Entrapment of Cyanase from Thermomyces lanuginosus Using Biomimetic Silica and Its Application for Cyanate Bioremediation.

Cyanate, a toxic product from the chemical oxidation treatment of highly toxic cyanide, can be converted to harmless ammonia and carbon dioxide by cyanase (EC 4.2.1.104). Cyanase from Thermomyces lanuginosus was entrapped in biomimetic silica to improve stability and reusability. After entrapment, the enzyme's activity increased by two-fold, and the residual activity after 30-min of incubation at 60 °C also increased by two-fold, compared to the free enzyme. After being stored at room temperature for 28 days, the entrapped cyanase retained 79% of the initial activity, while the free form retained 61%. The immobilized cyanase was successfully applied to cyanate detoxification; the co-entrapment of carbonic anhydrase from Sulfurihydrogenibium azorense decreased the amount of bicarbonate necessary for cyanate detoxification by 50%. The cyanate degradation retained 53% of the initial value after the co-entrapped cyanate and carbonic anhydrase were reused five times.

Application of Amyloid-Based Hybrid Membranes in Drug Delivery.

The properties of amyloid fibrils, e.g., unique structural characteristics and superior biocompatibility, make them a promising vehicle for drug delivery. Here, carboxymethyl cellulose (CMC) and whey protein isolate amyloid fibril (WPI-AF) were used to synthesize amyloid-based hybrid membranes as vehicles for the delivery of cationic and hydrophobic drugs (e.g., methylene blue (MB) and riboflavin (RF)). The CMC/WPI-AF membranes were synthesized via chemical crosslinking coupled with phase inversion. The zeta potential and scanning electron microscopy results revealed a negative charge and a pleated surface microstructure with a high content of WPI-AF. FTIR analysis showed that the CMC and WPI-AF were cross-linked via glutaraldehyde and the interacting forces between membrane and MB or RF was found to be electrostatic interaction and hydrogen bonding, respectively. Next, the in vitro drug release from membranes was monitored using UV-vis spectrophotometry. Additionally, two empirical models were used to analyze the drug release data and relevant rate constant and parameters were determined accordingly. Moreover, our results indicated that in vitro drug release rates depended on the drug-matrix interactions and transport mechanism, which could be controlled by altering the WPI-AF content in membrane. This research provides an excellent example of utilizing two-dimensional amyloid-based materials for drug delivery.

Studies of Protein Wastes Adsorption by Chitosan-Modified Nanofibers Decorated with Dye Wastes in Batch and Continuous Flow Processes: Potential Environmental Applications.

In this study, reactive green 19 dye from wastewater was immobilized on the functionalized chitosan nanofiber membranes to treat soluble microbial proteins in biological wastewater. Polyacrylonitrile nanofiber membrane (PAN) was prepared by the electrospinning technique. After heat treatment, alkaline hydrolysis, and chemically grafted with chitosan to obtain modified chitosan nanofibers (P-COOH-CS), and finally immobilized with RG19 dye, dyed nanofibers were generated (P-COOH-CS-RG19). The synthesis of P-COOH-CS and P-COOH-CS-RG19 are novel materials for protein adsorption that are not deeply investigated currently, with each of the material functions based on their properties in significantly improving the adsorption efficiency. The nanofiber membrane shows good adsorption capacity and great recycling performance, while the application of chitosan and dye acts as the crosslinker in the nanofiber membrane and consists of various functional groups to enhance the adsorption of protein. The dyed nanofibers were applied for the batch adsorption of soluble protein (i.e., lysozyme), and the process parameters including chitosan's molecular weight, coupling pH, chitosan concentration, dye pH, dye concentration, and lysozyme pH were studied. The results showed that the molecular weight of chitosan was 50 kDa, pH 5, concentration 0.5%, initial concentration of dye at 1 mg/mL dye and pH 12, lysozyme solution at 2 mg/mL at pH 8, and the maximum adsorption capacity was 1293.66 mg/g at a temperature of 318 K. Furthermore, thermodynamic, and kinetic studies suggested that the adsorption behavior of lysozyme followed the Langmuir adsorption isotherm model and the pseudo-second-order kinetic model. The optimal adsorption and desorption conditions based on batch experiments were directly applied to remove lysozyme in a continuous operation. This study demonstrated the potential of dyed nanofibers as an efficient adsorbent to remove approximately 100% of lysozyme from the simulated biological wastewater.

