Sodium Dodecyl Sulfate-polyacrylamide Gel Research Articles

Inhibition of fluorescent advanced glycation end-products by ferulic acid and chlorogenic acid: A fluorescence spectroscopy and molecular docking study

The effects of ferulic acid (FA) and chlorogenic acid (CGA) on fluorescent advanced glycation end-products (fAGEs) were investigated through the Maillard reaction. Model systems were incubated with different concentrations (0, 10, 20, 40, 80, and 160 μg/mL) of FA or CGA at 55 °C for 12 h, and fluorescence intensity of advanced glycation end-products (AGEs) was measured. After adding 160 μg/mL FA and CGA, the fluorescence intensity of AGEs was suppressed by 15.988% ± 0.556% and 16.544% ± 0.275%, respectively (P < 0.05). Compared with control group, free amino groups levels were the highest in model systems containing 160 μg/mL FA (0.096 ± 0.011 mg l-leucine equivalent/mL) and CGA (0.130 ± 0.000 mg l-leucine equivalent/mL) (P < 0.05). Carbonyls content decreased in a dose-dependent fashion with increasing concentrations of FA and CGA (P < 0.05). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results demonstrated that FA and CGA could impede the generation of bovine serum albumin (BSA)-glucose cross-linking substances. Spectroscopy results indicated that the binding constant of BSA-CGA was higher than that of BSA-FA. Meanwhile, molecular docking results suggested that the binding affinity of CGA to BSA was greater than to FA. These results suggested that CGA had a stronger inhibitory effect on the fluorescence intensity of AGEs. Therefore, FA and CGA may be potent natural antioxidants in mitigating the accumulation of AGEs during food thermal processing.

Proteolysis of dry-cured ham according to the type and temperature of starter mixed with Debaryomyces hansenii and Penicillium nalgiovense

This study analyzed the impact of yeast and mold on the proteolysis of dry-cured ham protein.Debaryomyces hansenii, isolated from Doenjang (DJ) and fermented sausage (FS), was used as a starter in the preparation. The experiment involved three repetitions, using 10 gluteus medius muscles each in the 2, 4, and 6-week aging periods. It assessed the following parameters: aging yield, moisture and crude protein content, pH, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) profile, non-protein nitrogen (NPN) content, shear force, and in-vitro digestibility. SDS-PAGE analysis reve protein aled that proteolysis occurred as the aging period increased, and the treatment groups DJ25, FS25, and DF25 displayed thicker bands at 25 kDa than the treatment groups DJ20, FS20, and DF20. The NPN tended to increase as the aging period increased, and treatment groups consistently exhibited higher NPN levels than the control at the same aging temperature. The shear force DJ25, FS25, and DF25 tended to have lower shear force values than the other treatments in the final week. The in-vitro digestion profile showed an increased digestion rate of pepsin and trypsin as the aging period increased. DJ25, FS25, and DF25 exhibited significantly higher pepsin and trypsin digestion rates than DJ20, FS20, and DF20 (P < 0.05). In conclusion, the findings indicate that aging dry-cured ham at 25 °C yields higher proteolysis, lower shear force, and higher digestion rates, suggesting that such a process would result in enhanced consumer preference and superior product quality.

A specific fluorescence resonance energy quenching–based biosensor for measuring thrombin activity in whole blood

BackgroundAt sites of vessel injury, thrombin acts as the central mediator of coagulation by catalyzing fibrin clot formation and platelet activation. Thrombin generation is most frequently measured in plasma samples using small-molecule substrates; however, these have low specificity for thrombin and limited utility in whole blood. Plasma assays are limited because they ignore the hemostatic contributions of blood cells and require anticoagulation and the addition of supraphysiological concentrations of calcium. ObjectivesTo overcome these limitations, we designed and characterized a fluorescence resonance energy quenching–based thrombin sensor (FTS) protein. MethodsThe fluorescence resonance energy quenching pair of mAmetrine and tTomato, separated by a thrombin recognition sequence, was developed. The protein was expressed using Escherichia coli, and purity was assessed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The cleavage of FTS was monitored by fluorescence using excitation at 406 nm and emission at 526 nm and 581 nm. ResultsCompared with small-molecule substrates, the FTS demonstrated high specificity for thrombin; it is not cleaved by thrombin or inhibited by α2-macroglobulin and interacts with thrombin’s anion-binding exosite I. The FTS can effectively measure thrombin generation in plasma and in finger-prick whole blood, which allows it to be developed into a point-of-care test of thrombin generation. The FTS does not inhibit standard thrombin-generation assays. Lastly, FTS-based thrombin generation in nonanticoagulated finger-prick blood was delayed but enhanced compared with that in citrated plasma. ConclusionThe FTS will broaden our understanding of thrombin generation in ways that are not attainable with current methods.

