Aspects of physical and chemical alterations to proteins during food processing - some implications for nutrition.

In this paper, we give an overview of our research exploring the impact of physical and chemical processing on food proteins. There are three themes, applied to the proteins of wheat, soya, egg and dairy foods. Firstly, the impact of the Maillard reaction on food proteins is discussed, with a particular focus on how the reactions might be harnessed to manipulate food texture. Secondly, the potential of enzymatic protein-protein crosslinking is considered, especially the enzyme transglutaminase. Thirdly, the broader question of how the aggregation of proteins within a food is altered by chemical and physical modification and how, in turn, this might impact on the overall nutritional quality of the food is considered.

Evaluation of protease resistance and toxicity of amyloid-like food fibrils from whey, soy, kidney bean, and egg white.

The structural properties of amyloid fibrils combined with their highly functional surface chemistry make them an attractive new food ingredient, for example as highly effective gelling agents. However, the toxic role of amyloid fibrils in disease may cause some concern about their food safety because it has not been established unequivocally if consumption of food fibrils poses a health risk to consumers. Here we present a study of amyloid-like fibrils from whey, kidney bean, soy bean, and egg white to partially address this concern. Fibrils showed varied resistance to proteolytic digestion in vitro by either Proteinase K, pepsin or pancreatin. The toxicity of mature fibrils was measured in vitro and compared to native protein, early-stage-fibrillar protein, and sonicated fibrils in two immortalised human cancer cell lines, Caco-2 and Hec-1a. There was no reduction in the viability of either Caco-2 or Hec-1a cells after treatment with a fibril concentration of up to 0.25 mg/mL.

Crystallin Nanofibrils: A Functionalizable Nanoscaffold with Broad Applications Manufactured from Waste.

Protein nanofibrils self-assembled from crystallin proteins, a waste material from the fishing industry, have been identified as a suitable material to be used as a bionanoscaffold. In this study, a successful method for the functionalization of crystallin protein nanofibrils by immobilizing several enzymes of industrial relevance (glucose oxidase, ß-galactosidase, pectinase, α-amylase, and laccase) through a glutaraldehyde cross-linking approach is reported. The extent of functionalization is evaluated by using gel electrophoresis, transmission electron microscopy, and thermostability studies. The functionalized fibrils are investigated further for reusability studies-the use of protein nanofibrils as nanoscaffolds results in a significant increase in enzyme thermostability and reusability relative to the free enzyme in solution under the same conditions. Finally, as an example and proof of concept, the use of the developed functionalization method in a biosensor platform for glucose and lactose detection is shown, by utilizing the crystallin protein nanofibrils as nanoscaffolds.

Stability and cytotoxicity of crystallin amyloid nanofibrils.

Previous work has identified crystallin proteins extracted from fish eye lenses as a cheap and readily available source for the self-assembly of amyloid nanofibrils. However, before exploring potential applications, the biophysical aspects and safety of this bionanomaterial need to be assessed so as to ensure that it can be effectively and safely used. In this study, crude crystallin amyloid fibrils are shown to be stable across a wide pH range, in a number of industrially relevant solvents, at both low and high temperatures, and in the presence of proteases. Crystallin nanofibrils were compared to well characterised insulin and whey protein fibrils using Thioflavin T assays and TEM imaging. Cell cytotoxicity assays suggest no adverse impact of both mature and fragmented crystallin fibrils on cell viability of Hec-1a endometrial cells. An IR microspectroscopy study supports long-term structural integrity of crystallin nanofibrils.

Amyloid fibrils as functionalizable components of nanocomposite materials.

Amyloid fibrils are a form of protein nanofiber that show promise as components of multifunctional bionanomaterials. In this work, native bovine insulin and bovine insulin that had been previously converted into amyloid fibrils were combined with poly(vinyl alcohol) (PVOH) via solution casting to determine the effect of fibrillization on the thermomechanical properties of the resulting composite. The synthesis method was found to preserve the amyloid fibril structure and properties of the resulting fibril-PVOH composite were investigated. At a filling level of 0.6 wt %, the fibril-reinforced PVOH was 15% stiffer than the PVOH control. Various properties of the films, including the glass transition temperature, degradation temperature, microstructure, and film morphology were characterized. Although more work is required to optimize the properties of the composites, this study provides proof of principle that incorporation of amyloid fibrils into a polymeric material can impart useful changes to the mechanical and morphological properties of the films.

