Euphorbia tirucalli β-Amyrin Synthase: Critical Roles of Steric Sizes at Val483 and Met729 and the CH–π Interaction between Val483 and Trp534 for Catalytic Action

The functions of Val483, Trp534, and Met729 in Euphorbia tirucalli β-amyrin synthase were revealed by comparing the enzyme activities of site-directed mutants against that of the wild type. The Gly and Ala variants with a smaller bulk size at position 483 predominantly afforded monocyclic camelliol C, which suggested that the orientation of the (3S)-2,3-oxidosqualene substrate was not appropriately arranged in the reaction cavity as a result of the decreased bulk size, leading to failure of its normal folding into the chair–chair–chair–boat–boat conformation. The Ile variant, with a somewhat larger bulk, afforded β-amyrin as the dominant product. Intriguingly, various variants of Trp534 exhibited significantly decreased enzymatic activities and provided no aberrantly cyclized products, although the aromatic Phe and Tyr residues were incorporated and the steric sizes of the aliphatic residues were altered. Therefore, the Trp534 residue does not stabilize the transient cation through a cation–π interaction. Furthermore, the Trp residue, with the largest steric bulk among all natural amino acids, is essential for high enzymatic activity. Robust CH–π complexation between the Val483 and Trp534 residues is proposed herein. Altering the steric bulk at the Met729 position afforded the pentacyclic skeletons. Thus, Met729 is positioned at the E-ring formation site. More detailed insights into the functions of the Val483, Trp534, and Met729 residues are provided by homology modeling.     

Polymeric protein formation during pasta-making with barley and semolina mixtures,and prediction of its effect on cooking behaviour and acceptability

Spaghetti were produced in a pilot plant from semolina and semolina blended with increasing amounts of barley flour. According to size exclusion high-performance liquid chromatography (SE-HPLC), barley proteins interact with semolina proteins during pasta making, forming polymers of high molecular weight. Of these, the unextractable polymeric proteins (UPP) were at significantly higher concentrations than in spaghetti made from semolina. The decrease of both S–S bonds and –SH free groups in barley semolina spaghetti, with respect to that made of semolina, suggested that polymerisation among the different classes of proteins involves a new bonding arrangement. Due to β-glucan hydrophilicity and competition with starch for water, the replacement of increasing amounts of semolina with barley flour was able to increase the optimal cooking time. The sensory properties of composite spaghetti were judged as better than the control because of the higher firmness and the lower bulkiness and stickiness of the former.     

Extraction,Composition and Functional Properties of Pennycress (Thlaspi arvense L.) Press Cake Protein

This study compared two methods for extracting the protein in pennycress (Thlaspi arvense L.) press cake and determined the composition and functional properties of the protein products. Proteins in pennycress press cake were extracted by using the conventional alkali‐solubilization–acid‐precipitation (AP) method or saline‐based (SE) procedure (0.1 M NaCl at 50 °C). The extraction method has a major influence on the purity and functional properties of press cake protein products. AP had a lower protein yield (23 %) but much higher purity (90 % crude protein) compared with SE (45 % yield, 67 % crude protein). AP protein isolate had high foam capacity (120 ml), high foam stability (96 % foam volume retention) and high emulsion stability (24–35 min), and it was resistant to heat denaturation (3 % loss of solubility at pH 2 and pH 10). On the other hand, SE protein concentrate showed remarkably high solubility (>76 %) between pH 2 and 10 and exceptional emulsifying activity (226–412 m²/g protein), but was more susceptible to heat denaturation at pH 7 and pH 10 (65–78 % loss of solubility). These results strongly demonstrate that higher purity pennycress press cake protein can be produced by either saline extraction or acid precipitation and have functional properties that are desirable for non‐food uses.     