Purification, Characterization, cDNA Cloning, and Bioinformatic Analysis of Zinc-Binding Protein from Magallana hongkongensis.

Oysters contain significant amounts of the zinc element, which may also be found in their proteins. In this study, a novel zinc-binding protein was purified from the mantle of the oyster Magallana hongkongensis using two kinds of gel filtration chromatograms. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that its molecular weight was approximately 36 kDa. The protein identified by the Q-Exactive mass spectrometer shared the highest sequence identity with carbonic anhydrase derived from Crassostrea gigas concerning amino acid sequence similarity. Based on homologous cloning and RACE PCR, the full-length cDNA of carbonic anhydrase from Magallana hongkongensis (designated as MhCA) was cloned and sequenced. The cDNA of MhCA encodes a 315-amino-acid protein with 89.74% homology to carbonic anhydrase derived from Crassostrea gigas. Molecular docking revealed that the two zinc ions primarily form coordination bonds with histidine residues in the MhCA protein. These results strongly suggest that MhCA is a novel zinc-binding protein in Magallana hongkongensis.

Role of pectin in the delivery of β-carotene embedded in interpenetrating emulsion-filled gels made with soy protein isolate

This study investigated the effects of different matrices on gel properties, lipid digestibility, β-carotene bioaccessibility, released free amino acids and gel network degradation. Microstructure studies have proven that sugar beet pectin/soy protein isolate-based emulsion-filled gel (SBP/SPI-E) with interpenetrating networks was formed. SBP/SPI-E exhibited higher hardness (2.67 N, p < 0.05) and released lesser free amino acids (269.48-μmol/g SPI) than soy protein isolate-based emulsion-filled gel (SPI-E) in simulated intestinal fluid (SIF); however, both had similar free amino acids contents in simulated colonic fluid. SBP has the potential to delay gel network degradation in SIF, as evidenced by the sugar stain strips of SDS-PAGE and microstructure observation. Furthermore, SBP/SPI-E and SPI-E exhibited similar β-carotene bioaccessibility in SIF, suggesting that SBP from composite gel could not affect the aforementioned bioaccessibility. The study provides useful information for the design of functional gels in the application of fat-soluble nutrient delivery.

Characterization and emulsion stability of soybean protein isolate/soybean peptide and ginseng polysaccharide conjugates

This study investigated glycosylation products via the Maillard reaction involving soy protein isolate (SPI)/soy peptide (SP) and ginseng polysaccharide (GP), examining their potential to enhance emulsion stability. SP-GP exhibited the highest grafting degree (55.69 ± 1.48%) in a notably brief reaction time (35 min) compared to SPI-GP (45 min). The amino acid compositions, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) imaging, scanning electron microscopy imaging, and circular dichroism spectroscopy verified the formation of SPI-GP/SP-GP conjugates. SDS-PAGE showed that SPI-GP and SP-GP formed new bands, with molecular weights exceeding 254 and 45 kDa, respectively. The highest grafting degree in SP-GP resulted in the largest proportion of random coil (10.97 ± 0.82%). SP-GP achieved the highest emulsifying activity (31.26 ± 0.68 m2/g) at pH 9. Notably, using SP-GP as an emulsifier resulted in the most homogeneous emulsion with the smallest particle size (2.10 ± 0.02 μm), smallest span (1.32 ± 0.02), and highest absolute Zeta potential (50.20 ± 0.87 mV). Its creaming index remained at 0.00% for 21 days. SP-GP's inclusion curbed oxidation reactions and significantly amplified the overall emulsion stability. Consequently, the remarkable attributes of SP-GP position it as a highly promising emulsifier for employment in emulsion preparation.

