Recent advances in the improvement of enzyme thermostability by structure modification.

Thermostability is considered to be an important parameter to measure the feasibility of enzymes for industrial applications. Generally, higher thermostability makes an enzyme more competitive and desirable in industry. However, most natural enzymes show poor thermostability, which restricts their application. Protein structure modification is a desirable method to improve enzyme properties. In recent years, tremendous progress has been achieved in protein thermostability engineering. In this review, we provide a systemic overview on the approaches of protein structure modification for the improvement of enzyme thermostability during the last decade. Structure modification approaches, including the introduction of non-covalent interactions and covalent bonds, increase of proline and/or decrease in glycine, reinforcement of subunit-subunit interactions, introduction of glycosylation sites, truncation and cyclization have been highlighted.

Modifying the Functional Properties of Egg Proteins Using Novel Processing Techniques: A Review.

Egg proteins can be used in a wide range of food products, owing to their excellent foaming, emulsifying, and gelling properties. Another important functional property is the susceptibility of egg proteins to enzymatic hydrolysis, as protein digestion is closely related to its nutritional value. These functional properties of egg proteins are likely to be changed during food processing. Conventional thermal processing can easily induce protein denaturation and aggregation and consequently reduce the functionality of egg proteins due to the presence of heat-labile proteins. Accordingly, there is interest from the food industry in seeking novel nonthermal or low-thermal techniques that sustain protein functionality. To understand how novel processing techniques, including high hydrostatic pressure, pulsed electric fields, ionizing radiation, ultraviolet light, pulsed light, ultrasound, ozone, and high pressure homogenization, affect protein functionality, this review introduces the mechanisms involved in protein structure modification and describes the structure-functionality relationships. Novel techniques differ in their mechanisms of protein structure modification and some have been shown to improve protein functionality for particular treatment conditions and product forms. Although there is considerable industrial potential for the use of novel techniques, further studies are required to make them a practical reality, as the processing of egg proteins often involves other influencing factors, such as different pH and the presence of other food additives (for example, salts, sugar, and polysaccharides).

Respiratory Syncytial Virus Vaccine Design Using Structure-Based Machine-Learning Models.

When designing live-attenuated respiratory syncytial virus (RSV) vaccine candidates, attenuating mutations can be developed through biologic selection or reverse-genetic manipulation and may include point mutations, codon and gene deletions, and genome rearrangements. Attenuation typically involves the reduction in virus replication, due to direct effects on viral structural and replicative machinery or viral factors that antagonize host defense or cause disease. However, attenuation must balance reduced replication and immunogenic antigen expression. In the present study, we explored a new approach in order to discover attenuating mutations. Specifically, we used protein structure modeling and computational methods to identify amino acid substitutions in the RSV nonstructural protein 1 (NS1) predicted to cause various levels of structural perturbation. Twelve different mutations predicted to alter the NS1 protein structure were introduced into infectious virus and analyzed in cell culture for effects on viral mRNA and protein expression, interferon and cytokine expression, and caspase activation. We found the use of structure-based machine learning to predict amino acid substitutions that reduce the thermodynamic stability of NS1 resulted in various levels of loss of NS1 function, exemplified by effects including reduced multi-cycle viral replication in cells competent for type I interferon, reduced expression of viral mRNAs and proteins, and increased interferon and apoptosis responses.

The Impact of High-Intensity Ultrasound-Assisted Extraction on the Structural and Functional Properties of Hempseed Protein Isolate (HPI).

Hempseed protein has become a promising candidate as a future alternative protein source due to its high nutritional value. In the current study, hempseed protein isolate (HPI) was obtained using ultrasonic-assisted extraction with the aim to improve the functionality of HPI via protein structure modification. The solubility of HPI could be improved twofold under 20 kHz ultrasound processing compared to conventional alkaline extraction-isoelectric point precipitation. The protein solubility was gradually enhanced as the ultrasonic power improved, whereas excessive ultrasound intensity would cause a decline in protein solubility. Ultrasonic processing was found to have beneficial effects on the other functionalities of the extracted HPI, such as emulsifying and foaming properties. This improvement can be ascribed to the physical effects of acoustic cavitation that changed the secondary and tertiary structures of the protein to enhance surface hydrophobicity and decrease the particle size of the extracted protein aggregates. In addition, more available thiols were observed in US-treated samples, which could be another reason for improved functionality. However, the results of this study also revealed that prolonged high-power ultrasound exposure may eventually have a detrimental impact on HPI functional properties due to protein aggregation. Overall, this study suggests that high intensity ultrasound can enhance the functionality of HPI, which may ultimately improve its value in HPI-based food products.

