Chymotrypsin Inhibitor Research Articles

Synergistic stabilization of a double mutant in chymotrypsin inhibitor 2 from a library screen in E. coli

Most single point mutations destabilize folded proteins. Mutations that stabilize a protein typically only have a small effect and multiple mutations are often needed to substantially increase the stability. Multiple point mutations may act synergistically on the stability, and it is often not straightforward to predict their combined effect from the individual contributions. Here, we have applied an efficient in-cell assay in E. coli to select variants of the barley chymotrypsin inhibitor 2 with increased stability. We find two variants that are more than 3.8 kJ mol−1 more stable than the wild-type. In one case, the increased stability is the effect of the single substitution D55G. The other case is a double mutant, L49I/I57V, which is 5.1 kJ mol−1 more stable than the sum of the effects of the individual mutations. In addition to demonstrating the strength of our selection system for finding stabilizing mutations, our work also demonstrate how subtle conformational effects may modulate stability.

Effects of Alpha-Connexin Carboxyl-Terminal Peptide (aCT1) and Bowman-Birk Protease Inhibitor (BBI) on Canine Oral Mucosal Melanoma (OMM) Cells.

Oral mucosal melanomas (OMM) are aggressive cancers in dogs, and are good models for human OMM. Gap junctions are composed of connexin units, which may have altered expression patterns and/or subcellular localization in cancer cells. Cell-to-cell communication by gap junctions is often impaired in cancer cells, including in melanomas. Meanwhile, the upregulated expression of the gap junction protein connexin 43 (Cx43) inhibits melanoma progression. The α-connexin carboxyl-terminal (aCT1) peptide reportedly maintains Cx43 expression and cell-cell communication in human mammary cells and increases the communication activity through gap junctions in functional assays, therefore causing decreased cell proliferation. The Bowman-Birk protease inhibitor (BBI), a component of soybeans, induces Cx43 expression in several tumor cells as a trypsin–chymotrypsin inhibition function, with antineoplastic effects. This study investigated the effect of aCT1 peptide and BBI treatment, alone or in combination, on TLM1 canine melanoma cell viability. Cell viability after treatment with aCT1, the reverse sequence peptide (R-pep), and/or BBI for 5 days was analyzed by PrestoBlue assay. Immunofluorescence was used to observe Cx43 localization and expression. aCT1 (200 μM) alone did not significantly decrease cell viability in TLM1 cells, whereas BBI (400 μg/ml) alone significantly decreased the TLM1 viability. Combined treatment with both aCT1 (200 μM) and BBI (400 μg/ml) significantly decreased cell viability in TLM1 cells. Cx43 expression, as identified by immunostainings in TLM1 cells, was increased in the cell membrane after the combination treatment with BBI and aCT1. This dual treatment can be combined to achieve the anticancer activity, possibly by increasing Cx 43 expression and affecting Cx43 migration to the cell membrane. In conclusion, a treatment strategy targeting Cx43 with BBI and aCT1 may possibly lead to new effective therapies for canine OMM.

Aggregation-enhanced emission of metal nanoclusters triggered by peptide self-assembly and application in chymotrypsin inhibitor screening

Herein, a new strategy is developed to induce the aggregation of glutathione-capped gold nanoclusters (GSH-AuNCs) by self-assembly of peptide. The diphenylalanine peptide (L-Phe-l-Phe, FF) self-assembles in aqueous solution into the nano-net structure (FF-Nanonet, FFN). With self-assembly of FF, GSH-AuNCs aggregate in the network of FFN to form a dual-emission probe, called FF-N@GSH-AuNCs. One emission is from the self-assembled FFN at 470 nm, and the other one is the aggregation-induced enhanced emission (AIEE) of the GSH-AuNCs at 575 nm. Due to the hydrolysis of FF by chymotrypsin (CHT), the peptide bond on the carboxyl side of the phenylalanine is cleaved and the AIEE from GSH-AuNCs becomes faint because the released nanoclusters are dispersed in the solution again. Therefore, FF-N@GSH-AuNCs can be applied to screen and evaluate the CHT inhibitors. The inhibitory effects of four ingredients from natural products (i.e. epigallocatechin gallate (EGCG), tannic acid, oleanolic acid, and ursolic acid) on CHT activity have been studied. Oleanolic acid and ursolic acid are invalid. The half maximal inhibitory concentration (IC 50) values of EGCG and tannic acid are 13.9 and 19.2 μM, respectively. The screening results show that these two compounds have significant inhibitory effects on CHT.

