Tryptophan extends the life of cytochrome P450.

Powerfully oxidizing enzymes need protective mechanisms to prevent self-destruction. The flavocytochrome P450 BM3 from Priestia megaterium (P450BM3) is a self-sufficient monooxygenase that hydroxylates fatty acid substrates using O2 and NADPH as co-substrates. Hydroxylation of long-chain fatty acids (≥C14) is well coupled to O2 and NADPH consumption, but shorter chains (≤C12) are more poorly coupled. Hydroxylation of p-nitrophenoxydodecanoic acid by P450BM3 produces a spectrophotometrically detectable product wherein the coupling of NADPH consumption to product formation is just 10%. Moreover, the rate of NADPH consumption is 1.8 times that of O2 consumption, indicating that an oxidase uncoupling pathway is operative. Measurements of the total number of enzyme turnovers before inactivation (TTN) indicate that higher NADPH concentrations increase TTN. At lower NADPH levels, added ascorbate increases TTN, while a W96H mutation leads to a decrease. The W96 residue is about 7 Å from the P450BM3 heme and serves as a gateway residue in a tryptophan/tyrosine (W/Y) hole transport chain from the heme to a surface tyrosine residue. The data indicate that two oxidase pathways protect the enzyme from damage by intercepting the powerfully oxidizing enzyme intermediate (Compound I) and returning it to its resting state. At high NADPH concentrations, reducing equivalents from the flavoprotein are delivered to Compound I by the usual reductase pathway. When NADPH is not abundant, however, oxidizing equivalents from Compound I can traverse a W/Y chain, arriving at the enzyme surface where they are scavenged by reductants. Ubiquitous tryptophan/tyrosine chains in highly oxidizing enzymes likely perform similar protective functions.

Tryptophan-96 in cytochrome P450 BM3 plays a key role in enzyme survival.

Flavocytochrome P450 from Bacillus megaterium (P450BM3 ) is a natural fusion protein containing reductase and heme domains. In the presence of NADPH and dioxygen the enzyme catalyses the hydroxylation of long-chain fatty acids. Analysis of the P450BM3 structure reveals chains of closely spaced tryptophan and tyrosine residues that might serve as pathways for high-potential oxidizing equivalents to escape from the heme active site when substrate oxidation is not possible. Our investigations of the total number of enzyme turnovers before deactivation have revealed that replacement of selected tryptophan and tyrosine residues with redox inactive groups leads to a twofold reduction in enzyme survival time. Tryptophan-96 is critical for prolonging enzyme activity, suggesting a key protective role for this residue.

Surface cysteines could protect the SARS-CoV-2 main protease from oxidative damage.

The SARS-CoV-2 main protease (Mpro) is responsible for cleaving twelve nonstructural proteins from the viral polyprotein. Mpro, a cysteine protease, is characterized by a large number of noncatalytic cysteine (Cys) residues, none involved in disulfide bonds. In the absence of a tertiary-structure stabilizing role for these residues, a possible alternative is that they are involved in redox processes. We report experimental work in support of a proposal that surface cysteines on Mpro can protect the active-site Cys145 from oxidation by reactive oxygen species (ROS). In investigations of enzyme kinetics, we found that mutating three surface cysteines to serines did not greatly affect activity, which in turn indicates that these cysteines could protect Cys145 from oxidative damage.

Copper(II) Binding to the Intrinsically Disordered C-Terminal Peptide of SARS-CoV-2 Virulence Factor Nsp1.

The first encoded SARS-CoV-2 protein (Nsp1) binds to the human 40S ribosome and blocks synthesis of host proteins, thereby inhibiting critical elements of the innate immune response. The final 33 residues of the natively unstructured Nsp1 C-terminus adopt a helix-turn-helix geometry upon binding to the ribosome. We have characterized the fluctuating conformations of this peptide using circular dichroism spectroscopy along with measurements of tryptophan fluorescence and energy transfer. Tryptophan fluorescence decay kinetics reveal that copper(II) binds to the peptide at micromolar concentrations, and electron paramagnetic resonance spectroscopy indicates that the metal ion coordinates to the lone histidine residue.