Thermal markers arising from changes in the protein component of milk

Classification of heat load applied to milk requires the detection of parameters appropriately related to the intensity of the heat treatment. Current analytical methods based on heat-induced changes in the protein component of milk have been directed either to determine the amount of protein-derived products arised from heat treatments or to evaluate the extent of thermal denaturation of milk proteins. Lately, a new analytical strategy has been developed according to the occurrence of three major whey proteins, namely bovine serum albumin (BSA), beta-lactoglobulin (βlg) and alfa-lactalbumin (αla), normally soluble at pH 4.6 in raw milk, in the pH 4.6 insoluble protein fraction recovered from heat-treated milk. The results have shown that pH 4.6 insoluble BSA, βlg and αla, as detected by ELISA in milk, can be regarded as thermal markers suited for either dairy process control or regulation purposes.     

Dynamic mechanical analysis of the multiple glass transitions of plasticized wheat gluten biopolymer

Glass transition of thermo‐molded biomaterials made from wheat gluten and its main protein classes is studied by dynamic mechanical analysis (DMA). The materials are plasticized with variable contents of glycerol (30–40 wt %) and water (0–20 wt %). For all materials, three successive relaxation phases are systematically detected. Their positions shift to lower temperature as the plasticizer content of materials increases. Composition in gluten, glycerol and water of each relaxation phase is estimated using the Couchman‐Karasz model. Irrespective of the plasticizer content or composition, the relaxation phases shows rather constant plasticizer volume fractions. The low‐, middle‐, and high relaxation phases include respectively around 30, 60 and 80 vol % of gluten protein. These relaxations are assigned to the segmental motion of the surface amino‐acid side groups, to the collective motion of packed gluten proteins, and to the gain in protein conformational mobility as a 2D network of interacting plasticizer molecules forms. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016 , 133, 43254.     

Effects of pH and storage time on the adhesive and rheological properties of cottonseed meal‐based products

To increase the basic knowledge of cottonseed meal (CSM)‐based adhesives and optimize the operational parameters for practical applications, in this study, we investigated the effects of pH and storage time on the adhesive performance, water resistance, and rheological properties of CSM, water‐washed cottonseed meal (WCSM), and cottonseed protein isolate (CSPI). We found all products possessed the highest dry, wet, and soaked adhesive strengths with the adhesive slurries prepared at pH 6.0. The effects of pH were smaller on WCSM than on CSM and CSPI slurries. Storage time (up to 8 days) did not greatly impact the adhesive performance of WCSM slurries prepared at pH 6.0, 7.5, and 9.0, but slightly reduced the adhesive strength of CSPI slurries with the same pH. The viscosity of WCSM slurries increased with storage over 8 days, but did not for CSPI slurries. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016 , 133, 43637.     

Application of the dry‐spinning method to produce poly(ε‐caprolactone) fibers containing bovine serum albumin laden gelatin nanoparticles

We designed and manufactured a polymeric system with combined hydrophilic–hydrophobic properties by loading gelatin nanoparticles (GNPs) containing bovine serum albumin (BSA) into poly(ε‐caprolactone) (PCL) fibers. Our ultimate goal was to create a device capable of carrying and releasing protein drugs. Such a system could find several biomedical applications, such as those in controlled release systems, surgical sutures, and bioactive scaffolds for tissue engineering. A two‐step desolvation method was used to produce GNPs, whereas PCL fibers were produced by a dry‐spinning method. The morphological, mechanical, and thermal properties of the produced system were investigated, and the distribution of nanoparticles both inside and on the surface of the fibers was examined. The effect of the particles on the biodegradability of the fibers was also evaluated. In vitro preliminary tests were performed to study the release of BSA from nanoparticle‐laden fibers and to compare this with its release from free nanoparticles. Our results indicate that the distribution of particles inside the fibers was quite homogeneous and only a few of them were present on the surface. The presence of the particles in the fibers did not affect the thermal properties of the PCL polymer matrix, although it created voids that affected the degradation characteristics so the PCL fibers favored faster erosion compared to the plain fibers. Preliminary results indicate that the release from GNP‐laden fibers occurred much more slowly compared to that in the free GNPs. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016 , 133, 44233.     