Photophysical and elecron-transfer reaction properties of tris(2,2’-bipyridine)ruthenium(II)-based inhibitors that covalently bound to the active site of chymotrypsin

We have prepared two tris(2,2’-bipyridine)ruthenium(II) complex-based new chymotrypsin (CHT) inhibitors, [Ru(bpy)2(bsfbpy)]Cl2 and [Ru(bsfbpy)3]Cl2 {bsfbpy = 4-methyl-2,2′-bipyridine-4′-carboxylic acid [2-(4-benzenesulfonylfluoride)-ethyl]-amide}. They were covalently modified with Ser195 in the active site of CHT. Photoinduced electron-transfer (ET) systems comprising a Ru-CHT complex and an electron acceptor such as methylviologen (MV2+) were studied. After photoirradiation, the photo-excited triplet state of 2CHT-3{[Ru(bsfbpy)3]2+}* and CHT-3{[Ru(bpy)2(bsfbpy)]}* were both quenched by MV2+ through an intermolecular photoinduced ET mechanism, forming oxidized 2CHT-[Ru(bsfbpy)3]3+ and CHT-[Ru(bpy)2(bsfbpy)]3+, respectively. The intramolecular ET from the amino acid residue of Tyr94 near the active site of CHT proceeded. Transient absorption spectra display the formation of a Tyr●+ radical in the 2CHT-[Ru(bsfbpy)3]2+-MV2+ and the CHT-[Ru(bpy)2(bsfbpy)]2+-MV2+ systems, having a lifetime of 2.24 ms for 2CHT-[Ru(bsfbpy)3]2+-MV2+. The present model system could be one of the available models for the further photochemical reaction of Tyr•+ formed by intramolecular photoinduced ET reactions within a protein scaffold. To clarify the photophysical properties of these Ru inhibitors and their multi step photoinduced ET reactions in the protein matrix, the detailed ET reaction kinetics are discussed.

Bowman-Birk inhibitor in soybean: Genetic variability in relation to total trypsin inhibitor activity and elimination of Kunitz trypsin inhibitor

Bowman-Birk inhibitor (BBI) is a soybean seed serine protease inhibitor whose nutraceutical properties far exceed its anti-nutritional property arising from its trypsin and chymotrypsin inhibitor activity. High BBI soybean genotypes are sought for the commercial preparation of this nutraceutical biomolecule while low BBI content in Kunitz trypsin inhibitor (KTI)-free genetic background is desirable in soymeal manufacturing. In the present investigation, monoclonal antibody assay of 95 soybean genotypes revealed 11-fold genetic variation for BBI concentration. The study led to the identification of 3 very high (>20 mg/g defatted soy flour) and 5 very low BBI (< 4 mg/g defatted soy flour) soybean genotypes. Contribution of BBI to total trypsin inhibition activity ranged from 2.2 to 53.5% with average value of 11.6%. Genotypes with varying level of BBI raised consecutively for two years exhibited non-significant (p>0.05) effect of growing year on the trait. BBI content in BC3F2 derived KTI-free lines was at par with the recurrent parent. Low and high BBI content genotypes with diverse genetic background identified in the study may be exploited to develop mapping population to identify genomic regions underlying BBI in soybean.

Stabilizing or Destabilizing: Simulations of Chymotrypsin Inhibitor 2 under Crowding Reveal Existence of a Crossover Temperature.