Controlling the dimensions of amyloid fibrils: toward homogenous components for bionanotechnology.

Amyloid fibrils have been recognized as having potential in a variety of bionanotechnological applications. However, realization of these applications is constrained by a lack of control over morphology and alignment, both crucial for potential end uses. This article focuses on the use of growth and storage conditions to control the length of amyloid fibrils formed from bovine insulin, with length distributions constructed from transmission electron microscopy (TEM) images. Growth temperature, pH, protein concentration, and storage conditions were examined and were seen to offer a range of conditions that favor different length distribution. The use of amyloid fibrils as nanowires is one area where control of fibril dimensions is desirable, for experimental setup and endpoint applications. The conductive properties of fibrils formed from bovine insulin are presented, with these insulin fibrils being shown to have high resistivity in their unmodified state, with current values in the nanoamp range. These low current values can be increased via modification, or the fibrils used in their native state in applications where low current values are desirable. These findings, coupled with the ability to predict and select for various insulin amyloid fibril dimensions, enhances their utility as nanomaterials.

Polymorphism and higher order structures of protein nanofibers from crude mixtures of fish lens crystallins: toward useful materials.

Protein nanofibers are emerging as useful biological nanomaterials for a number of applications, but to realize these applications requires a cheap and readily available source of fibril-forming protein material. We have identified fish lens crystallins as a feedstock for the production of protein nanofibers and report optimized methods for their production. Altering the conditions of formation leads to individual protein nanofibers assembling into much larger structures. The ability to control the morphology and form higher order structures is a crucial step in bottom up assembly of bionanomaterials. Cell toxicity assays suggest no adverse impact of these structures on mammalian cell proliferation. There are many possible applications for protein nanofibers; here we illustrate their potential as templates for nanowire formation, with a simple gold plating process.

Amyloid fibril formation from crude protein mixtures.

Amyloid fibrils have potential as bionanomaterials. A bottleneck in their commercial use is the cost of the highly purified protein typically needed as a starting material. Thus, an understanding of the role of heterogeneity in the mixtures from which amyloid fibrils are formed may inform production of these structures from readily available impure starting materials. Insulin, a very well understood amyloid-forming protein, was modified by various reagents to explore whether amyloid fibrils could still form from a heterogeneous mixture of insulin derivatives. Aggregates were characterized by thioflavin T fluorescence and transmission electron microscopy. Using acetylation, reduction carboxymethylation, reduction pyridylethylation, trypsin digestion and chymotrypsin digestion, it was shown that amyloid fibrils can form from heterogeneous mixtures of modified insulin. The modifications changed both the rate of reaction and the yield of the final product, but led to fibrillar structures, some with interesting morphologies. Well defined, long, unbranched fibrils were observed in the crude reduced carboxymethylated insulin mixture and the crude reduced pyridylethylated insulin revealed the formation of "wavy" fibrils, compared with the straighter native insulin amyloid fibrils. Although trypsin digestion inhibited fibrils formation, chymotrypsin digestion of insulin produced a mixture of long and short fibrils under the same conditions. We conclude that amyloid fibrils may be successfully formed from heterogeneous mixtures and, further, that chemical modification may provide a simple means of manipulating protein fibril assembly for use in bionanotechnological applications, enabling some design of overall morphology in the bottom-up assembly of higher order protein structures from amyloid fibrils.

Two new irreversible inhibitors of dihydrodipicolinate synthase: diethyl (E,E)-4-oxo-2,5-heptadienedioate and diethyl (E)-4-oxo-2-heptenedioate.

Dihydrodipicolinate synthase (DHDPS) is a key enzyme in lysine biosynthesis and an important antibiotic target. The enzyme catalyses the condensation of (S)-aspartate semialdehyde (ASA) and pyruvate to form dihydrodipicolinate. Two new irreversible inhibitors of dihydrodipicolinate synthase are reported, designed to mimic the acyclic enzyme-bound condensation product of ASA and pyruvate. These compounds represent an important new lead in the design of potent inhibitors for this enzyme.