Examining the effect of bovine serum albumin on the properties and drug release behavior of ß-lactoglobulin-derived amyloid fibril-based hydrogels.

Herein, we report the use of ß-lactoglobulin (ß-LG) combined with bovine serum albumin (BSA) for the preparation of amyloid-based hydrogels with aim of delivering riboflavin. The incorporation of BSA enhanced ß-LG fibrillogenesis and protected ß-LG fibrils from losing fibrillar structure due to the pH shift. The mechanical properties of hydrogels were observed to be positively correlated with the number of amyloid fibrils. While the addition of BSA induced amyloid fibril formation, its presence between the fibril chains interfered with the entanglement of fibril chains, thus adversely affecting the hydrogels' mechanical properties. Hydrogels' surface microstructure became more compact as the number of amyloid fibrils rose and the presence of BSA could improve hydrogels' surface homogeneity. In vitro riboflavin (RF) release rate was found to be correlated with the number of fibrils and BSA-RF binding affinity. However, when the digestive enzymes were present, the influence of BSA-RF affinity was alleviated due to enzymes' destructive and/or degradative effects on BSA and/or hydrogels, thus the release rate relied on the number of fibrils, which could be adjusted by the amount of BSA. Results indicate that the additional component, BSA, plays an important role in modulating the properties and functions of ß-LG fibril-based hydrogels.

Kinetic and Thermodynamic Studies of Lysozyme Adsorption on Cibacron Blue F3GA Dye-Ligand Immobilized on Aminated Nanofiber Membrane.

The polyacrylonitrile (PAN) nanofiber membrane was prepared by the electrospinning technique. The nitrile group on the PAN nanofiber surface was oxidized to carboxyl group by alkaline hydrolysis. The carboxylic group on the membrane surface was then converted to dye affinity membrane through reaction with ethylenediamine (EDA) and Cibacron Blue F3GA, sequentially. The adsorption characteristics of lysozyme onto the dye ligand affinity nanofiber membrane (namely P-EDA-Dye) were investigated under various conditions (e.g., adsorption pH, EDA coupling concentration, lysozyme concentration, ionic strength, and temperature). Optimum experimental parameters were determined to be pH 7.5, a coupling concentration of EDA 40 µmol/mL, and an immobilization density of dye 267.19 mg/g membrane. To understand the mechanism of adsorption and possible rate controlling steps, a pseudo first-order, a pseudo second-order, and the Elovich models were first used to describe the experimental kinetic data. Equilibrium isotherms for the adsorption of lysozyme onto P-EDA-Dye nanofiber membrane were determined experimentally in this work. Our kinetic analysis on the adsorption of lysozyme onto P-EDA-Dye nanofiber membranes revealed that the pseudo second-order rate equation was favorable. The experimental data were satisfactorily fitted by the Langmuir isotherm model, and the thermodynamic parameters including the free energy change, enthalpy change, and entropy change of adsorption were also determined accordingly. Our results indicated that the free energy change had a negative value, suggesting that the adsorption process occurred spontaneously. Moreover, after five cycles of reuse, P-EDA-Dye nanofiber membranes still showed promising efficiency of lysozyme adsorption.

Catalase immobilized in polypeptide/silica nanocomposites via emulsion and biomineralization with improved activities.

