Characterization of the Technofunctional Properties and Three-Dimensional Structure Prediction of 11S Globulins from Amaranth (Amaranthus hypochondriacus L.) Seeds.

Amaranth 11S globulins (Ah11Sn) are an excellent source of essential amino acids; however, there have been no investigations on the characterization of their techno-functional properties at different pH conditions and NaCl concentrations, which are necessary for food formulations. In this work, we report a new two-step purification method for native Ah11Sn with purity levels of ~95%. LC-MS/MS analysis revealed the presence of three different Ah11Sn paralogs named Ah11SB, A11SC, and Ah11SHMW, and their structures were predicted with Alphafold2. We carried out an experimental evaluation of Ah11Sn surface hydrophobicity, solubility, emulsifying properties, and assembly capacity to provide an alternative application of these proteins in food formulations. Ah11Sn showed good surface hydrophobicity, solubility, and emulsifying properties at pH values of 2 and 3. However, the emulsions became unstable at 60 min. The assembly capacity of Ah11Sn evaluated by DLS analysis showed mainly the trimeric assembly (~150-170 kDa). This information is beneficial to exploit and utilize Ah11Sn rationally in food systems.

Development of a rapid, high-sensitivity, low-cost fluorescence method for protein surface hydrophobicity determination using a Nanodrop fluorospectrometer.

A microvolumetric method for surface hydrophobicity (H0) determination of proteins using a Nanodrop fluorospectrometer was developed. This method reduces the protein and fluorophore quantities that are necessary for sample preparations and readings by two and three orders of magnitude, respectively, compared to conventional methods. In addition, readings can be obtained in just 2-6 s. Bovine serum albumin (BSA) and 1-anilino 8-naphthalene sulfonic acid (ANS) were used for the first optimization of appropriate fluorophore-protein conditions for H0 determination (20 µM ANS, 0.5-4 µM BSA, pH 5). Based on validation guidelines, the novel method shows linear behavior, good intraday precision, accuracy, and sensitivity. This method was robust against several factors, as determined by a Youden-Steiner test. Additional surface hydrophobicity determinations using several proteins demonstrate suitable method applicability. The present microvolumetric method provides a reliable technique to determine the H0 of proteins for pharmaceutical, biotechnological, and food applications.

Lactobacillus plantarum WCFS1 ß-Fructosidase: Evidence for an Open Funnel-Like Channel Through the Catalytic Domain with Importance for the Substrate Selectivity.

ß-Fructosidase, a glycoside hydrolase of a biotechnologically important strain, was studied for its biochemical, physicochemical, and three-dimensional structure characteristics. This enzyme was heterologously expressed in Escherichia coli as a C-terminal His-tagged protein (SacB). ß-Fructosidase catalyzes the cleavage of glycoside bonds toward certain carbohydrates with ß-fructofuranosyl linkages; however, SacB exhibited selectivity toward sucrose and an optimum activity at pH 6.0-6.5 and 37 °C. In such optimum enzymatic activity conditions, the SacB was commonly observed as a monodisperse protein by dynamic light scattering (DLS). As ß-fructosidase belongs to glycoside hydrolase family 32 (GH32), a ß-sandwich and a five-bladed ß-propeller domain are typical predicted folds in its structure. Docking and molecular dynamic simulations revealed for the first time a funnel-like channel perfectly exposed in the ß-propeller domain of the Lactobacillus plantarum ß-fructosidase (this allows the interaction between its entire catalytic triad and substrates that are larger than sucrose). In contrast, SacB showed a closed central tunnel collaterally induced by its His-tag.

Identification and Functional Characterization of a Fructooligosaccharides-Forming Enzyme from Aspergillus aculeatus.

Although fructosyltransferases from Aspergillus aculeatus have received a considerable interest for the prebiotics industry, their amino acid sequences and structural features remain unknown. This study sequenced and characterized a fructosyltransferase from A. aculeatus (AcFT) isolated by heat treatment of Pectinex Ultra SP-L. The AcFT enzyme showed two isoforms, low-glycosylated AcFT1 and high-glycosylated AcFT2 forms, with similar optimum activity at 60 °C. The purified heat-resistant AcFT1 and AcFT2 isoforms produced identical patterns of fructooligosaccharides (FOS; kestose, nystose and fructosylnystose) with a notable transfructosylation capability (~90 % transferase/hydrolase ratio). In contrast, the pI and optimum pH values exhibited discrete differences, attributable to their glycosylation pattern. Partial protein sequencing showed that AcFT enzyme corresponds to Aspac1_37092, a putative 654-residue fructosyltransferase encoded in the genome of A. aculeatus ATCC16872. A homology model of AcFT also revealed the typical fold common to members of the glycoside hydrolase family 32 (GH32), with an N-terminal five-blade ß-propeller domain enclosing catalytic residues D60, D191, and E292, linked to a C-terminal ß-sandwich domain. To our knowledge, this is the first report describing the amino acid sequence and structural features of a heat-resistant FOS-forming enzyme from A. aculeatus, providing insights into its potential applications in the prebiotics industry.