The effect of macromolecular crowding on the stability of proteins can change with temperature. This dependence might reveal a delicate balance between two factors: the entropic excluded volume and the stability-modulating quinary interactions. Here we computationally investigate the thermal stability of the native state of chymotrypsin inhibitor 2 (CI2), which was previously shown by experiments to be destabilized by protein crowders at room temperature. Mimicking experimental conditions, our enhanced-sampling atomistic simulations of CI2 surrounded by lysozyme and bovine serum albumin reproduce this destabilization but also provide evidence of a crossover temperature above which lysozyme is found to become stabilizing, as previously predicted by analysis of thermodynamic data. We relate this crossover to the different CI2-crowder interactions and the local packing experienced by CI2. In fact, we clearly show that the pronounced stabilization induced by lysozyme at high temperatures stems from the tight local packing created around CI2 by this smaller crowder.

Can 3D-Domain-Swapping Help Understand Polyq Sequences?

Can 3D-domain-swapping help understand polyQ sequences? Proteins with expanded polyQ repeats can form neurological disease-associated amyloids. The regions surrounding the polyQ sequences are often not resolved in 3D structures. Thus, understanding the structural details of polyQ stretches even in non-amyloid contexts may provide insight into the mechanism of amyloidogenesis. Previous work on the chymotrypsin inhibitor 2 (CI2) and ribonuclease A (RNase-A) has shown that insertion of polyQ stretches can induce 3D-domain-swapping in proteins.

The role of hydrogen bond in catalytic triad of serine proteases

In order to investigate the origin of catalytic power for serine proteases, the role of the hydrogen bond in the catalytic triad was studied in the proteolysis process of the peptides chymotrypsin inhibitor 2 (CI2), MCTI-A, and a hexapeptide (SUB), respectively. We first calculated the free energy profile of the proton transfer between His and Asp residues of the catalytic triad in the enzyme-substrate state and transition state by employing QM/MM molecular dynamics simulations. The results show that a low-barrier hydrogen bond (LBHB) only forms in the transition state of the acylation of CI2, while it is a normal hydrogen bond in the acylation of MCTI-A or SUB. In addition, the change of the hydrogen bond strength is much larger in CI2 and SUB systems than in MCTI-A system, which decreases the acylation energy barrier significantly for CI2 and SUB. Clearly, a LBHB formed in the transition state region helps accelerate the acylation reaction. But to our surprise, a normal hydrogen bond can also help to decrease the energy barrier. The key to reducing the reaction barrier is the increment of hydrogen bond strength in the transition state state, whether it is a LBHB or not. Our studies cast new light on the role of the hydrogen bond in the catalytic triad, and help to understand the catalytic triad of serine proteases.

Profiles of nutritional, bioactive compounds and cytotoxic activity of Dwarf date palm (Phoenix loureiroi) fruit

The wild edible fruits are basic food sources and these contain potential dietary compounds which provide a healthy diet. Such wild edible fruits play an important role in supplementing the diet and livelihood of rural/tribal communities. Thus, the present study determined the nutritive values of Phoenix loureiroi fruit by screening the phytochemical compounds, organic acids and polyphenolics along with the evaluation of cytotoxic activity. The nutritional composition revealed high contents of total proteins (36.30 mg BSAE/g dry fruit), total free amino acids (6.15 mg LE/g dry fruit), reducing sugars (85.40 mg GE/g dry fruit), vitamin E (2.50 mg TE/g dry fruit) and sodium (1.26 mg/g dry fruit) at Rutab stage of maturation. Anti-nutritional compositions of fruit were found to be chymotrypsin inhibitors (3.31 CIU/mg protein), trypsin inhibitors (0.01 TIU/mg protein) and phytate (0.42 g phytate/100 g dry fruit) contents. High-performance thin-layer chromatography (HPTLC) separation demonstrated that fruit contains a high amount of essential (tryptophan, 41.30 mg/g dry fruit) and non-essential (tyrosine, 25.70 mg/g dry fruit) amino acids. The total phenolics concentration was found to be 49.68 mg GAE/100 g dry fruit. Liquid chromatography analyses revealed that the fruit contained high amounts of fructose (0.12 mg/g dry fruit), ascorbic acid (2.80 mg/g dry fruit), catechin (3.78 mg/g dry fruit) and succinic acid (1.57 mg/g dry fruit). The bioactive compounds of fruit extract inhibited the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical (IC50 = 8.51 µg/mL). The cell viability value (65.87%) on fibroblast L929 cell lines indicates fruit extract was safe up to 1000 µg/mL concentration. The results revealed that the P. loureiroi fruit was rich in nutraceutical compounds and could be used for the development of safe natural products.

