Enzymatic cross-linking of β-casein and its impact on digestibility and allergenicity

Protein modification via enzymatic cross-linking is an attractive way for altering food structure so as to create products with increased quality and nutritional value. These modifications are expected to affect not only the structure and physico-chemical properties of proteins but also their physiological characteristics, such as digestibility in the GI-tract and allergenicity. Protein cross-linking enzymes such as transglutaminases are currently commercially available, but also other types of cross-linking enzymes are being explored intensively. In this study, enzymatic cross-linking of β-casein, the most abundant bovine milk protein, was studied. Enzymatic cross-linking reactions were performed by fungal Trichoderma reesei tyrosinase (TrTyr) and the performance of the enzyme was compared to that of transglutaminase from Streptoverticillium mobaraense (Tgase). Enzymatic cross-linking reactions were followed by different analytical techniques, such as size exclusion chromatography -Ultra violet/Visible multi angle light scattering (SEC-UV/Vis-MALLS), phosphorus nuclear magnetic resonance spectroscopy (31P-NMR), atomic force (AFM) and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF MS). The research results showed that in both cases cross-linking of β-casein resulted in the formation of high molecular mass (MM ca. 1 350 kg mol-1), disk-shaped nanoparticles when the highest enzyme dosage and longest incubation times were used. According to SEC-UV/Vis-MALLS data, commercial β-casein was cross-linked almost completely when TrTyr and Tgase were used as cross-linking enzymes. In the case of TrTyr, high degree of cross-linking was confirmed by 31P-NMR where it was shown that 91 % of the tyrosine side-chains were involved in the cross-linking. The impact of enzymatic cross-linking of β-casein on in vitro digestibility by pepsin was followed by various analytical techniques. The research results demonstrated that enzymatically cross-linked β-casein was stable under the acidic conditions present in the stomach. Furthermore, it was found that cross-linked β-casein was more resistant to pepsin digestion when compared to that of non modified β-casein. The effects of enzymatic cross-linking of β-casein on allergenicity were also studied by different biochemical test methods. On the basis of the research results, enzymatic cross-linking decreased allergenicity of native β-casein by 14 % when cross-linked by TrTyr and by 6 % after treatment by Tgase. It can be concluded that in addition to the basic understanding of the reaction mechanism of TrTyr on protein matrix, the research results obtained in this study can have high impact on various applications like food, cosmetic, medical, textile and packing sectors.

Architecture of Physalis Mottle Tymovirus as Probed by Monoclonal Antibodies and Cross-Linking Studies

Physalis mottle tymovirus (previously named belladonna mottle virus, Iowa strain) RNA was cross-linked to its coat protein by exposure of the intact virus to ultraviolet light. The site of cross-linking of the coat protein with the RNA was identified as Lys-10 by sequencing the oligonucleotide-linked tryptic peptide obtained upon HPLC separation subsequent to enzymetic digestion of the cross-linked and dissociated virus. Three monoclonal antibodies PA3B2, PB5G9, and PF12C9, obtained using denatured coat protein as antigen, cross-reacted effectively with the intact virus indicating that the epitopes recognized by these monoclonals are on the surface of the virus. Using the peptides generated by digestion with CNBr, clostripain, V-8 protease, or trypsin and a recombinant protein lacking the N-terminal 21 residues expressed from a cDNA clone, it was shown that PA3B2 recognizes the sequence 22-36 on the coat protein while PB5G9 and PF12C9 recognize region 75-110. These results suggest that Lys-10 is one of the specific sites through which the RNA interacts in the intact virus. The polypeptide segment (region 22-36) following this buried portion as well as the epitope within the region 75-110 are exposed in the intact virus. These observations are consistent with the canonical β-barrel structure observed in certain other plant viruses.

Characterization of the DNA-binding domain of beta protein, a component of phage lambda Red-pathway by UV catalyzed cross-linking

beta protein, a key component of Red-pathway of phage lambda is necessary for its growth and general genetic recombination in recombination-deficient mutants of Escherichia coli. To facilitate studies on structure-function relationships, we overexpressed beta protein and purified it to homogeneity. A chemical cross-linking reagent, glutaraldehyde, was used to stabilize the physical association of beta protein in solution. A 67-kDa band, corresponding to homodimer, was identified after separation by SDS-polyacrylamide gel electrophoresis. Stoichiometric measurements indicated a site-size of 1 monomer of beta protein/5 nucleotide residues. Electrophoretic gel mobility shift assays suggested that beta protein formed stable nucleoprotein complexes with 36-mer, but not with 27- or 17-mer DNA. Interestingly, the interaction of beta protein with DNA and the stability of nucleoprotein complexes was dependent on the presence of MgCl2, and the binding was abolished by 250 mM NaCl. The K-d of beta protein binding to 36-mer DNA was on the order of 1.8 x 10(-6) M. Photochemical cross-linking of native beta protein or its fragments, generated by chymotrypsin, to 36-mer DNA was performed to identify its DNA-binding domain. Characterization of the cross-linked peptide disclosed that amino acids required for DNA-binding specificity resided within a 20-kDa peptide at the N-terminal end. These findings provide a basis for further understanding oi the structure and function of beta protein.

