability. The objective of the current study was to stabilize the enzyme through entrapment within porous silicon (pSi) nanoparticles. pSi was prepared by an electrochemical etch of p-type silicon using hydrofluoric acid/ethanol. The size of nanoparticles were determined 180nm by dynamic light scattering and the mean pore diameter was in the range of 40-60nm obtained by scanning electron microscopy. Enzymes were immobilized on porouse silicon nanoparticles by entrapment. The capacity of matrix was 35µg enzyme per 1mg of silicon. The immobilized enzyme displayed lower Vmax values compared to the free enzyme, but Km values were the same for both enzymes. Immobilization significantly increased the enzyme stability at various temperatures (-20, 4, 25 and 37°C). For example, at 4°C, the free enzyme (in 10mM imidazole) retained 20% of its activity after 100min, while the immobilized one retained 50% of its initial activity. Nanoparticles loading capacity and the enzyme release rate showed that the selected particles could be a pharmaceutically acceptable carrier for chondroitinase.

Investigations on possible roles of C-terminal propeptide of a Ca-independent α-amylase from bacillus.

Previously, an extracellular α-amylase (BKA) had been purified from the culture of Bacillus sp. KR8104. Subsequently, the crystal structure of the active enzyme revealed a 422 amino acids polypeptide. In this study, the bka was cloned into E. coli, which encoded a polypeptide of 659 amino acids including two additional fragments: one 44 residues N-terminal fragment and another 193 residues C-terminal fragment. In order to investigate the role of the C-terminal fragment, two constructs with and without this region [BKAΔ(N44) and BKAΔ(N44C193)] were designed and expressed in E. coli BL21. The optimum pH, thermal stability, and the end-products of starch hydrolysis were found to be similar in both constructs. The Km and V(max) values for BKAΔ(N44) were lower than BKAΔ(N44C193), using either starch or ethylidene-blocked 4-nitrophenylmaltoheptaoside as a substrate.

Effect of ionic liquids on the structure, stability and activity of two related α-amylases.

Ionic liquids are recognized as green solvents for carbohydrates dissolution. However, only a limited number of studies have been carried out to investigate their effect on carbohydrate hydrolyzing enzymes. We have investigated the influence of two water miscible ionic liquids on the activity, stability and structure of two related α-amylases from Bacillus amyloliquefaciens and Bacillus lichiniformis. Upon changes in ionic liquids concentrations, both enzymes activity and stability were reduced. Associated thermodynamic and conformational changes were observed using differential scanning calorimetry and fluorescence techniques. Thermal denaturation was accompanied by aggregation in both aqueous buffer and [BMIm][Cl] but [HMIm][Cl] significantly suppressed aggregation.