Polypeptide-mediated silica mineralization is an attractive approach to prepare polypeptide/silica nanocomposites for enzyme immobilization. Herein, a facile approach for in situ immobilization of catalase (CAT) in polypeptide/silica nanocomposites is developed via the preparation of cross-linked polypeptide/enzyme microgels using an emulsion process followed by silica mineralization. The efficient protein immobilization under benign condition (25-28 °C, pH 7.0, 0.05 N) was evidenced by high immobilization yield (> 99%) and no protein leakage. Our data showed that the immobilized CAT exhibited prolonged reusability and storage stability compared to free one, suggesting that the composite networks not only provide suitable microenvironments to facilitate enzymatic reactions but also confine the enzyme macromolecules to prevent subunit dissociation. Star-shaped topology exhibited better coverage onto the enzyme than linear counterpart, leading to the superior reusability (relative activity >95% for 30 cycling number) and storage stability (relative activity >95% for 60 days) of the immobilized CAT (~ 14 mg/g of support). The substrate affinity and enzymatic reaction rate for the immobilized CAT were also influenced by silica content and polypeptide topology. This strategy may provide a feasible and inexpensive approach to fabricate polypeptide/silica nanocomposites, which would be promising materials in biotechnological fields such as enzyme immobilization.

Investigating the effect of sugar-terminated nanoparticles on amyloid fibrillogenesis of ß-lactoglobulin.

In vivo tissue deposition of fibrillar protein aggregates is the cause of several degenerative diseases. Evidence suggests that interfering with the pathology-associated amyloid fibrillogenesis by inhibitory molecules is envisaged as the primary therapeutic strategy. Amyloid fibril formation of proteins has been demonstrated to be influenced by nanoparticles/nanomaterials. As compared with their molecular form counterpart, this work examined the effect of sucrose-terminated nanoparticles on the in vitro amyloid fibrillogenesis and structural properties of ß-lactoglobulin at pH 2.0 and 80 °C. ThT binding and electron microscopy results demonstrated that sucrose-terminated nanoparticles were able to suppress ß-lactoglobulin fibrillogenesis in a concentration-dependent fashion. Importantly, sucrose-terminated nanoparticles showed better ß-lactoglobulin fibril-inhibiting ability than sucrose molecules. ANS fluorescence and right-angle light scattering results showed reduced solvent exposure and decreased aggregation, respectively, in the ß-lactoglobulin samples upon treatment with sucrose-terminated nanoparticles. Moreover, fluorescence quenching analyses revealed that the static quenching mechanism and formation of a non-fluorescent fluorophore-nanoparticle complex are involved in the nanoparticle-ß-lactoglobulin interaction. We believe that the results from this study may suggest that the nanoparticle form of biocompatible sugar-related osmolytes may serve as effective inhibiting/suppressing agents toward protein fibrillogenesis.

Exploring the influence of brilliant blue G on amyloid fibril formation of lysozyme.

Evidence suggests that amyloid fibril mitigation/inhibition is considered a promising approach toward treating amyloid diseases. In this work, we first examined how amyloid fibrillogenesis of lysozyme was affected by BBG, a safe triphenylmethane compound with nice blood-brain-barrier-permeability, and found that shorter fibrillar species were formed in the lysozyme samples treated with BBG. Next, alterations in the features including the secondary as well as tertiary structure, extent of aggregation, and molecular distribution of lysozyme triggered by the addition of BBG were examined by various spectroscopic techniques, right-angle light scattering, dynamic light scattering, and SDS-PAGE. In addition, we have investigated how BBG affected the lysozyme fibril-induced cytotoxicity in SH-SY5Y cells. We found that a large quantity of shorter fibrillar species and more lysozyme monomers were present in the samples treated with BBG. Also, the addition of BBG rescued SH-SY5Y cells from cell death induced by amyloid fibrils of lysozyme. Finally, information about the binding sites and interacting forces involved in the BBG-lysozyme interaction was further explored using synchronous fluorescence and molecular docking approaches. Molecular docking results revealed that, apart from the hydrophobic interaction(s), hydrogen bonding, electrostatic interactions, and van der Waal forces may also be involved in the binding interaction.

Brilliant blue R dye is capable of suppressing amyloid fibril formation of lysozyme.