Recent advances in design and applications of biomimetic self-assembled peptide hydrogels for hard tissue regeneration.

ABSTRACT: The development of natural biomaterials applied for hard tissue repair and regeneration is of great importance, especially in societies with a large elderly population. Self-assembled peptide hydrogels are a new generation of biomaterials that provide excellent biocompatibility, tunable mechanical stability, injectability, trigger capability, lack of immunogenic reactions, and the ability to load cells and active pharmaceutical agents for tissue regeneration. Peptide-based hydrogels are ideal templates for the deposition of hydroxyapatite crystals, which can mimic the extracellular matrix. Thus, peptide-based hydrogels enhance hard tissue repair and regeneration compared to conventional methods. This review presents three major self-assembled peptide hydrogels with potential application for bone and dental tissue regeneration, including ionic self-complementary peptides, amphiphilic (surfactant-like) peptides, and triple-helix (collagen-like) peptides. Special attention is given to the main bioactive peptides, the role and importance of self-assembled peptide hydrogels, and a brief overview on molecular simulation of self-assembled peptide hydrogels applied for bone and dental tissue engineering and regeneration.

Monitoring the heme iron state in horseradish peroxidase to detect ultratrace amounts of hydrogen peroxide in alcohols.

Despite the importance of hydrogen peroxide (H2O2) in initiating oxidative damage and its connection to various diseases, the detection of low concentrations of H2O2 (<10 µM) is still limited using current methods, particularly in non-aqueous systems. One of the most common methods is based on examining the color change of a reducing substrate upon oxidation using UV/Vis spectrophotometry, fluorophotometry and/or paper test strips. In this study, we show that this method encounters low efficiency and sensitivity for detection of ultratrace amounts of H2O2 in non-aqueous media. Thus, we have developed a simple, fast, accurate and inexpensive method based on UV/Vis spectrophotometry to detect H2O2 in non-aqueous systems, such as alcohols. In this regard, we demonstrate that monitoring the Soret and Q-band regions of high-valent iron-oxo (ferryl heme) intermediates in horseradish peroxidase (HRP) is well suited to detect ultratrace amounts of H2O2 impurities in alcohols in the range of 0.001-1000 µM using UV/Vis spectrophotometry. We monitor the optical spectra of HRP solution for the red shift in the Soret and Q-band regions upon the addition of alcohols with H2O2 impurity. We also monitor the reversibility of this shift to the original wavelength over time to check the spontaneous decay of ferryl intermediates to the ferric state. Thus, we have found that the ferryl intermediates of HRP can be used for the detection of H2O2 in alcohols at µg L-1 levels through via UV/Vis spectrophotometric method.

l-Histidine Crystals as Efficient Vehicles to Deliver Hydrophobic Molecules.

l-Histidine (l-His) molecules can form highly ordered fluorescent crystals with tunable size and geometry. The polymorph A crystal of l-His contains hydrophobic domains within the structure's interior. Here, we demonstrate that these hydrophobic domains can serve as vehicles for highly efficient entrapment and transport of hydrophobic small molecules. This strategy shows the ability of l-His crystals to mask the hydrophobicity of various small molecules, helping to address issues related to their poor solubility and low bioavailability. Furthermore, we demonstrate that we can modify the surface of these crystals to define their function, suggesting the significance of l-His crystals in designing site-specific and bioresponsive platforms. As a demonstration, we use l-His crystals with loaded doxorubicin, featuring hyaluronic acid covalently bonded on the crystal surface, controlling its release in response to hyaluronidase. This strategy for entrapment of hydrophobic small molecules suggests the potential of l-His crystals for targeted drug-delivery applications.

Ultrastable Water-in-Oil High Internal Phase Emulsions Featuring Interfacial and Biphasic Network Stabilization.