Effects of Ultrasonic Power on the Structure and Rheological Properties of Skin Collagen from Albacore (Thunnus alalunga).

The effects of ultrasonic power (0, 150, 300, 450, and 600 W) on the extraction yield and the structure and rheological properties of pepsin-soluble collagen (PSC) from albacore skin were investigated. Compared with the conventional pepsin extraction method, ultrasonic treatment (UPSC) significantly increased the extraction yield of collagen from albacore skin, with a maximum increase of 8.56%. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that peptides of low molecular weight were produced when the ultrasonic power exceeded 300 W. Meanwhile, secondary structure, tertiary structure, and X-ray diffraction analyses showed that the original triple helix structure of collagen was intact after the ultrasonic treatment. The collagen solutions extracted under different ultrasonic powers had significant effects on the dynamic frequency sweep, but a steady shear test suggested that the collagen extracted at 150 W had the best viscosity. These results indicate that an ultrasonic power between 150 and 300 W can improve not only the extraction yield of natural collagen, but also the rheological properties of the collagen solution without compromising the triple helix structure.

Ultrasound-Mediated Technique for Facilitating Collagen Recovery from Yellowfin (Thunnus albacares) Skin: Insight into the Structure, Functional and Antioxidant Specification

ABSTRACT The highest yield (34%) and imino acid (18.7%) of collagen were obtained from yellowfin tuna (Thunnus albacares) skin using 20 min ultrasound. The collagen was identified as type I collagen according to the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and its structure remained intact, proved by ultraviolet spectroscopy (UV) and fourier transform infrared spectroscopy (FTIR). Collagen solubility was superior in acidic and low salt environments. Ultrasound cavitation for 20 min improved collagens’ emulsifying (EAI: 78.8 m2/g and ESI: 110.3 min) and foam properties (FA: 151.8% and FS: 133.3%) and water holding capacity (51.9%) by enhancing internal hydrophobic interactions and surface hydrophobicity. 20 min ultrasound resulted in desirable DPPH (61.1%) and ABTS (53.1%) radical scavenging and ferric-reducing antioxidant power (0.078 ABS).

A protocol for cloning, expression, and purification of Lysine exporter (LysE) gene of Mycobacterium tuberculosis

Background Tuberculosis (TB) is among the deadliest diseases and a significant cause of illnessacross the globe. Several studies on mycobacterial proteins, such as proteases and transporters that are essential for survival and pathogenesis have aimed to develop an efficient anti-tubercular agent. In mycobacterium, lysine exporter (LysE) is an amino acid transporter and a probable target for an anti-tubercular agent as it is responsible for bacterial growth inhibition and is also absent in the widely used Bacillus Calmette-Guérin (BCG) vaccine. Methods Some studies have purified LysE using different protocols. This study describes a protocol for purifying different constructs of LysE, focusing on its hydrophobic region using immobilized metal affinity chromatography (IMAC) after expressing LysE gene in a bacterial expression system. pET vector (pET28a) is used as an expression vector. Amplified LysE gene is ligated with the pET28a vector, and the resultant plasmid is then transformed into E. coli cells. The vector has a histidine tag that makes the purification process convenient. After IMAC, the samples will be subjected to size-exclusion chromatography for further purification. Results Cloning and amplification findings will be analyzed using 1% agarose gel, and protein expression and purification outcomes will be examined using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Domain-specific constructs of LysE can be further analyzed as an anti-tubercular agent. Conclusions Despite being a potential anti-tubercular target, research is quite limited on this protein. Therefore, we aim to purify LysE protein for further analysis. Similar protocols have already been implemented to purify several other bacterial proteins with &gt;95% purity.

Construction of a g-C3N4/Bi(OH)3 Heterojunction for the Enhancement of Visible Light Photocatalytic Antibacterial Activity.