Evaluation of the inhibitory potential of pro-peptide region as the inhibitor of the digestive chymotrypsin of cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae), based on in silico studies

The cotton bollworm, Helicoverpa armigera (Hubner),is a wide host range pest that causes severe economic damages to agricultural crops in Iran and all around the world. During recent years, chemical insecticides have been used as the most effective strategy in control of this pest, but due to their hazardous effects, most of the researches are being conducted to offer an alternative approach for chemical control. In this regard, digestive systems, in particular inhibition of insect digestive enzymes, are considered as a target for pest control. Here, we used the original pro-region of H. armigera chymotrypsin as a potent and specific inhibitor of the pest enzyme. The structural model of the insect chymotrypsin was predicted based on homology modeling and the crystal structure of Bos taurus L. as template. The reliability of the model was assessed using VERIFY_3D, ERRAT, PROCHECK, WHAT-IF and Z-scores, and the results confirmed that the predicted structural model has an appropriate quality. Moreover, molecular docking simulations between the predicted structural model of enzyme and designed peptide showed that the inhibitor peptide has the most appropriate docking score and total binding energy for interactions with the insect chymotrypsin’s active site. However, it showed a weak potential for interaction with Sus scrofa L. chymotrypsin, as a representative of the mammalian enzyme. The results of this report indicate the importance of computational studies in design and selection of the favored inhibitor pro-peptides against the target enzymes. Such inhibitors can be further suggested as a replacement of chemical pesticides for controlling of H. armigera as well as the other pests in future.

Effect of high-temperature, short-time cooking conditions on in vitro protein digestibility, enzyme inhibitor activity and amino acid profile of selected legume grains

African yam beans (Sphenostylis stenocarpa), Bambara groundnut (Vigna subterranean) and Pigeon pea (Cajanus cajan) flours were extruded in a single screw extruder at two extrusion temperatures; 100 °C and 140 °C, and the effect of extrusion cooking temperature on the chemical composition; crude protein, crude fibre, ether extract and nitrogen-free extracts, protein digestibility, enzyme inhibitor activity and amino acid profiles was investigated. The crude protein, amino acid profile and ether extract of the grain legumes were negatively affected (p < 0.05) by the extrusion cooking process, with a significant increase in nitrogen-free extracts for all grain legumes, and increased crude fibre of Bambara groundnut and Pigeon pea extrudates. Extrusion cooking of African yam beans and Pigeon pea produced extrudates with significantly lower trypsin, chymotrypsin and amylase inhibitor activity as well as improved protein digestibility. However, extrusion cooking did not modify the chymotrypsin and amylase inhibitor activity of Bambara groundnut extrudates. Extrusion cooking at 140 °C compared to 100 °C significantly reduced the protein quality of extrudates resulting in 22.94–51.27%, 5.11–25.18%, and 7.78–38.42% reduction in amino acid concentration of African yam beans, Bambara groundnut and Pigeon pea, respectively.

P.417 The emotional disturbances in young patients with type 1 diabetes using various methods of insulin therapy by four-dimensional symptom questionnaire

The southern green stink bug, Nezara viridula is a polyphagous pest of commercially important crops during both nymph and adult stages. This insect has recently transitioned from a secondary agricultural pest to one of primary concern. Novel management solutions are needed due to the limited effectiveness of current control strategies. We performed biochemical and transcriptomic analyses to characterize digestive enzymes in the salivary glands and along midgut tissues of N. viridula nymphs and adults fed on sweet corn. The digestive profiles were more distinct between midgut regions (M1 to M3) than between life stages. Aminopeptidase and chymotrypsin activities declined from the M1 (anterior) toward the M3 midgut region. Cysteine protease activity was higher in the M2 and M3 regions than in M1. Differences in sensitivity to chymotrypsin inhibitors between midgut regions suggest that distinct genes or isoforms are expressed in different regions of the gut. In nymphs, DNA and RNA degradation was higher in M1 than in M3. Adult nuclease activity was low across all midgut regions, but high in salivary glands. The differences in protease activities are reflected by transcriptomic data and functional enrichment of GO terms. Together, our results show that different regions of the digestive tract of N. viridula have specific and distinct digestive properties, and increase our understanding of the physiology of this organism.