In this work, we present gel-in-gel water-in-oil (W/O) high internal phase emulsions (HIPEs) that feature high stability by structuring both phases of the emulsion. Compared to significant advances made in oil-in-water (O/W) HIPEs, W/O HIPEs are extremely unstable and difficult to generate without introducing high concentrations of surfactants. Another main challenge is the low viscosity of both water and oil phases which promotes the instability of W/O HIPEs. Here, we demonstrate ultrastable W/O HIPEs that feature biphasic structuring, in which hydrogels are dispersed in oleogels, and self-forming, low-concentration interfacial Pickering crystals provide added stability. These W/O HIPEs exhibit high tolerance toward pH shock and destabilizing environments. In addition, this novel ultrastable gel-in-gel W/O HIPE is sustainable and made solely with natural ingredients without the addition of any synthetic stabilizers. By applying phase structuring within the HIPEs through the addition of various carrageenans and beeswax as structurants, we can increase the emulsion's stability and viscoelastic rheological properties. The performance of these gel-in-gel W/O HIPEs holds promise for a wide range of applications. As a proof of concept, we demonstrated herein the application as a gelled delivery system that enables the co-delivery of hydrophilic and hydrophobic materials at maximized loads, demonstrating high resistance to gastrointestinal pHs and a controlled-release profile.

Catalyzed Oxidation of Carotenoids by Lactoperoxidase in the Presence of Ethanol.

The discovery of the lactoperoxidase system as a biocatalyst in milk was a landmark finding. The activation of this system using hydrogen peroxide (H2O2) raised hopes for oxidation of various organic substrates. The involvement of lactoperoxidase system in the catalyzed-oxidation of carotenoids in the whey proteins, and the effect of various solvents on carotenoids' oxidation reaction rate has been studied. However, there is no evidence for this reaction without the addition of oxidizing agents, such as peroxides. Here, we reveal that carotenoids are oxidized through the addition of just ethanol in the presence of lactoperoxidase. The oxidation of carotenoids through this exquisite strategy is ∼360 times faster than harnessing the lactoperoxidase system in whey proteins via the addition of hydrogen peroxide. Bearing in mind that ethanol is not an oxidizing agent, this observation suggests a potential paradigm shift in our understanding of lactoperoxidase and catalyzed oxidation in biochemical systems.

Solvent-mediated pressure-treated bixin-casein complexation for targeted color delivery.

Carryover color in the whey fluid is one of the major challenges faced by the cheese manufacturing industry. In this study, we describe a solvent-mediated high-pressure process to complexate bixin and casein micelles as a novel strategy for color delivery. High pressures (120 and 240 MPa) and added ethanol resulted in change in casein hydrophobicity by exposure of tryptophan residues, as confirmed by spectroscopic methods. The incorporation of bixin resulted in a marked quenching of the fluorescence peak associated with tryptophan. A simulated coagulation study has shown that pressure-treated complexes resulted in whey powder with significantly lower a* values than unbleached whey, whereas no significant differences were observed for b* values. The results suggest that complexes can produce curds with a color similar to that using commercial annatto powder and whey powder with equal or superior color quality than obtained with chemical bleaching.

Modulation of whey protein-kappa carrageenan hydrogel properties via enzymatic protein modification.

Treatment of whey protein isolate (WPI; 1 to 25% w/w) in heated κ-carrageenan (KC; 2% w/w) slurries with protease and/or transglutaminase modulated the properties of the hydrogels formed after cooling. Observation of peak compression stress and strain at gel rupture showed WPI incorporation at 1, 5 and 10% (w/w) significantly reduced the strength and deformability of 2% (w/w) KC gels. Treatment of WPI solutions in KC slurries with Alcalase 2.4L was shown by both SDS-Page and size exclusion HPLC to reduce protein/peptide molecular weight distributions below 10 kDa, with large portions below 1 kDa. This peptide size reduction within the KC matrix produced more translucent gels with a more organized wall and cell structure as observed by SEM, which resulted in gels with observed rupture stress/strain levels similar to 2% KC alone. Transglutaminase treatment of WPI-KC slurries showed the reverse behavior, reducing gel translucency, strength and deformability. At these loadings, WPI-KC gel strength/deformability appears to relate decreasing peptide size to gel behavior trending towards KC-only gels; suggesting peptide size modulation in protein-carbohydrate complexes will allow significant tailoring of texture for the delivery of protein/peptide rich gelled products.