Skimmed milk structural dynamics during high hydrostatic pressure processing from in situ SAXS.

Understanding the changes in milk at a nanostructural level during high-pressure (HP) treatment can provide new insights to improve the safety and functionality of dairy products. In this study, modifications of milk nanostructure during HP were studied in situ by small-angle X-ray scattering (SAXS). Skimmed milk was pressurized to 200 or 400 MPa at 25, 40 or 60 °C and held for 5 or 10 min, and the effect of single- and double-HP treatment was also investigated. In most cases, the SAXS patterns of skimmed milk are well fitted with a three-population model: a low-q micellar feature reflecting the overall micelle size (~0.002 Å-1), a small casein cluster contribution at intermediate-q (around 0.01 Å-1) and a high-q (0.08-0.1 Å-1) population of milk protein inhomogeneities. However, at 60 °C a scattering feature of colloidal calcium phosphate (CCP) which is normally only seen with neutron scattering, was observed at 0.035 Å-1. By varying the pressure, temperature, holding and depressurization times, as well as performing cycled pressure treatment, we followed the dynamic structural changes in the skimmed milk protein structure at different length scales, which depending on the processing conditions, were irreversible or reversible within the timescales investigated. Pressure and temperature of the HP process have major effects, not only on size of casein micelles, but also on "protein inhomogeneities" within their internal structure. Under HP, increasing processing time at 200 MPa induced re-association of the micelles, however, the changes in the internal structure were more pressure-dependent than time dependent.

High hydrostatic pressure-assisted enzymatic hydrolysis improved protein digestion of flaxseed protein isolate and generation of peptides with antioxidant activity.

Exploration of innovative high hydrostatic pressure (HHP)-assisted enzymatic hydrolysis of plant based food proteins may help improve peptide yield and bioactivity of hydrolysates. In this study, we performed enzymatic hydrolysis of flaxseed proteins using trypsin under HHP (100 and 300 MPa for 5 and 10 min) to evaluate the effect of presurization on protein denaturation, degree of hydrolysis (DH), and peptide profile and bioactivity of hydrolysate. Spectrofluorimetric analyses showed that 300 MPa induced the maximum destablization of flaxseed protein structures. The same pressure level drastically improved the DH by 1.7 times as compared to that of control. Applying HHP did not modify the peptide profiles of flaxseed protein hydrolysates but their concentrations increased with severity of treatment. Similarly, peptide molecular weight distributions were affected by pressurization parameters, increasing mainly the relative abundance of 500-1500 Da peptides. Finally, pressurization at 300 MPa for 5 and 10 min improved the antioxidant activity of flaxseed protein hydrolysates by 39 and 55%, respectively, compared to the control.

The effect of high pressure on the functional properties of pork myofibrillar proteins.

Complementary methodologies were used to analyse the pressure-induced modification and functionality of myofibrillar proteins from pork meat pressurised at 200, 400, 600, or 800 MPa (10 min, 5 or 20 °C). Pressure at 400 MPa was found to be the threshold for loss of solubility, and the structural proteins, myosin and actin, lost their native solubility due to aggregation. The results from the extraction of proteins with different reagents targeting the disruption of specific molecular interactions suggested that pressure-induced aggregation was caused mainly by hydrogen bonding during pressurisation and not hydrophobic interactions nor disulphide cross-links. Furthermore, the soluble proteins were exposed to remarkable structural changes already at 200 MPa and lost their native functionality. The modification of the proteins in pressurised meat affected the water binding sites of the myofibrillar proteins and, thereby, the interactions between proteins and water molecules, and distribution between myofibrillar and extra-myofibrillar compartments.