The first Kunitz-type proteins from a viperid venom that potentiate neuromuscular transmission

Kunitz-type proteins that interfere with neuronal transmission have been thus far exclusively detected in venoms of elapid snakes. Here, we report for the first time that such proteins are also present in the venom of a viperid snake. From the venom of the nose-horned viper (Vipera ammodytes ammodytes; Vaa), we isolated Kunitz-type chymotrypsin inhibitors (VaaChi) and demonstrated that these molecules also significantly increase the amplitudes of an indirectly evoked simple muscle contraction of the mouse hemidiaphragm, the end-plate potential and the miniature end-plate potential. By facilitating neuromuscular transmission, these proteins resemble structurally homologous dendrotoxins from mamba (Dendroaspis spp.) venoms, which are blockers of voltage-dependent K+ channels at the presynaptic site of the neuromuscular junction. What is the mechanism behind facilitation of neuromuscular transmission by VaaChi has not been established yet, however, blocking of K+ channels does not seem to be the most probable option.

Evidence for Many Unique Solution Structures for Chymotrypsin Inhibitor 2: A Thermodynamic Perspective Derived from vT-ESI-IMS-MS Measurements.

Chymotrypsin inhibitor 2 (CI-2) is a classic model for two-state cooperative protein folding and is one of the most extensively studied systems. Alan Fersht, a pioneer in the field of structural biology, has studied the wild-type (wt) and over 100 mutant forms of CI-2 with traditional analytical and biochemical techniques. Here, we examine wt CI-2 and three mutant forms (A16G, K11A, L32A) to demonstrate the utility of variable-temperature (vT) electrospray ionization (ESI) paired with ion mobility spectrometry (IMS) and mass spectrometry (MS) to map the free energy folding landscape. As the solution temperature is increased, the abundance of each of the six ESI charge states for wt CI-2 and each mutant is found to vary independently. These results require that at least six unique types of CI-2 solution conformers are present. Ion mobility analysis reveals that within each charge state there are additional conformers having distinct solution temperature profiles. A model of the data at ∼30 different temperatures for all four systems suggests the presence of 41 unique CI-2 solution conformations. A thermodynamic analysis of this system yields values of ΔCp as well as ΔG, ΔH, and ΔS for each state at every temperature studied. Detailed energy landscapes derived from these data provide a rare glimpse into Anfinsen's thermodynamic hypothesis and the process of thermal denaturation, normally thought of as a cooperative two-state transition involving the native state and unstructured denatured species. Specifically, as the temperature is varied, the entropies and enthalpies of different conformers undergo dramatic changes in magnitude and relative order to maintain the delicate balance associated with equilibrium.

Fasciola hepatica serine protease inhibitor family (serpins): Purposely crafted for regulating host proteases.

Serine protease inhibitors (serpins) regulate proteolytic events within diverse biological processes, including digestion, coagulation, inflammation and immune responses. The presence of serpins in Fasciola hepatica excretory-secretory products indicates that the parasite exploits these to regulate proteases encountered during its development within vertebrate hosts. Interrogation of the F. hepatica genome identified a multi-gene serpin family of seven members that has expanded by gene duplication and divergence to create an array of inhibitors with distinct specificities. We investigated the molecular properties and functions of two representatives, FhSrp1 and FhSrp2, highly expressed in the invasive newly excysted juvenile (NEJ). Consistent with marked differences in the reactive centre loop (RCL) that executes inhibitor-protease complexing, the two recombinant F. hepatica serpins displayed distinct inhibitory profiles against an array of mammalian serine proteases. In particular, rFhSrp1 efficiently inhibited kallikrein (Ki = 40 nM) whilst rFhSrp2 was a highly potent inhibitor of chymotrypsin (Ki = 0.07 nM). FhSrp1 and FhSrp2 are both expressed on the NEJ surface, predominantly around the oral and ventral suckers, suggesting that these inhibitors protect the parasites from the harmful proteolytic effects of host proteases, such as chymotrypsin, during invasion. Furthermore, the unusual inhibition of kallikrein suggests that rFhSrp1 modulates host responses such as inflammation and vascular permeability by interfering with the kallikrein-kinin system. A vaccine combination of rFhSrp1 and rFhSrp2 formulated in the adjuvant Montanide ISA 206VG elicited modest but non-significant protection against a challenge infection in a rat model, but did induce some protection against liver pathogenesis when compared to a control group and a group vaccinated with two well-studied vaccine candidates, F. hepatica cathepsin L2 and L3. This work highlights the importance of F. hepatica serpins to regulate host responses that enables parasite survival during infection and, coupled with the vaccine data, encourages future vaccine trials in ruminants.