Annatto-entrapped casein-chitosan complexes improve whey color quality after acid coagulation of milk.

A fraction of annatto is often transferred to the whey fluid during Cheddar cheese processing, which negatively impacts the visual and sensory attributes of the resultant whey powder. Alternatives to reduce the color in the powder are still needed. In this study, casein-chitosan complexes were prepared to deliver annatto preferentially to the curd and reduce the amount of carryover colorant in whey powder. These complexes were relatively spherical, with a mean complex diameter of 8.3 ±â€¯1.9 µm, zeta-potential of +39.4 ±â€¯1.3 mV, and entrapment efficiency of 38.2 ±â€¯3.1%. FT-IR spectroscopy confirmed the electrostatic interaction between casein and chitosan. Complexes and commercial annatto powder were incorporated into homogenized, reduced-fat, and fat-free milk, and subjected to acid coagulation. Whey powder produced from casein-chitosan-complex-treated samples exhibited better color quality than that prepared with annatto powder, indicating that the approach considered in this study was efficient in preventing the migration of colorant to the whey.

Water-in-oil-in-water emulsion obtained by glass microfluidic device for protection and heat-triggered release of natural pigments.

Anthocyanins and norbixin are natural pigments used in food; however, they are unstable. The aim of this study was to evaluate the microencapsulation technique to protect these pigments. Elderberry extract (source of anthocyanins) and norbixin were encapsulated using a microfluidic device with palm oil as middle phase in a water-in-oil-in-water emulsion. The formulations were characterized for morphology, particle size, encapsulation efficiency, zeta potential, color release under heating, Fourier transform infrared spectrophotometry, and color stability under different conditions. Spherical, mononucleated microcapsules, with particle size of 187-190 µm (elderberry) and 164-184 µm (norbixin), and with encapsulation efficiencies values of 47.80-54.87% (elderberry) and 49.18-74.73% (norbixin) were obtained. The formulations showed high color retention, with the encapsulated elderberry extract stored at pH 3.0 being the most stable. This study shows that the microencapsulation of these pigments using a microfluidic device provided protection, and represents a new method for anthocyanins and norbixin delivery in foods.

Enhancing the physicochemical stability of ß-carotene solid lipid nanoparticle (SLNP) using whey protein isolate.

ß-Carotene is a nutraceutical that acts as a coloring agent and as pro-vitamin A, but its incorporation into foods is limited because of its hydrophobicity and low chemical stability. The aim of this study was to improve the physicochemical stability of ß-carotene by encapsulating into solid lipid nanoparticles (SLNPs) containing palmitic acid and corn oil, stabilized using whey protein isolate (WPI). The palmitic acid crystals covered the surface of the oil droplets and formed a solid shell to protect the encapsulated ß-carotene. Corn oil decreased the exclusion of ß-carotene from the solid lipid matrix to the surface of SLNPs. WPI increased the colloidal stability of the system, and improved ß-carotene oxidative stability. The rate of color fading due to ß-carotene degradation increased with increasing temperature and was faster at lower pH. Lower ionic strengths had a slight impact on ß-carotene degradation, while higher ionic strengths accelerated ß-carotene breakdown.

Controlling the Release from Enzyme-Responsive Microcapsules with a Smart Natural Shell.

We design a natural and simple core-shell-structured microcapsule, which releases its cargo only when exposed to lipase. The cargo is entrapped inside a gel matrix, which is surrounded by a double-layer shell containing an inner solid lipid layer and an outer polymer layer. This outer polymer layer can be designed according to the intended biological system and is responsible for protecting the microcapsule architecture and transporting the cargo to the desired site of action. The lipid layer contains natural ester bonds, which are digested by lipase, controlling the release of cargo from the microcapsule core. To demonstrate the feasibility of this approach, our model system includes a colorant bixin entrapped inside a κ-carrageenan gel matrix. This core is surrounded by an inner beeswax-palmitic acid layer and an outer casein-poloxamer 338 layer. These fabricated microcapsules are then applied into Cheddar cheese, where they selectively color the cheese matrix.