Azide and Alkyne Terminated Polybutadiene Binders: Synthesis, Cross-linking, and Propellant Studies

In composite solid propellants, the fuel and oxidizer are held together by a polymer binder. Among the different types of polymeric binders used in solid propellants, hydroxyl terminated polybutadiene (HTPB) is considered as the most versatile. HTPB is conventionally cured using isocyanates to form polyurethanes. However, the incompatibility of isocyanates with energetic oxidizers such as ammonium dinitramide and hydrazinium nitroformate, the short pot life of the propellant slurry, and undesirable side reactions with moisture are limiting factors which adversely affect the mechanical properties of HTPB based propellant. With an aim of resolving these problems, HTPB was chemically transformed to azidoethoxy carbonyl amine terminated polybutadiene and propargyloxy carbonyl amine terminated polybutadiene by adopting appropriate synthesis strategies and characterizing them by spectroscopic and chromatographic techniques. This is the first report on 1,3-dipolar addition reaction involving azide and alkyne end groups for cross-linking HTPB. The blend of these two polymers underwent curing under mild temperature (60 degrees C) conditions through 1,3-dipolar cycloaddition reaction resulting in triazoletriazoline networks. The curing parameters were studied using differential scanning calorimetry. The kinetic parameter, viz., activation energy, was computed to be 107.6 kJ/mol, the preexponential factor was 2.79 x 10(12) s-(1), and the rate constant at 60 degrees C was computed to be 3.64 x 10-(5) s-(1). The cure profile at a given temperature was predicted using the kinetic parameters. Rheological studies revealed that the gel time for curing through the 1,3-dipolar addition is 280 min compared to 120 min for curing through the urethane route. The mechanical properties of the resultant cured polybutadiene network were superior to those of polyurethanes. The cured triazolinetriazole polymer network exhibited biphasic morphology with two glass transitions (T-g) at -56 and 42 degrees C in contrast to the polyurethane which exhibited a single transition at -60 degrees C. This was corroborated by associated morphological changes observed by scanning probe microscopy. The propellant processed using this binder has the advantages of improved pot life as indicated by the end of the mix viscosity which is 165 Pas as compared with 352 Pas for the polyurethane system along with a slow build- up rate. The mechanical properties of the propellant are superior to polyurethane with an improvement of 14% in tensile strength, 22% enhancement in elongation at break, and 12% in modulus.

Three-dimensional plotted hydroxyapatite scaffolds with predefined architecture: comparison of stabilization by alginate cross-linking versus sintering

Scaffolds for bone tissue engineering are essentially characterized by porous three-dimensional structures with interconnected pores to facilitate the exchange of nutrients and removal of waste products from cells, thereby promoting cell proliferation in such engineered scaffolds. Although hydroxyapatite is widely being considered for bone tissue engineering applications due to its occurrence in the natural extracellular matrix of this tissue, limited reports are available on additive manufacturing of hydroxyapatite-based materials. In this perspective, hydroxyapatite-based three-dimensional porous scaffolds with two different binders (maltodextrin and sodium alginate) were fabricated using the extrusion method of three-dimensional plotting and the results were compared in reference to the structural properties of scaffolds processed via chemical stabilization and sintering routes, respectively. With the optimal processing conditions regarding to pH and viscosity of binder-loaded hydroxyapatite pastes, scaffolds with parallelepiped porous architecture having up to 74% porosity were fabricated. Interestingly, sintering of the as-plotted hydroxyapatite-sodium alginate (cross-linked with CaCl2 solution) scaffolds led to the formation of chlorapatite (Ca9.54P5.98O23.8Cl1.60(OH)(2.74)). Both the sintered scaffolds displayed progressive deformation and delayed fracture under compressive loading, with hydroxyapatite-alginate scaffolds exhibiting a higher compressive strength (9.5 +/- 0.5MPa) than hydroxyapatite-maltodextrin scaffolds (7.0 +/- 0.6MPa). The difference in properties is explained in terms of the phase assemblage and microstructure.

Role of transglutaminase-mediated cross-linking of protein in the temperature-dependent setting of Alaska pollack surimi

During the low temperature setting of fish paste, myosin heavy chain (MHC) is polymerized to cross-linked myosin heavy chain (CMHC), which is considered to occur by the action of endogenous transglutaminase (TGase). In this study the contribution of TGase on the setting of Alaska pollack surimi at different temperatures was studied. Alaska pollack surimi was ground with 3% NaCl, 30% h2o and with or without ethylene glycol bis (β-aminoethylether) N, N, N¹,N¹- tetra acetic acid (EGTA), an inhibitor of TGase. Among the pastes without EGTA, highest TGase activity was observed at 25°C but breaking force of the gel set at 25°C was lower than that set at 30°, 35°, and 40°C. Addition of EGTA (5m mol/kg) to the paste suppressed TGase activity at all setting temperatures from 20° to 40°C. Gelation of the pastes and cross-linking of MHC on addition of EGTA were suppressed completely at 20° and 25°C, partially at 30° and 35°C, and not at all at 40°C. The findings suggested that during the setting of Alaska pollack surimi TGase mediated cross-linking of MHC was strong at around 25°C but the thermal aggregation of MHC by non-covalent bonds was strong at above 35°C. Setting of surimi at 40°C and cross-linking of its MHC did not involve TGase.