Global analysis of protein stability by temperature and chemical denaturation

The stability of a protein is a fundamental property that determines under which conditions, the protein is functional. Equilibrium unfolding with denaturants requires preparation of several samples and only provides the free energy of folding when performed at a single temperature. The typical sample requirement is around 0.5–1 mg of protein. If the stability of many proteins or protein variants needs to be determined, substantial protein production may be needed. Here we have determined the stability of acyl-coenzyme A binding protein at pH 5.3 and chymotrypsin inhibitor 2 at pH 3 and pH 6.25 by combined temperature and denaturant unfolding. We used a setup where tryptophan fluorescence is measured in quartz capillaries where only 10 μl is needed. Temperature unfolding of a series of 15 samples at increasing denaturant concentrations provided accurate and precise thermodynamic parameters. We find that the number of samples may be further reduced and less than 10 μg of protein in total are needed for reliable stability measurements. For assessment of stability of protein purified in small scale e.g. in micro plate format, our method will be highly applicable. The routine for fitting the experimental data is made available as a python notebook.

Partial Characterization of Protease Inhibitors of Ulva ohnoi and Their Effect on Digestive Proteases of Marine Fish

This piece of research evaluates the presence of protease inhibitors in the macroalga Ulva ohnoi and provides an initial overview of their mode of action. The ability of Ulva protease inhibitors to inhibit digestive proteases of three marine fish species, as well as their capacity to hamper the hydrolysis of a reference protein by those fish proteases, were assessed. In addition, thermal stability and the mode of inhibition on trypsin and chymotrypsin were also studied. Dose-response inhibition curves and in vitro protein hydrolysis assays revealed a noticeable inhibition of fish enzymes when Ulva concentration increased in the assay. The thermal treatment of Ulva reduced markedly the inhibitory effect on fish digestive protease. Finally, Lineweaver–Burk plots indicated that trypsin and chymotrypsin inhibition consisted of a mixed-type inhibition mechanism in which the inhibitory effect depends on Ulva concentration. Overall, the results confirmed the presence of protease inhibitors in Ulva, though heat treatment was enough for inactivating these compounds.

Phytochemical analysis, antimicrobial, antioxidant and enzyme inhibitory activities of ethanolic extract ofCentaureasolstitialis L. and its different fractions

Resistance to conventional antimicrobial regimes is one of the issues of concern in healthcare and it drives the need for development of new antimicrobial agents. Medicinal plants, as rich source of biochemical and bioactive compounds, serve as potential source for new drugs. Here, we evaluated the ethanolic extract of Centaureasolstitialis L.and its different fractions (n-hexane, choloroform and n-butanol soluble fraction) for antimicrobial, antioxidant and enzymes inhibitory activities. Antibacterial activity against two gram-positive and three gram-negative bacteria species were determined by using agar well diffusion and 96-wells microplate methods. Similarly, antifungal activity against two fungal strains was also evaluated by agar well diffusion method. Antioxidant activity analyzed by measuring the scavenging activity of DPPH radicals and acetylcholinesterase, butrylcholinesterase and chymotrypsin inhibitory activity was determined at 10 µg/mL and 1.0 mg/mL concentrations. Results revealed that the ethanolic extract of C. solstitialis and its different fractions possesses significant ( p &lt;0.05) antibacterial activity and effective against fungi Aspergillus niger and Macrophominaphaseolina . Significant ( p &lt;0.05) DPPH scavenging activity (88.52±0.23%) among all fractions was noted. n-butanol fraction showed significant acetyl-cholinesterase (78.55±0.76%) and butrylcholinesterase inhibitory activity (78.1±0.41%) with IC 50 values of 54.6±0.39 µg/mL and 211.9±0.15 µg/mL, respectively. Maximum chymotrypsin inhibition activity was shown by crude ethanolic extract (87.76±1.17) with IC 50 valueof 38.23±0.75 µg/mL. It is concluded that C. solstitialis extract and its fractions possess significant antimicrobial, antioxidant and enzyme inhibitory activity