Optimization of microcapsules shell structure to preserve labile compounds: A comparison between microfluidics and conventional homogenization method.

A new technique is presented to optimize the formulation of microcapsules loaded with labile compounds. Fish oil was loaded into the microcapsule core and protected with a shell composed of whey protein microgel/beet pectin complexes. The microcapsules were formed using two different methods: microfluidics and homogenization. The microcapsules were further classified into three sub-groups. The first group was the microcapsules cross-linked with laccase (MCL), the second group was the microcapsules cross-linked with divalent cationic CaCl2 salt (MCS), and the third group consisted of control microcapsules (CM), with no cross-linking. The microfluidics method enabled tracking of the effect of the shell cross-linking ability of laccase, or CaCl2, on microcapsules. It was demonstrated that MCL obtained by microfluidics are more physicochemically stable than those produced via a homogenizer. The effect of cross-linking agents on the microcapsules were more significant when the microcapsules were produced by microfluidics.

Improving oxidative stability of echium oil emulsions fabricated by Microfluidics: Effect of ionic gelation and phenolic compounds.

Echium oil is rich in omega-3 fatty acids, which are important because of their benefits to human health; it is, however, unstable. The objective of this work was the coencapsulation of echium oil and quercetin or sinapic acid by microfluidic and ionic gelation techniques. The treatments were analyzed utilizing optical and scanning electron microscopy, encapsulation yield, particle size, thermogravimetry, Fourier transform infrared spectroscopy, stability under stress conditions, and oil oxidative/phenolic compound stability for 30days at 40°C. High encapsulation yield values were obtained (91-97% and 77-90% for the phenolic compounds and oil) and the encapsulated oil was almost seven times more stable than the non-encapsulated oil (0.34 vs 2.42mgMDA/kg oil for encapsulated and non-encapsulated oil, respectively). Encapsulation was shown to promote oxidative stability, allowing new vehicles for the application of these compounds in food without the use of solvents and high temperature.

Preservation of anthocyanins in solid lipid nanoparticles: Optimization of a microemulsion dilution method using the Placket-Burman and Box-Behnken designs.

Anthocyanins are the main polyphenol components from red cabbage (Brassica oleracea L. Var. Capitata f. Rubra) extracts that have inherent antioxidant activities. Anthocyanins are effectively stable in acidic gastric digestion conditions, with nearly 100% phenol content recovery. However, the total phenol content recovery after simulated pancreatic digestion was approximately 25%. To protect anthocyanins against harsh environmental conditions (e.g., pH and temperature), solid lipid nanoparticles were prepared by the dilution of water in oil (w/o) microemulsions containing anthocyanins in aqueous media. The formulations were characterized for particle size and encapsulation efficiency. The formulation parameters (e.g., volume of the internal aqueous phase, homogenization time and the percentages of total lipid, total surfactant or stabilizer) were optimized using the Placket-Burman and Box-Behnken experimental designs. Entrapment efficiency (89.2 ± 0.3%) was calculated when the mean particle size was 455 ± 2 nm. A scanning electron microscopy study revealed the spherical morphology of the particles.

Optimization of ultrasound assisted extraction of anthocyanins from red cabbage using Taguchi design method.

There is a growing demand for developing suitable and more efficient extraction of active compounds from the plants and ultrasound is one of these novel methodologies. Moreover, the experimental set up to reach an appropriate condition for an optimum yield is demanding and time consuming. In the present study, Taguchi L9 orthogonal design was applied to optimize the process parameters (output power, time, temperature and pulse mode) for ultrasound assisted extraction of anthocyanins from red cabbage and the concluding yield of anthocyanin was measured by pH differential method. The statistical analysis revealed that the most important factors contributing to the extraction efficiency were time, temperature and power, respectively and the optimum condition was at 30 min, 15 °C and 100 W which could result the maximum anthocyanin yield of about 20.9 mg/L. The theoretical result was confirmed experimentally by carrying out the trials at the optimum condition and evaluating the actual yield.