Electroactive Aniline Pentamer Cross-Linking Chitosan for Stimulation Growth of Electrically Sensitive Cells

A new kind of electroactive polymers was synthesized by using aniline pentamer (AP) cross-linking chitosan (CS) in acetic acid/DMSO/DMF solution. UV-vis and CV confirmed the electroactivity of polymers in acidic aqueous solution. The amphiphilic polymers self-assembled into 200-300 nm micelles by dialysis against deionized water from the acetic acid buffer solution. Three samples with different weight percentages of AP were used to identify the relationship between the content of AP and the differentiation of rat neuronal pheochromocytoma PC-12 cells without external stimulation.

Synthesis and characterization of photo-cross-linked hydrogels based on biodegradable polyphosphoesters and poly(ethylene glycol) copolymers

Novel biodegradable hydrogels by photo-cross-linking macromers based on polyphosphoesters and poly(ethylene glycol) (PEG) are reported. Photo-cross-linkable macromers were synthesized by ring-opening polymerization of the cyclic phosphoester monomer 2-(2-oxo-1,3,2-dioxaphospholoyloxy) ethyl methacrylate (OPEMA) using PEG as the initiator and stannous octoate as the catalyst. The macrorners were characterized by H-1 NMR, Fourier transform infrared spectroscopy, and gel permeation chromatography measurements. The content of polyphosphoester in the macromer was controlled by varying the feed ratio of OPEMA to PEG. Hydrogels were fabricated by exposing aqueous solutions of macromers with 0.05% (w/w) photoinitiator to UV light irradiation, and their swelling kinetics as well as degradation behaviors were evaluated. The results demonstrated that cross-linking density and pH values strongly affected the degradation rates. The macromers was compatible to osteoblast cells, not exhibiting significant cytotoxicity up to 0.5 mg/mL. "Live/dead" cell staining assay also demonstrated that a large majority of the osteoblast cells remained viable after encapsulation into the hydrogel constructs, showing their potential as tissue engineering scaffolds.

Advanced research work on radiation cross-linking of polymers in CIAC

Recent research carried out at the Chinese Institute of Applied Chemistry has contributed significantly to the understanding of the radiation chemistry of polymers. High energy radiation has been successfully used to cross-link fluoropolymers and polyimides. Here chain flexibility has been shown to play an important role, and T-type structures were found to exist in the cross-linked fluoropolymers. A modified Charlesby-Pinner equation, based upon the importance of chain flexibility, was developed to account for the sol-radiation dose relationship in systems of this type. An XPS method has been developed to measure the cross-linking yields in aromatic polymers and fluoropolymers, based upon the dose dependence of the aromatic shake-up peaks and the F/C ratios, respectively. Methods for radiation cross-linking degrading polymers in polymer blends have also been developed, as have methods for improving the radiation resistance of polymers through radiation cross-linking.

RADIATION CROSS-LINKING OF POLYTETRAFLUOROETHYLENE

Polytetrafluoroethylene (PTFE) is considered to be a typical radiation degradative polymer. In this work the polymer was crosslinked by means of radiation at 335 +/- 5-degrees-C under vacuum. Crosslinked PTFE showed a great improvement in high temperature

EFFECTS OF STRUCTURE MULTIPLICITY ON MECHANISM OF RADIATION CROSS-LINKING OF POLYMERS

In an attempt to explore the effects of structural multiplicity of polymers on the mechanism of radiation crosslinking, the adaptability of the Charlesby-Pinner's equation and its various modified versions are examined. It is recognized that both chemical

MECHANISM OF RADIATION CROSS-LINKING OF POLYMERS AND ITS RELATIONSHIP WITH STRUCTURAL MULTIPLICITY

In an attempt to explore the effect of structural multiplicity of polymers on the mechanism of radiation crosslinking, the adaptability of the Charlesby-Pinner's equation and its various modified versions are examined. It is recognized that both chemical and morphological multiplicity of polymer structure results in the multiplicity of crosslinking mechanism, and that any single equation can only be applicable to a certain step of the whole radiation process.

STUDIES ON RADIATION-INDUCED CROSS-LINKING OF POLYISOBUTYLENE

Polyisobutylene can be crosslinked under irradiation as to be expected by either introducing radiation sensitive groups into its macromolecules or blending with a proper radiation crosslinkable polymer.