Charged groups synergically enhance protein imprinting in amphoteric polyacrylamide cryogels

Proteins are amphoteric biopolymers with unevenly charged exterior surfaces. Taking this point fully into account could accomplish ingenious recognition systems for the biological macromolecues. Molecularly imprinted polymers (MIPs) are good tools to study the interactions between polymeric matrices and template molecules. Here different protein imprinted cryogels were prepared. Imprinting factors (IFs) were determined with bovine serum albumin (BSA) as the template. The IF of the polymeric cryogel made from only acrylamide (AM) and N,N′‐methylenebisacrylamide (BisAM) is about 1.38. The introduction of charged monomers, either acrylic acid or diallylamine, would increase IFs obviously. One of the basic cryogels gave the maximum IF (about 2.0) of that type. As both acrylic acid and diallylamine were involved, IFs were further increased. An amphoteric cryogel with a suitable acid‐base ratio gave a high IF of about 3.7. Whatever used alone or both, too many added acidic or basic monomers resulted in IF reduction. Taking full advantage of charged groups in MIPs could be a good way to manipulate protein–polymer interactions. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016 , 133, 43851.     

Formulation of a novel soybean protein‐based wood adhesive with desired water resistance and technological applicability

A novel soybean protein‐based wood adhesive with good bond strength, excellent water resistance, and the desired technological applicability was formulated by combining thermal alkali degradation, thermal acid treatment, and crosslinking. The characterization results indicated that thermal alkali degradation could effectively improve the technological applicability, thermal acid treatment could positively improve the water resistance, and appropriate crosslinking modification could significantly enhance the bond strength and water resistance of the soybean protein adhesive. The crosslinker species, crosslinker level, and ratio of thermal alkali‐degraded soybean protein (DSP) to thermal acid‐treated soybean protein (TSP) had important effects on the primary properties of the soybean protein adhesives. The modified polyamide aqueous solution was the most preferable crosslinker because of its low viscosity, good crosslinking efficiency, and excellent miscibility with soybean protein solution. The optimal soybean protein adhesive that was formulated from 20 wt % modified polyamide as the crosslinker and a DSP/TSP ratio of 1:3 had a solid content of more than 35 wt %, suitable viscosity (～2180 mPa s), a long work life (>16 h), good dry bond strength (2.94 MPa), and 28 h of boiling–dry–boiling cycled wet strength (1.29 MPa) that met the required values for structural use according to JIS K6806‐2003 commercial standards. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016 , 133, 43586.     

Isoprenoid Biosynthesis Inhibitors Targeting Bacterial Cell Growth

We synthesized potential inhibitors of farnesyl diphosphate synthase (FPPS), undecaprenyl diphosphate synthase (UPPS), or undecaprenyl diphosphate phosphatase (UPPP), and tested them in bacterial cell growth and enzyme inhibition assays. The most active compounds were found to be bisphosphonates with electron‐withdrawing aryl‐alkyl side chains which inhibited the growth of Gram‐negative bacteria (Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa) at ～1–4 μg mL^?1 levels. They were found to be potent inhibitors of FPPS; cell growth was partially “rescued” by the addition of farnesol or overexpression of FPPS, and there was synergistic activity with known isoprenoid biosynthesis pathway inhibitors. Lipophilic hydroxyalkyl phosphonic acids inhibited UPPS and UPPP at micromolar levels; they were active (～2–6 μg mL^?1) against Gram‐positive but not Gram‐negative organisms, and again exhibited synergistic activity with cell wall biosynthesis inhibitors, but only indifferent effects with other inhibitors. The results are of interest because they describe novel inhibitors of FPPS, UPPS, and UPPP with cell growth inhibitory activities as low as ～1–2 μg mL^?1.