Photocatalytic technology has been recently conducted to remove microbial contamination due to its unique features of nontoxic by-products, low cost, negligible microbial resistance and broad-spectrum elimination capacity. Herein, a novel two dimensional (2D) g-C3N4/Bi(OH)3 (CNB) heterojunction was fabricated byincorporating Bi(OH)3 (BOH) nanoparticles with g-C3N4 (CN) nanosheets. This CNB heterojunction exhibited high photocatalytic antibacterial efficiency (99.3%) against Escherichia coli (E. coli) under visible light irradiation, which was 4.3 and 3.4 times that of BOH (23.0%) and CN (28.0%), respectively. The increase in specific surface area, ultra-thin layered structure, construction of a heterojunction and enhancement of visible light absorption were conducive to facilitating the separation and transfer of photoinduced charge carriers. Live/dead cell staining, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) assays and scanning electron microscopy (SEM) have been implemented to investigate the damage to the cell membrane and the leakage of the intracellular protein in the photocatalytic antibacterial process. The e-, h+ and O2•- were the active species involved in this process. This study proposed an appropriate photocatalyst for efficient treatment of bacterial contamination.

Extraction, Purification, and Characterization of Olive (Olea europaea L., cv. Chemlal) Polyphenol Oxidase.

Among fruits susceptible to enzymatic browning, olive polyphenol oxidase (OePPO) stood out as being unisolated from a natural source until this study, wherein we successfully purified and characterized the enzyme. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of heated and nonheated OePPO revealed distinct molecular weights of 35 and 54 kDa, respectively, indicative of its oligomeric nature comprising active and C-terminal subunits. OePPO displayed latency, fully activating with 5 mM SDS under optimal conditions of pH 7.5 and 15 °C. The enzyme demonstrated monophenolase activity and showcased the highest efficiency toward hydroxytyrosol. Despite its low optimal temperature, OePPO exhibited high thermal resistance, maintaining stability up to 90 °C. However, beyond this threshold, the oligomeric enzyme disassociated, yielding a denatured main subunit and C-terminal fragments. Six OePPO genes were found in the fruits. Tryptic digestion identified the enzyme as mature OePPO1 (INSDC OY733096), while mass spectrometry detected the active form mass alongside several C-terminal fragments, revealing potential cleavage sites (Gly407, Tyr408).

The RNA-binding protein Cpeb4 regulates splicing of the Id2 gene in osteoclast differentiation.

Cytoplasmic polyadenylation element-binding protein 4 (Cpeb4) is an RNA-binding protein that regulates posttranscriptional regulation, such as regulation of messengerRNA stability and translation. In the previous study, we reported that Cpeb4 localizes to nuclear bodies upon induction of osteoclast differentiation by RANKL. However, the mechanisms of the localization of Cpeb4 and osteoclastogenesis by Cpeb4 remain unknown. Here, we show that Cpeb4 localizes to the nuclear bodies by its RNA-binding ability and partially regulates normal splicing during osteoclast differentiation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresisanalysis with Phos-tag® revealed that the phosphorylation levels of Cpeb4 were already high in the RAW264.7 cells and were not altered by RANKL treatment. Immunofluorescence showed that exogenous Cpeb4 in HEK293T cells without RANKL stimulation localized to the same foci as shown in RANKL-stimulated RAW264.7 cells. Furthermore, when nuclear export was inhibited by leptomycin B treatment, Cpeb4 accumulated throughout the nucleus. Importantly, RNA recognition motif (RRM) 7 of Cpeb4 was essential for the localization. In contrast, the intrinsically disordered region, RRM1, and zinc finger domain CEBP_ZZ were not necessary for the localization. The mechanistic study showed that Cpeb4 co-localized and interacted with the splicing factors serine/arginine-rich splicing factor5 (SRSF5) and SRSF6, suggesting that Cpeb4 may be involved in the splicing reaction. RNA-sequencing analysis revealed that the expression of genes related to cell proliferation processes, such as mitotic cell cycle and regulation of cell cycle processes, was elevated in osteoclasts depleted of Cpeb4. Interestingly, the splicing pattern of the inhibitor of DNA binding 2 (Id2) gene, which suppresses osteoclast differentiation, was altered by the depletion of Cpeb4. These results provide new insight into the role of Cpeb4 as a player of normal splicing of Id2 in osteoclast differentiation.