Amyloid fibril formation is associated with an array of degenerative diseases. While no real cure is currently available, evidence suggests that suppression of amyloid fibrillogenesis is an effective strategy toward combating these diseases. Brilliant blue R (BBR), a disulfonated triphenylmethane compound, has been shown to interact with fibril-forming proteins but exert different effects on amyloid fibrillogenesis. These inconsistent findings prompted us to further evaluate BBR's effect on the inhibition/suppresion of protein fibrillogenesis. Using 129-residue hen lysozyme, which shares high sequence homology to human lysozyme associated with hereditary non-neuropathic systemic amyloidosis, as a model, this study is aimed at thoroughly examining the influence of BBR on the in vitro protein fibrillogenesis. We first showed that BBR dose-dependently attenuated lysozyme fibril formation probably by affecting the fibril growth rate, with the value of IC50 determined to be ~4.39 µM. Next, we employed tryptophan fluorescence quenching method to determine the binding constant and number of binding site(s) associated with BBR-lysozyme binding. In addition, we further conducted molecular docking studies to gain a better understanding of the possible binding site(s) and interaction(s) between lysozyme and BBR. We believe some of the information and/or knowledge concerning the structure-function relationship associated with BBR's suppressing activity obtained here can be applied for the future work in the subject matter related with the therapeutic strategies for amyloid diseases.

Exploring the effects of methylene blue on amyloid fibrillogenesis of lysozyme.

The 129-residue lysozyme has been shown to form amyloid fibrils in vitro. While methylene blue (MB), a compound in the phenothiazinium family, has been shown to dissemble tau fibril formation, its anti-fibrillogenic effect has not been thoroughly characterized in other proteins/peptides. This study examines the effects of MB on the in vitro fibrillogenesis of lysozyme at pH 2.0 and 55 °C. Our results demonstrated that, upon 7-day incubation, the plateau ThT fluorescence of the sample was found to be ~8.69% or ~2.98% of the control when the molar ratio of lysozyme to MB was at 1:1.11 or 1:3.33, respectively, indicating that the inhibitory potency of MB against lysozyme fibrillogenesis is positively correlated with its concentration. We also found that MB is able to destabilize the preformed lysozyme fibrils. Moreover, molecular docking and molecular dynamics simulations results revealed that MB's mechanism of fibril formation inhibition may be triggered by binding with lysozyme's aggregation-prone region. Results reported here provide solid support for MB's effect on amyloid fibrillogenesis. We believe the additional insights gained herein may pave way to the discovery of other small molecules that may have similar action toward amyloid fibril formation and its associated diseases.

Effects of glycation on human γd-crystallin proteins by different glycation-inducing agents.

Human γd-crystallin (Hγd-crystallin), a major protein component of the human eye lens, is associated with the development of juvenile- and mature-onset cataracts. Evidence suggests that nonenzymatic protein glycation plays an important role in the aetiology of cataract and diabetic sequelae. This research compared the effects of various glycation modifiers on Hγd-crystallin aggregation, by treating samples of Hγd-crystallin with ribose, galactose, or methylglyoxal using several biophysical techniques. To measure advanced glycation end products, an Nε-(carboxyethyl)lysine enzyme-linked immunosorbent assay was performed on the glycating agent-treated Hγd-crystallin samples. Fructosamine production detection was performed for both ribose-treated and galactose-treated samples. Methylglyoxal-treated samples had the highest level of aggregation and the greatest extent of unfolding, and upon incubation for a minimum of 12 days, exhibited a marked enhancement in the amount of Nε-(carboxyethyl)lysine. The molecular profiles and morphological features of the glycated samples were highly correlated to the type of glycation agent used. These findings highlight a close connection between the type of glycation modifier and the various aggregation species that form. Thus, these results may facilitate deciphering of the molecular mechanism of diabetic cataractogenesis.

Examining the effects of dextran-based polymer-coated nanoparticles on amyloid fibrillogenesis of human insulin.