Thermodynamics, Kinetics and Mechanisms of Noncompetitive Allosteric Inhibition of Chymotrypsin by Dihydrolipoic Acid-Coated Gold Nanoclusters.

Enzymes are an important class of biomacromolecules which catalyze many metabolic processes in living systems. Nanomaterials can be synthesized with tailored sizes as well as desired surface modifications, thus acting as promising enzyme regulators. Fluorescent gold nanoclusters (AuNCs) are a representative class of ultrasmall nanoparticles (USNPs) with sizes of ∼2 nm, smaller than most of proteins including enzymes. In this work, we chose α-chymotrypsin (ChT) and AuNCs as the model system. Activity assays and inhibition kinetics studies showed that dihydrolipoic acid (DHLA)-coated AuNCs (DHLA-AuNCs) had a high inhibitory potency (IC50 = 3.4 μM) and high inhibitory efficacy (>80%) on ChT activity through noncompetitive inhibition mechanism. In distinct contrast, glutathione (GSH)-coated AuNCs (GSH-AuNCs) had no significant inhibition effects. Fluorescence spectroscopy, agarose gel electrophoresis and circular dichroism (CD) spectroscopy were conducted to explore the underlying mechanisms. A two-step interaction model was proposed. First, both DHLA-AuNCs and GSH-AuNCs might be bound to the positively charged sites of ChT through electrostatic forces. Second, further hydrophobic interactions occurred between three tyrosine residues of ChT and the hydrophobic carbon chain of DHLA, leading to a significant structural change thus to deactivate ChT on the allosteric site. On the contrary, no such interactions occurred with GSH of zwitterionic characteristic, which explained no inhibitory effect of GSH-AuNCs on ChT. To the best of our knowledge, this is the first example of the allosteric inhibition of ChT by nano regulators. These findings provide a fundamental basis for the design and development of nano regulators.

Cooking Effect on the Bioactive Compounds, Texture, and Color Properties of Cold-Extruded Rice/Bean-Based Pasta Supplemented with Whole Carob Fruit.

Pasta is considered as the ideal vehicle for fortification; thus, different formulations of gluten-free pasta have been developed (rice 0–100%, bean 0–100%, and carob fruit 0% or 10%). In this article, the content of individual inositol phosphates, soluble sugars and α-galactosides, protease inhibitors, lectin, phenolic composition, color, and texture were determined in uncooked and cooked pasta. The highest total inositol phosphates and protease inhibitors contents were found in the samples with a higher bean percentage. After cooking, the content of total inositol phosphates ranged from 2.12 to 7.97 mg/g (phytic acid or inositol hexaphosphate (IP6) was the major isoform found); the protease inhibitor activities showed values up to 12.12 trypsin inhibitor (TIU)/mg and 16.62 chymotrypsin inhibitor (CIU)/mg, whereas the competitive enzyme-linked immunosorbent assay (ELISA) showed the elimination of lectins. Considering the different α-galactosides analyzed, their content was reduced up to 70% (p < 0.05) by the cooking process. The total phenols content was reduced around 17–48% after cooking. The cooked samples fortified with 10% carob fruit resulted in darker fettuccine with good firmness and hardness and higher antioxidant activity, sucrose, and total phenols content than the corresponding counterparts without this flour. All of the experimental fettuccine can be considered as functional and healthy pasta mainly due to their bioactive compound content, compared to the commercial rice pasta.