Development of a New Affinity Gold Polymer Membrane with Immobilized Protein A.

New and highly selective stationary phases for affinity membrane chromatography have the potential to significantly enhance the efficiency and specificity of therapeutic protein purification by reduced mass transfer limitations. This work developed and compared different immobilization strategies for recombinant Protein A ligands to a gold-sputtered polymer membrane for antibody separation in terms of functionalization and immobilization success, protein load, and stability. Successful, functionalization was validated via X-ray photoelectron spectroscopy (XPS). Here, a recombinant Protein A ligand was coupled by N-hydroxysuccinimide (NHS)/N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) chemistry to carboxy-functionalized, gold-sputtered membranes. We achieved a binding capacity of up to 104 ± 17 mg of the protein ligand per gram of the gold-sputtered membrane. The developed membranes were able to successfully capture and release the monoclonal antibody (mAb) Trastuzumab, as well as antibodies from fresh frozen human blood plasma in both static and dynamic setups. Therefore, they demonstrated successful functionalization and immobilization strategies. The antibody load was tested using bicinchoninic acid (BCA), ultraviolet-visible spectroscopy (UV-vis) measurements, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The outcome is a fully functional affinity membrane that can be implemented in a variety of different antibody purification processes, eliminating the need for creating individualized strategies for modifying the surface to suit different substrates or conditions.

Effect of ultrasound combined with pineapple protease treatment on the tenderness of dried shrimp.

In order to improve the tenderness of dried shrimp products as well as to reduce the hardness of the meat during the drying process, shrimp were treated with ultrasound combined with pineapple protease and the tenderization condition was optimized by measuring the texture and shear force of dried shrimp. In addition, the sulfhydryl content, myofibril fragmentation index (MFI) and microstructure were also examined to clarify the mechanisms of shrimp tenderization. The results showed UB1 group withultrasonic power of 100 W, heating temperature of 50 °C and pineapple protease concentration of 20 U mL-1 were the optimum tenderization conditions, where shrimp showed the lowest hardness (490.76 g) and shear force (2006.35 gf). Microstructure as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis results suggested that during the tenderization process the muscle segments of shrimps were broken, degradation of myofibrillar proteins occurred, and MFI values and total sulfhydryl content increased significantly (P < 0.05) (MFI value = 193.6 and total sulfhydryl content = 93.93 mmol mg-1 protein for UB 1 group). Ultrasound combined with bromelain could be used as a simple and effective tenderization method for the production of tender dried shrimp. The best conditions were 100 W ultrasonic power, 50 °C ultrasonic temperature, and 20 U mL-1 bromelain. © 2024 Society of Chemical Industry.

The Eastern Bandy Bandy Vermicella annulata, expresses high abundance of SVMP, CRiSP and Kunitz protein families in its venom proteome

The Australian elapid snake radiation (Hydrophiinae) has evolved in the absence of competition from other advanced snakes. This has resulted in ecological specialisation in Australian elapids and the potential for venom proteomes divergent to other elapids. We characterised the venom of the Australian elapid Vermicella annulata (eastern bandy bandy). The venom was analysed using a two-dimensional fractionation process consisting of reverse-phase high-performance liquid chromatography then sodium dodecyl sulphate polyacrylamide gel electrophoresis, followed by bottom-up proteomics. Resulting peptides were matched to a species-specific transcriptome and 87% of the venom was characterised. We identified 11 toxins in the venom from six families: snake venom metalloproteinases (SVMP; 24.2%; two toxins) that are class P-III SVMPs containing a disintegrin-like domain, three-finger toxins (3FTx; 21.6%; five toxins), kunitz peptides (KUN; 19.5%; one toxin), cysteine-rich secretory proteins (CRiSP; 18%; one toxin), and phospholipase A2 (PLA2; 4%; two toxins). The venom had low toxin diversity with five protein families having one or two toxins, except for 3FTx with five different toxins. V. annulata expresses an unusual venom proteome, with high abundances of CRiSP, KUN and SVMP, which are not normally highly expressed in elapid venoms. This unusual venom composition could be an adaptation to its specialised diet. Biological significanceAlthough the Australian elapid radiation represents the most extensive speciation event of elapids on any continent, with 100 terrestrial species, the venom composition of these snakes has rarely been investigated, with only five species currently characterised. Here we provide the venom proteome of a sixth species, Vermicella annulata. The venom of this species could be particularly informative from an evolutionary perspective, as it is an extreme dietary specialist, only preying on blind snakes (Typhlopidae). We show that V. annulata expresses a highly unusual venom for an elapid, due to the high abundance of the protein families SVMP, CRiSP, and KUN, which together make up 61% of the venom. When averaged across all species, a typical elapid venom is 82% PLA2 and 3FTx. This is the second recorded instance of an Australian elapid having evolved highly divergent venom expression.