More than thirty human proteins and/or peptides can aggregate to form amyloid deposits that are linked to several amyloid diseases including clinical syndrome injection-localized amyloidosis, which is correlated with the aggregation of the 51-residue polypeptide insulin. While no cure is currently available toward tackling amyloid diseases, prevention or suppression of amyloid fibrillization is considered as the primary therapeutic strategy. Nanomaterials have been demonstrated to possess great potential in the fields of biomedical diagnosis and drug delivery, they are also able to affect the amyloid aggregation of proteins. This work explores the effects of three different magnetic nanoparticles coated with dextran-based polymers on the in vitro amyloid fibrillogenesis of human insulin. Surface modification of nanoparticles with dextran-based polymers was used to improve the biocompatibility of maghemite nanoparticles. We demonstrated that insulin fibrillization may be mitigated by the studied nanoparticles in a concentration-dependent fashion as verified by ThT binding assay and transmission electron microscopy. The extent of inhibitory activity against human insulin fibril formation was found to be associated with the physico-chemical properties of nanoparticles, with the highest inhibitory activity observed for diethylaminoethyl-dextran-coated nanoparticles. Using circular dichroism spectroscopy, ANS fluorescence spectroscopy, and right-angle light scattering, we probed the structural/conformational changes and investigated the aggregating behavior of insulin upon treatment with nanoparticles. This work demonstrates that nanoparticles with an appropriate surface modification can be utilized to suppress or even inhibit amyloid fibril formation of proteins.

Cell-targeted, dual reduction- and pH-responsive saccharide/lipoic acid-modified poly(L-lysine) and poly(acrylic acid) polyionic complex nanogels for drug delivery.

A cell-targeted, reduction-/pH-responsive polyionic complex (PIC) nanogel system was developed by simply mixing cationic lactobionolatone/lipoic acid-modified poly(L-lysine) (PLL-g-(Lipo-Lac)) and anionic poly(acrylic acid) (PAA), followed by disulfide cross-linking. The nanogels with sizes smaller than 150nm can be prepared at certain mixing ratio via forming interchain disulfide cross-link and helical PAA/PLL complexes. In vitro drug release study showed that Doxorubicin (Dox) release from the nanogels was significantly enhanced by increasing acidity and/or introducing disulfide cleaving agent. Carbohydrate-lectin binding and cellular uptake studies confirmed that Lac-conjugated nanogels can effectively bind to the cells bearing asialoglycoprotein receptors and subsequently afford efficient cell internalization. Cytotoxicity assays showed that Dox-loaded, Lac-conjugated nanogels exhibited efficient cell proliferation inhibition toward HepG2 cells, whereas the nanogels exhibited excellent biocompatibility. Furthermore, TUNEL assay was employed to detect apoptosis pertaining to the mechanism of cell death. This study highlights that polyionic complexation with subsequent cross-linking can be a simple approach to prepare multifunctional nanogels as drug delivery vehicles.

Investigating the effects of erythrosine B on amyloid fibril formation derived from lysozyme.

Formation of amyloid fibrils has been associated with at least 30 different protein aggregation diseases. The 129-residue polypeptide hen lysozyme, which is structurally homologous to human lysozyme, has been demonstrated to exhibit amyloid fibril-forming propensity in vitro. This study is aimed at exploring the influence of erythrosine B on the in vitro amyloid fibril formation of hen lysozyme at pH 2.0 and 55°C using ThT binding assay, transmission electron microscopy, far-UV circular dichroism absorption spectroscopy, 1-anilinonaphthalene-8-sulfonic acid fluorescence spectroscopy, and synchronous fluorescence study. We found that lysozyme fibrillogenesis was dose-dependently suppressed by erythrosine B. In addition, our far-UV CD and ANS fluorescence data showed that, as compared with the untreated lysozyme control, the α-to-ß transition and exposure of hydrophobic clusters in lysozyme were reduced upon treatment with erythrosine B. Moreover, it could be inferred that the binding of erythrosine B occurred in the vicinity of the tryptophan residues. Finally, molecular docking and molecular dynamics simulations were further employed to gain some insights into the possible binding site(s) and interactions between lysozyme and erythrosine B. We believe the results obtained here may contribute to the development of potential strategies/approaches for the suppression of amyloid fibrillogenesis, which is implicated in amyloid pathology.

Amyloid fibrillogenesis of lysozyme is suppressed by a food additive brilliant blue FCF.