Crystal structure of a major seed storage protein, 11S proglobulin, from Amaranthus hypochondriacus: insight into its physico-chemical properties.

Amaranth is a crop known for its high quality proteins. 11S Globulin is one of the most abundant and important storage proteins of the amaranth grain. Here, we report the crystal structure of amaranth 11S proglobulin at a final resolution of 2.28 Å. It belonged to the space group P6(3) with cell dimensions a=b=96.6, c=75.0 Å. It contains one asymmetric unit consisting of 372 residues and 100 water molecules. Disordered regions in the model approximately correspond to the variable regions of the 11S globulins. The structure has an extended α-helix and ß-barrel domains at both N-terminal and C-terminal regions, which are characteristic of the 11S and 7S globulins. The three dimensional structure suggests that its high thermal stability is due to the cumulative effects of many factors and its good emulsifying property depended on the balance between its surface hydrophobicity and hydrophilicity.

Proteomic analysis of differentially accumulated proteins during ripening and in response to 1-MCP in papaya fruit.

Papaya (Carica papaya L.) is a climacteric fruit susceptible to postharvest losses due to the ethylene-induced ripening. The inhibitor of ethylene action, 1-methylcyclopropene (1-MCP), has been used worldwide as a safe postharvest non-toxic agent, but the physiological and biochemical modifications induced by 1-MCP are not well understood. Using the 2-DE analysis, we report the changes in the protein profiles after 6 and 18 days of postharvest and the effect of the effect of 1-MCP treatment on fruits. Twenty seven protein spots showing differences in abundance during ripening were successfully identified by nano-LC-ESI/MS/MS. Some spots corresponded to the cell wall degrading enzymes related to fruit ripening; others were involved in oxidative damage protection, protein folding, and cell growth and survival that were induced by 1-MCP. This is the first proteomic report analyzing the effect of 1-MCP in papaya ripening. The present data will help to shed light on papaya fruit ripening process.

Expression, purification and preliminary crystallization of amaranth 11S proglobulin seed storage protein from Amaranthus hypochondriacus L.

11S globulin is one of the major seed storage proteins in amaranth. Recombinant protein was produced as up to approximately 80% of the total bacterial protein using Escherichia coli Rosetta-gami (DE3) containing pET21d with amaranth 11S globulin cDNA. The best expression condition was at 302 K for 20 h using LB medium containing 0.5 M NaCl. The recombinant protein was easily separated from most of the Escherichia coli proteins by precipitation with 0-40% ammonium sulfate solution. It formed aggregates at low temperature and at low salt concentrations. This behaviour may imply that it has a more hydrophobic nature than other 11S seed globulins. The crystals diffracted to 6 A resolution and belonged to space group P6(3), with unit-cell parameters a=b=97.6, c=74.8 A, gamma=120.0 degrees. One subunit of a trimer was estimated to be present in the asymmetric unit, assuming a Vsol of 41%. To obtain the complete structure solution, experiments to improve crystallization and flash-cooling conditions are in progress.

Dystrophin Dp71f associates with components of the beta1-integrin adhesion complex in PC12 cell neurites.

Dystrophin Dp71 has been implicated with cognitive impairment shown by Duchenne muscular dystrophy patients. To study Dp71 neural role, we used PC12 cell line, since these cells differentiate into sympathetic like neurons when stimulated with nerve growth factor Previously in undiferentiated PC12 cells, it was demonstrated that dystrophin Dp71f is a key component of the beta1-integrin adhesion complex that confers proper complex assembly. Since integrin based mediated adhesion is important during neuronal differentiation, it was important to know if dystrophin Dp71f was a structural component of the beta1-integrin adhesion complex in neurites of nerve growth factor stimulated PC12 cells. In the present work, by performing immunofluorescence assays, we determined the association of dystrophin Dp71f with some components of the beta1-integrin adhesion complex such as beta1-integrin subunit, talin, alpha-actinin and vinculin in neurites of nerve growth factor stimulated PC12 cells seeded onto the extracellular matrix protein laminin. The association was stronger in neural growth cones suggesting that dystrophin Dp71f is important for the function that the beta1-integrin complex has during neurite outgrowth.

Chemical and biochemical changes in prickly pears with different ripening behaviour.

Chemical and biochemical changes were studied in ripening prickly pears from three Opuntia morphospecies with different ripening behaviour: Naranjona (O. ficus-indica), Blanca Cristalina (Opuntia sp.), and Charola (O. streptacantha), of early, intermediate, and late ripening, respectively. At fullyripe stage (commercial maturity), Blanca Cristalina showed the biggest fruits, the hardest texture, and its pulp had the highest protein content. There were no significant differences among morphospecies in pH or total soluble solids in fully ripe fruits. The three species exhibited considerable levels of vitamin C, dietary fibre, and minerals such as calcium, iron, and zinc. Protein expression was analysed in pulp and skin from every species at physiological and commercial maturity. Some proteins appeared at both stages, while many others expressed differentially. This study evaluated prickly pear components important for human nutrition and health, and provided basic information on pricky pear ripening, with a view to its control and to improving shelf life.