Covalent conjugation of Inca peanut albumin and polyphenols with different phenolic hydroxyl numbers through laccase catalysis to improve functional properties.

Enzymatic crosslinking is a method that can be used to modify Inca peanut albumin (IPA) using polyphenols, and provides desirable functionalities; however, the effect of polyphenol structures on conjugate properties is unclear. In this study, we selected four polyphenols with different numbers of phenolic hydroxyl groups [para-hydroxybenzoic acid (HBA), protocatechuic acid (PCA), gallic acid (GA), and epigallocatechin gallate (EGCG)] for covalent modification of IPA by enzymatic crosslinking, and explored the structure-function changes of the IPA-polyphenol conjugates. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis showed that laccase successfully promoted covalent crosslinking of IPA with polyphenols, with the order of degree of conjugation as EGCG > GA > PCA > HBA, the IPA-EGCG conjugate showed the highest polyphenol binding equivalents (98.35 g kg-1 protein), and a significant reduction in the content of free amino, sulfhydryl, and tyrosine group. The oxidation of polyphenols by laccase forms quinone or semiquinone radicals that are covalently crosslinked to the reactive groups of IPA, leading to significant changes in the secondary and tertiary structures of IPA, with spherical structures transforming into dense lamellar structures. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging ability and emulsification stability of IPA-EGCG conjugates improved by almost 6-fold and 2.7-fold, respectively, compared with those of unmodified IPA. These data suggest that the higher the number of polyphenol hydroxyl groups, the higher the degree of IPA-polyphenol conjugation; additionally, enzymatic crosslinking can significantly improve the functional properties of IPA. © 2024 Society of Chemical Industry.

Physicochemical, structural, and functional characterization of guar meal protein isolate (Cyamopsis tetragonoloba)

Guar korma and churi protein isolates were assessed for their physicochemical, nutritional, functional, structural, and digestibility properties for their application in the food industry. The water extracted protein isolate of guar korma showed a protein content of 89.7 % and a yield of 48.7 %. Water extracted protein isolate of guar korma showed an excellent protein efficiency ratio, essential amino acid/total amino acids (34.35 %), amino acid score, and protein digestibility corrected amino acid score values, suggesting the existence of high-quality proteins. Water extracted protein isolate of guar korma contains all the essential amino acids except Methionine and Cysteine, according to World Health Organization recommendations for children and adults. The protein profiling of water extracted protein isolate of guar korma was analyzed using 12 % sodium dodecyl sulfate polyacrylamide gel electrophoresis and indicated the presence of eight major protein bands in the range of 17–100 kDa. In vitro digestibility of water extracted protein isolate of guar korma showed the complete digestion of the abundant protein bands within 15 min. Further, the foaming capacity, water/oil holding capacity, and emulsifying stability of water extracted protein isolate of guar korma were comparable with soy protein isolate. Fourier Transform Infrared and Circular Dichroism spectral analysis revealed the presence of several aromatic groups and β-sheets, random coils respectively in water extracted protein isolate of guar korma. The morphological nature of the guar protein isolate was characterized by Scanning Electron Microscopy. Overall, these findings support that water extracted protein isolate of guar korma has excellent functional and nutritional properties and could be a potential alternative plant protein in food industries.