At least 30 different human proteins can fold abnormally to form the amyloid deposits that are associated with a number of degenerative diseases. The research presented here aimed at understanding the inhibitory potency of a food additive, brilliant blue FCF (BBF), on the amyloid fibril formation of lysozyme. Our results demonstrated that BBF was able to suppress the formation of lysozyme fibrils in a dose-dependent fashion. In addition, the structural features and conformational changes in the lysozyme samples upon the addition of BBF were further characterized using circular dichroism spectroscopy, nile red fluorescence spectroscopy, turbidity assay, and sodium dodecyl sulfate electrophoresis. Through molecular docking and molecular dynamics simulations, BBF's mechanism of action in lysozyme fibrillogenesis inhibition was found to be initiated by binding with the aggregation-prone region of the lysozyme. We believe the results from this research may contribute to the development of effective therapeutics for amyloidoses.

Examining the inhibitory potency of food additive fast green FCF against amyloid fibrillogenesis under acidic conditions.

More than thirty human proteins and/or peptides can fold incorrectly to form amyloid deposits associated with several protein aggregation diseases. No cure is currently available for treating these diseases. This work is aimed at examining the inhibitory potency of fast green FCF, a biocompatible dye, toward the fibrillogenesis/aggregation of lysozyme. As verified by ThT binding assay along with transmission electron microscopy, fast green FCF was observed to suppress the generation of lysozyme fibrils in a concentration-dependent manner. We next used circular dichroism absorption spectroscopy, ANS fluorescence spectroscopy, and SDS-PAGE to characterize the structural alterations in lysozyme samples upon the addition of fast green FCF. Furthermore, experiments with the addition of fast green FCF at different time points of incubation showed that fast green FCF also exhibited disaggregating activity against the preformed/existing lysozyme fibrils. We believe that the results from this study suggest a potential therapeutic role of biocompatible molecules in treating or preventing protein aggregation diseases.

Bioactive saccharide-conjugated polypeptide micelles for acid-triggered doxorubicin delivery.

The synthesis and self-assembly of lactobionolactone-conjugated poly(l-glutamic acid)-b-poly(l-phenylalanine) amphiphilic block copolypeptides (Lac-PGA-b-PPhe) and their evaluation for anticancer drug doxorubicin (DOX) delivery have been investigated. Lactobionolactone was functionalized with the azide group and successfully conjugated with the terminal alkyne groups on the polypeptides through click reaction and these amphiphilic glycopolypeptides self-assembled to form micelles with bioactive galactose units on the particle surface as confirmed by selective lectin binding experiments. Drug release experiments showed that DOX released faster from saccharide-conjugated micelles under acidic conditions than under neutral conditions. The DOX-loaded, saccharide-conjugated micelles exhibited higher cytotoxicity toward HepG2 tumor cells than free DOX and saccharide-free micelles loaded with DOX at low concentrations, suggesting that the saccharide-conjugated micelles can effectively bind to the cells through specific recognition and subsequently the higher uptake of saccharide-conjugated micelles led to higher drug release and cytotoxicity under pH-sensitive conditions.

Carnosine's effect on amyloid fibril formation and induced cytotoxicity of lysozyme.

Carnosine, a common dipeptide in mammals, has previously been shown to dissemble alpha-crystallin amyloid fibrils. To date, the dipeptide's anti-fibrillogensis effect has not been thoroughly characterized in other proteins. For a more complete understanding of carnosine's mechanism of action in amyloid fibril inhibition, we have investigated the effect of the dipeptide on lysozyme fibril formation and induced cytotoxicity in human neuroblastoma SH-SY5Y cells. Our study demonstrates a positive correlation between the concentration and inhibitory effect of carnosine against lysozyme fibril formation. Molecular docking results show carnosine's mechanism of fibrillogenesis inhibition may be initiated by binding with the aggregation-prone region of the protein. The dipeptide attenuates the amyloid fibril-induced cytotoxicity of human neuronal cells by reducing both apoptotic and necrotic cell deaths. Our study provides solid support for carnosine's amyloid fibril inhibitory property and its effect against fibril-induced cytotoxicity in SH-SY5Y cells. The additional insights gained herein may pave way to the discovery of other small molecules that may exert similar effects against amyloid fibril formation and its associated neurodegenerative diseases.