Characterization of mung bean endogenous proteases and globulins and their effects on the production of mung bean protein

Mung bean protein possesses several health benefits, and aqueous processing methods are used for its production. However, mung bean protein yields are different with different methods, which are actually different in conditions (e.g., pH, temperature, and time). Herein, liquid chromatography tandem mass spectrometry identified 28 endopeptidases and exopeptidases in mung bean protein extract, and the positions of 8S and 11S globulins on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel were confirmed in our experimental conditions. The SDS–PAGE, trichloroacetic acid–nitrogen solubility index, and free amino acid analysis revealed that (1) 8S globulins showed strong resistance to the endopeptidases (optimal at pH 5 and 50 °C) at pH 3–9, and 11S globulin exhibit strong resistance expect at pH 3–3.5; (2) the exopeptidases (optimal at pH 6 and 50 °C) preferred to liberate methionine and tryptophan. These proteases negatively affected protein yield, and short production time and low temperature were recommended.

Coprecipitated Keratin/hydroxyapatite nanocomposites assisted by microwave/ultrasound irradiation, and its cytotoxic evaluation on NIH/3T3 fibroblast cells

This work is aimed to synthesize Hydroxyapatite (Hap) and Keratin/Hydroxyapatite (KerHap) nanocomposites by coprecipitation using the simultaneous microwave/ultrasound (Mw/Us) method to confer specific textural and morphological properties to the materials using keratin (Ker) extracted from human hair. Four materials were synthesized applying different powers of Mw/Us irradiation: Hap (1100 W Mw, 40 kHz Us), KerHap A (700 W Mw, 40 kHz Us), KerHap B (900 W Mw, 40 kHz Us) and KerHap C (1100 W Mw, 40 kHz Us) at 120 °C for 10 min. The materials were characterized according to their structure (FTIR, XRD, Raman spectroscopy), morphology and size (TEM), and thermal properties (TG). Their cytotoxic effects were studied at different concentrations (25, 50, 100, 200, and 400 μg/mL) using the fibroblast cell line (NIH/3T3) by MTT assay at 24, 48, and 72 h. The Ker extract exhibited a major molecular weight band between 11–10 kDa in SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Morphology, particle, and crystallite sizes were dependent on the Mw irradiation power applied and keratin added during the synthesis. The MTT assay at 48 h demonstrated a significant increase in cell viability of 39% for Hap at 50 µg/mL, for KerHap A did not show a significant difference, while KerHap B-C showed a significant increase at 200 µg/mL of 30% in cell viability. It means that fibroblast presented better cell adhesion on Hap than on KerHap A-C composites; it may be due to keratin blocking Hap pores because of electrostatic interactions between keratin and Hap.

Purification and characterization of α-galactosidase isolated from Klebsiella pneumoniae in the human oral cavity

The enzyme α-Galactosidase (α-D-galactoside galactohydrolase [EC 3.2.1.22]) is an exoglycosidase that hydrolyzes the terminal α-galactosyl moieties of glycolipids and glycoproteins. It is ubiquitous in nature and possesses extensive applications in the food, pharma, and biotechnology industries. The present study aimed to purify α-galactosidase from Klebsiella pneumoniae, a bacterium isolated from the human oral cavity. The purification steps involved ammonium sulfate precipitation (70 %), dialysis, ion exchange chromatography using a DEAE-cellulose column, and affinity monolith chromatography. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis was used to determine the molecular weight of the purified enzyme. The kinetic constants, Michaelis constant (Km) and maximal velocity (Vmax), for this enzyme were determined by using p-nitrophenyl-α-D-galactopyranoside as substrate. The results showed that the purification fold, specific activity, and yield were 126.52, 138.58 units/mg, and 21.5 %, respectively. The SDS-PAGE showed that the molecular weight of the purified enzyme was 75 kDa. The optimum pH and temperature of the purified α-galactosidase were detected at pH 6.0 and 50 °C, respectively. The kinetic constants, Michaelis constant (Km) and maximal velocity (Vmax), for this enzyme were 4.6 mM and 769.23 U/ml, respectively. α-galactosidase from Klebsiella pneumoniae was purified and characterized. (SDS-PAGE) analysis showed that the purified enzyme appeared as single band with a molecular weight of 75 kDa.